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Dairy Science and Technology Handbook

1 Principles and Properties


Y. K Hui
EDITOR

VCH

Dairy Science and Technology Handbook


2 Product Manufacturing
Y. H. Hui
EDITOR

VCH

Dairy Science and Technology Handbook


3 Applications Science, Technology, and Engineering
Y. K Hui
EDITOR

VCH

Dr. Y. H. Hui 3006 4 4 S " Street Eureka, California 95501 U.S.A.

A NOTC TO THE READER:


This book has been electronically reproduced from digital information stored at John Wiley & Sons, Inc. We are pleased that the use of this new technology will enable us to keep works of enduring scholarly value in print as long as there is a reasonable demand for them. The content of this book is identical to previous printings.

Copyright O 1993 by Wiley-VCH, Inc. Originally published as ISBN 1 -56081 -078-5 No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, scanning or otherwise, except as permitted under Sections 107 and 108 of the 1976 United States Copyright Act, without either the prior written permission of the Publisher, or authorization through payment of the appropriate per-copy fee to the Copyright Clearance Center, 222 Rosewood Drive, Danvers, MA 01923, (978) 750-8400, fax (978) 750-4744. Requests to the Publisher for permission should be addressed to the Permissions Department, John Wiley & Sons, Inc., 605 Third Avenue, New York, NY 10158-0012. (212) 850-6011, fax (212) 850-6008, e-mail PERMREQ@WILEY.COM for ordering, call 1-800-CALL-WILEY. Printed in the United States of America. 10 9 8 7 6 5 4
Library of Congress Cataloging-in-Publication Data Dairy science and technology handbook / editor, Y.H. Hui. p. cm. Includes bibliographical references and index. ISBN 1-56081-078-5 1. Dairy processing. 2. Dairy products. I. Hui, Y. H. (Yiu H.) SF250.5.D35 1992 637dc20 92-30191

PREFACE

Although there are many professional reference books on the science and technology of processing dairy products, this 3-volume set is unique in its coverage (topics selected, emphasis, and latest development) and its authors (experts with diversified background and experience). Volume I discusses four important properties and applications of milk and dairy ingredients: chemistry and physics, analyses, sensory evaluation, and protein. Each chapter is not a comprehensive treatment of the subject, since more than one reference book has been written on each of the four disciplines. Rather, each chapter discusses the basic information in reasonable details that are supplemented by new research data and advances. This assures that each chapter contributes new information not available in many reference books already published. Volume II discusses the manufacture technology for yogurt, ice cream, cheese, and dry and concentrated dairy products. The direction of each chapter is carefully designed to provide two types of information. Each chapter details the currently accepted procedures of manufacturing the product and then explores new advances in technology and their potential impact on the processing of such products in the future. The fifth chapter in this volume discusses microbiology and associated health hazards for dairy products. The goal of this chapter is obvious, since there are so much new information on this topic in the last few years. The authors have done an excellent job in reviewing available data on this highly visible field. Volume III is unique because it covers five topics not commonly found in professional reference books for dairy manufacture: quality assurance, biotechnology, computer application, equipment and supplies, and processing plant designs. The length

of each chapter is limited by the size of the book. As a result, I assume full responsibility for any missing details since I assigned a fixed length to each chapter. The appendix to Volume I alphabetically lists products and services in the dairy industry. Under each product or service, the appendix describes the names of companies that provide those products and services. In Volume III, the appendix provides information for each company listed in Volume I. This includes contact data and the types of products and services for each company. The appendixes for Volumes I and III are not repeated in Volume II in order to assure a reasonable price for the books. As for the expertise of the authors, you are the best judge since most of them are known among scientists, technologists, and engineers in the dairy discipline. This three-volume set is a reference book and will benefit dairy professionals in government, industry, and academia. The information is useful to individuals engaged in research, manufacturing, and teaching. In general, the texts form an excellent background source for professionals who just enter the field. For expert dairy professionals, these books serve as a subject review as well as a summary of what is new. Any chapter in the three volumes can be used as a supplement material for a class teaching a specific topic in or an overview of the science and technology of processing diary products. Y.H. Hui October 1992

Contributors

Genevieve L. Christen, Department of Food Science and Technology, University of Tennessee, Knoxville, TN 37901-1071, U.S.A. H. D. Goff, Department of Food Science, University of Guelph, Guelph, Ontario NlG 2Wl, Canada A. R. Hill, Department of Food Science, University of Guelph, Guelph, Ontario NlG 2Wl, Canada Lynn V. Ogden, Department of Food Science and Nutrition, Brigham Young University, Provo, UT 84602, U.S.A. Paul Paquin, Department of Food Science and Technology, University of Laval, Quebec, Province of Quebec, GlK 7P4, Canada Olivier Robin, Department of Food Science and Technology, University of Laval, Quebec, Province of Quebec, GlK 7P4, Canada Sylvie Turgeon, Department of Food Science and Technology, University of Laval, Quebec, Province of Quebec, GlK 7P4, Canada

Contributors

Marijana Caric, Faculty of Technology, University of Novi Sad, 2100 Novi Sad, Bulevar, Yugoslavia Ramesh C. Chandan, James Ford Bell Technical Center, General Mills, Inc., 9000 Plymouth Avenue North, Minneapolis, MN 55427, U.S.A. Maribeth A. Cousin, Department of Food Science, Purdue University, Lafayette, IN 47906, U.S.A. Rafael Jimenez-Flores, Agricultural Bioprocessing Laboratory, University of Illinois, Urbana, IL 61801-4726, U.S.A. Norman J. Klipfel, Baskin-Robbins International Company, Glendale, CA, U.S.A. K. Rajinder Nath, Kraft General Foods, 801 Waukegan Road, Glenview, IL 60025, U.S.A. Khem Shahani, Department of Food Science and Technology, Food Industry Complex, University of Nebraska, Lincoln, NE 68583-0919, U.S.A. Joseph Tobias, Agricultural Bioprocessing Laboratory University of Illinois, Urbana, IL 61801-4726, U.S.A. P.C. Vasavada, Department of Animal and Food Science, University of Wisconsin, River Falls, WI 54022

Contributors

Jeffrey R. Broadbent, Department of Nutrition and Food Science, Utah State University, Logan, UT 84322-8100, U.S.A. Vance Caudill, Lockwood Greene Engineers, Inc., Spartanburg, SC 29304, U.S.A. Thomas Gilmore, Dairy and Food Industries Supply Association, 6245 Executive Boulevard Drive, Rockville, MD 20852-3938, U.S.A. Jeffrey K. Kondo, Marschall Products, Rhone-Poulenc, Inc., 601 Science Drive, Madison, WI 53711, U.S.A. Robert L. Olsen, Department of Research and Development, Schreiber Foods, Inc., Green Bay, WI 54307-9010, U.S.A. Jim Shell, Consultant, Ellicott City, MD 21043, U.S.A. John E. Stauffer, Stauffer Technology, 6 Pecksland Road, Greenwich, CT 06831, U.S.A.

Contents

Preface ............................................................................. Contributors (Volume 1.) .................................................. Contributors (Volume 2.) .................................................. Contributors (Volume 3.) .................................................. Volume 1. Principles and Properties 1. Chemistry and Physics ..............................................
1.1 1.2 Introduction ................................................................... Composition ................................................................. 1.2.1 1.2.2 1.2.3 1.2.4 1.3 1.3.1 1.3.2 1.4 1.4.1 1.4.2 1.4.3 Proteins ....................................................... Lipids ........................................................... Lactose ........................................................ Minor Components ...................................... Casein Micelles ........................................... Fat Globules ................................................ Density ........................................................ Viscosity ...................................................... Freezing Point .............................................

vii ix x xi

1:1
1:2 1:5 1:9 1:18 1:26 1:28 1:30 1:30 1:41 1:49 1:49 1:50 1:52

Structure .......................................................................

Physical Properties ......................................................

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Contents
1.4.4 1.4.5 1.4.6 1.4.7 1.4.8 1.5 1.6 1.7 Electrochemistry .......................................... Surface Tension .......................................... Acid-Base Equilibria ..................................... Heat Capacity and Thermal Conductivity ................................................. Optical Properties ........................................ 1:54 1:56 1:57 1:60 1:60 1:61 1:62 1:62

Summary ...................................................................... Future Developments ................................................... References ...................................................................

2.

Analyses ....................................................................
2.1 Introduction ................................................................... 2.1.1 2.1.2 2.1.3 2.2 2.2.1 2.2.2 2.2.3 2.2.4 2.2.5 2.3 2.3.1 2.3.2 2.3.3 2.3.4 2.3.5 2.3.6 2.3.7 Purpose of Analysis of Dairy Products ...................................................... Sources of Additional Information ................ Types of Analyses ....................................... General Comments ...................................... Sampling of Liquid Products ........................ Sampling of Dry Products ............................ Sampling of Butter ....................................... Sampling of Cheese .................................... Fat ............................................................... Total Solids .................................................. Protein ......................................................... Lactose ........................................................ Ash .............................................................. Vitamins ....................................................... Minerals .......................................................

1:83
1:85 1:85 1:86 1:86 1:86 1:86 1:87 1:88 1:88 1:88 1:89 1:89 1:96 1:98 1:99 1:101 1:101 1:102

Sampling ......................................................................

Tests for Milk Composition ...........................................

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Contents
2.4 Tests for Milk Quality .................................................... 2.4.1 2.4.2 2.4.3 2.4.4 2.4.5 2.4.6 2.4.7 2.4.8 2.5 2.5.1 2.5.2 2.5.3 2.6 2.6.1 2.6.2 2.6.3 2.6.4 2.7 Titratable Acidity .......................................... Added Water ................................................ Sediment ..................................................... Antibiotics .................................................... Acid Degree Value ....................................... Iodine and Hypochlorites ............................. Aflatoxins ..................................................... Pesticides .................................................... Cow-Side Tests ......................................... Wisconsin Mastitis Test ............................... Somatic Cell Count ...................................... Aerobic Plate Count ..................................... Coliform Count ............................................. Tests for Specific Spoilage Bacteria ............ Tests for Specific Pathogenic Bacteria .......................................................

vii
1:102 1:102 1:105 1:106 1:107 1:112 1:113 1:113 1:114 1:115 1:115 1:116 1:117 1:120 1:121 1:126 1:131 1:135 1:139 1:139 1:141 1:142 1:144 1:145 1:146 1:146

Tests for Abnormal Milk ...............................................

Microbiological Methods ..............................................

Selected Analytical Techniques for Dairy Products ....................................................................... 2.7.1 2.7.2 2.7.3 2.7.4 2.7.5 Assurance of Adequate Pasteurization .............................................. Total Solids in Butter and Cheese ................ Salt in Butter and Cheese ............................ Sorbic Acid in Cheese ................................. Overrun in Frozen Dairy Desserts ................ Sensory vs. Chemical and Microbiological Methods ..............................

2.8

Sensory Analysis .......................................................... 2.8.1

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Contents
2.9 Summary ...................................................................... 1:148 1:148 1:149 2.10 Future Developments ................................................... 2.11 References ...................................................................

3.

Sensory Evaluation of Dairy Products .......................


3.1 The Senses .................................................................. 3.1.1 3.1.2 3.1.3 3.1.4 3.1.5 3.1.6 3.2 3.2.1 3.2.2 3.2.3 3.2.4 3.3 Introduction .................................................. Taste ........................................................... Smell ........................................................... Sight ............................................................ Hearing ........................................................ Touch .......................................................... Introduction .................................................. Affective Testing .......................................... Discrimination Testing ................................. Descriptive Analysis .....................................

1:157
1:158 1:158 1:159 1:162 1:163 1:165 1:166 1:166 1:166 1:168 1:170 1:171 1:174 1:175 1:175 1:175 1:185 1:198 1:214 1:229 1:243 1:254 1:267 1:274

Sensory Evaluation Techniques ..................................

Application of Sensory Analysis to Dairy Products ....................................................................... 3.3.1 The Philosophy of Judging of Dairy Products ...................................................... Fluid Milk and Cream ................................... Cottage Cheese ........................................... Butter ........................................................... Ice Cream and Related Products ................. Cheese ........................................................ Cultured Products ........................................ Yogurt .......................................................... Dry Milk .......................................................

3.4

Descriptive Sensory Defects of Dairy Products ........... 3.4.1 3.4.2 3.4.3 3.4.4 3.4.5 3.4.6 3.4.7 3.4.8

3.5

References ...................................................................

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Contents 4. Functional Properties of Milk Proteins .......................


4.1 4.2 Introduction ................................................................... Composition and Principal Physicochemical Properties of Major Milk Proteins ................................. 4.2.1 4.2.2 4.3 Major Protein Components in Milk ............... Principal Physicochemical Properties of Milk Proteins ............................................

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1:277
1:278 1:280 1:280 1:281 1:282 1:282 1:292 1:302 1:325 1:325 1:329 1:332 1:333 1:334

Major Functional Properties of Milk Proteins ........................................................................ 4.3.1 4.3.2 4.3.3 Water-Protein Interactions ........................... Protein-Protein Interactions ......................... Protein-Surface Interactions ........................

4.4

Some Selected Processing Effects on the Functional Properties of Major Milk Proteins ............... 4.4.1 4.4.2 Effects of Heat Treatments .......................... Membrane Separation Processes ................

4.5 4.6 4.7

Conclusion .................................................................... Acknowledgments ........................................................ References ...................................................................

Appendix: Product Listing .................................................


Advertising to Instantizers/Agglomerators ............................ Instruments to X-Ray Inspection ...........................................

1:355
1:355 1:385

Volume 2. Product Manufacturing 1. Yogurt ........................................................................


1.1 1.2 Introduction ................................................................... Definition of Yogurt ....................................................... 1.2.1 Standard of Identity and Regulatory Aspects of Yogurt ........................................

2:1
2:2 2:7 2:8

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Contents
1.2.2 1.2.3 1.3 1.3.1 1.3.2 1.4 1.4.1 1.4.2 1.4.3 1.4.4 1.4.5 1.4.6 1.5 1.5.1 1.5.2 1.5.3 1.6 1.6.1 1.6.2 1.7 National Yogurt Association Criteria for Live and Active Culture Yogurt ............... Frozen Yogurt .............................................. Taxonomy of Yogurt Bacteria ...................... Production of Yogurt Starters ....................... Ingredients and Equipment .......................... Mix Preparation ........................................... Heat Treatment ............................................ Homogenization ........................................... Fermentation ............................................... Packaging .................................................... Yogurt Ingredients and Flavor, Texture, and Rheological Aspects ............... Yogurt Starter and Its Contribution to Texture and Flavor ....................................... Manufacturing Procedures ........................... Refrigerated Yogurt ..................................... Frozen Yogurt .............................................. 2:10 2:11 2:13 2:15 2:20 2:22 2:22 2:25 2:25 2:27 2:27 2:27 2:28 2:28 2:31 2:32 2:36 2:36 2:39 2:39 2:39 2:41 2:45 2:45 2:54

Yogurt Starters .............................................................

General Principles of Manufacture ..............................

Yogurt Production ........................................................

Yogurt Quality Control ..................................................

Physicochemical, Nutritional, and Health Properties of Yogurt ..................................................... 1.7.1 1.7.2 1.7.3 1.7.4 Prefermentation Changes ............................ Changes During Fermentation ..................... Postfermentation Changes .......................... Prophylactic and Therapeutic Properties ....................................................

1.8

References ...................................................................

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Contents 2. Ice Cream and Frozen Desserts ................................


2.1 Introduction ................................................................... 2.1.1 2.1.2 2.1.3 2.1.4 2.2 2.2.1 2.2.2 2.2.3 2.2.4 2.2.5 2.2.6 2.2.7 2.2.8 2.2.9 Steps in the Manufacture of Ice Cream .......................................................... Ice Cream as a "Generic" Name .................. Government Regulations ............................. Types of Frozen Desserts ............................ Sources of Dairy Products ........................... Nonconcentrated Milk Products ................... Concentrated Milk Products ......................... Perishable Concentrated Milk Products ...................................................... Dehydrated Concentrated Milk Products ...................................................... Dry Whey ..................................................... Dried Buttermilk ........................................... Other Dry Ingredients .................................. Preserved Fluid Concentrated Milk Products ......................................................

xi 2:57
2:59 2:59 2:60 2:60 2:61 2:61 2:62 2:63 2:67 2:67 2:69 2:73 2:73 2:74 2:74 2:75 2:75 2:76 2:79 2:80 2:81 2:82 2:82 2:87 2:90 2:92

Selection of Ingredient .................................................

2.2.10 Frozen Concentrated Milk Products ............. 2.2.11 Substitutes for Dairy Products ..................... 2.2.12 Sweetening Agents ...................................... 2.2.13 Sucrose ....................................................... 2.2.14 Dextrose ...................................................... 2.2.15 Corn Syrups ................................................. 2.2.16 Honey .......................................................... 2.2.17 Stabilizers .................................................... 2.2.18 The Mode of Stabilizer Action ...................... 2.2.19 Emulsifiers ................................................... 2.2.20 Miscellaneous Ingredients ...........................
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Contents
2.3 Calculations and Mix Standardization ......................... 2.3.1 2.3.2 2.3.3 2.3.4 2.3.5 2.3.6 2.3.7 2.4 Calculating MSNF in Skim Milk and Cream .......................................................... Standardization of Ice Cream Mixes the Simplest Case ........................................ The Serum Point Method of Mix Standardization ............................................ Algebraic Method of Mix Standardization ............................................ Restandardizing a Mix of Erroneous Composition ................................................. Mix Made in a Vacuum Pan ......................... Calculating Density and Degrees Baume (Be) ................................................. Premium and Superpremium Products ...................................................... The "All-Natural" Designation ...................... Formulations for a Plain (White) Ice Cream Mix ................................................... Formulations for a Chocolate Ice Cream Mix ................................................... Fruit Ice Cream ............................................ Products Containing 2 to 7% Fat ................. Products Containing 0 to 2% Fat ................. Sherbets and Ices ........................................ Direct-Draw Shakes ..................................... 2:92 2:92 2:93 2:94 2:100 2:104 2:108 2:109 2:110 2:112 2:113 2:114 2:114 2:115 2:116 2:117 2:117 2:118 2:119 2:119 2:120 2:121 2:121

Formulation .................................................................. 2.4.1 2.4.2 2.4.3 2.4.4 2.4.5 2.4.6 2.4.7 2.4.8 2.4.9

2.4.10 Frozen Yogurt .............................................. 2.4.11 Other Frozen Desserts ................................ 2.4.12 Nonstandardized Products ........................... 2.5 Mix Processing ............................................................. 2.5.1 Pasteurization ..............................................

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Contents
2.5.2 2.5.3 2.6 2.6.1 2.6.2 2.6.3 2.6.4 2.6.5 2.7 2.8 2.9 2.7.1 Homogenization ........................................... Mix Cooling and Storage .............................. Flavor Character and Intensity ..................... Quantity of Flavoring .................................... Propriety Flavorings ..................................... Vanilla Flavor ............................................... Chocolate Flavor ......................................... Amount of Water Frozen ..............................

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2:125 2:127 2:129 2:132 2:133 2:134 2:134 2:135 2:136 2:138 2:142 2:145 2:146 2:146 2:146 2:146 2:147 2:147 2:147 2:148 2:149 2:149 2:149 2:149 2:150 2:151 2:153 2:153

Flavoring of Frozen Desserts .......................................

Freezing of the Mix ....................................................... Ice Cream Hardening ................................................... Defects of Ice Cream ................................................... 2.9.1 2.9.2 2.9.3 2.9.4 2.9.5 2.9.6 2.9.7 2.9.8 2.9.9 Defects Identified by Sight ........................... Defective Container ..................................... Product Appearance .................................... Meltdown Characteristics of Ice Cream .......................................................... Defects of Texture ....................................... Defects in Body ........................................... Flavor Defects ............................................. Defects Contributed by the Dairy Ingredients ................................................... Defects Due to Mix Processing and Storage ........................................................

2.9.10 Defects Due to Flavoring Materials .............. 2.9.11 Defects Due to Sweetening Agents ............. 2.9.12 Defects Due to Storage of Ice Cream .......... 2.9.13 Defects of Frozen Dessert Novelties ............ 2.10 Plant Management ....................................................... 2.11 Active Areas of Research in Ice Cream ....................... 2.11.1 Ice Cream Mix .............................................
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Contents
2.11.2 Ice Cream Structure ..................................... 2.11.3 Processing and Freezing ............................. 2.12 References ................................................................... 2:155 2:156 2:157

3.

Cheese ......................................................................
3.1 Introduction ................................................................... 3.1.1 3.1.2 3.2 3.3 Classification ............................................... Cheese Production and Composition ...........

2:161
2:163 2:164 2:165 2:169 2:173 2:174 2:178 2:178 2:179 2:179 2:180 2:180 2:181 2:182 2:182 2:187 2:188 2:191 2:191 2:191 2:192 2:194

Heat Treatment of Milk for Cheesemaking .................. Cheese Starter Cultures .............................................. 3.3.1 3.3.2 3.3.3 3.3.4 3.3.5 3.3.6 3.3.7 3.3.8 Types of Cultures ........................................ Leuconostoc ................................................ Streptococcus salivarius subsp. Thermophilus ............................................... Lactobacilli ................................................... Lactobacilli Found During Cheese Ripening ...................................................... Propionibacteria ........................................... Pediococci ................................................... Molds ........................................................... Inhibitors of Starter Bacteria ........................ Culture Systems ..........................................

3.4 3.5 3.6

Growth of Starter Bacteria in Milk ................................ 3.4.1 3.5.1 Starter Culture Systems ............................................... Culture Production and Bulk Starter Propagation .................................................................. 3.6.1 3.6.2 3.6.3 3.6.4 History ......................................................... Concentrated Cultures ................................. Bulk Starter Propagation .............................. pH-Controlled Propagation of Cultures .......................................................

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Contents
3.6.5 3.6.6 3.7 3.7.1 3.7.2 3.7.3 3.7.4 3.7.5 3.7.6 3.7.7 3.7.8 3.8 3.9 General Comments ...................................... Helpful Points to Phage-Free Starters ......... Cheddar Cheese .......................................... Stirred Curd or Granular Cheddar Cheese ........................................................ Colby Cheese .............................................. Swiss Cheese .............................................. Parmesan Cheese ....................................... Mozzarella and Provolone Cheese .............. Brick Cheese ............................................... Mold-Ripened Cheese .................................

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Manufacture of Cheese ................................................

Cheese from Ultrafiltered Retentate ............................ Salting of Cheese ......................................................... 3.10.1 Proteolysis of Caseins ................................. 3.10.2 Proteolysis in Cheese .................................. 3.10.3 Amino Acid Transformations ........................ 3.10.4 Flavor Development .....................................

3.10 Cheese Ripening and Flavor Development .................

3.11 Microbiological and Biochemical Changes in Cheddar Cheese .......................................................... 3.11.1 Fate of Lactose ............................................ 3.11.2 Fate of Casein ............................................. 3.11.3 Microbiological Changes .............................. 3.11.4 Fate of Fat ................................................... 3.11.5 Flavor of Cheddar Cheese ........................... 3.12 Microbiological and Biochemical Changes in Swiss Cheese .............................................................. 3.12.1 Fate of Lactose ............................................ 3.12.2 CO2 Production ............................................

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3.12.3 Eye Formation ............................................. 3.12.4 Fate of Proteins ........................................... 3.12.5 Flavor of Swiss Cheese ............................... 3.13 Microbiological and Biochemical Changes in Gouda Cheese ............................................................. 3.13.1 Fate of Lactose ............................................ 3.13.2 Fate of Proteins ........................................... 3.13.3 Fate of Fat ................................................... 3.13.4 Microbiological Changes .............................. 3.13.5 Flavor of Gouda Cheese .............................. 3.14 Microbiological and Biochemical Changes in Mold-Ripened Cheese ................................................. 3.14.1 Blue Cheese ................................................ 3.14.2 Camembert and Brie Cheese ....................... 3.15 Microbiological and Biochemical Changes in Bacteria Surface-Ripened Cheese .............................. 3.15.1 Brick Cheese ............................................... 3.16 Microbiological and Biochemical Changes in Mozzarella Cheese ...................................................... 3.17 Microbiological and Biochemical Changes in Parmesan and Romano Cheese ................................. 3.18 Accelerated Cheese Ripening ..................................... 3.19 Processed Cheese Products ....................................... 3.19.1 Advantages of Process Cheeses over Natural Cheese ............................................ 3.19.2 Processing ................................................... 3.19.3 Emulsifiers ................................................... 3.19.4 Heat Treatment ............................................ 3.19.5 pH and Microbiological Stability ................... 3.20 References ................................................................... 2:221 2:222 2:222 2:222 2:223 2:223 2:224 2:224 2:224 2:224 2:224 2:226 2:227 2:227 2:227 2:228 2:229 2:229 2:231 2:231 2:231 2:234 2:234 2:235

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Contents 4. Concentrated and Dried Dairy Products ....................


4.1 4.2 History and Definitions ................................................. Unsweetened Condensed Milk .................................... 4.2.1 4.2.2 4.2.3 4.2.4 4.3 4.3.1 4.3.2 Processing Chart and Preparing Raw Milk .............................................................. Preheating and Evaporation ........................ Homogenization and Second Standardization ............................................ Packaging, Sterilization, and Storage .......... Processing Chart and Raw Milk to First Standardization .................................... Heat Treatment, Evaporation, Sugar Addition, and Second Standardization ............................................ Cooling with Crystallization ..........................

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2:258 2:259 2:259 2:259 2:265 2:266 2:267 2:267

Sweetened Condensed Milk ........................................

2:267 2:270 2:270 2:271 2:271 2:278 2:282 2:285 2:286 2:286 2:289 2:290 2:296 2:299

4.3.3 4.4 4.5

Other Concentrated Dairy Products ............................ Dried Dairy Products .................................................... 4.5.1 4.5.2 4.5.3 4.5.4 Milk Powder ................................................. Instant Milk Powder ..................................... Infant Formulas ............................................ Other Products ............................................ Whey Powder .............................................. Whey Protein Concentrates ......................... Casein Products .......................................... Lactose ........................................................

4.6

Dried Dairy Ingredients ................................................ 4.6.1 4.6.2 4.6.3 4.6.4

4.7

References ...................................................................

5.

Dairy Microbiology and Safety ...................................


5.1 Introduction ...................................................................

2:301
2:303

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5.2 General Dairy Microbiology .......................................... 5.2.1 5.2.2 5.3 Morphological Features ............................... Microorganisms Associated with Milk .......... 2:304 2:305 2:305 2:321 2:321 2:323 2:324 2:326 2:326 2:327 2:330 2:331 2:331 2:332 2:335 2:336 2:336 2:338 2:338 2:338 2:339 2:341 2:341 2:341

Growth of Dairy Microbes in Milk and Dairy Products ....................................................................... 5.3.1 5.3.2 5.3.3 Relative Growth Rates of Psychrotrophs .............................................. Sources of Psychrotrophs in Milk ................. Significance of the Presence and Growth of Psychrotrophs .............................

5.4

Inhibition and Control of Microorganisms in Milk and Dairy Products ....................................................... 5.4.1 5.4.2 5.4.3 5.4.4 5.4.5 5.4.6 5.4.7 5.4.8 5.4.9 Natural Antimicrobial Systems ..................... Lactoperoxidase .......................................... Lactoferrin ................................................... Lysozyme .................................................... Xanthine Oxidase ........................................ Lactic Acid Bacteria and Bacteriocins .......... Potassium Sorbate ...................................... Carbon Dioxide ............................................ Removal of Microorganisms by Physical Methods ........................................ Effect on Milk Composition .......................... Economic Losses ......................................... Common Mastitis Pathogens ....................... Uncommon Mastitis Pathogens ................... Factors Affecting the Incidence of Mastitis ........................................................ Detection and Diagnosis ..............................

5.5

Mastitis ......................................................................... 5.5.1 5.5.2 5.5.3 5.5.4 5.5.5 5.5.6

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Contents
5.6 Pathogenic Bacteria in Milk and Dairy Products ....................................................................... 5.6.1 5.6.2 5.6.3 5.6.4 5.6.5 5.6.6 5.6.7 5.6.8 5.7 5.7.1 5.7.2 5.7.3 5.7.4 5.8 5.8.1 5.8.2 5.8.4 5.8.5 5.9 Listeria Monocytogene ................................. Yersinia Enterocolitica ................................. Campylobacter Jejuni .................................. Escherichia Coli ........................................... Escherichia Coli 0157:H7 ............................. Bacillus Cereus ............................................ Economic Significance of Pathogens ........... Mycotoxins and Amines ............................... Presence of Mycotoxins in Milk and Dairy Products ............................................. Fate of Aflatoxin M1 in Dairy Product Manufacture and Storage ............................ Elimination of Mycotoxins ............................ Regulation of Mycotoxins in Foods .............. Terminology ................................................. Function of Starter Cultures ......................... Inhibition of Starter Cultures ........................ Genetic Engineering for Improving Starter Cultures ...........................................

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Mycotoxins in Milk and Dairy Products ........................

Microbiology of Starter Cultures ..................................

Methods for Microbiological Analysis of Milk and Dairy Products .............................................................. 5.9.1 5.9.2 5.9.3 Conventional Methods ................................. Rapid Methods and Automation in Dairy Microbiology ....................................... Microbiological Tests for Assessing Sanitation and Air Quality in Dairy Plant ............................................................

2:377

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Contents
5.9.4 Shelf-Life Tests ............................................ 2:378 2:378 2:379 2:381 2:381 2:382 2:382 2:383 2:384 2:385 2:385 2:386 2:386 2:389 2:391 2:392 5.10 Microbiology of Milk and Dairy Products ..................... 5.10.1 Pasteurized Milk and Cream ........................ 5.10.2 Dried Milk Powder ........................................ 5.10.3 Evaporated Milk ........................................... 5.10.4 Cottage Cheese ........................................... 5.10.5 Mold-Ripened Cheeses ............................... 5.10.6 Hard Cheese ............................................... 5.10.7 Yogurt and Cultured Milks ............................ 5.10.8 Butter ........................................................... 5.10.9 Ice Cream and Frozen Dairy Desserts ...................................................... 5.11 Microbiological Considerations of New Processing Technologies ............................................. 5.11.1 Ultrafiltration and Reverse Osmosis ............. 5.11.2 Ultrahigh Temperature Sterilization of Milk and Dairy Products ............................... 5.11.3 Low-Dose Irradiation of Milk ........................ 5.11.4 Microwave Processing of Milk and Dairy Products ............................................. 5.11.5 Use of Carbon Dioxide and Supercritical Carbon Dioxide for Reduction of Microbial Populations .............. 5.12 Assuring Microbiological Quality and Safety of Milk and Milk Products: HACCP Approach ................. 5.12.1 HACCP Principle ......................................... 5.12.2 Elements of the HACCP System .................. 5.13 Conclusion .................................................................... 5.14 References ...................................................................

2:392 2:393 2:394 2:394 2:395 2:395

Appendix: Food and Drug Administration, Part 135 Frozen Desserts, April 1, 1992 .................
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2:427

Contents Volume 3. Applications Science, Technology, and Engineering 1. Quality Assurance and Dairy Processing ...................
1.1 Introduction ................................................................... 1.1.1 1.1.2 1.1.3 1.2 1.2.1 1.2.2 1.2.3 1.2.4 1.2.5 1.2.6 1.2.7 1.2.8 1.3 1.3.1 1.3.2 1.3.3 1.3.4 1.3.5 1.3.6 1.4 1.4.1 1.4.2 1.4.3 1.4.4 Definition of Quality ..................................... Quality Assurance Versus Quality Control ......................................................... Organization and Management .................... Basic Concepts ............................................ Food Hazards .............................................. Critical Control Points .................................. Pasteurization .............................................. Cheese Processes ....................................... Ice Cream Processes .................................. Yogurt Processes ........................................ Butter and Milk Processes ........................... Food Additives and GRAS Substances .................................................. Unavoidable Contaminants .......................... Standards of Identity .................................... USDA Grades .............................................. Analytical Methods ....................................... Codex Alimentarius ...................................... Regulatory Requirements ............................ Sanitation .................................................... Plants and Grounds ..................................... Employee Training .......................................

xxi

3:1
3:3 3:3 3:3 3:4 3:4 3:4 3:5 3:8 3:12 3:20 3:23 3:25 3:27 3:30 3:30 3:33 3:33 3:35 3:37 3:39 3:40 3:40 3:41 3:47 3:49

Hazard Analysis and Critical Control Points ................

Product Specifications .................................................

Good Manufacturing Practice ......................................

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xxii

Contents
1.5 Product Labeling .......................................................... 1.5.1 1.5.2 1.5.3 1.5.4 1.5.5 1.5.6 1.6 1.6.1 1.6.2 1.6.3 1.6.4 1.6.5 1.7 1.7.1 1.7.2 1.7.3 1.8 1.8.1 1.8.2 1.9 1.9.1 1.9.2 1.9.3 Ingredient Labeling ...................................... Nutritional Labeling ...................................... Fortification .................................................. Imitation and Substitute Foods .................... Open Date Labeling ..................................... Kosher Certification ..................................... Functional Needs ......................................... Materials Testing ......................................... Tamper-Evident Closures ............................ Aseptic Packaging ....................................... Packaged Weight Control ............................ Shelf Life ..................................................... Warehousing and Shipping .......................... Product Recall ............................................. Importance of Process Controls ................... Need to Avoid Recontamination ................... The Promise of Biotechnology ..................... Internationalization of the Dairy Industry ........................................................ Proliferation of New Products ...................... 3:50 3:50 3:52 3:55 3:57 3:59 3:59 3:60 3:60 3:62 3:63 3:63 3:64 3:65 3:65 3:65 3:66 3:67 3:67 3:68 3:68 3:68 3:69 3:69 3:70

Packaging .....................................................................

Distribution ...................................................................

Summary ......................................................................

Future Developments ...................................................

1.10 References ...................................................................

2.

Biotechnology of Dairy Starter Cultures .....................


2.1 2.2 Introduction ................................................................... Applications and Successes ........................................ 2.2.1 Low-Fat Dairy Products ...............................

3:77
3:77 3:78 3:79

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Contents
2.2.2 2.2.3 2.2.4 2.3 Bacteriocins as Food Preservatives ............. Bacteriophage Resistance ........................... Accelerated Cheese Maturation ...................

xxiii
3:80 3:83 3:84 3:85 3:85 3:88 3:91 3:92 3:95 3:95

Yesterday and Tomorrow: Tools for Biotechnology ............................................................... 2.3.1 2.3.2 2.3.3 Conjugation and Cell Fusion ........................ Transformation and Gene Delivery Systems ....................................................... Manufacture of Heterologous Proteins .......................................................

2.4 2.5 2.6

Regulatory Aspects of Dairy Biotechnology ................ Summary ...................................................................... References ...................................................................

3.

Computer Applications: Expert Systems ....................


3.1 Introduction ................................................................... 3.1.1 3.1.2 3.2 Artificial Intelligence and Expert Systems ....................................................... Relationship to Traditional Programming ............................................... Knowledge Representation .......................... Searching and Inference Strategies .................................................... Uncertainty .................................................. Feasibility .................................................... Knowledge Acquisition ................................. Tool Selection .............................................. Preexpert System Developments .................

3:105
3:106 3:106 3:108 3:109 3:109 3:113 3:116 3:117 3:117 3:118 3:120 3:121 3:121

Knowledge-Based Architecture ................................... 3.2.1 3.2.2 3.2.3

3.3

Building Expert Systems .............................................. 3.3.1 3.3.2 3.3.3

3.4

Expert Systems and Process Control .......................... 3.4.1

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Contents
3.4.2 3.4.3 3.4.4 3.5 3.5.1 3.5.2 3.5.3 3.6 3.6.1 3.6.2 3.6.3 3.7 3.7.1 3.7.2 3.7.3 3.8 3.9 Expert System Applications ......................... Knowledge Representation in Process Control ......................................................... Commercial Examples ................................. Physical Goods Management ...................... Time Management: Planning and Scheduling ................................................... Computer Integrated Manufacturing ............ Quality Control Programs ............................. Laboratory Systems ..................................... Quality Defect Analysis ................................ Simulation .................................................... Research and Development ........................ Training ....................................................... 3:123 3:126 3:127 3:128 3:128 3:130 3:132 3:138 3:138 3:140 3:142 3:143 3:143 3:146 3:149 3:150 3:151

Business and Manufacturing Operations ....................

Quality Management Applications ...............................

Strategic Operations ....................................................

Future Trends ............................................................... References ...................................................................

4.

Dairy Equipment and Supplies ..................................


4.1 4.2 Dairy Equipment and Supplies .................................... Equipment Common to all Dairies ............................... 4.2.1 4.2.2 4.2.3 4.2.4 4.2.5 4.2.6 4.2.7 Tanks ........................................................... Heat Exchangers ......................................... Pumps ......................................................... Pipe, Valves, and Fittings ............................ Centrifuges .................................................. Homogenizers ............................................. Cleaning Dairy Processing Systems ............

3:155
3:156 3:160 3:160 3:171 3:179 3:195 3:203 3:213 3:217

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Contents
4.3 Specialty Equipment .................................................... 4.3.1 4.3.2 4.3.3 4.3.4 4.3.5 4.3.6 4.3.7 Ice Cream and Frozen Dessert Equipment ................................................... Butter Manufacture ...................................... Cheesemaking Systems .............................. Concentration and Drying ............................ Cottage Cheese and Other Cultured Products ...................................................... High-Temperature Processes ...................... Membrane Separation .................................

xxv
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5.

Engineering: Plant Design, Processing, and Packaging ..................................................................


5.1 5.2 Introduction ................................................................... Plant Construction and Arrangement .......................... 5.2.1 5.2.2 5.3 5.3.1 5.3.2 5.3.3 5.3.4 5.3.5 5.3.6 5.4 5.4.1 5.4.2 5.5 5.5.1 5.5.2 5.6 Construction Considerations ........................ Plant Layout ................................................ Dimensions and Units .................................. Fluid Flow Characteristics ............................ Heat Transfer ............................................... Principles of Homogenization ...................... Material Handling ......................................... Preventative Maintenance Program ............. Fluid Milk Packaging .................................... Aseptic Packaging ....................................... Plant and Equipment ................................... Product ........................................................

3:295
3:296 3:296 3:297 3:303 3:307 3:307 3:309 3:310 3:316 3:318 3:319 3:320 3:320 3:321 3:326 3:326 3:327 3:327

Processing Engineering ...............................................

Product Packaging .......................................................

Regulations ..................................................................

Summary ......................................................................

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xxvi

Contents
5.7 5.8 Future Developments ................................................... References ................................................................... 3:327 3:328

Appendix: Company Listing ..............................................


A & B Process Systems Corp. to FrigoTech ......................... Fristam Pumps, Inc. to Quest International .......................... Quest International Flavors, Inc. to Zurn Industries, Inc. ..............................................................

3:331
3:331 3:356 3:385

Index ................................................................................

3:409

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CHAPTER

1
Chemistry and Physics
H. D. GoffandA. R. Hill
1.1 Introduction, 2 1.2 Composition, 5 1.2.1 Proteins, 9 1.2.1.1 Caseins, 9 1.2.1.2 Whey Proteins, 14 1.2.1.3 Enzymes, 15 1.2.2 Lipids, 18 1.2.2.1 Chemical Properties, 18 1.2.2.2 Physical Properties, 19 1.2.2.3 Lipolysis, 22 1.2.2.4 Oxidation, 24 1.2.3 Lactose, 26 1.2.3.1 Biochemical Properties, 26 1.2.3.2 Physicochemical Properties, 26 1.2.4 Minor Components, 28 1.2.4.1 Vitamins, 28 1.2.4.2 Minerals, 29 1.3 Structure, 30 1.3.1 Casein Micelles, 30 1.3.1.1 Properties, 30 1.3.1.2 Stability, 35 1.3.1.3 Aggregation, 38 1.3.2 Fat Globules, 41 1.3.2.1 Native Fat Globule Membrane, 41 1.3.2.2 Recombined Membranes, 44 1.3.2.3 Stability, 46 1.3.2.4 Destabilization, 48 1.4 Physical Properties, 49 1.4.1 Density, 49 1.4.2 Viscosity, 50 1.4.3 Freezing Point, 52 1.4.4 Electrochemistry, 54 1.4.4.1 Electrical Conductivity, 54 1.4.4.2 Oxidation-Reduction Potentials, 55

1.4.5 Surface Tension, 56 1.4.6 Acid-Base Equilibria, 57 L4.7 Heat Capacity and Thermal Conductivity, 60 1.4.8 Optical Properties, 60 1.5 Summary, 61 1.6 Future Developments, 62 1.7 References, 62

1.1 Introduction
A characteristic unique to mammals is their ability to secrete milk as a source of nutrients and immunological protection for their young. Milk from domesticated species has also been recognized since prehistoric times as a food source for humans.1 Some of the properties of milk that are still under study today, such as its ability to clot with chymosin and the ability to turn milk into products such as cheese and butter, have been known to humans for centuries.2 Consequently, the applications of chemistry and physical chemistry to milk are probably among the oldest scientific disciplines and are still recognized as very important and integral parts of the field of food science. Today, the majority of milk for human consumption is secreted by the domesticated cow, genus Bos, although milk from goats, buffaloes, and sheep, in addition to human milk, is also consumed in significant quantity. Milk is defined by the United States Code of Federal Regulations as "the lacteal secretion, practically free from colostrum, obtained by the complete milking of one or more healthy cows, which contains not less than 8.25% of milk solids-not-fat and not less than 3.25% of milkfat".3 Reviews of the composition of goat's milk,4-5 ewe's milk,6 buffalo's milk,7 camel's milk,8 human milk,9 and the milk of other species 1011 are available in the literature. This chapter is limited to a discussion of cow's milk. Milk is synthesized in the mammary gland. An average cow in North America produces 5400 kg of milk in a 305-day lactation period. The components of the mammary gland at various magnifications are shown in Figure 1.1. The alveolus is the milk-producing unit within the gland. In the alveolus, a single layer of epithelial secretory cells surrounds a central storage area, the lumen, which is connected to a duct system. These secretory cells are, in turn, surrounded by a layer of myoepithelial cells and blood capillaries. The raw materials for milk production are transported via the bloodstream to the secretory cell. Within the cell, components are synthesized mainly by the endoplasmic reticulum and its attached ribosomes, which are supplied with energy from the mitochondria and then passed along to the Golgi apparatus, which is responsible for their eventual movement out of the cell. Vesicles containing many of the aqueous nonfat components are released

SECMETOW TISSUE

ONt QUARTER
LOOD VESSEL

CAMlAMES CtSTEWf

touct LARGE OUC f OUCT CAPILLARIES


VENOUS BLOOO

CONNCCTlVg TISSUE

LUMEN

MVOEPrrMEUAL CELL ARTEMAL BLOOO ALVEOLUS I1WUTUN. LMD OtKWLET MCtKMLU ^ytfrtl - S W NUCLEUS
ENDOPIASMC RETCULUM ,

LUMEN

MfTOCHQNOfOON

SECRETORY CELL

Figure 1.1 Bovine mammary gland at various magnifications. (Reprinted from ref. 12, p. 794, by courtesy of Marcel Dekker.)

by the Golgi apparatus, pass through the cytoplasm and the apical plasma membrane, and are deposited in the lumen of the alveolus. Lipid droplets, synthesized by the endoplasmic reticulum, also pass through the cytoplasm and the apical plasma membrane and are deposited in the lumen. As is discussed further in Section 1.3.2.1, it is believed that the milk fat globule membrane (FGM) is comprised of the apical plasma membrane of the secretory cell, which continually envelops lipid droplets as they pass into the lumen. The apical cell membrane is continually being replaced from endomembrane material synthesized in the endoplasmic reticulum and transported from the Golgi in the form of vesicles containing aqueous nonfat components. The vesicle membrane fuses with the apical cell membrane as the contents of the vesicle are released. Milk components stored in the lumen of the alveolus are released into the duct system as a result of hormonal stimulation. The duct systems within the mammary gland, a complex network, flow into the teat cistern from which they are milked. Further details of milk biosynthesis and mammary physiology are beyond the scope of this chapter and have been reviewed extensively elsewhere. 13 " 15 Milk is estimated to contain more than 100,000 molecular species. However, the average gross composition of milk can be simplified to 4.1% fat, 3.6% protein (75% casein protein and 25% whey protein), 4.9% lactose, and 0.7% ash, with the balance

consisting of water.16 (Details of the composition of milk are covered in Section 1.2.) Variation in milk composition can be caused by inherited characteristics (breed), physiological characteristics (stage of lactation, pregnancy, age, nutritional balance, season, and udder health), and milking procedure (within milkings and between milkings).3 Although milk is a fluid food, it has considerable structural organization (described in further detail in Section 1.3). Milk can be described as: an emulsion of milkfat globules which contain the milk lipids, fat soluble vitamins, and the components of the FGM; a colloidal suspension of casein micelles (which contain casein proteins, calcium, phosphate, citrate and water), globular proteins, and lipoprotein particles; and a solution of lactose, soluble proteins, minerals, vitamins, acids, enzymes, and other components. Milk plasma is defined as milk minus the milkfat globules, which is close in composition to separated or skim milk, although separation is never complete. Milk serum is defined as milk plasma minus casein micelles, which is close to the composition of whey, except for the presence of some proteolytic products from chymosin.16 The casein micelles and the milkfat globules are the principal structureforming constituents that form the basic structural elements of most dairy products.17'18 Dairy foods make a significant contribution to the total nutrient intake of the North American population, supplying, for example, one-fourth or more of individuals' protein, calcium, phosphorus, and riboflavin requirements. Dairy foods are an excellent source of vitamin B 12 as well as an adequate source of vitamin A, thiamine, niacin, and magnesium. Vitamin D is added to most liquid dairy products; vitamin A is added to most low-fat fluid products. Only iron, vitamin C, and folacin are present in somewhat deficient amounts.1219 The nutrient composition of whole milk is listed in Table 1.1. From a nutritional viewpoint, milk has been described as nature's most nearly perfect food, owing mainly to its biological role as the only source of nutrition for the infant mammal. Milk proteins are slightly deficient in methionine and cysteine, the sulfur amino acids. Milk lipids are slightly high in saturated fats and cholesterol and thus may have an impact on cardiovascular disease. The nutritional significance of milk proteins and lipids has recently been reviewed.19"21 A small but significant part of the population, particularly among African and Asian peoples, produce less than average intestinal /3-galactosidase. This leads to lactose intolerance, or malabsorption, which causes diarrhea, abdominal cramps, and intestinal gas if dairy products are consumed. Lactose intolerance has recently been reviewed.22 The purpose of this chapter is to serve as a reference for many of the processes and technologies described in other chapters and volumes of this set. In this chapter, we review the basics of milk composition and milk structure as they affect the utilization of milk in industrial practice and provide a comprehensive bibliography for further reading. This chapter is not designed to be a comprehensive review of

Table 1.1 NUTRIENT COMPOSITION OF WHOLE MILK (3.3% FAT)


Nutrient Protein Vitamin A Vitamin C Thiamine Riboflavin Niacin Vitamin B 6 Folacin Vitamin B 12 Calcium Phosphorus Magnesium Iron Zinc Amount in 100 g 3.29 g 31RE b 0.94 mg 0.038 mg 0.162 mg 0.85 NEC 0.042 mg 5 |xg 0.357 jig 119 mg 93 mg 13 mg 0.05 mg 0.38 mg %RDAa in 250 ml 17.2 8.9 4.2 8.2 30.0 13.9 5.4 3.2 30.7 32.0 25.0 10.2 0.9 6.5

From ref. 12, p. 822. Reprinted courtesy of Marcel Dekker. Average Recommended Dietary Allowances for all males and females above age 11. Retinol Equivalents: 1 u,g retinol or 6 u,g ^-carotene. c Niacin Equivalents: 1 mg niacin or 60 mg dietary tryptophan. Only 10% of the NE in milk corresponds to niacin.
b a

the tremendously growing fields of dairy chemistry and physics. Several very recent excellent reviews and monographs of aspects of dairy chemistry are available and recommended for those seeking more detail.16^23"28

1.2 Composition
The gross composition of milk is defined as the fat, protein, lactose, ash, and total solids content. Gross composition for large numbers of samples is determined by indirect methods calibrated against chemical methods.29 The most common chemical methods for milkfat determination are gravimetric (solvent extraction by the Mojonnier or Roese-Gottlieb procedure) or volumetric (the Babcock or Gerber procedure).30 For raw milks, the Babcock procedure produces slightly higher results (0.021% fat) than does the Mojonnier procedure and has significantly lower interand intralaboratory repeatability.30 Total protein is generally determined as Kjeldahl nitrogen multiplied by the factor 6.38. This factor is still in common use, although a more representative one is 6.34.31 It is also common to report protein as crude protein (total N X 6.38), which overestimates true protein content (protein N X 6.38) by about 4 to 8%.3 The most

Table 1.2 GROSS COMPOSITION OF MILK OF VARIOUS BREEDS, g/100 g3


Breed Holstein Ayrshire Guernsey Jersey Brown Swiss Fat 3.54 3.95 4.72 5.13 3.99 Protein 3.29 3.48 3.75 3.98 3.64 Lactose 4.68 4.60 4.71 4.83 4.94 Ash 0.72 0.72 0.76 0.77 0.74 Total Solids 12.16 12.77 14.04 14.42 13.08

common method of lactose analysis is polarimetric determination of lactose in a clarified milk extract.32 Lactose is frequently reported (especially in the older literature) as lactose monohydrate, which overestimates the amount of lactose by 5.26%.3 Total solids of milk are most frequently determined by an oven method involving initial drying on a steam bath followed by further drying in a forced air oven at 98 to 1000C,32 although a longer drying time in the oven without initial boiling off on the steam bath may be more accurate.33 Ash content is normally determined by dry ashing at about 5500C.32 Ash content is not equivalent to the total content of salts. Milk salts are discussed in Section 2.4. In the determination for payment purposes of the gross composition of producer milk, the largest source of error is bulk tank sampling error. Standard deviations associated with bulk tank sampling error of 0.01% for milk protein and 0.093% for milk fat have been reported.34 Corresponding standard deviations associated with laboratory analyses were 0.01% for both fat and protein. Milk analysis is discussed in detail in Chapter 3. Many factors affect the gross composition of milk. The factors most significant to the processing of milk and milk products are breed, feed, season, region, and herd health.35 In the short term, the only factors available to the farmer to alter milk composition are selection of breed and feed.36 The gross composition of milk of various breeds is listed in Table 1.2. Note that breeds producing high-fat milk also produce milk with lower ratios of protein to fat. This is certainly significant to multiple component pricing37"42 and suggests that genetic selection can achieve relatively rapid increases in the ratio of milk protein to fat, provided the change is achieved by lowering fat content.43 A large negative correlation between fat content and protein/fat ratio but a small correlation between protein content and protein/fat ratio have also been reported.44 Heritabilities (based on milk records of 32,000 firstlactation cows) of percent composition of milk fat, protein, and protein/fat ratio were 0.61, 0.59, and 0.58.44 The effects of feed on milk composition have been reviewed.45'46 The most important dietary factors are the amount and type of roughage, the forage/concentrate ratio, and the carbohydrate composition of the concentrates and lipids.46"49 Feeding frequency does not affect milk composition, provided the total feed intake is constant.50 The greatest effects of feeding are on the concentration of milkfat, with smaller changes in protein concentration.

Percent

Protein Fat

Jan. Feb. Mar. Apr. May June July Aug.Sept.Oct. Nov. Dec. 1988 Figure 1.2 Seasonal variation of protein and fat content of Ontario milk. Primary standard methods were Mojonnier for fat and semi-micro-Kjeldahl for protein. Protein is total nitrogen X 6.38. Data represent means of 10,000 herds tested four times each month at the Ontario Central Milk Testing Laboratory.

In the Northern Hemisphere, maximum annual fat contents occur during the winter months, usually peaking in November or December; minimum fat contents occur in August as shown in Figure 1.2.51 Seasonal trends in protein contents follow a similar trend, with some significant differences: the seasonal variation is not as great, the minimum occurs in July, and the maximum occurs in October (Fig. 1.2).51 These differences cause seasonal variation of the protein/fat ratio of milk, which is of significant economic consequence, especially to cheese manufacturing.51 Small seasonal variations in lactose content have also been reported.52 Although there is some evidence that climatic conditions affect milk composition, the principal effect of climatic factors is on milk production.53 It is likely that the observed seasonal effects on milk composition are primarily due to variations in feed and stage of lactation.3'54 Variations in feed and stage of lactation probably also account for most regional variations in milk composition. Regional variations in the Ontario, Canada, milkfat composition for the years 1978 to 1988 are shown in Figure 1.3. These data and earlier unpublished data (Ontario Central Milk Testing Laboratory, Guelph) going back to 1971 show a continual increase in average fat content of Ontario milk over time, with little or no increase in protein content. The result is a significant decrease in the protein/fat ratio of Ontario milk. There has also been a gradual increase in average lactose content of Ontario producer milks, from 4.80% lactose monohydrate (w/v) in 1970 to 5.2% (w/v) in 1988. With respect to herd health, yield and compositional effects of greatest economic

Fat

WESTERN SOUTHERN NORTHERN EASTERN CENTRAL ONTARIO 1978 1979 1980 1981 1982 1983 1984 1985 1986 1987 1988

Year
Figure 1 3 Regional and annual variation of fat content of Ontario milk. Primary standard method was Mojonnier. Data represent annual means within each region. Herds were tested four times per month.

significance are due to mastitis.55 Average yield losses due to udder infection may exceed 1 kg of milk per cow per day. 56 Somatic cell counts in excess of 300,000 indicate subclinical mastitis.57 In 1989, average somatic cell counts for all Ontario producer milks were 350,000/mL. (Ontario Central Milk Testing Laboratory, Guelph, Ontario, Canada). In the United Kingdom, the national average was 390,000/ mL. 58 Elevated somatic cell counts are correlated with reduced lactose content 52 and a corresponding increase in mineral content to maintain osmotic equilibrium. Casein content is reduced, but total protein content increases with increasing somatic cell counts due to increased whey protein content.59 Modest levels of somatic cells may affect cheese yield 60 due to increased proteolysis, 61 but effects of somatic cell counts <2,000,000 ml" 1 on cheese texture and flavor are probably more significant than yield effects. 58 Production aids may also affect milk composition. Supplementation of dairy rations with the antibiotic Flavomycin increases feed conversion efficiency, milk production, and the percent composition of both fat and protein. 62 Like other factors affecting milk composition, the effect on fat content is greater than on protein content. Numerous authors have reported minimal or no effects of bovine somatotropin (BST) on gross composition of milk. 63 " 66

1.2.1 Proteins
The nitrogen content of milk is distributed among caseins, whey proteins, and nonprotein nitrogen (NPN), excepting some minor proteins that are associated with the FGM (Section 1.2.2). Nitrogen distribution is normally determined by the classical Rowland fractionation,67 which separates caseins from whey nitrogen by precipitation at pH 4.6 and separates total proteins from whey NPN by precipitation with sodium acetate and acetic acid at pH 5.0. Based on this procedure, average milk nitrogen distribution is about 76% casein, 18% whey protein, and 6% NPN. This operational classification of proteins is still used for both research and process control. However, a classification system of milk proteins based on their amino acid sequences (Table 1.3) has been developed by the American Dairy Science Association's (ADSA) Committee on Milk Protein Nomenclature, Classification and Methodology.68 The amino acid distributions of the principal milk proteins are summarized in Table 1.4.

1.2.1.1 Caseins
The casein content of milk is about 26 g/kg, representing about 80% of milk protein. The principal casein fractions are asl-casein (10 g/kg), as2-casein (2.6 g/kg), /3-casein (9.3 g/kg), y-casein (0.8 g/kg), and /c-casein (3.3 g/kg).16 These fractions are all included in the pH 4.6 precipitate from milk, but y-caseins are now reclassified as carboxyl terminal fragments of /3-casein. The corresponding N-terminal fragments,formerly classified as proteose-peptones70are also classified as casein subfractions.68 These fractions result from cleavage of ^-casein by the milk protease, plasmin. The carboxyl terminal fragments (y-caseins) remain associated with the casein micelle and are recovered by rennet coagulation and by pH 4.6 precipitation. The N-terminal fragments are hydrophilic and appear as heat-stable fractions in both cheese whey and the pH 4.6 supernatant. Carboxyl terminal fragments correspond to /3-casein subfractions 2, 3, and 4; and the N-terminal fractions correspond to /3-casein subfractions 5 to 9, as listed in Table 1.3. The N-terminal fractions do not contain aromatic amino acids (Table 1.4) and, therefore, show no absorbency at 280 nm. The nomenclature used for the caseins consists of a Greek letter with or without a numerical subscript to identify the family of proteins; and an uppercase Latin letter to indicate the genetic variant. Post-translational modifications such as phosphorylation or formation of subfractions are indicated after the genetic variant.68 For example, the notation /3-casein B-5P (fl-105) indicates that the protein belongs to the /3-family of caseins, is the B genetic variant, contains five phosphate groups, and represents an N-terminal fragment of /3-casein B from amino acid residues 1 to 105.69 In most breeds of dairy cattle, a sl -casein is >90% variant B. Exceptions are Guernsey and Jersey cattle, which produce about 75% variant B and 25% variant C.71 The A variant of /3-casein occurs with nearly 100% frequency in most dairy breeds, excepting Jersey and Brown Swiss, which produce significant levels of /3-casein B. Significant effects of milk protein genetic variants on heat stability,72

TaWe 1.3 CLASSIFICATION AND DISTRIBUTION OF THE MILK PROTEINSGENUS BOS (30-35 g/L)69
I. Caseins (24-28 g/L) A. ctsl-Caseins (12-15 g/L) 1. asl-Casein Xa-8P (genetic variantsA, B, C, D-9P, and E) 2. ctsl-Casein Xa-9P (genetic variantsA, B, C, D-10P, and E) 3. asl-Casein fragments0 B. <xs2-Caseins (3-4 g/L) 1. ots2-Casein XMOP (genetic variantsA, B, C-9P, and D-7P) 2. as2-Casein X M l P (genetic variantsA, B, C-10P, and D-8P) 3. as2-Casein XM2P (genetic variantsA, B, C-IlP, and D-9P) 4. as2-Casein XM3P (genetic variantsA, B, C-12P, and D-10P) C. P-Caseins(9-ll g/L) 1. P-Casein Xa-5P (genetic variantsA1, A 2 , A3, B, C-4P, D-4P, and E) 2. p-Casein XMP (f 29-209) (genetic variantsA1, A2, A3, and B) 3. p-Casein Xa-(f 106-209) (genetic variantsA2, A3, and B) 4. P-Casein Xa-(f 108-209) (genetic variantsA and B) 5. p-Casein Xa-4P (f l - 2 8 ) b 6. P-Casein Xa-5P (f l-105) b 7. P-Casein Xa-5P (f l-107) b 8. p-Casein XMP (f 29-105) b 9. p-Casein XMP (f 29-107) b D. K-Caseins (2-4 g/L) 1. K-Casein XMP (genetic variantsA and B) 2. Minor K-Casein X M , -2, -3, etc. (genetic variantsA and B) Whey proteins (5-7 g/L) A. p-Lactoglobulins (2-4 g/L) 1. P-Lactoglobulins X a (genetic variantsA, B, C, D, Dr, E, F, and G) B. ct-Lactalbumins (0.6-1.7 g/L) 1. a-Lactalbumin Xa (genetic variantsA and B) 2. Minor a-Lactalbumins C. Bovine serum albumin (0.2-0.4 g/L) (Continued)

n.

renneting properties,73'74 and concentration and distribution of milk components have been reported.75-76 The potential for genetic engineering of the caseins to modify the behavior of milk during processing has been reviewed.77 The milk proteins of other species in comparison to bovine milk proteins have also been reviewed.11 Caseins are conjugated proteins with phosphate groups esterified to serine residues. The exceptions are some /3-casein fragments (Table 1.3) which contain no phosphate. Phosphate groups are important to casein association and the structure of the casein micelle (Section 1.3.3). Calcium binding by individual caseins is proportional to phosphate content.71 In addition to phosphorylation, about one-third of /c-casein monomers are glycosylated at threonine 133 (Fig. 1.4).69 Caseins contain high numbers of proline residues, which are distributed relatively uniformly throughout the polypeptide chains (Fig. 1.4). Proline gives rise to a particular bending of the protein chain and inhibits formation of an ordered, stable a-helix structure.78 Early literature suggests that caseins have little secondary struc-

Table 1.3 (Continued)


D. Immunoglobulins (0.5-1.8 g/L) 1. IgG immunoglobulins a. IgG1 immunoglobulins b. IgG2 immunoglobulins c. IgG fragments 2. IgM immunoglobulins 3. IgA immunoglobulins a. IgA immunoglobulins b. Secretory IgA immunoglobulins 4. IgE immunoglobulins 5. J-chain or component 6. Free secretory component in. Milk fat globule membrane (MFGM) proteins A. Zone A (MFGM) proteins B. Zone B (MFGM) proteins C. Zone C (MFGM) proteins D. Zone D (MFGM) proteins IV. Minor proteins A. Serum transferrin B. Lactoferrin C. P2-Microglobulin D. Mrglycoproteins E. M2-glycoproteins F. OL1-AcId glycoprotein or orosomucoid G. Ceruloplasmin H. Trypsin inhibitor I. Kininogen J. Folate-binding protein (FBP) K. Vitamin B12-binding protein V. Enzymes (See Table 1.5)
a b c

X represents the genetic variant. Genetic variants of these fragments have not been specifically identified. Nomenclature has not been established for these fragments.

ture.71 However, it has been reported that specified secondary structure in K-caseins is in the range of about 50 to 75%,79 and there is evidence that native micellar caseins may have as much as 14% helical structure, 27% /3-structure, and 41% turns, leaving only 18% unspecified.80 However, there is little evidence of tertiary structure of caseins, which accounts for the stability of caseins against heat denaturation because there is little tertiary structure to unfold. Lack of tertiary structure also requires considerable exposure of hydrophobic residues to water. This accounts for the strong association reactions of caseins and their insolubility in water. Both as2-casein and K-casein contain two cysteine residues, but other caseins have no cysteine. Disulfide linked polymers of K-casein monomers, ranging from trimers to very large polymers, exist naturally.71 Some covalent dimers (disulfide linked) of as2- caseins also exist.16 Caseins differ greatly in charge distribution (Fig. 1.4) and can be distinguished by their sensitivity to calcium precipitation.

Table 1.4 CHEMICAL COMPOSITION OF THE MAJOR PROTEINS OCCURRING IN MILK68 P<*s2" K-

Ot-

PCasein A2 4 5 9 11 5 18 21 35 5 5 0 19 6 10 22 4 9 1 11 5 4 0

Acid Asp Asn Thr Ser SerP GIu GIn Pro GIy Ala ViCys VaI Met lie Leu Tyr Phe Trp Lys His Arg Pyr or GIu

Casein B 7 8 5 8 8 24 15 17 9 9 0 11 5 11 17 10 8 2 14 5

Casein A 4 14 15 6 11 25 15 10 2 8 2 14 4 11 13 12 6 2 24 3 6 0

Casein B 4 7 14 12 1 12 14 20 2 15 2 11 2 13 8 9 4 1 9 3 5 1

7r Casein A2 4 3 8 10 1 11 21 34 4 5 0 17 6 7 19 4 9 1 10 5 2 0

T2Casein A2 2 1 4 7 0 4 11 21 2 2 0 10 4 3 14 3 5 1 4 4 2 0

Casein A 2 1 4 7 0 4 11 21 2 2 0 10 4 3 14 3 5 1 3 3 2 0

Lactoglobulin A 11 5 8 7 0 16 9 8 3 14 5 10 4 10 22 4 4 2 15 2 3 0

Lactalbumin B 9 12 7 7 0 8 5 2 6 3 8 6 1 8 13 4 4 4 12 3 1 0

6 0

The following summary of association characteristics and calcium sensitivites of casein fractions is based largely on the discussion in ref. 16. The primary structure of asl-casein consists of two hydrophobic regions (residues 1 to 44 and 90 to 199). These regions contain all the proline residues, separated by a polar region (residues 45 to 89) that contains all but one of eight phosphate groups (Fig. 1.4). Association of asl-casein at neutral pH is dependent on both ionic strength and temperature and is mainly due to hydrophobic interactions and hydrogen bonding. asl-Casein can be precipitated at very low levels of Ca 2+ (7 mM). as2-Casein has a concentration of negative charges near the N-terminus and of positive charges near the C-terminus (Fig. 1.4). It is similar to asl-casein with respect to association at neutral pH and sensitivity to calcium precipitation. /3-Casein has a highly charged N-terminal region and a hydrophobic C-terminal region (Fig. 1.4), causing it to behave like a detergent. It is less sensitive to calcium precipitation than are the a s l - and as2-caseins, and its association is very temperature dependent, suggesting that hydrophobic interactions are most important. Association does not occur if the hydrophobic portion of the molecule is cleaved. /3-Caseins are the most water soluble of all caseins, especially at lower temperatures. /3-Caseins in

a si - en B SS as2-cn

p-cnA2 S
K-en B

Residue Sequence Number


Figure 1.4 Location, magnitude (right ordinate), and direction ( ) of charged residues (pH 6-7), Pro (.), and Cys (s), in caseins, (a) Location of glucide residue, (b) Point of cleavage by chymosin. (Reprinted from ref. 16 by permission of John Wiley & Sons.)

milk also have a higher isoelectric point (about 5.2) than a sl -caseins (about 4.8), which is important to the formation of acid gels (Section 1.3.1.3).81 Disulfide-linked polymers of K-casein further associate by noncovalent bonding to form large polymers with molecular weights of 600,000 to 650,000. These polymers are very stable at physiological pH and cannot be dissociated by changes in ionic strength or temperature.71 K-Casein is extremely resistant to calcium precipitation and is able to stabilize up to 10 times its own weight of a s - or /3-caseins against calcium precipitation.16 This stabilizing ability is lost after rennet cleavage of /c-casein at the Phe 1 0 5 -Met 1 0 6 bond, which results in the formation of a hydrophobic portion called para-K-casein (residues 1 to 105) and a hydrophilic portion (residues 106 to 169). The hydrophilic fragment is referred to as K-casein glycomacropeptide (GMP), or caseinomacropeptide (CMP). The latter is a better term because the predominant variants of K-casein are not glycosylated. 71 ' 82 CMP has an apparent molecular weight of 33,000 by size exclusion chromatography, but dispersion and analysis of aggregates by sodium dodecyl sulfate-polyacrylamide gel elec-

trophoresis (SDS-PAGE) revealed size-heterogeneous peptides with molecular weight <18,000. 83 In summary, all caseins self-associate and interspecies aggregation occurs in the presence and absence of calcium. The order of decreasing sensitivity to calcium precipitation is as2-, a sl -, /3-, and, finally, /c-, which stabilizes the casein system against calcium precipitation. Caseins do not form aggregates with whey proteins except during heat treatment (Chapter 2).

1.2.1.2 Whey Proteins


Proteins appearing in the pH 4.6 supernatant of milk are collectively referred to as whey proteins. As noted earlier, this operational definition includes N-terminal fragments of casein (Table 1.3), formerly known as proteose-peptone components 8-fast, 8-slow, and 5. A fourth proteose-peptone fraction, component 3, appears in whey but is the antigenic equivalent of protein fractions isolated from the fat globule membrane.84'85 Rennet whey also contains the CMP (C-terminal fragment of /c-casein) which is cleaved by rennet. The distribution of the principal whey protein fractions (/3-lactoglobulins, a-lactalbumins, bovine serum albumin, and immunoglobulins) and the identification of their genetic variants are listed in Table 1.3. Bovine /3-lactoglobulins of Western breeds are almost exclusively A and B variants, with <i predominance of the B variant in the most common breeds.71 In Western breeds, a-lactalbumins are almost exclusively variant B. The total concentration of whey proteins in milk is 5 to 7 g/L, not including about 1.1 g/L of proteose peptones. /3-Lactoglobulin has two internal disulfide bonds and one free thiol group (Fig. 1.5). It has considerable a-helix and /3-structure and exists naturally as a noncovalently linked dimer. The dimers are further associated to octamers in the isoelectric region (pH 3.5 to 5.2) but are dissociated to monomers at pH below 3.4.71 The predominant B variant, which contains one more charged group (aspartic acid residue 64) than the A variant,68 forms octomers less readily, possibly due to increased electrostatic repulsion. Association in the isoelectric region is preceded by a conformational change, which results in the binding of one proton group per monomer.71 A conformational change, known as the Tanford transition,86 takes place near pH 7.5 and results in the exposure of one previously untitrated carboxyl group and increased reactivity of the free cysteine group,71 which is important to thermal interactions of milk proteins.87"89 /3-Lactoglobulins may have a physiological role in vitamin A transport.90 a-Lactalbumin contains eight cysteine groups, all of which are involved in disulfide bonds (Fig. 1.5), and four tryptophan residues (Table 1.4), which account for its high absorbency at 280 nm. Differences in aromatic amino acid contents permit rapidfingerprintingof whey proteins by UV scanning spectroscopy.91 a-Lactalbumin has a highly ordered secondary structure, and hydrodynamic data indicate a compact, spherical tertiary structure.71 At pH <4.0, a-lactalbumin undergoes a conformational change, which can be observed by circular dichroism92 and is accompanied by the release of bound calcium.93 A similar conformational change was

S P-IgB

S SH

S a-Ia B

SS

S S

Residue Sequence Number


Figure 1.5 Location, magnitude (right ordinate), and direction ( ) of charged residues (pH 6-7), Pro (.), and Cys (s), in /3-lactoglobulin and a-lactalbumin. (Reprinted from ref. 16 by permission of John Wiley & Sons.)

observed by circular dichroism after removal of calcium from a-lactalbumin at pH 7.5. 93 Magnesium 94 and other metal ions 93 - 95 are also bound by a-lactalbumin. The conformational change at pH < 4 . 0 results in temperature- and concentrationdependent aggregation of a-lactalbumin.96 Thermal denaturation of a-lactalbumin is also accompanied by a release of bound calcium, and a-lactalbumin is stabilized against heat denaturation and aggregtion in the presence of calcium. 97 ' 98

1.2.1.3 Enzymes
Milk contains both indigenous and exogenous enzymes, the latter being mainly bacterial. With respect to dairy processing, the most significant bacterial enzymes occurring in milk are heat-stable Upases and proteinases elaborated by psychrotrophic bacteria.99"101 Indigenous enzymes of milk, the reactions they catalyze, and their location in milk are summarized in Table 1.5. With respect to dairy processing and quality control, the most significant enzymes are several hydrolases, namely, lipoprotein lipase, plasmin, and alkaline phosphatase. The functions and significance of these enzymes are briefly described in this section. Properties and functions of other indigenous milk enzymes have been reviewed. 1 6 1 0 2 Most milk enzymes have pH and temperature optima near physiological values, with the notable exceptions of alkaline phosphatase and phosphoprotein phosphatase, which have pH optima of 9.8 and 4.0 to 5.5, respectively. 16 Alkaline phosphatase activity is used to distinguish raw milk from pasteurized milk because its heat sta-

Table 1.5
ECNo. 1.1.1.27 1.1.1.37 1.2.3.2 1.4.3.6 1.6.4.3 1.6.99.3 1.8 1.11.1.6 1.11.1.7 1.15.1.1 2.3.2.2 2.4.1.22 2.4.99.1

ENZYMES OF BOVINE MILK


Enzyme Lactate dehydrogenase Malate dehydrogenase Xanthine oxidase Amine oxidase (Cu containing) Lipoamide dehydrogenase (NAD + ) (diaphorase) NADH dehydrogenase (cytochrome c reductase) Sulfhydryl oxidase (not 1.8.3.2 thiol oxidase) Catalase Lactoperoxidase Superoxide dismutase 7-Glutamyl transferase Lactose synthase CMP-A^-acetylneuraminategalactosyl-glycoprotein sialyl transferase Aspartate aminotransferase Alanine aminotransferase Riboflavin kinase Glycerol kinase FMN adenyltransferase Thiosulfate sulfur transferase (Rhodanase) Carboxylesterase (B-esterase) Arylesterase (A-esterase) Acetylcholine esterase Cholinesterase Lipoprotein lipase Alkaline phosphatase Acid phosphatase Reaction L-lactate + NAD ^ pyruvate + NADH 4- H + L-malate 4- NAD + ^ oxaloacetate 4NADH 4- H + Xanthine 4- H2O 4- 2O2 ^ uric acid 42O2 + 2H + RCH2NH2 + H2O 4- O2 ; RCHO 4H2O2 4- NH3 NADH 4- H + 4- lipoamide ? NAD + + dehydrolipoamide NADH 4- H + + acceptors NAD + 4reduced acceptor 2RSH + O2 ^ R-S-S-R 4- H2O2 2H2O2 ^ O2 4- 2H2O Donor 4- H2O2 ^ oxidized donor + 2H2O 2O2 + 2 H + ^ O 2 + H2O2 L-7-Glutamyl-peptide + amino acid ^ peptide 4- L-glutamyl-amino acid UDP-galactose 4- D-glucose ^ UDP + lactose CMP-7V-acetylneuraminate + D-galactosyl-glycoprotein ? CMP + N-acetylneuraminyl-D-galactosyglycoprotein L-Aspartate + 2-oxoglutarate ^ oxaloacetate + L-glutamate L-Alanine + 2-oxoglutarate ^ pyruvate + L-glutamate ATP + riboflavin ^ ADP + FMN ATP 4- glycerol ^ ADP 4- glycerol-3phosphate ATP 4- FMN ^ FAD 4- pyrophosphate S 2 Oi" 4- CN- 5 SOf" + SCNR-COOR' 4- H2O ^ ROH + RCOOH A phenyl acetate 4- H2O ^ a phenol + acetate acetylcholine 4- H2O ^ choline 4- acetate An acylcholine 4- H2O ^ choline + carboxylate anion Triglyceride 4- H2O ^ diglyceride 4fatty acid R-O-PO3H2 + H2O ^ ROH + H3PO4 R-O-PO3H2 + H2O ^ ROH + H3PO4
+

Location Plasma

MFGM

MFGM Serum Leukocytes Serum

MFGM Serum

2.6.1.1 2.6.1.2 2.7.1.26 2.7.1.30 2.7.7.2 2.8.1.1 3.1.1.1 3.1.1.2 3.1.1.7 3.1.1.8 3.1.1.34 3.1.3.1 3.1.3.2

Plasma

MFGM Serum Casein MFGM MFGM (Continued)

Table 1.5 (Continued)


ECNo. 3.1.3.5 3.1.3.9 3.1.3.16 3.1.4.1 3.1.27.5 Enzyme 5'-Nucleotidase Glucose-6-phosphatase Phosphoprotein phosphatase Phosphodiesterase Ribonuclease (pancreatic) Reaction A 5' ribonucleotide + H2O ^ a ribonucleoside + H3PO4 D-Glucose-6-phosphate + H2O ^ Dglucose 4- H3PO4 Protein phosphate -I- H2O ^ protein + H3PO4 A phosphoric diester + H2O ^ a phosphoric monoester + alcohol Endonucleolytic cleavage to 3' phosphomono- and oligonucleotides ending in Cp or Up Hydrolyzes a-1-4 glucan links in polysaccharides at random Hydrolyzes a-1-4 glucan links in polysaccharides by removing successive maltose units from the nonreducing end Hydrolyzes the p-1-4 glycosidic bond between N-acetylgucosamine and Nacetylmuraminic acid units in mucopolysaccharides Hydrolyzes a-r>mannosides by removing a-D mannose from the nonreducing end Hydrolyzes chitobiose and higher analogs and protein derivatives by removing JV-acetyl-D-glucosamine from the nonreducing end A p-D-glucuronide + H2O ^ alcohol + D-glucuronic acid Hydrolyzes peptide bond, preferentially at Lys > Arg Hydrolyzes peptide bond H4P2O7 + H2O ; 2H3PO4 ATP + H2O ^ ADP + H3PO4 A dinucleotide + H2O ^ 2 mononucleotides r>Fructose-l,6-phosphate ^ dihydroxyacetone-phosphate + D-glyceraldehyde-3-phosphate H2CO3 s CO2 + H2O D-Glucose-6-phosphate ^ D-fructose6-phosphate Location MFGM MFGM Plasma MFGM Serum

3.2.1.1 3.2.1.2

a-Amylase p-Amylase

Serum

3.2.1.17

Lysozyme

Serum

3.2.1.24

a-D Mannosidase

3.2.1.30

p-W-AcetylD-glucosaminidase

3.2.1.31 3.4.21.7 3.4 3.6.1.1 3.6.1.3 3.6.1.9 4.1.2.13

p-Glucuronidase Plasmin Acid protease Inorganic pyrophosphatase Adenosine triphosphatase (Mg 2+ activated) Nucleotide pyrophosphatase Fructose-biphosphate aldolase Carbonic dehydratase (carbonic anhydrase) Glucose phosphate isomerase

Casein

MFGM

4.2.1.1 5.3.1.9

From ref. 16. Reprinted by permission of John Wiley & Sons.

bility is similar to the minimum conditions used for milk pasteurization.103 Two isozymes, a- and /3-phosphatase, occur in milk. The latter is more abundant and occurs mainly in the fat globule membrane. Interference by the heat-stable acid phosphatase is avoided by performing the assay at pH near 10. The /3-isozyme of alkaline phosphatase is also subject to renaturation, especially in creams, where the enzyme is more concentrated.16 Residual phosphatase can be distinguished from reactivated phosphatase by increased activity of the latter in the presence of magnesium.104105 It was reported that heat inactivation of alkaline phosphatase was more rapid in highly concentrated, ultrafiltered milk rententates.106 Milk lipoprotein lipase (LPL) has been well characterized.16'107"109 Milk LPL is present mainly in the plasma in association with casein micelles. It does not attack the fat globule unless the FGM has been damaged or if certain blood serum lipoproteins are present. These lipoproteins, acting as cofactors, enable LPL to attack the lipoproteins of the FGM. Further discussion of lipolytic breakdown of dairy products is presented in Section 1.2.2.3. The principal milk protease is an alkaline serine protease, which is apparently identical to blood plasmin.16'102 Plasmin is present mainly as plasminogen in fresh milk, but, with time, it is converted to active plasmin. It has been indicated that the plasmin content of milk is associated with the process of involution (i.e., the declining phase of milk production) and that administration of BST reduced levels of plasmin and plasminogen in late lactation.61110 It is well known that increased proteolytic activity is associated with high somatic cell counts, 111112 but the protease associated with somatic cells is apparently not plasmin.113 Plasmin attacks both /3-casein and as2-casein. As indicated previously, protein fractions formerly known as y-caseins and proteose-peptones are plasmin produced fragments of /3-casein. Plasmin has optimal activity at slightly alkaline pH and 37C. The enzyme is extremely heat stable114 and is responsible for the development of bitterness in pasteurized and ultra-high-temperature processed milk. The distribution of plasmin between cheese and whey is dependent on the pH of whey separation; higher-running pH causes increased retention of plasmin in the cheese.115

1.2.2 lipids 1.2.2.1 Chemical Properties


The milkfat of ruminants is very complex, due to the diversity of lipid species that are produced by microbial activity in the rumen and are transported to the milk secretory cells in the blood stream. 15116 " 118 Other lipids are produced by synthesis in the secretory cells. 116119 Fatty acids found in milk fat include: (1) saturated even and odd n-chain acids from 2 to 28; (2) at least 50 branched chain fatty acids; (3) cis monoenoic fatty acids of 12 and 14 to 24 -chain acids; (4) trans 16 to 24 nchain fatty acids; (5) various positional and geometric isomers of dienes and trienes of 18, 20, 22, and 24 -chain acids; and (6) small amounts of tetra- and pentanoic acids (Tables 1.6 and 1.7).116 Short-chain fatty acids (butyric, caproic, caprylic, and capric) comprise about 11 % by weight of total methyl esters. Quantitatively, the

Table 1.6 FATTY ACID COMPOSITION OF BUTTER OIL AS DETERMINED BY GLC-MASS SPECTROMETRY (WEIGHT PERCENT) OF TOTAL METHYL ESTERS116
Methyl Ester Carbons 4 6 8 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 Monoenes Saturates 3.25 2.32 1.85 4.02 0.16 4.15 0.03 11.05 0.95 26.15 0.70 9.60 0.11 0.19 0.06 0.10 0.07 0.06 0.01 0.04 trans Iso Anteiso Branched Other

0.03 0.47 0.08 1.25 0.32 20.40 0.10 0.15 0.03 0.02 0.01 0.02 0.01 0.08 0.23 0.32 0.33 0.15 0.06 0.04 Trace Trace Trace 0.42 0.40 0.09 0.01 DDL pristanate, 0.01 DDD pristanate, 0.01 DDL, DDD phytanates, 0.04

0.03 0.01 5.34 0.01 0.01 Trace Trace 0.01 0.01

major fatty acids of milk fat are myristic (11%), palmitic (26%), stearic (10%), and oleic (20%). Saturated fatty acids account for about two-thirds of milk fatty acids (Table 1.8), with larger quantities of unsaturated fatty acids present during the summer months. The summer and winter mean iodine values of Finnish butter fat have been reported as 36.1 and 27.6, respectively.120 Estimates of total trans fatty acids vary widely within the range of 2 to 11%, expressed as elaidic acid {trans 18:1).121 The distribution in milk and some properties of the major milk lipids are summarized in Table 1.8. Triglycerides account for 98.3% of milkfat. It is not known whether small amounts of free fatty acids occurring in fresh milk are secreted from the epithelial cell or are the product of early lipolysis. Phospholipids comprise about 0.8% of milk lipids and are mainly associated with FGM. About 0.3% of milk lipid is sterols, mainly cholesterol, which is located mostly in the core of the fat globule.

1.2.2.2 Physical Properties


The physical properties of milkfat have been summarized as follows: density at 200C is 915 kg m~ 3 ; refractive index (589 nm) is 1.462 and decreases with increasing temperature; solubility of water in fat is 0.14% (w/w) at 200C and increases with increasing temperature; thermal conductivity is about 0 . 1 7 J m - 1 S - 1 K " 1 at 200C;

Table 1.7 FATTY ACID COMPOSITION OF BUTTER OIL AS DETERMINED BY GLC-MASS SPECTROMETRY (A CONTINUATION OF TABLE 1.6)116
Weight Percent of Total Methyl Esters Methyl Ester Carbons 18 Positional isomers Conjugated cis, trans trans, trans 20 Positional isomers 0.03 Trace 0.01 0.13 0.02 0.06 0.02 0.01 0.03 0.02 0.10 0.02 Dienes Trienes Tetraenes Pentaenes

0.14 2.30 0.70 0.05

0.02 0.60 di-0.03 tri-0.01

22 Positional isomers 24 Positional isomers

0.04 Trace Trace

0.02 0.02

specific heat at 400C is about 2.1 kJ k g " 1 K" 1 ; electrical conductivity is <10~ 1 2 ohm" l cm" *; and the dielectric constant is about 3.1. 27 Crystallization of lipids and the resulting effects on fat structure, melting range, and rheological properties have been reviewed.122"124 The most complete review of the crystallization behavior of milkfats is ref. 27. The following discussion is based largely on that reference. The native mixture of milk lipids is solid at room temperature and is, therefore, properly described as milk "fat" as opposed to "oil," which is liquid at room temperature. The melting points of individual triglycerides in milk ranges from 75C for tributyric glycerol to 72C for tristearin. However, the final melting point of milkfat is about 37C because higher melting triglycerides are dissolved in liquid fat.16 Milkfat crystals occur in a, /3^, /3J and /3 forms, although for slowly cooled fat, the least stable a form is too transient to be observed.125 Crystal behavior and melting curves of milkfat are complicated by the diverse lipid composition: trans unsaturation increases melting points; odd-numbered and branched chains decrease melting points because they are unable to form dense crystal structures; and variations in chain length also contribute to softer fats. Typical melting curves for summer and winter milkfat are shown in Figure 1.6. Crystal structure and properties are also dependent on the state of dispersion, so globular fat behaves very differently from bulk fat and the crystal behavior of globular fat is affected by globule size. Homogenized recombined milkfat behaves differently because of its uniform lipid composition as opposed to natural fat, which shows wide variation in lipid composition

Table 1.8 AN OVERVIEW OF THE LIPIDS OF FRESH MILK


Component Fatty Acids Alcohol Residue Glycerol Glycerol Glycerol Phospho group Choline Ethanolamine Serine Inositol Choline6 Choline Hexose Hexose8 Other Constituent Percentage in Milk Fat (w/w) 98.7 98.3 0.3 0.03 0.1 0.8 0.26 0.28 0.03 0.04 0.02 0.16 0.1 0.01 0.32 0.30 0.02? 0.002 Percentage of the Lipid In Core of Globule -100 90? 60 Globule Membrane Milk Plasma

Lipid Class Neutral glycerides Triglycerides Diglycerides Monoglycerides Free fatty acids Phospholipidsa Lecithin Ph. ethanolamineb Ph. serineb Ph. inositide6 Plasmalogens Sphingomyelind Cerebrosidescd Gangliosidescd Sterols Cholesterol Cholesteryl esters Carotenoids + vitamin A

MW 728 536 314 253

Number 3 2 1

y 0.35 0.38 0.36 0.36

14.4 14.9 15.0 15.8

10?

+
10? 65 35

Glycerol Glycerol Glycerol Glycerol Glycerol Sphingosine Sphingosine Sphingosine

764 742 784 855 -700 770 770 -1600 387 642

2 2 2 2 lf 1 1 1

17.2 17.9 17.8

0.60 1.00 0.80

19.0 20.0

0.20 0.20

80

70 70? 10

30 30? 10

Cholesterol

16.0

0.40

95?

5?

From ref. 16. Reprinted by permission of John Wiley & Sons. Note: Not complete: approximate average values from various sources, x number of carbon atoms; y number of double bonds. a A small fraction (e.g., 1%) is present as lysophosphatides. b Phosphatidyl ethanolamine + serine = cephalin. c Glycolipids. d Sphingolipids. e Or ethanolamine. f Also a fatty aldehyde residue. 8 Also neuraminic acid.

1 Solid Fat (%)

Temperature ( 0 C ) Figure 1.6 Melting curves of milk fat, determined by dilatometry. 1, summer fat, slowly cooled before the experiment; 2, the same fat, rapidly cooled; 3, winter fat, rapidly cooled. (From ref. 27 with permission of Pudoc, Wageningen, the Netherlands.)

and melting ranges of individual globules. For example, fat dispersed in natural globules has a lower mean melting point than bulk fat but, because of widely varying composition between globules, some dispersed fat has a melting point that is much higher than the final melting point of bulk fat. These effects are summarized in Table 1.9.

1.2.2.3 Lipolysis Hydrolysis of fatty acid esters by the action of lipases results in the common flavor defect known as lipolytic or hydrolytic rancidity and is distinct from oxidative rancidity. A comprehensive review of flavor impairment of milk and milk products due to lipolysis has been published by the International Dairy Federation.126 Lipases are unique among enzymes in that they are active at the lipidserum interface. In milk, lipases are ineffective unless the FGM is damaged or weakened in some way. Lipolysis may be caused by the lipoprotein lipase (LPL), which is endogenous to milk, or by bacterial lipases. The principal bacterial lipases that occur in milk are heat-stable exocellular lipases of psychotrophic bacteria.99'127 However, the principal psychotrophic bacteria of milk, Pseudomonas sp., do not elaborate significant quantities of proteases or lipases until cell counts exceed 106 to 108 mL~ ] . 128 In practice, this means that significant elaboration of Pseudomonas lipases is unlikely to occur except in improperly cleaned raw-milk-handling equipment; and psychotrophic lipases should not be a serious problem, except possibly in ultra-high-

Table 1.9 FACTORS INFLUENCING CRYSTALLIZATION OF MILK FAT


Effect on Crystallization Characteristics
9

Factors Fat composition Lower temp, of crystallization Rapid cooling Cooling in steps Preliminary cooling to low temp. Prolonged at not too low temp. Fat in natural globules as compared to bulk fat Smaller globules

Melting Range Yes Main melting at lower temp. Main melting at higher temp. More than one melting max. Main melting at lower temp. More even Somewhat higher final melting point

Amount of Solid Fat Yes More Usually more Usually less More Usually more Less at high temp, more at low temp. Still less at high temp.

Smaller Smaller Often larger; spherulites Smaller Larger; solid networks Smaller; no networks

Smaller

From ref. 27 with permission of Pudoc, Wageningen, Netherlands.

temperature processed milk, where low levels of heat-stable proteases and lipases may cause deterioration.129 Cow's milk contains sufficient total lipase activity (mainly LPL) to release about 2 /imol of free fatty acids (FFA) per minute at 37C, but the actual activity during storage of raw milk at 4C may be as low as 0.002 /imol of FFA min~ l 13 The following conditions affect the rate of lipolysis in fresh milk: the pH of milk (6.6 to 6.8) and the storage temperature of raw milk (normally <4C) are less than the LPL optima of pH 8.3 and 37C; about 80% of milk LPL is bound to micellar casein, 10 to 20% is present in the serum, and only 0 to 10% is associated with the fat globule; milk plasma contains at least two inhibitors of lipolysis; and a lipoprotein is present in milk, which acts as a cofactor to increase LPL activity.130 The inhibitory effect of milk plasma is probably due to its effect on the distribution of LPL and can be reversed by addition of heparin, which causes dissociation of LPL from the casein micelles.131-132 The properties of the FGM are most important to lipolysis. The observed lactation effects may be due to reduced contents of phospholipids in the FGM in late lactation.107 Mastitis, which alters milk composition, also increases sensitivity of the fat globule to lipolysis.133 The lipoprotein cofactor, which is derived from blood, apparently enables LPL to hydrolyse lipoproteins of the FGM and gives LPL access to triglycerides in the fat globule.107 Other factors that destabilize the FGM, especially agitation and foaming, also promote lipolysis. Churned fat does not appear to be a good substrate for LPL,134 but lipolysis is accelerated by the replacement of the native membrane with surface active material (mainly casein micelles and whey

proteins) from the plasma.130 This effect is at least partly due to redistribution of LPL from the plasma to the FGM and accounts for greatly increased lipolysis after homogenization. Similarly, experiments with on-farm ultrafiltration demonstrate that milk must be heated after ultrafiltration to inactivate LPL.135 Milking systems will promote lipolysis to greater or lesser degrees, depending on the amount of agitation and aeration that takes place during milking and milk transfer.130 Lipolysis can also be induced in fresh milk by a temperature cycle of cooling to <5C, warming to 25 to 35C, and recooling to <10C. 107 Such an effect may occur if a large amount of warm milk is added to a small amount of cooled milk. About 20% of cows produce milk in which LPL is activated by cooling to < 15C soon after milking. Lipolysis proceeds without subsequent thermal or mechanical activation. This effect, frequently referred to as spontaneous lipolysis, is unlikely to occur in herd milks or in pooled milks because it is prevented by mixing affected milk with three to five times its volume of normal milk.136 The major conditions that affect spontaneous lipolysis are: late-lactation milk is more susceptible than early-lactation milk;137 fresh forage reduces the incidence of spontaneous lipolysis; more lipolysis occurs during the winter months, but this effect may be related to feed and lactation effects; and low-yielding cows are more likely to produce spontaneous milk. 107138139 Spontaneous vs. nonspontaneous milk may be determined by differences in contents of lipolytic inhibitors and activators. Sensory perception of lipolytic rancidity is strongly affected by the pH of the product because, at low pH, more free fatty acids are present in the aqueous phase, where they are more readily tasted.16 In fresh milk, sensory threshold values corresponded to acid degree values (ADV) of 4.1 to 4.5 mmol per 100 g of fat as estimated by the Frankel and Tarassuk solvent extraction method and 1.85 to 2.05 as estimated by the Bureau of Dairy Industries (BDI) detergent extraction method.140

1.2.2.4 Oxidation
Oxidation of milk and other fats proceeds by the well-known autoxidation reaction124 in three stages: initiation, propagation, and termination. During propagation, antioxidant compounds such as tocopherols and ascorbic acid are depleted while peroxide derivatives of fatty acids accumulate. Peroxides, which have little flavor, undergo further reactions to form a variety of carbonyls, some of which are potent flavor compounds, especially some ketones and aldehydes. Most methods available to monitor lipid oxidation are unsuitable as an early index of oxidized flavor development in milk: measurement of peroxides is not useful because peroxides are unstable intermediates; tests based on colorimetric reaction of thiobarbituric acid with malonaldehyde show some correlation to sensory values141 but are rather insensitive; and direct measurement of oxygen uptake is suitable only for controlled experimental conditions.142 In milk, the initiation reactions involve phospholipids present in the FGM. Free radicals formed from phospholipids are then able to initiate oxidation of triglycerides, especially in the presence of copper and proteins.16 During the winter months (October to May), when cattle are on dry feed, the incidence of oxidized flavor in

raw producer milks in Ontario is about 20% as determined for 2- to 3-day-old milk by expert graders (unpublished data). The following summary of conditions affecting oxidation of lipids and the development of oxidized flavor in milk is based mainly on several significant reviews.16-128'142"146 Although all milk probably requires some extrinsic factor such as added copper or light to initiate lipid oxidation, the milk of some cows and herds is said to oxidize spontaneously, implying that oxidation results from factors intrinsic to this milk whereas lipid oxidation in normal milk requires activation by extrinsic factors. Significant intrinsic factors affecting milkfat oxidation include (1) metalloproteins such as milk peroxidase and xanthine oxidase: (2) endogenous ascorbic acid, which acts as a cocatalyst with copper to promote oxidation; (3) endogenous copper content; and (4) endogenous antioxidants, mainly tocopherols. Fresh forage is well known to control spontaneous oxidation, as indicated by obvious seasonal effects on the incidence of oxidized flavor. This effect is probably due to increased levels of endogenous antioxidants. However, attempts to supplement dry forage with tocopherols have had limited success because only about 2% of added tocopherol is transmitted to the milk. 147148 Most managers, therefore, have concentrated on the control of extrinsic factors to minimize the extent of oxidation. Important extrinsic factors include contamination with metals, temperature of storage, oxygen tension, heat treatment, agitation, light, and acidity. Both copper and iron may catalyze lipid oxidation, but probably only copper is significant in milk. Added copper is much more potent than natural copper because a significant portion of added copper goes directly to the fat globule.16 In some milks, 5 /jig k g " 1 of added copper is sufficient to induce lipid oxidation. Fortification149 or contamination150 of milk with iron is reported to induce oxidized flavor development. This seems surprising because iron should be inactivated by proteins in milk.16 Autoxidation of fats is generally increased with higher temperatures; but in raw milk and low pasteurized milk, this trend is reversed,128 in spite of copper migration from the fat globule to the plasma at low temperatures.16 Effects of low temperature on oxidation are normal for processed dairy products. Heating milk causes migration of copper from the plasma to the fat globule, indicating that oxidation of butter can be reduced by separating cream before heat treatment and by separating to high fat contents.16 Heating can also denature metalloproteins and increase the availability of metals to catalyze oxidation; however, high-heat treatments (in excess of 800C) stabilize milk against both copper- and light-induced oxidation.151 This effect is probably due to exposure of sulfhydryl groups of denatured proteins and the release of hydrogen sulfide, which binds copper as cupric sulfide. The oxygen tension required to permit lipid oxidation in milk is low (0.1 atmosphere oxygen pressure), and bacterial respiration in normal fresh milk is of no consequence in decreasing oxidation.152 De-aeration significantly reduces oxidation of packaged whole milk powder.153 Homogenization drastically reduces the sensitivity of milkfat to both copper- and light-induced oxidation, probably because oxidation-sensitive membrane phospholipids are displaced. Ultrafiltered milks are also more resistant to oxidation.135

Photooxidation of milk fat is accompanied by depletion of riboflavin, ascorbic acid, and some amino acids.154"15 So-called sunlight flavor induced by photooxidation is due to oxidation of methionine to methional by photoactivated riboflavin. Sunlight flavor is, therefore, due to oxidation of proteins but is often confused with lipid oxidation because the flavor notes are similar. Nonfat retail milks are reported to show high incidence of oxidized flavor,145 but this effect is most likely sunlight flavor.

1.2.3 Lactose 1.2.3.1 Biochemical Properties


The lactose content of normal milk is relatively constant at 4.8 to 5.2% lactose monohydrate. Lower levels occur in colostrum and mastitic milk to offset high mineral levels and maintain osmotic balance.159 Lactose comprises about 52% of milk solids-not-fat, about 70% of whey solids, and >90% of the solids in milk ultrafiltrate. Several reviews have appeared on the utilization of lactose.160"162 In addition to lactose (4-O-/3-D-galactopyranosyl-D-glucopyranose), fresh milk contains other carbohydrates in small amounts, including glucose (1 mg/ml), galactose (1 mg/ml), and oligosaccharides (0.1 mg/ml).163 Lactose is synthesized in the mammary gland, where the final step is the transfer of D-galactose to D-glucose by galactosyltransferase in the presence of a-lactalbumin, which acts as an enzyme modifier.164 Lactose is a reducing sugar with the aldehydic group on the glucose residue. It exists in both a and /3 forms, which are indicated by interchanging the OH and H on the reducing group. Lactose is optically active because of its asymmetry, and the a form can be distinguished from the /3 form by its greater rotation of polarized light in the dextro direction.163 Optical activity is the basis for polarimetric determination of lactose in fresh, nonfermented dairy products.165 Polarimetry is not useful for fermented products due to interference from lactic acid, which rotates light to the levo direction. The most important function of lactose in milk and dairy products is its utilization as a fermentation substrate. Lactose can be hydrolyzed to glucose and galactose by /3-D-galactosidase (lactase). Elaboration of this enzyme gives lactic acid bacteria a competitive advantage over many pathogenic and spoilage organisms. It is this property that makes naturally fermented milk a relatively safe product and is the basis for controlled fermentations in the production of cultured dairy products. Enzymatic hydrolysis of lactose is used to reduce lactose crystallization in certain products and to produce lactose-reduced products for persons who do not possess sufficient lactase.166'167

1.2.3.2 Physicochemical Properties


There are several literature reviews on physicochemical properties of lactose.16'160'163-166'168 Only those properties most important to dairy processing are discussed here.

Figure 1.7 a-Lactose crystals prepared by scanning electron microscopy. (Courtesy of A. Smith.)

The a and /3 forms of lactose exist in solution in a temperature-dependent equilibrium, according to Eq. 1.1, where T is temperature in 0 C. [/3]/[a] = 1.64 - 0.00277 (1.1)

Supersaturated solutions of lactose at temperatures >93.5C crystallize as anhydrous /3-lactose, which is sweeter than a-lactose monohydrate (a-hydrate).163 At temperatures <93.5C, supersaturated solutions crystallize as a-lactose monohydrate. Mutarotation from /3-lactose to a-lactose occurs as a-lactose is crystallized from solution. Crystals of a-lactose monohydrate form many different shapes, but all are crystallographically equivalent to the most common "tomahawk" shape (Fig. 1.7).163 The different shapes arise from differential rates of crystallization on the various crystal faces. The most important variables affecting the rate of crystal growth and crystal shape is the degree of supersaturation (the ratio of actual concentration to the solubility) and the presence of inhibitors. In milk, the most important inhibitor is /3-lactose, which inhibits crystallization on some faces more than others and is largely responsible for the characteristic tomahawk shape of crystalline a-lactose monohydrate.16 The final solubility of lactose is 15.1 g/100 g of water at 100C and 11.9 g/100 g of water at 00C. In practice, a-hydrate is likely to crystallize in refrigerated dairy products containing more than about 13 g lactose per 100 g of water. Amorphous anhydrous lactose (lactose glass) is formed by rapid drying (e.g., spray-drying of milk or whey) or by rapid freezing. Lactose glass is extremely hygroscopic and accounts for the caking of skim milk powder, which occurs at moisture contents greater than about 8%.163 When sufficient

Table 1.10 SOME MINOR COMPONENTS IN FRESH MILK. RESULTS ARE EXPRESSED AS CONTENTS PER LITER 3'16'169>170 Minerals Sodium (mg) Potassium (mg) Chloride (mg) Calcium (mg) Magnesium (mg) Phosphorus (mg) Iron (|xg) Zinc (jig) Copper (|xg) Manganese (|xg) Iodine (u,g) Fluoride (|xg) Selenium (|xg) Cobalt ([Lg) Chromium (|xg) Molybdenum (jig) Nickel (jLg) Silicon ([ig) Vanadium (jxg) Tin (jig) Arsenic (|xg) 350-900 1100-1700 900-1100 1100-1300 90-140 900-1000 300-600 2000-6000 100-600 20-50 260 30-220 5-67 0.5-1.3 8-13 18-120 0-50 750-7000 tr-310 40-500 20-60 Vitamins A (jig RE) D(IU) E(JJLg) K(JJLg) B 1 (jig) B 2 (JJLg) Niacin (jig) B 6 (M^g) Pantothenic acid (jxg) Biotin (jig) Folic acid (jxg) B 12 (^g) C(IiIg) NPN Compounds Total NPN (mg) Urea-N (mg) Creatine N (mg) Uric acid-N (mg) Orotic acid N (mg) Peptides N (mg) Ammonia N (mg) Amino acid N (mg) Choline (mg) Carnitine (mg) Af-Acetylneuraminic acid (mg) 229-308 84-134 6-20 5-8 12-13 32 3-14 39-51 43-285 10-17 120-270 400 40 1000 50 450 1750 900 500 3500 35 55 4.5 20

Selected Miscellaneous Compounds Ethanol (mg) Formic acid (mg) Acetic acid (mg) Lactic acid (mg) Citric acid (mg) Phosphoric esters (mg) Nucleic acids and Nucleotides (mg) 3 10-85 3-50 34-104 1750 300 555

moisture is available, lactose glass forms a-hydrate crystals, which bind powder particles together.

1.2.4 Minor Components


The contents of some minor components of milk are listed in Table 1.10, including some vitamins, minerals, nonprotein nitrogenous compounds, phosphoric esters, ethanol, and some acids.

1.2.4.1 Vitamins
The physiological functions and the activity in milk of vitamins have been reviewed. 1 7 0 1 7 1 Milk contains fat-soluble vitamins A, D, E, and K (Table 1.10). Milk

is an important source of dietary vitamin A; many Western countries require supplementation of skim milk to replace vitamin A removed with the cream. U.S. Government Regulations require 2000IU of vitamin A per quart (1 U.S. quart = 0.95 L). The actual amounts, however, have been reported to be less and are extremely variable.172 Natural vitamin A activity is derived from retinol and /3-carotene and varies with the season, due to seasonal variation in /3-carotene,170 which also accounts for the seasonal variation in the color of milk fat. Vitamin D activity in milk is derived from cholecalciferol (D3) and ergocalciferol (D2). Vitamin E occurs in milk as atocopherol, an important natural antioxidant. Activities of vitamins D and E in milk vary with the season or, more directly, with type of forage. Milk contributes a rather small proportion of the dietary vitamin K in Western diets. Milk is an important dietary source of water-soluble vitamins B 1 (thiamine), B 2 (riboflavin), B 6 (pyridoxine), B 12 (cyanocobalamin), niacin (nicotinic acid), andpantothenic acid (Table 1.10). All the water-soluble vitamins are quite stable to milk processing treatments, although riboflavin is extremely sensitive to degradation by light of wavelengths <610 nm. Light-activated riboflavin is an agent in the development of sunlight flavor in milk (Section 1.2.2.4) and also catalyzes the photodegradation of ascorbic acid. Ascorbic acid is the most heat-labile vitamin in milk, but this is of little consequence because milk is not an important source of dietary vitamin C.

1.2.4.2 Minerals
Twenty-two minerals are considered essential to human nutrition. All of these are present in milk, confirming milk's nutritional excellence. 169173174 However, negative factors may also exist. In particular, there is currently concern about iodine concentrations, which may be elevated by disinfectant iodophors.175"180 There have also been numerous recent investigations on the presence of radionuclides in milk, especially 90Sr and 131 L 3 ' 181 Three families of salt constituents may be considered in milk.159 The first includes sodium (Na), potassium (K), and chloride (Cl), which exist almost entirely as free ions in milk and are readily diffusible (i.e., are present in milk ultrafiltrate). The concentrations of these three ions are negatively correlated to lactose, as required to maintain osmotic equilibrium of milk with blood. Thus, as compared to midlactation, in early lactation, Na and Cl concentrations of milk are higher and lactose concentration is lower. A second family includes colloidal calcium (Ca), magnesium (Mg), inorganic phosphorus (P1), and citrate. Total concentrations of Ca, Mg, P1, and citrate in milk plasma are 30.3, 5.2, 21.4, and 9.5 mM, respectively, by calculation.159 On a molar basis, about two-thirds of the calcium, one-third of the magnesium, one-half of the inorganic phosphorus, and less than one-tenth of citrate in milk are colloidal (i.e., nondiffusible) and mainly present in the casein micelle (Section 1.3.1). Therefore, concentrations of colloidal Ca, Mg, P1, and citrate are strongly correlated to the casein content of milk.

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A third family includes salts, whose concentrations are affected by the natural pH of milk, namely, diffusible Ca, diffusible Mg, diffusible citrate, Ca2+ , and HPO2TAbout 20 to 30% of diffusible Ca and Mg is present as free ions; the remainder exists as citrate and phosphate salts. Diffusible Ca, Mg, and citrate concentrations are positively correlated because Ca and Mg form strong complexes with citrate at the pH of milk. A negative correlation between Ca 2+ concentration and pH and a positive correlation between Ca 2+ and HPO 2 - relate to the solubility product of micellar calcium phosphate. The degree of saturation of micellar calcium phosphate and the concentration of H2POX are relatively constant. For example, increased Ca 2+ causes formation of colloidal calcium phosphate, and the level of H2PO^ is maintained by reaction of HPO2," with H + . Further discussion of acid-base equilibria is presented in Section 1.4.6.

1.3 Structure 1.3.1 Casein Micelles 1.3.1.1 Properties


In their classic monograph on dairy chemistry, Jenness and Patton in 1959 stated that "Many of the problems of dairy technology revolve around the behaviour of the (calcium caseinate-phosphate complex) and particularly the aggregation of the particles by heat, salts, acid, and rennin. Therefore, a study of its composition and properties is a most important phase of dairy chemistry".182 In the 30 years since this statement was made, considerable effort has been put into studying the properties of the casein micelle. The progress to date has recently been reviewed.183"185 The intricacy of the micelles may be related to their biological functions in milkto carry a large amount of highly insoluble calcium phosphate to the mammalian young in liquid form, and to form a clot in the stomach for more efficient nutrition.16'184 The micelle is extremely stable under some conditions of processing, for example, concentration, ultrafiltration, pelleting, drying,183 but very unstable under others, for example, acid, chymosin.16 A manipulation of micelle stability gives rise to many traditional and nontraditional dairy products.17 A summary of the properties and structure of the casein micelle will be presented here. About 75% of the proteins in milk are classified as casein protein, that which precipitates at pH 4.6.68 Most, but not all, of this casein protein exists in a colloidal particle known as the casein micelle, which contains other components as well as casein, including calcium, phosphate, citrate, minor ions, lipase and plasmin enzymes, and entrapped milk serum.16 This particle is a calcium-caseinate-calciumphosphate complex, and not a true micelle in the colloidal sense.16 The principal casein proteins have been identified as asl~, as2-, /3-, and K-casein.184 Their properties and primary structure have been discussed in Section 1.2.1.1. The identification of two classes of milk proteins, y-casein and part of the proteose peptone fraction, has been determined from primary sequencing to be degradation products or incompletely synthesized precursors of /3-casein.68 The molar ratio of proteins within the

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A third family includes salts, whose concentrations are affected by the natural pH of milk, namely, diffusible Ca, diffusible Mg, diffusible citrate, Ca2+ , and HPO2TAbout 20 to 30% of diffusible Ca and Mg is present as free ions; the remainder exists as citrate and phosphate salts. Diffusible Ca, Mg, and citrate concentrations are positively correlated because Ca and Mg form strong complexes with citrate at the pH of milk. A negative correlation between Ca 2+ concentration and pH and a positive correlation between Ca 2+ and HPO 2 - relate to the solubility product of micellar calcium phosphate. The degree of saturation of micellar calcium phosphate and the concentration of H2POX are relatively constant. For example, increased Ca 2+ causes formation of colloidal calcium phosphate, and the level of H2PO^ is maintained by reaction of HPO2," with H + . Further discussion of acid-base equilibria is presented in Section 1.4.6.

1.3 Structure 1.3.1 Casein Micelles 1.3.1.1 Properties


In their classic monograph on dairy chemistry, Jenness and Patton in 1959 stated that "Many of the problems of dairy technology revolve around the behaviour of the (calcium caseinate-phosphate complex) and particularly the aggregation of the particles by heat, salts, acid, and rennin. Therefore, a study of its composition and properties is a most important phase of dairy chemistry".182 In the 30 years since this statement was made, considerable effort has been put into studying the properties of the casein micelle. The progress to date has recently been reviewed.183"185 The intricacy of the micelles may be related to their biological functions in milkto carry a large amount of highly insoluble calcium phosphate to the mammalian young in liquid form, and to form a clot in the stomach for more efficient nutrition.16'184 The micelle is extremely stable under some conditions of processing, for example, concentration, ultrafiltration, pelleting, drying,183 but very unstable under others, for example, acid, chymosin.16 A manipulation of micelle stability gives rise to many traditional and nontraditional dairy products.17 A summary of the properties and structure of the casein micelle will be presented here. About 75% of the proteins in milk are classified as casein protein, that which precipitates at pH 4.6.68 Most, but not all, of this casein protein exists in a colloidal particle known as the casein micelle, which contains other components as well as casein, including calcium, phosphate, citrate, minor ions, lipase and plasmin enzymes, and entrapped milk serum.16 This particle is a calcium-caseinate-calciumphosphate complex, and not a true micelle in the colloidal sense.16 The principal casein proteins have been identified as asl~, as2-, /3-, and K-casein.184 Their properties and primary structure have been discussed in Section 1.2.1.1. The identification of two classes of milk proteins, y-casein and part of the proteose peptone fraction, has been determined from primary sequencing to be degradation products or incompletely synthesized precursors of /3-casein.68 The molar ratio of proteins within the

micelle at the time of secretion is approximately a s l : a s2 : (j8 + y): K = 4:1:4:1.3 185 or about 38% a s l -, 10% a s2 -, 36% )8-, and 13% /c-casein.184 The K-casein content requires special consideration. It differs from other caseins in that it is soluble over a very broad range of calcium ion concentrations. 186 It plays a unique role during clotting by rennet in that it is cleaved by chymosin at the Phe 1 0 5 -Met 1 0 6 bond into para-K-casein (residues 1 to 105) and the hydrophilic caseinomacropeptide (residues 106 to 169) fractions. 187 ' 188 On either side of this bond lies an unusual proline sequence that may account for this unique specificity. 184 An inverse relationship between /c-casein content and micelle size has been reported. 189 " 191 Micelles in the 154-nm diameter range may contain as little as 4% K-casein whereas those in the 62-nm diameter range contained up to 12% /c-casein.191 Decreases in a s l -, a s2 -, and /3-casein were noted as K-casein increased. Artificial micelles can be prepared if a s l - or /3-casein is absent, but not if K-casein is absent. 192 Thus is plays an important role in casein micelle stabilization. The macropeptide portion, once cleaved, is readily soluble and migrates away from the micelle. The para-casein micelle, devoid of the CMP, coagulates readily with other para-casein micelles. The action of rennet on the micelle is discussed further in Section 1.3.1.3. However, it is important to recognize that K-casein must exist at or near the surface of the micelle, oriented with easy access of the CMP to cleavage by rennet. Thus, this macropeptide is thought to exist in a hairlike manner covering the outer surface of the micelle. 193 " 201 As little as 10% of the K-casein may exist in the hairy layer 200 and the rest of the K-casein may be quite inflexible. 201 About 7% of the dry matter of the micelles consists of inorganic material, principally calcium and phosphate.185 Milk contains approximately 117 mg of calcium per 100 g of milk. Milk serum contains 40 mg per 100 g of serum, only 32% of the calcium content. The rest is associated with the casein micelle, approximately 31 mg per gram of dry casein. Likewise, of 203 mg of inorganic phosphate per 100 g of milk, only 53% is present in the serum and the rest, 37 mg per gram of dry casein, is associated with the micelle. 16 The micelle also contains 5.6 mg of citrate, 3.3 mg of K, 1.5 mg of Mg, and 0.9 mg of Na per gram of dry casein. 16 The approximate composition of the casein micelle is given in Table 1.11. The micelles may contain as high as 7.9 g of water, 202 or milk serum depleted of large solute molecules such as globular proteins, 16 per gram of protein. The water sorption properties of milk proteins have been reviewed. 203 The micelle is more highly solvated than most globular proteins and therefore has a rather porous structure.184 This hydration water is probably not bound but is characteristic of the packing density of hydrophobic side chains. 202 The voluminosity of the micelles may range from about 2 ml per gram of casein up to 4 ml per gram of casein, depending on consideration of the outer hairy layers of the micelle, representing 6 to 12% of the volume fraction of milk. 194 Measured size from electron microscopy and molecular weight measurements from light scattering studies also indicate a very low density packing of the casein monomers into the micelle structure.184 Many studies of the size distribution of the micelles have been made using electron microscopy, light scattering, and controlled pore glass chromatography methods. 204 " 206 An approximate distribution is shown in Figure 1.8. The number average

T a b l e 1.11

COMPOSITION OF CASEIN MICELLES IN COW'S MILK AT ROOM TEMPERATURE, g/100 g OF MICELLES, DRY BASIS 183 Content 35.6 9.9 33.6 11.9 2.3 93.3 2.87 0.11 0.11 0.26 2.89 0.40 6.6

Component a sr Casein cts2-Casein p-Casein K-Casein Casein fragments Whole casein Calcium Magnesium Sodium Potassium Inorganic phosphate (PO4) Citrate Total inorganic material

Number frequency (%) N V Volume

frequency (%)

Diameter (nm)
Figure 1.8 Examples of the size frequency distribution of casein micelles. Number (Af, lefthand ordinate) and volume frequency (V, right hand ordinate), both in percentage of total per 50-nm class width, against micelle diameter. (From ref. 16. Reprinted by permission of John Wiley & Sons.)

diameter has been reported as 25 nm,207 up to 140 run,184 and the volume surface average diameter as 86 nm.207 The population may be bimodal with a volume surface average diameter peak at 90 to 180 nm, with a lesser peak at >200 nm,205 although results vary according to technique used. A small number of very large particles, up to 800 nm in diameter, and a large number of small particles, maybe representing submicelles, have been reported.16 The size distribution of the micelles may have an impact on both nutritional and technological considerations.205'208 The molar ratios of the casein fractions of the micelle given above refer to the micelle at the time of secretion. However, it has been recognized that some of the casein fractions, particularly /3-casein, are able to migrate out of the micelle to the serum phase in a reversible manner without causing collapse of the micellar structure. 209 " 212 This migration is temperature dependent.211 As much as 60% of the /3-casein has been found in the serum phase after 48 h at 40C.210 This serum /3-casein is free to interchange with micellar casein.211 These changes, however, are reversible on rewarming to 37C. This cold dissociation phenomenon has pedagogical impact on a model for the casein micelle, and also has technological impact, particularly on the cheese industry, as cheesemaking parameters may be altered210 and enhanced proteolysis of the /3-casein by plasmin and proteinases of the bacterial microflora may occur in the serum phase211'213 and may lead to the formation of y-caseins.212 All of the above considerations have led to numerous proposed models for the casein micelle. The earlier models have been reviewed183'184'192-214"218 and the relevant portions of each have been combined. The most accepted model seems to be that elaborated by Schmidt. 183 " 185192 Casein micelles consist of a number of smaller units, referred to as submicelles, which may be 8 to 20 nm in diameter.207'219"221 These have been differentiated by electron microscopy which shows the micelle to have a "raspberry-like" structure. 183222 " 224 This is shown in Figure 1.9. The size of the submicelles is not uniform but is governed by concentration, pH, ionic strength, and temperature.220 They may contain between 15 and 25 casein molecules,185 with molecular weight 250,000 to 2,000,000. The submicelles contain a hydrophobic core and are covered by a hydrophilic coat much less dense than globular proteins,202 which is at least partly comprised of the polar moieties of I O C /c-casein. The stoichiometric ratio of caseins as noted above at 37C, varies between individual submicelles, some being rich in K-casein and others depleted in /c-casein.226'227 The hydrophilic C-terminus end of /c-casein, the CMP, exists as a flexible hair, behaving more or less as a random-coil polymer chain.16 The effective thickness of the hairy layer is at least 7 nm.185 It must be recognized that this Kcasein probably exists as an oligomer of, on average, six molecules.185 Consequently, steric repulsion would prohibit further aggregation of submicelles to the surface rich in CMP.228 The recognition that /c-casein exists at the surface of the micelles implies that submicelles rich in /c-casein occupy a surface position whereas those with less K-casein are buried in the interior, although K-casein has been isolated from within the micelle whereas a sl - and /3-casein are scattered throughout the micelle including the surface. 190192196 ' 202 ' 229 Many hydrophobic patches probably exist on the surface of the micelle.184

Figure 1.9 Casein micelles (A and B) from yogurt prepared by the fixation method of ref. 225 and examined by scanning electron microscopy with field emission. (Courtesy of A. Smith.)

B hydrophobic core

GMP "hairy" layer K - casein enriched surface

cluster

Figure 1.10 Schematic representation of the casein micelle (A) and submicelle (B).16'183'184

Colloidal calcium phosphate (CCP), probably in the form of C a ^ P O ^ clusters,183 although this remains unclear, 185 ' 230231 acts to aggregate submicelles into micelles 232 and thus plays a very important role in maintaining the integrity of the micelles. The micelle may contain hundreds or even thousands of submicelles. 233 Micellar growth would come to an end when the whole surface consists of K-casein.183'185 The high voluminosity of the micelles implies a loose spongelike structure with much interstitial water and highly hydrated hydrophilic groups at the surface,194*234 which probably allows for migration of /3-casein in and out of the micelle as a function of temperature.211'212 This may also imply a structural role for a s l - casein. 235 " 237 The model for the casein micelle based on the above discussion is illustrated in Figure 1.10. Association of submicelles may occur in the Golgi vesicles within the secretory cell. Calcium and phosphate pass through the membrane into the Golgi vesicles and when their concentration is sufficient to form Ca9(PO4)S clusters, micellar aggregation may occur. When the Golgi vesicles fuse to the apical plasma membrane, micelles are emptied into the alveolar lumen. 183 Micellar growth may continue in the lumen; micelles of 1 /urn diameter have been observed in this area.205

1.3.1.2 Stability
It should be recognized from the above discussion that a precise mechanism of formation and ultrastructure of the casein micelle is still uncertain; nevertheless, a great deal is known about this complex colloidal particle. A number of factors are responsible for holding this micelle together and giving it stability, including the role of colloidal calcium phosphate, disulfide bonding, hydrogen bonding, hydro-

phobic interactions, electrostatic interactions, van der Waals forces, and steric forces, 184 ' 185 - 217 ' 238 and these will be discussed here. More than 90% of the calcium content of skim milk is either associated in some way with other ions or found within the casein molecules. 184 There are two distinct forms of ions associated with the casein micelle: an outer system, perhaps in the form of a charged double layer, and an inner system not easily washed away. 184 An amount of calcium approximately equivalent to the number of ester phosphate groups appears to be bound to the casein monomers. 192 A small amount of calcium is bound to micellar citrate. The balance of the calcium in the micelle may be in the form of amorphous tertiary calcium phosphate, Ca 3 (PO 4 ) 2 , found in clusters of Ca 9 (PO 4 )S. 183 It must be prevented from being transformed into a more stable form such as hydroxyapatite by the casein, although small ions such as magnesium, which has also been isolated from the micelle, may play a role. 231 Casein may also prevent sedimentation of tertiary calcium phosphate. The exact nature of the colloidal calcium phosphate complex in the micelle is undetermined, yet its role in casein micelle stabilization is well documented. 185 ' 230 The removal of calcium ions from the micelle causes reversible dissociation of /3and /c-casein from the micelles without micellar disintegration, whereas the addition of excess calcium favors micellar component aggregation. 232 ' 235 Mineral solubilization at low temperature may be responsible for the dissociation of /3-casein, which indicates its responsibility for micelle stabilization. 209 ' 235 ' 239 The mineral complex found in the micelle can be reproduced only when calcium, phosphate, and citrate are present. 224 The nature of the binding between CCP and casein has been the subject of much speculation. Various types of covalent bonding have been proposed. 183 The phosphoserine residues of the caseins are potential sites for interaction. Binding may also be electrostatic, between the negatively charged ester phosphate group of casein and the Ca 9 (POJ 6 clusters which, with the adsorption of two Ca 2 + ions, are positively charged. 183 In addition to binding submicelles together, the CCP may be responsible for a fairly rigid conformation of caseins within the submicelle. 201 Hydrogen bonding between casein monomers in the casein micelle may occur and hydrogen bonding between ionizable side chains or residues and solvent, water, exist within the micelle. 184 Many bioproteins, including the milk serum proteins, exhibit considerable secondary and tertiary structure, in the form of a-helix or j8-sheet configurations.71 These structures are stabilized by hydrogen bonds along the primary backbone of the protein. Spectral investigations such as circular dichroism or infrared spectroscopy of the isolated caseins have shown that these proteins possess little tertiaiy structure. Also, if a secondary structure is present, it is somewhat disorganized, possibly due to high levels of proline residues 16 which tend to inhibit the formation of secondary and tertiary structure. It has been stated that at least 75% of the three major caseins, a s l -, /3-, and K-, exist in an aperiodic conformation. 184 Because little periodic structure occurs in the individual components, the degree of stabilization of the micelle by a-helix or jS-structure is probably quite low. However, it has recently been demonstrated by Raman spectroscopy that 40% of whole casein in submicellar form may have a /3-turn structure.202 The role of hy-

drogen bonding between the various casein components with the micelle is still unclear. Disulfide bonds between cysteine residues normally accounts for the development of, or at least the stabilization of predeveloped, tertiary and possibly quaternary structure within bioproteins. Both a sl - and /3-casein contain no cysteine residues and therefore do not enter into disulfide bonding.16 However, as2- and K-casein contain two cysteine residues each, and therefore the potential for disulfide bridges cannot be excluded.16 Disulfide-linked aggregates of /c-casein have been isolated from the micelle, and it is thought that many molecules of /c-casein must exist contiguous to each other in the micelle in order to form such aggregates.184 It would appear, however, that if disulfide bridges appear within the micelle, they are not the driving force for micelle formation. Hydrophobic interactions result from the presence of apolar amino acid residues within a protein molecule. These residues are forced out of the solvent, water, and into the interior of the protein molecule, where they can interact with other apolar groups. A small gain in entropy and stabilization energy results. Hydrophobic interactions are highly temperature sensitive and are minimized at 5C or less. Hydrophobic interactions are also reduced by increased pressure.184 The caseins rank among the most hydrophobic proteins71 and thus it should be expected that the micelle is at least in part stabilized by hydrophobic interactions. Increased pressure tends to dissociate casein and disrupt casein micelle structure.219'240 Low temperatures also have a disruptive effect on the micelle as evidenced by the cold dissociation of /3-casein from the micelle212 and by its sensitivity to freezing.184 However, micelles are quite stable to moderate to high temperatures.185 These factors are evidence for some role of hydrophobic interactions in the stability of the micelle. Electrostatic interactions between amino acid side chains and ions in solution can impart reasonable structural stability to a protein. The role of inter- and intramolecular ionic bonds among the casein in stabilization of the micelle is unclear; however, there are a number of sites for potential ionic bonding within the casein molecules, and these may play a role in subunit interactions.184 Both calcium and phosphate are critical for micelle stability, as discussed above. Binding between colloidal calcium phosphate may be electrostatic in nature, as CCP is positively charged and the casein is negatively charged.183 Due to the number of charged groups on casein monomers, not all of them can exist at the surface. However, due to the open structure generally recognized for the micelle, solvent may be available within the micelle itself.184 Ethanol, for example, tends to decrease solvent interactions and is known to lower the stability of the micelle.185 Van der Waals forces are always attractive and are formed from the establishment of a dipole moment as a result of the fluctuating electron cloud about an atom or molecule. The interaction falls off proportionally to an inverse power of the interparticle distance; however, the proportionality coefficient, the Hamaker constant, is uncertain.16 The DLVO theory of interparticle stability for lyophobic colloids (after Deiyagin, Landau, Verwey, and Overbeek) relates London-van der Waals attraction to electrostatic double layer repulsion as a function of interparticle distance.241 Many attempts have been made to relate DLVO theory to micelle stability and aggregation

with limited success.185'217'233 Although the casein micelles act in many cases as lyophobic colloidal particles, their behavior deviates in other cases making stability and aggregation of micelles very difficult to explain in terms of DLVO principles.233 Steric stabilization results from the presence of adsorbed macromolecules onto a colloidal particle and the protrusion of molecular chains (called "hairs") from the particle. This may interfere with interparticle approach through either compression and thus restriction of freedom of motion and loss of conformational entropy or through interpenetration of the hairs resulting in either osmotic repulsion or interparticle attraction, depending on the solvation properties and hydrophilic nature of the hairs.241 As is noted in the preceding discussion, the role of /c-casein at the surface of the micelle acts to give the micelle a hairy layer associated with the protrusion of the caseinomacropeptide into solution. Thus it follows that much of the stability of the micelle to flocculation must be associated with steric stabilization.* The stability of the micelles usually correlates well with their voluminosity, corresponding to a more extensive hairiness and a stronger steric repulsion. However, voluminosity also affects the Hamaker constant and consequently van der Waals forces, and also the electrokinetic or ^-potential, which makes a precise determination of forces difficult.16 At cold temperatures, the dissociation of /3-casein from the micelle212 may give rise to another source of hairiness and steric stability. This makes it difficult to assess the relative role of hydrophobic versus steric forces in describing the stability of the micelles to the action of various environmental factors including a lack of aggregation by chymosin at cold temperatures.185'245 However, the reduction in hydrophobic bonds may be responsible for the release of the /3-casein from the micelle, assuming that they play some role in holding the micelle together. The importance of steric stabilization is discussed further in Section 1.3.1.3. with regard to aggregation processes, particularly the action of chymosin on the micelle.

1.3.1.3 Aggregation
Although casein micelles are fairly stable, there are at least four technologically significant ways in which aggregation of casein micelles can be made to occur.185 These include the action of the proteolytic enzyme chymosin on the micelle, such as in the cheese industry; the aggregation of casein by acid, as in the manufacture of some types of cheeses and fermented products; aggregation caused by heating; and the age gelation of micelles, which is important in the preparation of sterilized, shelf-stable milk products. Each of these will be discussed in some detail. There are many commercially available milk-clotting enzymes. Of these, calf rennet, whose active principle is chymosin, rennin (EC 3.4.23.4) is the most common. Also used are bovine pepsin, porcine pepsin, and microbial aspartic proteinases from Mucor miehei, M. pusillus, and Endothia parasitica.246 The milk clotting ability of aspartic proteinases results from the cleavage of a specific linkage (Phe105-Met106) of /c-casein.187'188'247 There are three distinct but overlapping stages during the enzymatic coagulation of milk.217'248~250 Enzymatic cleavage of the
Refs. 185, 193-195, 197, 198, 200, 228, 242-244.

Phe 105 -Met 106 linkage of K-casein results in the formation of the soluble CMP which diffuses away from the micelle and para-/c-casein, a distinctly hydrophobic peptide that remains on the micelle.251 This is a relatively quick reaction, the turnover being 100 s~ l in milk, pH 6.7, 300C,16 and seems to be independent of micelle size.208 The loss of the CMP results in decreased steric stabilization of the paracasein,195 and may also result in a reduction in electrostatic repulsive forces and increased micellar hydrophobicity,187 and leads to the formation of small aggregates and chains consisting of destabilized paracasein micelles of various lengths, complexed with calcium.252 Paracasein micelles have much reduced voluminosity and potential, compared to native casein micelles, but otherwise are not disintegrated.208 The action of the enzyme on the micelle is unclear.242253 It probably cleaves CMP molecules fairly randomly,244 although it may cleave a patch or an entire micelle of CMP before moving on. However, the formation of a reactive site or patch on the micelle is necessary before aggregation of paracasein micelles can begin. This requires cleavage of > 85% of the CMP,187'188 although this is pH dependent.254-255 Enzymatic cleavage of CMP through the use of immobilized enzymes has been very difficult to achieve,256 probably due to the proximity of the Phe-Met bond to the micelle surface and the orientation of the bond to the enzyme.185 The second stage involves coagulum or curd formation following enzymatic action.257 As a result, the lag time before clotting depends on both the time for enzyme action and the production of appreciable concentrations of aggregatable material, namely paracasein micelles.233 The forces of attraction between paracasein micelles include van der Waals, possibly electrostatic interactions in the form of salt bridges between positively and negatively charged regions of paracasein micelles, possibly mediated by Ca 2 + ions or through CCP-like linkages,185 and possibly hydrophobic bonding. It has been observed that renneted micelles will not aggregate at 5C and this is often used as evidence for the importance of hydrophobic bonding, although the temperature dependence of aggregation may also be related to /3-casein dissociation and resulting steric stabilization from /3-casein hairs (see Section 1.3.1.2). The Ca 2+ concentration and the colloidal/soluble calcium phosphate balance are critical, as calcium is needed for continued aggregation.245 Renneted micelles nearly always lead to the formation of a gel rather than a precipitate due to the steric retardation of the clotting as a result of a restricted number of reactive sites on the micelle surface and thus a large number of unsuccessful collisions.233'258'259 This must be related to the action of the enzyme in cleaving a portion of the CMP hairs. This is illustrated in Figure 1.11. The final stage in the clotting of milk is not well defined and includes syneresis and firming of the curd,260""263 a loss of paracasein micelle identity, and nonspecific proteolysis of caseins in the coagulum.187 The paracasein micelles fuse into larger units as CCP rearranges throughout the micellar region,264'265 and may be analogous to binding between submicelles in a casein micelle.266 Micelles can also be destabilized or aggregated by a reduction in pH,267 independent of enzymatic action, as in the manufacture of directly acidified cheeses or in fermented products. The first consequence of a lowering of the pH below 6.7 is partial dissolution of CCP and a decrease in micelle voluminosity.254'268 Dissolution

Figure 1.11 Clotting of casein micelles. (A) Surface completely reactive: all collisions will lead to sticking. (B) Surface only partly reactive: unsuccessful collosion. (C) Surface only partly reactive: successful collosion. (D) Total surface reactive: dense precipitate. (E) Surface only partly reactive: loose network. (From ref. 233 with permission of Cambridge University Press.)

of some of the micelles into submicelles may also occur and has been reported at pH 5.6. 254 At pH < 5 . 5 , micelles may fuse as the surface potential is lowered. The potential approaches zero at near pH 5.2. 16 Most of the CCP will be lost at this pH. Near pH 4.8, nearly all of the CCP is dissolved, and at pH 4.6, the isoelectric point, the solubility of casein is negligible. 71 - 186 Casein micelles have disintegrated and casein precipitates.81 Aggregation occurs as a result of entropically driven hydrophobic interactions.268 A further method of micellar destabilization is through heating to temperatures above the boiling point.269""271 Although casein is not denaturable, casein micelles irreversibly aggregate. Chemical changes of the casein must occur because the addition of agents that break H bonds, reduce disulfide linkages, and dissolve calcium phosphate leave the aggregates intact.16 The coagulation time is very pH dependent, and two distinct behaviors for lots of milk have been shown. 272 Most samples of milk require a maximum time for heat coagulation at 1400C when adjusted to pH 6.7 and a minimum time at pH 6.9 (type A behavior). However, some milk samples fail to show minimum and maximum points but instead increase in coagulation time

as pH is increased from 6.2 to 7.4 (type B behavior).185-272 The pH change caused by heating may be primarily responsible for the heat coagulation phenomena.273 On heating, the buffer capacity of milk salts change, carbon dioxide is released, organic acids are produced, and tricalcium phosphate and casein phosphate may be precipitated with the release of hydrogen ions. The pH at the time of coagulation, when measured at room temperature, is always low, <6.2. Heat coagulation can be prevented by adding alkali to maintain the native pH. In addition, /3-lactoglobulin precipitates onto micelle surfaces274'275 and a /c-casein-/3-lactoglobulin complex that associates and dissociates from the micelle as a function of pH may be responsible for the erratic behavior of casein to heat coagulation.269 The effects of heat on milk proteins will be considered more fully in Chapter 2. Age gelation of casein micelles is another aggregation phenomenon with both pedagogical implications about the casein micelle and technological implications in the production of shelf-stable, sterilized products. Destabilization of casein in milk which has been treated under UHT conditions, for example, 142C for 5 s, followed by aging at ambient temperatures for weeks or months, leads to the formation of age thickening and age gelation.129'276 Usually a sudden sharp increase in viscosity accompanied by visible gelation and irreversible aggregation of the micelles into long chains forming a three-dimensional network occurs.192 The action of heat-resistant proteinases, either bacterial in origin or native plasmin enzymes, may be responsible for proteolysis of casein during storage. However, plasmin may cause dissolution of casein rather than gelation.16 Proteolysis activity in UHT-treated products has been reported to be very low, but it has been pointed out that a very limited number of reactive sites would lead to the formation of a loose gel after considerable time. However, proteolysis may not be required for gelation. Changes in the protein during heat treatment, a polymerization of both casein and whey proteins due to Maillard type or other chemical reactions, or the formation of K-casein-/3-lactoglobulin complexes might also be responsible for age gelation.129-276

1.3.2 Fat Globules

1.3.2.1 Native Fat Globule Membrane


Lipid droplets in milk are covered by a thin membrane, 8 to 10 nm in thickness, that reduces the lipid serum interfacial tension to very low values, 1 to 2.5 mN/m, preventing the globules from immediate flocculation and coalescence, and protecting them from enzymatic action.16 The surface area of this membrane in milk is considerable, approximately 80 m2/L of milk.16 Consequently, this membrane has a large impact on the technical aspects of milk processing.277 The FGM has been extensively studied and reviewed.27'277""291 Fat droplets appear to originate in the secretory cell of the mammary gland as small precursor "lipovesicles" in the endoplasmic reticulum and to migrate through the cytoplasm to apical regions of the cell. These droplets appear to grow during migration through the cytoplasm by the fusion of lipovesicles with larger droplets. It is now widely accepted that these lipid droplets acquire the native FGM by budding

directly from the apical cell membrane. 284 The compositional similarity between milkfat globule membranes and apical cell membranes 286 and electron micrographs illustrating this budding process within the secretory cell 281 - 284 are strong evidence for this process. Therefore, the milk FGM is in part, or at least derived from, a lipid bilayer membrane. The fat globule may acquire an inner coat of adsorbed molecules as it passes through the cell cytoplasm before it is enveloped in the cell membrane. 284 Two principal components of this inner coat material are the enzyme xanthine oxidase and the glycoprotein butyrophilin, which may have specific functions in the recognition and envelopment of lipid droplets in apical plasma membrane. 281 This tightly bound inner coat has been observed by electron microscopy and is found as paracrystalline arrays covering only limited areas of and slightly pressed into the triglyceride core. 292 It is possible that other cytoplasmic molecules such as sterols, phospholipids, gangliosides, and proteins may also be adsorbed to the triglyceride core prior to envelopment by the cell membrane. 16 Considerable rearrangement of this membrane is thought to occur shortly after its release into the lumen. 277 - 287 The lipid bilayer membrane of the cell was bordered on each side by an aqueous environment; however, one side has now been brought into close contact with the lipid droplet. Part of the more apolar substances of the membrane may dissolve into the core. Polar substances may dissolve into the serum. Amphiphilic substances from the milk plasma may adsorb onto the fat globule. Enzymatic changes of both the lipid and protein portions of the membrane may also occur. 16 Several methods are available for isolation of the milk FGM, and depending on the procedure, compositional changes have been found to occur, making quantification of the membrane more difficult. 284 ' 293 - 294 Some authors have reported the presence of a significant quantity of high melting point glycerides on the innermost edge of the membrane that are closely associated with the membrane.27-278 This, in addition to evidence of fat globule birefringence under a polarizing microscope, 295 has led to speculation as to the structural nature of the lipid core and the crystallization characteristics of the globule. 27 - 125 - 296 " 300 It appears that the high melting glyceride (HMG) fraction and the possibility of a partially crystalline fat globule existing as a solid shell of HMG and an inner liquid core may be artefacts of the preparation procedure. 295 ' 301 " 305 On the outermost edge of the membrane, isolation procedures can also lead to either adsorption of plasma material or desorption of membrane material, leading to a high degree of variability in the reported composition of the FGM. 294 The estimated average composition of the natural FGM is given in Table 1.12. The composition of milk lipids has been discussed in Section 1.2.2.1. More than 95% of the total milk lipid is found in the globule fraction. Approximately 1% of the total lipids of the globule are associated with the membrane, whereas the remainder are found in the core. The vast majority of the globule core, 98 to 99%, is comprised of glycerides, mostly triglycerides. 284 The presence of diglycerides in the core varies, and may be due to either incomplete triglyceride synthesis or to lipolytic cleavage of fatty acid from the triglyceride.27 In addition to glycerides, the fat globule

Table 1.12 ESTIMATED AVERAGE GROSS COMPOSITION OF NATURAL MILK UPID GLOBULE MEMBRANES
Components Protein Phospholipids Cerebrosides Gangliosides Cholesterol Neutral glycerides Hydrocarbons Ribonucleic acid Carotenoids Iron Molybdenum Copper Water Total mg per 100 g Fat Globules 900 600 80 20 40 + 20? + 0.04? 0.3 0.05 0.01 200? >1860 mg per m2 Fat Surface 4.5 3.0 0.4 0.1 0.2 + 0.1 + 2 X 10" 4 15 X 10" 4 2 X 10" 14 0.5 X 10~ 4 1.0 >9.3 Percent of Membrane 48 33 4 1 2 ? 1 7

11 100

From ref. 16. Reprinted by permission of John Wiley & Sons.

core contains some free fatty acids; sterols; phospholipids; glycolipids; carotenoids; vitamins A, D, E, and K; water; and other miscellaneous components.16 It is highly probable that these components, including a very large number of different triglycerides, are randomly distributed throughout the core. Crystallization of the globule may cause a stratification of the HMG to an outer layer, but this is questionable.302*304 It may be that the adsorbed layer acts as the nucleating site for lipid crystallization to begin.27'297 Some of the lipid in milk cannot be separated by centrifugation, and this has been termed serum lipid.284 This may be due in part to the presence of very small globules with dense membraneous layers of a density high enough to cause them to sediment rather than cream during centrifugation.278 The FGM is responsible for giving the fat globule many of its characteristics in milk. Consequently, its behavior during processing is of great interest.277 The conditions of the fat globule change greatly after milking, owing primarily to cooling and crystallization of fat, and agitation. Cooling leads to a migration from fat globules to milk plasma of about 20% of its phospholipid content, and about 30% of its copper, xanthine oxidase, and other substances.16 When membrane material is lost due to damage, other amphiphilic molecules in the milk plasma become adsorbed to the fat globule.16 Foaming can lead to considerable loss of membrane material as it spreads over the air plasma interface. Action of bacterial enzymes, for example, phospholipases, may also lead to changes in the membrane. The membrane is responsible for the separation of natural milk lipases from the lipids of the fat globule. If it is damaged, lipase action can cause lipolytic rancidity to occur in milk. Disruption of globules leads to a greatly increased surface area, causing some desorption of natural FGM, and considerable adsorption of plasma proteins. This is certainly the case during homogenization of milk, as will be discussed in the next section.

Due to the amphiphilic nature of many of the membrane components, for example, proteins and phospholipids, isolated FGM material exhibits greatly increased functional properties such as emulsification and foaming. 306 Dairy products that are enhanced in membrane material are thus known to have improved functional characteristics. However, this material may oxidize rapidly under improper storage conditions. 307 In the churning of butter, for example, membrane material is lost to the buttermilk,27 and buttermilk powder can greatly increase whipping and foaming properties of foods to which it has been added, for example ice cream mix, 308 more so than skim milk powder. Likewise, recombined milk from butter will be nearly devoid of natural FGM material, and will form a much different adsorbed layer on the fat globule as a result of homogenization. 309

1.3.2.2 Recombined Membranes


The natural FGM in raw milk is of tremendous interest; however, in most milk products, processing steps such as homogenization have greatly changed the characteristics of adsorbed layers onto fat globules. Recombined membranes are much different than native membranes, and their presence, composition, structure, and behavior are thus of great interest in dairy processing and dairy products. In discussing size distributions of fat globules, volume surface-weighted average diameters (dvs) are preferred to number average (d) as the latter are skewed by the large number of relatively small particles. Volume surface-weighted average diameters relate total volume to total surface area of the dispersed phase, dvs = 2 N1 d, 3 /2 N1 d\ where N1 and dt represent number of globules of diameter i. 16 Homogenization creates fat globules of dys < 0 . 5 ^m, depending on the conditions of homogenization. 310 The surface area is increased by four- to sixfold or more as a result of this disruption.27'280 Some of the native FGM will remain adsorbed to the fat; 311 ' 312 however, there is not nearly enough present to cover this newly created surface area. On immediate disruption, the fat plasma interfacial tension raises to a high level, 15 mN/m, 16 and amphiphilic molecules in the plasma will quickly be adsorbed to the lipid droplet to lower this value. This material consists mainly of plasma proteins, and may diffuse and adsorb within 10 to 100 ms after disruption.302 Figure 1.12 shows an homogenized fat globule with considerable adsorption of casein micelles to its surface. Several studies have been conducted on recombined milk products, those made by homogenizing a mixture of skim milk and butterfat,309'313"315 and on homogenized milks 312 - 316 and creams. 317 Adsorbed layers consist mainly of serum proteins and casein micelles, 313 ' 317 although other molecules such as phospholipid 16 and lipoprotein complexes different from the native FGM are also found. 312 Spreading of the casein micelles on the surface of the fat droplet can occur. 313 ' 318 The disruption of the micelle into constituent subunits may also occur as a result of surface adsorption.317 The surface excess is defined as the protein adsorbed per surface area of fat in an emulsion. Values reported for fat globules in various aqueous phases include the following: 20 mg/m 2 in skim milk; 40 mg/m 2 in casein micelle suspensions; 5 mg/m 2 in suspensions of casein subunits; and 1 mg/m 2 whey protein solutions. 309

Figure 1.12 Homogenized milkfat globules (F) prepared by transmission electron microscopy thin sectioning showing adsorbed casein micelles (P) and evidence of internal fat crystallization (arrow).

Surface excess is higher for smaller fat globules in milk plasma, for example, 15 mg/m 2 for globules of diameter 0.4 ^m, and 3 mg/m 2 for d ** 1.6 /x.m 3 1 4 3 1 9 Casein particles may be preferentially adsorbed over serum proteins. 317 Serum proteins have been reported to cover 25% of the globule surface of fat homogenized in milk solids, but account for only 5% by weight of the membrane protein due to the different molecular conformations of the two classes of proteins. 314317 This protein adsorption is irreversible within a limited time frame (hours) but considerable rearrangement of the adsorbed layer occurs as molecules compete for surface space and as surface denaturation occurs. 302 However, small molecule surfactants can displace proteins readily from the surface, probably due to a lowering of the interfacial tension. 309 ' 320 ' 321 Heat can also affect the adsorbed layer of recombined fat globules. A higher temperature during adsorption, up to about 70 0 C, causes a thinner protein layer, 10.7 mg/m 2 at 40 0 C versus 6.0 mg/m 2 at 60 0 C, probably due to a faster rate of casein spreading.309 However, a heat treatment of the skim milk prior to homogenization leads to a thicker adsorbed layer, 15 mg/m 2 at a preheating temperature of 95 0 C for 10 min. 309 A significant quantity of /3-lactoglobulin was recovered from the membranes of pasteurized cream. 316 UHT products also exhibited enhanced adsorption of caseins and serum proteins, particularly /3-lactoglobulin.312 The adsorption of higher levels of serum proteins after heating results from the partial denaturation of the proteins87 and the association of serum proteins and caseins as a result of heat treatments.71'275 Consequently, fat globules can be created with a variety of mem-

N Number/ ml (xlOexp-9) % % Lipid

Diameter (jim)
Figure 1.13 Size distribution of lipid globules in milk of a Holstein cow. The number of globules (N) of various diameters and the percentage of the total lipid present in globules at indicated diameters are plotted. (From ref. 27 with permission of Pudoc, Wageningen, Netherlands.)

branes depending on the protein composition of the homogenizing solution and on the processing conditions. 322

1.3.2.3 Stability
Milk is an emulsion of fat droplets, and no emulsion is thermodynamically stable. 306 Thus the stability of the fat emulsion is a kinetic time-dependent phenomenon. Milk is known to separate or cream spontaneously and rapidly,302 and many processes and products involve manipulation of the creaming phenomenon. Emulsion stability is largely dependent on the size distribution of the globules. In raw milk, fat globules range in size from 0.1 to 15.0 fjum. The milk emulsion has been found to contain three distinct populations of fat globules. 323 " 325 These populations may be synthesized differently within the secretory cell. 16 About 75% of the number of globules in milk are < 1 /nn in diameter, and represent only a small percentage of total milk fat. Methods for determination of globule size distribution must account for this population for accuracy of results. 326 A few globules, representing about 2 to 3% of the fat, are > 12 fxm in diameter. These may arise by coalescence within the lumen or mammary cistern. 16 Ninety percent of the fat is found in globules in the 1 to 8 /nm range. 284 The size distribution profile for Holstein milk is shown in Figure 1.13. A calculation of the "average" diameter is difficult as a result of this trimodal distribution, and many values have been reported. The volume surface average diameter, dvs, calculated as dvs = S N1 d]lX N1 dj, where N1 and dt represent number of globules of diameter z, is about 3.4 /xm for milk from Holstein cattle.27 This value relates the surface area of the fat to its volume. The surface area, A, in cm 2 /ml, can then be calculated as A = 670 G/dvs, where G is the gravimetric fat percentage. The relative standard deviation of the surface weighted distribution has been found to be

remarkably constant, around 0.5.302 Thus the fat emulsion for raw milk can generally be characterized by two factors, the gravimetric fat percentage and the average globule diameter, dys. The main factors affecting globule size are breed, individual cow, and stage of lactation. Milk from breeds with higher fat contents, Jerseys and Guernseys, has been found to have larger fat globules, dvs = 4.5 /xm, than milk from animals with lower fat content, Holsteins.27 Average globule diameter is reduced as lactation progresses.284 Stokes' Law predicts that fat globules will cream, due to the differences in densities, p, between the fat, / , and plasma, p, phases of milk (pf 920 kg/m3 at 25C, pp ** 1030 kg/m3).16 However, the fat globules in cold, raw milk will cream much faster than is predicted from Stokes' Law based on their size distribution alone.284'302 Sampling of milk for determination of fat content must take this into account. This fast rate of rise results from the formation of fat globule clusters which may exceed 800 jjim in diameter.280 One of the immunoglobulins in milk, IgM, acts as an agglutinin, for example, flocculating bacteria. IgM forms a complex with lipoproteins and possibly other components known as cryoglobulin that precipitates onto fat globules to an increasing extent as temperature is lowered.16'280'302 Once flocculation of fat globules as a result of cryoglobulin precipitation begins, the speed of globule rise increases. Smaller globules are thus swept out of the milk plasma by these large globule clusters whose speed of rising continues to increase.306 The cream layer forms very rapidly, within 20 to 30 min, in cold milk.27'280 This can easily be redispersed, however, by agitation which causes the cryoglobulin to become associated with individual globules.16 This agglutinin factor is inactivated by heat or homogenization.302 The stabilization of the fat emulsion in milk is principally achieved through homogenization, which causes the fat globules to become disrupted to form much smaller globules. Homogenization is performed at temperatures that render the fat globule completely liquid, a prerequisite for disruption.27 Lipid globules in homogenized milk typically have fat globule diameters of 1 /im or less, and the size distribution profile is greatly narrowed, causing the speed of globule rise to be similar for the majority of globules.310 In addition, the formation of the adsorbed layer onto the nascent globule immediately after homogenization brings the density of the globule closer to that of the continuous phase, again slowing down of the rate of globule rise.311-318 Creaming rate after pasteurization is much slower than would be predicted from the Stokes equation. This results from the destruction of the IgM factor (which is not considered in the Stokes equation), the enhanced adsorption of partially denatured serum proteins to the fat surface as discussed in the next section, and for other reasons.325 Fat crystallization has also been shown to greatly affect the stability of the fat globule.327 The coalescence stability of oil-in-water emulsions with crystals in the disperse phase was decreased by six orders of magnitude over a noncry stalline emulsion. It was hypothesized that the crystals were protruding into the aqueous phase, causing surface distortion of the globule which led to rapid coalescence during shear.328

1.3.2.4 Destabilization
The stability of the milkfat emulsion is an important criterion for the manufacture of many dairy products. An emulsion is not in a thermodynamic equilibrium because it is not at its lowest energy state, energy being stored at an interface.241 The forces acting on a particle in solution include: electrostatic repulsion from the formation of an electrical double layer around a charged particle; attraction forces, mainly van der Waals; steric forces from adsorbed macromolecules; hydrophobic forces; and applied external fields. These must all be accounted for in determining emulsion stability.241 However, an activation energy for flocculation and coalescence must be overcome.302 Flocculation generally refers to a reversible aggregation process in which the individual identities of the particles have been maintained. Coalescence is the flowing together of two emulsion droplets into one. In such products as fluid milk or coffee cream, the emulsion must be very stable and disruption of the natural emulsion through high-pressure homogenization is often performed to add stability to the fat emulsion.16-329 On the other end of the scale, flocculation and coalescence are necessary to bring about a complete churning or separation of the fat phase necessary for butter making.27 The third group of products, represented by heavy cream for whipping or ice cream, requires an emulsion that is stable in the liquid form but that will undergo flocculation, clumping, and partial coalescence but not to the point of complete churning when a shear force is applied.16'27-306'328 The applied force must be sufficient to cause the flocculation to occur. This requires an input of energy to overcome thermodynamic repulsion between the globules. However, the applied force must not be large enough to complete the phase inversion. Different kinds of aggregates of fat globules are recognized. Floccules are easily redispersed as the fat globules that flocculate keep their identity and are held together only by weak forces. Fat floccules are formed, for example, in the creaming of raw milk.328 Clusters are bound together by stronger forces, because the globules may share part of their interfacial layers. They are more difficult to redisperse. Examples are clusters that are formed during single-stage homogenization of a milkfat emulsion.330 Clumps of fat globules can form when partially solid/partially liquid globules are brought into contact.328 If the globules were solid, they would not clump; if the globules were liquid, they could coalesce into one larger droplet. Clumps of fat globules are important in ice cream and whipped cream destabilization, and also in the initial stages of coalescence during buttermaking.27 In all of these products, a partially crystalline fat globule is necessary for successful manufacture of the product.280 Three types of aggregation processes can occur: (1) weak attractive forces as described, for example, by DLVO theory and that may play only a small role in milkfat emulsion stability or instability, (2) polymer bridging where a macromolecule, such as a protein or polysaccharide, adsorbs onto more than one droplet to form a particle-particle bridge, and (3) an aggregation process where any part of the membrane material between two adjacent droplets is disrupted and the aggregate becomes fat continuous at the site of membrane rupture.328 This clumping phenom-

enon is possible only in partially crystalline emulsions, 302 which accounts at least in part for the role of temperature in aggregation processes. Polymer bridging via milk proteins is common in fat protein aggregates found in many dairy products such as homogenized milk where a casein micelle 330 or a composite of milk proteins 317 can adsorb onto two or more fat globules simultaneously. Clustering of fat globules subsequent to homogenization is increased as the fat surface area is increased, either by an increased fat content as in cream, or by an increased homogenization pressure.27 Clustering is also increased if protein is limiting. 16 The third process is important in terms of milkfat destabilization in ice cream 331 or whipped cream 332 manufacture. Fat destabilization refers to the process of clustering and clumping of the fat globules which leads to the development of a continuous internal fat network or matrix or structure in the product.306 The interaction of partially crystalline fat globules with air bubbles is responsible for the formation of structure in whipped cream and ice cream, for flotation churning of fat in the manufacture of butter, and for the undesirable formation of a foaminduced fat layer in products such as cream. 16 ' 333 Liquid fat may be disrupted by the presence of air as a result of spreading and subsequent rupture of the bubble. 16 Electron microscopy techniques have been used to study the whipping of heavy cream 296 - 306 - 332334 " 337 and the destabilization process in ice cream. 308 ' 320 It was reported that the proteinaceous membrane that envelops the air bubble during the whipping of heavy cream is penetrated by fat globules as the process proceeds, and this fat penetration offers foam stability to the whipped product. 335 During the initial stages of whipping, air bubbles are stabilized primarily by /3-casein and whey proteins with little involvement of fat. 338 ' 339 Adsorption of fat or fat crystals to air bubbles occurred when the fat globule membrane coalesced with the air-water interface.332'337 Only rarely did fat spread at the air-water interface. The final cream was stabilized by a cross-linking of fat globules surrounding each air cell to adjacent air cells thus building an infrastructure in the foam. Fat destabilization is responsible for the formation of the dryness and smoothness associated with ice cream, 308 and is promoted by the presence of small molecule surfactants that displace proteins from the surface of the fat globule, rendering them more susceptible to flocculation and coalescence. 320 ' 331

1.4 Physical Properties 1.4.1 Density


The density of milk and milk products is used to convert volumetric measurements to gravimetric or vice versa, to estimate total solids content (e.g., the use of hydrometers to monitor total solids of concentrated milk), and to calculate other physical properties (e.g., kinematic viscosity). Density is designated p and is expressed as kg m ~ 3 (SI units). Specific gravity is a dimensionless property defined by density product/density water where the temperature of both product and water must be specified. Specific gravity is practically equivalent to density if the water temperature is 4C

where its density is 1.000 g ml" 1 (999.972 kg m"3). The density of milk at 200C is on average about 1030 kg m~ 3 and normally varies within the range of 1027 to 1033 kg m~ 3 . 16 The density of milk is dependent on composition and can be calculated from the density and mass fraction of individual components. The following equations have been cited to approximate the density of milk, skim milk, creams, and concentrated milks.16 Equation 1.2 can be used to estimate density of the product (P) given the apparent density of each component (Px) and the mass fraction of each component (mx). At 200C the densities of water, milk fat, protein, lactose, and other components are 998.2, 918, 1400, 1780, and 1850 kg m"3, respectively.16 Equation 1.3 can be used to estimate the density of concentrated products (P0) given the density of the initial unconcentrated milk (P0), the density of water (Pw), and the concentration ratio (R = total solids of concentrated milk/total solids initial milk). (1.2) (13) The coefficient of thermal expansion of fresh milk of 4.0% fat and 8.95% solidsnot-fat is on average about 0.335 cm3/kg/C in the temperature range of 5 to 400C but is dependent on temperature340 and temperature history. This value is similar to the coefficient for water and, therefore, the specific gravity of milk is nearly constant over this temperature range with a slight decrease in the order of 5 X 10 ~ 5 due to greater coefficient of thermal expansion for fat than for water.16 At temperatures >40C there is a slight increase in specific gravity.341 Milk density is also affected by temperature history which determines the state of the fat. Complete solidification of milkfat causes a contraction of 70 cm3/kg.16 Frequently, milk density is determined by warming to 400C and then cooling to the specified temperature. This results in more liquid fat (due to super cooling) and, therefore, lower density values than if the milk was warmed to the specified temperature. Table 1.13 shows averages of empirically determined specific gravity values of some common fluid milk products at several temperatures. The data represent 8000 raw and processed samples analyzed over a 12-month period. Included in Table 1.13 are regression coefficients and intercepts that have been calculated from these data and can be used to calculate (approximate estimates only) the densities of milks and creams at the specified temperature given the contents of fat and solids-not-fat in the product.

1.4.2 Viscosity
Viscosity (or fluidity, which is the reciprocal of viscosity) is an important factor in determining the rate of creaming, rates of mass and heat transfer, and flow conditions in dairy processes. For example, recent calculations suggest that viscosity of ice cream mix may be sufficiently high to maintain laminar flow conditions during

Table L13 DENSITY OF VARIOUS FLUID DAIRY PRODUCTS AS A FUNCTION OF FAT AND SOUDS-NOT-FAT (SNF) COMPOSITION
Product Composition Product Producer milk Homogenized milk Skim milk, packaged Fortified skim milk Half and half Half and half, fort. Light cream Heavy cream Regression3 Intercept Fat coefficient SNF coefficient Fat (%) 4.00 3.60 0.02 0.02 12.25 11.30 20.00 36.60 SNF (%) 8.95 8.60 8.90 10.15 7.75 8.90 7.20 5.55 4.4C 1.035 1.033 1.036 1.041 1.027 1.031 1.021 1.008 1.0027 -0.00042 0.00373 Density (kg/m2) at: 1O0C 1.033 1.032 1.035 1.040 1.025 1.030 1.018 1.005 0.9991 -0.00047 0.00403 2O0C 1.030 1.029 1.033 1.038 1.020 1.024 1.012 0.994 1.0017 -0.00075 0.00351 38.90C 1.023 1.022 1.026 1.031 1.010 1.014 1.000 0.978 0.9955 -0.00102 0.00348

Calculated from data in ref. 342.


a

Density = intercept + (fat coeff. X fat content) + (SNF coeff. X SNF content)

pasteurization with the result that heat transfer may be too slow to ensure adequate heat treatment.343 The literature on the viscosity of milk has been reviewed.16'341 Viscosity (rf) is the ratio of shearing stress (T = force per unit area) to shear rate (y = velocity difference divided by distance in reciprocal seconds) assuming laminar flow with parallel stream lines. For reviews of the principles of viscosity and its measurement see refs. 118, 344. The c.g.s. or metric unit for viscosity is the poise (dynes s cm" 2 ) which is the force in dynes c m " 2 required to maintain a relative velocity of 1 cm s " l between two parallel planes 1 cm apart. The SI unit for viscosity is N s m~ 2 which is equivalent to Pa s. Ten N s m " 2 equals one poise. With respect to dairy products, the most commonly used units are centipoise (poise X 10 ~ 2 ) and mPa s. Milk and skim milk, excepting cooled raw milk, exhibit Newtonian behavior. For Newtonian fluids at constant temperature and pressure the viscosity is independent of the rate of shear, and a plot of shearing stress versus shearing rate is a straight line passing through the origin. The coefficient or slope of this line is the dynamic viscosity or simply, viscosity. The viscosity of a Newtonian fluid containing particles of diverse sizes is described by the Eilers equation345 and is a function of the hydrodynamic volume fraction of the dispersed particles, including all particles at least an order of magnitude larger than water and the viscosity of the liquid in which the particles are suspended. In milk the dispersed particles include lactose, whey proteins, casein micelles, and fat globules which are suspended in water with other small molecules. See ref. 16 for a discussion of the hydrodynamic volumes of milk components. There are many confounding interactions making generalizations difficult. For example, cooling from 30 to 5C causes increased viscosity of skim milk

due to increased voluminosity of casein micelles whereas at temperatures above 65C, denaturation of whey proteins causes increased viscosity. Voluminosity of casein micelles is also increased by a decrease or increase in the pH of milk (Section 1.3.1). Useful nomograms that can be used to estimate the density and viscosity of milks and creams in the ranges of 0 to 50% fat and 40 to 800C have been presented in ref. 346. For non-Newtonian or pseudoplastic fluids the apparent viscosity is dependent on shear rate. Cooled raw milk and creams which are subject to cold agglutination (Section 1.3.2.4) exhibit reduced viscosity when the globule aggregates are dispersed by agitation (shear thinning). Shear thinning is also observed if homogenization clusters are present. Agitation of heavy cream causes increased viscosity (shear thickening) due to partial coalescence of fat globules (partial churning).

1.4.3 Freezing Point


Freezing point is a colligative property that is determined by the molarity of solutes rather than by the percentage by weight or volume. The ideal molal depression constant for water as defined by Raoult's law is 1.86 for dilute solutions (i.e., each mole of solute will decrease the freezing point of water by 1.86C). Freezing point therefore can be used to estimate the molecular weight of pure solutes or the average molecular weight of mixed solutes. In the dairy industry, freezing point is used mainly to determine added water but it can also be used to determine lactose content in milk,347 estimate whey powder contents in skim milk powder,348 and to determine water activity of cheese.349 Although milk is not an ideal solution, the molal depression constant of 1.86 can be used to approximate the contribution of milk components to freezing point depression. Lactose accounts for about 55% of freezing point depression, chloride accounts for about 25%, and the remaining 20% is due to other soluble components including calcium, potassium, magnesium, lactates, phosphates, and citrates.350 Freezing point determinations may be done by the Hortvet procedure351 which uses a mercury in glass thermometer or, as in most modern instruments, by using a thermistor cryoscope.352 For many years most cryoscopes were calibrated in degrees Hortvet because Hortvet's procedure produced freezing points about 3.7% lower than the correct values in degrees Celsius.353 A formula given by the Association of Official Analytical Chemists32354'355 for the conversion of 0H to 0C gives lower values than the true values.353 An alternate formula published by the International Dairy Federation350 is: C = 0.96418 H - 0.00085. (1.4)

Added water may also be estimated from changes in osmotic pressure as measured by vapor pressure osmometry.356 Vapor pressure is measured as a function of dewpoint depression. A thermocouple detector senses the temperature of a milk sample at vapor pressure equilibrium in a sample chamber headspace. The results expressed as milliosmoles per kilogram of water are highly correlated to freezing points and

the procedure356 has been approved by the AOAC for the determination of added water in milk. 355 The freezing point of milk is usually in the range of - 0 . 5 1 2 to - 0 . 5 5 0 0 C with an average of about 0.522 0 C. 341 Freezing points of goat's and ewe's milk are generally lower than that of cow's milk whereas the freezing point of buffalo milk is similar to that of cow's milk. 350 If the freezing point of unwatered milk is known, the relationship between added water and freezing point depression is given by Eq. 1.5. If the actual freezing point of the unwatered milk is not known a reference value can be used. (1.5) where W = percent (w/w) extraneous water in the suspect milk C = actual or reference freezing point of genuine milk D = freezing point of suspect milk 5 = the percent (w/w) of total solids in the suspect milk. For routine added water determinations it is of course important to have a reliable reference point. Based on a United Kingdom study, it was concluded that fewer than 1 in 1000 samples of genuine or authentic milk (i.e., milk produced under supervised conditions and certified free of added water) will have a freezing point higher than - 0 . 5 0 8 0 C and that samples with freezing points higher than this reference point may be considered to contain added water. 350 The reference point recommended in 1970 by the Association of Official Analytical Chemists is - 0 . 5 0 5 0 C ( - 0 . 5 2 5 H). 3 5 4 This value is based on a North American study of genuine milks 357 and is still used by most milk testing laboratories in North America. Freezing point results obtained for Minnesota and Wisconsin herds from 1979 to 1988 showed that the average freezing point had decreased significantly during this time. 358 The same authors conducted a comprehensive freezing point survey of herds in Minnesota and recommended that the reference point for that state should be decreased from the AOAC value of - 0 . 5 0 5 0 C ( - 0 . 5 2 5 H) to - 0 . 5 1 2 0 C ( - 0 . 5 3 0 H). 3 5 9 In a study of freezing points of milks in the Netherlands, it was suggested that the reference point should not be fixed but should vary with season and region. 360 Correct interpretation of freezing point data with respect to added water depends on a good understanding of the factors affecting freezing point depression. It is frequently necessary to conduct repeat sampling and/or obtain genuine samples (supervised sampling) from herds showing freezing points near the reference point in order to eliminate natural causes of abnormally high freezing points. If a repeat sample has been taken from a herd within 48 h, the suspect milk should not be considered to have contained added water unless the freezing point of the repeat sample is at least 0.007 0 C lower than that of the suspect sample. 350 This difference in freezing point depression corresponds to about 1.2% of added water for milk of typical total solids content. Numerous references are available on factors affecting freezing points. 341 ' 361 ' 362 The following summary of these factors is based mainly on the discussion in ref. 361.

There are small differences in freezing points between breeds (in the order of 0.002 to 0.0070C), with Holstein milks generally having the lowest freezing points. There is a slight tendency toward lower freezing points in late lactation but it is not clear whether this effect is independent of feed effects. Similarly, seasonal differences in freezing points are probably due to feed effects. The freezing point of morning milk tends to be 0.003 to 0.0070C lower than that of evening milk. Larger differences may be observed if the cattle do not have free access to water at all times. Variations in the proportions of grains to roughage and fresh versus dry forage have significant but small effects on freezing point. Udder health (mastitis) also has slight effects on freezing point. With respect to interpretation of freezing points for added water determination, the most significant variables are the nutritional status of the herd and the access to water. Under-feeding causes increased freezing points. Large temporary increases in freezing point occur after consumption of large amounts of water because milk is isoosmotic with blood. The primary sources of nonintentional added water in milk are residual rinse water and condensation in the milking system. Leaky coolers used to precool milk before it enters the bulk tank may also be a problem. Recommended procedures to avoid added water, to determine residual water in milking systems, and to obtain authentic milk samples for interpretation of freezing points have been reported.350 Soured or fermented milk is unsuitable for added water testing because the freezing point is lowered by lactic acid and increased concentrations of soluble minerals. Several reports suggest that heat treatment of milk, including UHT and retort sterilization, causes little permanent effect on freezing points350 but it has also been suggested that freezing points are not a reliable index of added water in processed milk.363

1.4.4 Electrochemistry

1.4.4.1 Electrical Conductivity


Specific electrical conductivity measured in ohm " l cm " l is the reciprocal of specific conductance (ohm cm). Electrical conductivity has been considered as an index for mastitic infections, added water, added neutralizes, and milk concentration during evaporation.341 The main application of interest in recent years has been its use as an index of mastitic infection.364"366 Changes in electrical conductivity can also be used to detect the initial stages of micelle aggregation during rennet coagulation of milk.367 Electrical conductivity begins to decrease at about 60% of clotting time and continues to decrease for several hours. The principal ions contributing to the electrical conductivity of milk are sodium, chloride, and potassium. At 25C the specific conductivity of milk is on avereage about 0.005 ohm" 1 c m " ! and the normal range is 0.0040 to 0.0055 ohm" 1 Cm" 1 3 4 1 The following conditions affect conductivity.341 Conductivity decreases with increasing fat content so that skim milk has higher conductivity than milk. Whey and ultrafiltrate have greater conductivity than skim milk. Conductivity changes with concentration or dilution of milk but the relationship is not simple because of the effects of concentration on the distribution of minerals between colloidal and dia-

Eh, Eo (V)

raw mik

ascorbate methylene blue glutathione

riboflavin

cysteine hydrogen electrode

Figure 1.14 The redox potential (h) of milk and the standard potentials () of various systems in relation to pH. (From ref. 16. Reprinted by permission of John Wiley & Sons.)

lysable phases. Production of lactate ions and solubilization of colloidal minerals during lactic fermentation increases conductivity.

1.4.4.2 Oxidation-Reduction Potentials


A molecular species is oxidized when it loses electrons and is reduced when it gains electrons. Loss or gain of electrons may or may not include the transfer of oxygen or hydrogen. Oxidation-reduction (redox) potential is expressed in volts and designated as Eh. The standard potential when the oxidized (Ox) and reduced (Red) forms are at equal activity is designated E. Redox potential is measured relative to the potential of the standard hydrogen electrode which is assigned a value of O V at pH O. At 25C and one electron transfer Eh is defined as: !Red]) (1.6)

By convention a larger ratio of [Ox]/[Red] indicates a positive potential. The redox capacity of the system is determined by the total amount of reactants ([Ox] + [Red]). E is an index of the potential of the system relative to other systems. When the value of h is near E the system exhibits poising or a resistance to change in potential similar to the buffering that occurs in an acid-base system near its pK value. E values are pH dependent as illustrated for several redox systems of milk

in Figure 1.14. The principles of oxidation-reduction systems and their measurement are described in many chemical texts and a monograph on oxidation-reduction potentials of biological systems has been prepared.368 The redox potential of milk is in the range of + 0.2 to + 0.3 V and is mainly determined by dissolved oxygen.341 Milk is essentially oxygen free when excreted but about 0.3 mM O 2 is present after equilibrium with air is established. Removal of oxygen by nitrogen sweeping lowers the E of milk to about 0.12 V.16 Decreased oxygen tension by bacterial respiration is the basis of the methylene blue reduction test for milk bacterial quality. The other redox systems of significance in milk are ascorbate (0.25 mEq L" 1 ) and riboflavin. Ascorbate in freshly drawn milk is all in the reduced form but during refrigerated storage is reversibly oxidized to dehydroascorbate which is irreversibly changed by hydrolysis of the lactone ring to 2,3-diketo-L-gulonate. Oxidation of ascorbate in the presence of copper and oxygen produces superoxide anion which in the presence of peroxide is converted to singlet oxygen. Singlet oxygen is probably responsible for the initiation of lipid oxidation.16 (See also Section 1.2.2.4.) The small quantity of riboflavin in milk contributes little to redox capacity but is important in photooxidation of milk. When excited to a triplet state by exposure to light near 450 nM, riboflavin oxidizes the methioine residues in the whey proteins to methional which is the principal component of "sunlight" flavor in milk. Excited riboflavin can also oxidize ascorbate, and reduced riboflavin can react with triplet oxygen to produce singlet oxygen.16 Heat treatment is well known to increase the reducing capacity of milk, mainly due to activation of protein thiol groups and products of Maillard browning reactions. Activated thiol groups cause cooked flavor which decreases as cysteine bonds reform on standing.

1.4.5 Surface Tension


Interfacial tension is the work required to increase the area of contact between two phases expressed as force per unit length i n N m ' 1 or dynes cm" 1 which is equivalent to mN m~ 1 . Interfacial energy can also be expressed as energy per unit area in J m~ 2 which is numerically equivalent to N m~ *. If the interface is liquid-solid, air-liquid, or air-solid the interfacial tension is referred to as surface tension. The principal interfaces in milk are the fat globule-plasma interface and the air-plasma interface. Excellent reviews are available on the fat globule-plasma interface of
m i l k

16,369 (

S e e

8 0S e c t i o n

L3-2.)

Factors affecting the surface tension of milk, that is the interfacial tension between milk and air, have been reviewed341 and there is little new information in the literature. The surface tension of milk is about 50 mN m~ l compared to water which is 72 mN m" 1 (Table 1.14). Surface tension is increased by about 10% in skim milk and is reduced in cream. AU of the principal milk proteins are strong depressants and are present in excess so that considerable dilution is necessary to significantly reduce the surface tension of skim milk; the surface tension of rennet whey is similar to that of skim milk. The gross composition of buttermilk is similar to skim milk but its surface tension is decreased (Table 1.14) by higher levels of phospholipids.

Table 1.14 INTERFACIAL TENSIONS (7) OF VARIOUS INTERFACES IN MILK COMPARED TO OTHERS
Interface Between Phases Water-air 22 mM Na laurate in water-air 0.3 mM stearate in water-air /i-Octane-air Water-rt-octane Milk plasma-air Sweet-cream buttermilk-air Liquid milk fat-air Liquid milk fat-water Liquid milk fat-milk plasma Liquid milk fat-protein solutions Milk fat globule-milk plasma Liquid fat-fat crystal (a modification) 7 (mN m l) 72 43 43 22 51 48 40 34 20 15 10-15 2a 10

From ref. 16. Reprinted by permission of John Wiley & Sons. Note: Approximate values at 20 to 4O0C.
a

Measured values range from 1. to 2.5 mN m ~'.

Lipolysis decreases surface tension due to the release of surface active free fatty acids. Homogenization increases surface tension possibly due to adsorption of surface active substances onto the enlarged fat globule-plasma interface. Cold storage of milk apparently activates some surface active substance in milk because it effectively lowers surface tension. Normal heat treatments of milk have no effect on surface tension. The importance of interfacial tension in fat destabilization processes has been discussed in Section 1.3.2.4.

1.4.6 Acid-Base Equilibria


Both titratable acidity and pH are used to measure milk acidity. pH is a measure of the activity of the hydronium ion (H 3 O + ) which, according to the Debye-Hiickel expression, is a function of the concentration of the hydronium ion [H 3 O + ], the effective diameter of the hydrated ion and the ionic strength (/A) of the solvent. For solutions of low ionic strength (/x <0.1) hydronium ion activity is nearly equivalent to [H 3 O + ] which is normally abbreviated to [H + ]. Then, for a weak acid (HA) dissociating to H + and A~ with a dissociation constant, Kz and p/a equal to log10 K&, the most important relationships are defined by Eqs. 1.7 and 1.8. (1.7) (1.8)

Table 1.15 BUFFERING GROUPS IN MILK


Group Protein-bound residues Aspartic acid Glutamic acid Histidine Tyrosine Lysine Ester-phosphate iV-acetylneuraminic acid Terminal groups Salts Phosphate8 Citrate3 Phosphate esters3 Carbonate Various carboxylic acids Various amines Lactic acid Concentration (mM)

19 50 6 12 20 7 0.5 1.5 21 b 9 2.5 2 2 1.5 50-120 c

4.1 4.6 6.5 9.7 10.5 2.0, 6? 5 3.7, 7.9 3.0, 5.8, 6.6 3.0,4.1,4.8 1.7, 5.9 6.4, 10.1 4.8 7.6? 3.95

From ref. 16. Reprinted by permission of John Wiley & Sons. Note: Approximate average concentration, and their estimated (stoichiometric) pK values in milk.
a b c

pA" values from titration with Ca(OH)2. About 10 mM colloidal phosphate, 11 mM in solution. In sour milk products.

The buffer capacity (dB/dpH) is the amount of strong acid or base in moles per liter (strong meaning completely dissociated in the experimental pH range) required per unit change in pH. Because pH is a dimensionless quantity the units of buffer index are simply mol/L. When pH equals pK& the weak acid is half dissociated and the buffer capacity is maximum. For species such as proteins which have numerous acidic and basic groups, maximum buffering occurs in the region of isoelectric pH (pi). The principles of pH and its measurement can be found in many chemistry texts. Titratable acidity is a measure of the total buffer capacity of milk for the pH range between the pH of milk and the phenolphthalein end point (about 8.3). The pH of milk at 25C normally varies within a relatively narrow range of 6.5 to 6.7.341 The normal range for titratable acidity of herd milks is 13 to 20 mmol L~" *. This corresponds to 0.12 to 0.18% lactic acid but there is practically no lactic acid in fresh milk and there is no good reason for the North American convention of reporting titratable acidity as percent lactic acid. Because of the large inherent variation, the measure of titratable acidity has little practical value except to measure changes in acidity (e.g., during lactic fermentation) and even for this purpose, pH is a better measurement.

mM

1 2

dpH

1 3 HCI NaOH 3 2

PH

PH

Figure 1.15 Examples of titration curves (mAf HCl or NaOH needed to obtain a certain pH) of milk (1), and of sweet whey (2) and ultrafiltrate (3) made from that milk; the same results also expressed as buffer index dB/dpH (in mmol L~ l ). (From ref. 16. Reprinted by permission of John Wiley & Sons.)

The major buffering groups of milk and their p a values are listed in Table 1.15 but the actual pKA values in milk are different due to interactions with other ions. 16 The two most important buffer components of milk are caseins (buffer maximum near pH 4.6) and phosphate (buffer maximum near pH 7.0). The titration curve for sweet whey (rennet whey with no culture) indicates a small buffer maximum due to whey proteins in the range of pH 4.0 to 5.0 (Fig. 1.15). The morphologies of the titration and buffer capacity curves of milk and milk products are dependent on the rate of titration because of sluggish equilibrium reactions between colloidal and dialyzable salts, especially phosphate salts. The rate of titration should, therefore, be given when titration data are reported. In the region of the phosphate buffer maximum several days are required to obtain final equilibrium between dialyzable and colloidal calcium phosphates. The most important effects are: (1) Formation of colloidal calcium phosphates greatly increases the buffer capacity of phosphates. (2) The presence of citrate and caseins promotes the formation of tricalcium phosphates at pH levels where mono- and dicalcium phosphates would otherwise predominate. 370 " 372 This broadens the phosphate buffer range by moving the calcium phosphate saturation point to higher pH levels. (3) Lactic acid has a pKa near 4.0 so that fermented dairy products have a large buffer maximum near pH 4.0. (4) Formation of colloidal calcium phosphates during concentration of milk causes the pH to decrease. This effect does not occur during concentration by ultrafiltration.373 (5) Heating also causes pH reduction due to formation of colloidal phosphate salts. The pH of milk decreases by about 0.4 units over the range of 20 to 60 0 C. 3 4 1 (6) Concentration of milk salts during freezing causes pH to decrease. 341 (7) The acid-base

properties of cheese whey are largely determined by the pH at the time of draining. 374375 Greater amounts of calcium phosphates and larger calcium/phosphate ratios in acid wheys cause greater buffer capacity and a shift in the phosphate buffer maximum to lower pH.

1.4.7 Heat Capacity and Thermal Conductivity


Heat capacity is expressed as J kg" l K~~l (SI units) which is equivalent to 1/4186 cal g" 1 0 C" 1 (c.g.s. units). Specific heat is the unit-less quantity of heat capacity divided by the heat capacity of water. It is nearly equivalent to heat capacity because the heat capacity of water varies only slightly from 4186 J kg" 1 K" ! (1.000 cal g~ 1 0 1 C' ) over the range of 0 to 1000C. Thermal conductivity is the rate of heat transfer by conduction in Jm"1 s" 1 K"1 which is equivalent to 1/420 cal cm" 1 s" 1 0 C" 1 . There are many reviews of the principles and methodology of thermal analysis.376"381 Thermal properties of milk have been reviewed recently.341 Values for skim milk of 3906 J kg - l K " 2 at 00C and 3993 J kg " 1 K " 1 at 500C have been reported.377 There is a linear increase in the heat capacity of skim milk from 3965 J kg" l K" l at 500C to 4218 J kg" l K"l at 1400C according to Eq. 1.9 where T is temperature in 0 C. 379 Heat capacity = 2.8147 + 3824 (1.9)

Allowing for variations in data reported by various workers, the heat capacity of skim milk increases with fair linearity over the entire temperature range of 1 to 1400C. For example, extrapolation of the above equation to 200C gives an estimate very close to the literature value of 3890 J kg" 1 K"1.16 Heat capacity decreases with increasing total solids but normal variations in composition of skim milk should not cause large differences.379 The variation of heat capacity of whole milk and cream with temperature is more complex than skim milk because of the effect of milk fat. Milk fat has a heat capacity of about 2177 J kg"J K"1 and a heat of fusion of about 8.37 Jg" 1 . Over the range of 50 to 1400C where milk fat is liquid the heat capacity of milk can be estimated approximately by Eq. IA(P79: Heat capacity = 2.976 X temperature + 3692 (1.10) Thermal conductivity of water increases from 2 1 8 J m - 1 S - 1 K " 1 at 00C to 244 Jm" 1 S - 1 K " 1 at 1000C. Thermal conductivity of milk increases with temperature and decreases with increasing total solids. Typical values are 193 Jm""1 S - 1 K " 1 at 37C and 223 J m" ! s" l K" ! at 800C.341

1.4.8 Optical Properties


Optical properties provide the basis for many rapid, indirect methods of analysis such as proximate analysis by infrared absorbency or light scattering. These aspects are reviewed in Chapter 3. Optical properties also determine the appearance of milk and milk products. Light scattering by fat globules and casein micelles causes milk

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to appear turbid and opaque. Light scattering is maximal when the wavelength of light is near the same magnitude as the particle. Thus, smaller particles scatter light of shorter wavelengths.16-382"384 Skim milk appears slightly blue because casein micelles scatter the shorter wavelengths of visible light (blue) more than the red. The carotenoid precursor of vitamin A, /3-carotene, contained in milk fat, is responsible for the "creamy" color of milk. Riboflavin imparts a greenish color to whey and its concentration can be measured in whey by its fluorescent emission at 530 nm when exposed to light at 440 to 500 nm.341 Refractive index (RI) is normally determined at 200C with the D line of the sodium spectrum and is designated 7^0. The refractive index of milk is 1.3440 to 1.3485341 and can be used to estimate total solids (Chapter 3). Contributions of plasma components to RI are additive. The RI of milk fat is 1.4537 to 1.4552 at 400C but fat globules do not contribute to RI because refraction occurs at the interface between the air and the plasma.341'385

1.5 Summary
Milk is a very complex liquid consisting of over 100,000 different molecules. It serves a biological function as the food of the infant mammal and particularly the milk of the domesticated cow, genus Bos, serves an important role in human feeding and nutrition. The gross composition of cow's milk is 4.1% fat; 3.6% protein; 4.9% lactose; 0.7% miscellaneous components including minerals, vitamins, and gases; and the balance in water. The fat in milk is comprised mainly of triglycerides containing a wide range of fatty acids, which in turn contain a relatively high proportion of short-chain and saturated fatty acids. The melting range of the fat extends from 37C to -40 0 C. The fat exists in milk in the form of a globule of 3 to 8 /im in diameter which is coated by a protective membrane. The origin of this membrane is thought to be the apical cell membrane of the mammary secretory cell. In the raw state, the membrane acts to protect the milk fat from deleterious reactions such as the action of lipase enzymes in creating rancidity. However, during processing the membrane is largely replaced by a layer of amphiphilic molecules that adsorb to the fat surface. There are many dairy products that derive their structure from milkfat, including whipped cream, ice cream, and butter. There are two main categories of milk proteins: the caseins, about 75 to 80% of the total, and the serum or whey proteins. The four principal casein proteins, a sl -, a s2~> -> and /c-casein, are found complexed with tertiary calcium phosphate in a spherical particle 100 to 300 nm in diameter known as the casein micelle. These proteins precipitate at pH 4.6. Interactions of micelles are responsible for the formation of structure in many dairy products such as cheeses or fermented products. The whey proteins, including /3-lactoglobulin, a-lactalbumin, bovine serum albumin, immunoglobulins, numerous enzymes, and the proteose-peptone fraction (see Section 1.2.1.1), are found in the milk serum and are soluble at pH 4.6. Lactose, a carbohydrate virtually unique to milk, is a disaccharide of glucose and galactose. It has two crystalline forms, a and /3. The a monohydrate form can cause problems in

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to appear turbid and opaque. Light scattering is maximal when the wavelength of light is near the same magnitude as the particle. Thus, smaller particles scatter light of shorter wavelengths.16-382"384 Skim milk appears slightly blue because casein micelles scatter the shorter wavelengths of visible light (blue) more than the red. The carotenoid precursor of vitamin A, /3-carotene, contained in milk fat, is responsible for the "creamy" color of milk. Riboflavin imparts a greenish color to whey and its concentration can be measured in whey by its fluorescent emission at 530 nm when exposed to light at 440 to 500 nm.341 Refractive index (RI) is normally determined at 200C with the D line of the sodium spectrum and is designated 7^0. The refractive index of milk is 1.3440 to 1.3485341 and can be used to estimate total solids (Chapter 3). Contributions of plasma components to RI are additive. The RI of milk fat is 1.4537 to 1.4552 at 400C but fat globules do not contribute to RI because refraction occurs at the interface between the air and the plasma.341'385

1.5 Summary
Milk is a very complex liquid consisting of over 100,000 different molecules. It serves a biological function as the food of the infant mammal and particularly the milk of the domesticated cow, genus Bos, serves an important role in human feeding and nutrition. The gross composition of cow's milk is 4.1% fat; 3.6% protein; 4.9% lactose; 0.7% miscellaneous components including minerals, vitamins, and gases; and the balance in water. The fat in milk is comprised mainly of triglycerides containing a wide range of fatty acids, which in turn contain a relatively high proportion of short-chain and saturated fatty acids. The melting range of the fat extends from 37C to -40 0 C. The fat exists in milk in the form of a globule of 3 to 8 /im in diameter which is coated by a protective membrane. The origin of this membrane is thought to be the apical cell membrane of the mammary secretory cell. In the raw state, the membrane acts to protect the milk fat from deleterious reactions such as the action of lipase enzymes in creating rancidity. However, during processing the membrane is largely replaced by a layer of amphiphilic molecules that adsorb to the fat surface. There are many dairy products that derive their structure from milkfat, including whipped cream, ice cream, and butter. There are two main categories of milk proteins: the caseins, about 75 to 80% of the total, and the serum or whey proteins. The four principal casein proteins, a sl -, a s2~> -> and /c-casein, are found complexed with tertiary calcium phosphate in a spherical particle 100 to 300 nm in diameter known as the casein micelle. These proteins precipitate at pH 4.6. Interactions of micelles are responsible for the formation of structure in many dairy products such as cheeses or fermented products. The whey proteins, including /3-lactoglobulin, a-lactalbumin, bovine serum albumin, immunoglobulins, numerous enzymes, and the proteose-peptone fraction (see Section 1.2.1.1), are found in the milk serum and are soluble at pH 4.6. Lactose, a carbohydrate virtually unique to milk, is a disaccharide of glucose and galactose. It has two crystalline forms, a and /3. The a monohydrate form can cause problems in

dairy products such as ice cream and condensed milk due to its relative insolubility. Of the miscellaneous components, milk contains a number of minerals, including Ca, Mg, K, Na, Cl, citrate, sulfate, phosphate, and bicarbonate; vitamins, mainly A, the B vitamins, D, E, and K; and acids, including citrate, formate, acetate, lactate, and oxalate.

1.6 Future Developments


The title of this chapter reflects the modern trend to study milk as a structured physical system. Milk chemistry with respect to chemical composition is now quite advanced (e.g., primary sequences of quantitatively significant proteins are well defined). However, this is only a preliminary step toward understanding the properties of milk. Future work will increasingly focus on the physciochemical properties of the structural components of milk and their behavior in milk and milk products during processing and storage. Current models of casein micelles will be challenged or refined by advanced microscopy, rheological data, and a greater understanding of casein molecular biology and the interaction of caseins with milk salts. Much work has been done to describe the structure of the milk fat globule membrane and the interactions that occur at the fat globule surface, but more study is needed to adequately describe the factors determining the stability of the milk fat globule to physical, chemical, and enzymatic changes. There will be increased study of minor components such as enzymes and oxidative activators or inhibitors. Study of milk salts and the factors affecting their distribution in milk will increase our understanding of milk properties such as heat stability and enzymatic coagulation. One only needs to peruse the last several volumes of abstracts presented at the American Dairy Science Association annual meetings, and the numbers of excellent papers presented at the International Dairy Federation meetings to appreciate the effort being expended into dairy chemistry research. This can only lead to enhanced understanding of the complex milk system.

1.7 References
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6. Anifantakis, E. M. 1986. Comparison of the physico-chemical properties of ewe's and cow's milk. Bull. Int. Dairy Fed. 202:42-53. 7. LaI, D., and K. M. Narayanan. 1986. Frequency distribution of fat and solids-not-fat contents of the milk of different breeds of cows and Murrah buffaloes. Indian Vet. J. 63:923-926. 8. Bachmann, M. R., and W. Schulthess. 1987. Lactation of camels and composition of camel milk in Kenya. Milchwissenschaft 42:766-768. 9. Jenness, R. 1979. The composition of human milk. Sent. Perinatol. 3:225. 10. Jenness, R., and R. E. Sloan. 1970. The composition of milks of various species. A review. Dairy Sci. Abstr. 32:599-612. 11. Jenness, R. 1982. Inter-species comparison of milk proteins. In P. F. Fox (ed.), Developments in Dairy Chemistry I. Proteins, pp. 87-114. Applied Science Publishers, London. 12. Swaisgood, H. 1985. Characteristics of edible fluids of animal origin: milk. In O. R. Fennema (ed.), Food Chemistry, 2nd edit., pp. 791-827. Marcel Dekker, New York. 13. Larson, B. L. (ed.). 1978. LactationA Comprehensive Treatise, Vol. IV. Academic Press, New York. 14. Larson, B. L., and V. R. Smith (eds.). 1974. Lactation-A Comprehensive Treatise, VoIs. I, II, III. Academic Press, New York. 15. Patton, S., and R. G. Jensen. 1976. Biomedical Aspects of Lactation. Pergamon Press, New York. 16. Walstra, P., and R. Jenness. 1984. Dairy Chemistry and Physics. John Wiley & Sons, New York. 17. Kalab, M. 1979. Microstructure of dairy foods. 1. Milk products based on protein. / . Dairy Sci. 62:1352-1364. 18. Kalab, M. 1985. Microstructure of dairy foods. 2. Milk products based on fat. J. Dairy Sci. 68:3234-3248. 19. McBean, L. D., and E. W. Speckmann! 1988. Nutritive value of dairy foods. In N. P. Wong, R. Jenness, M. Keeney, and E. H. Marth (eds.), Fundamentals of Dairy Chemistry, 3rd edit., pp. 343-407. Van Nostrand Reinhold, New York. 20. Hambraeus, L. 1982. Nutritional aspects of milk proteins. In P. F. Fox (ed.), Developments in Dairy Chemistry. 1. Proteins, pp. 289-313. Applied Science Publishers, London. 21. Gurr, M. I. 1983. The nutritional significance of lipids. In P. F. Fox (ed.), Developments in Dairy Chemistry. 2. Lipids, pp. 365-417. Applied Science Publishers, London. 22. Houts, S. S. 1988. Lactose intolerance. Food Tech. 42:110-113. 23. Fox, P. F. (ed.). 1982. Developments in Dairy Chemistry. I. Proteins. Applied Science Publishers, London. 24. Fox, P. F. (ed.). 1983. Developments in Dairy Chemistry. 2. Lipids. Applied Science Publishers, London. 25. Fox, P. F. (ed.). 1985. Developments in Dairy Chemistry. 3. Lactose and Minor Constituents. Elsevier, London. 26. Fox, P. F. (ed.). 1989. Developments in Dairy Chemistry. 4. Functional Milk Proteins. Elsevier, London. 27. Mulder, H., and P. Walstra. 1974. The Milk Fat Globule. Emulsion Science as Applied to Milk Products and Comparable Foods. Pudoc, Wageningen, the Netherlands. 28. Wong, N. P., R. Jenness, M. Keeney, and E. H. Marth (eds.). 1988. Fundamentals of Dairy Chemistry, 3rd edit. Van Nostrand Reinhold,'New York.

29. Grappin, R. 1987. Monograph on rapid indirect methods, for measurement of the major components of milk. Bull. Int. Dairy Fed. No. 208. 30. Barbano, D. M., J. L. Clark, and C. E. Dunham. 1988. Comparison of Babcock and Ether extraction methods for determination of fat content of milk: collaborative study. J. Assoc. Off. Anal. Chem. 71:898-914. 31. Karman, A. H., and M. van Boekel. 1986. Evaluation of the Kjeldahl factor for conversion of the nitrogen content of milk and milk products to protein content. Netherlands Milk Dairy J. 40:315-336. 32. Assoc. Official Analytical Chemists. 1990. Methods of Analysis, 15th edit. Washington, D.C. 33. Clark, J. L., D. M. Barbano, and C. E. Dunham. 1989. Comparison of two methods for determination of total solids content of milk: collaborative study. /. Assoc. Off. Anal. Chem. 72:712-718. 34. Hemingway, R. G., and T. Aitchison. 1988. Sources of variation in the assessment of milk composition based on the mean values of four consecutive weekly samples of bulked farm milk. / . Dairy Res. 55:109-112. 35. Moore, J. H., and J. A. F. Rook. Eds. 1980. Factors affecting the yields and contents of milk constituents of commercial importance. Bull. Int. Dairy Fed. No. 125. 36. Banks, W. 1987. Opportunities for varying the composition of cow's milk. /. Soc. Dairy Technol. 40:96-99. 37. Emmons, D. B., D. Tullock, and C. A. Ernstrom. 1990. Product yield pricing system. 1. Technological considerations in multiple component pricing of milk. / . Dairy Sci. 73:1712-1723. 38. Emmons, D. B., D. Tullock, C. A. Ernstrom, M. Morisset, and D. Barbano. 1990. Product-yield pricing system. 2. Plant considerations in multiple component pricing of milk. / . Dairy Sci. 73:1724-1733. 39. Gibson, J. P. 1989. Selection on the major components of milk: alternate methods of deriving economic weights. / . Dairy ScL 72:3176-3189. 40. Gibson, J. P. 1989. The effect of pricing systems, economic weights and population parameters on economic response to selection on milk components. / . Dairy Sci. 72:3314-3326. 41. Gruebele, J. W. 1982. Alternative milk pricing programs. / . Dairy Sci. 65:460-468. 42. Hillers, J. K., V. H. Nielsen, A. E. Freeman, J. Dommerholt, and R. E. Deiter. 1980. Value of fat and protein in producer milk. / . Dairy Sci. 63:322-327. 43. Gibson, J. P. 1989. Altering milk composition through genetic selection. J. Dairy Sci. 72:2815-2825. 44. de Jager, D., and B. W. Kennedy. 1987. Genetic parameters of milk yield and composition and their relationships with alternative breeding goals. J. Dairy Sci. 70:1258-1266. 45. Kirchgessner, M., H. Friesecke, and G. Koch. 1967. Nutrition and the Composition of Milk. Crosby Lockwood, London, England. 46. Sutton, J. D. 1989. Altering milk composition by feeding. / . Dairy Sci. 72:2801-2814. 47. Grant, R. J., V. F. Colenbrander, and D. R. Mertens. 1990. Milk fat depression in dairy cows: role of particle size of alfalfa hay. / . Dairy Sci. 73:1823-1833. 48. Grant, R. J., V. F. Colenbrander, and D. R. Mertens. 1990. Milk fat depression in dairy cows: role of silage particle size. J. Dairy ScL 73:1834-1842. 49. Keown, J. F. 1988. Relationship between herd management practices in the midwest on milk and fat yield. J. Dairy ScL 71:3154-3165.

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299. Precht, D. 1988. Electron microscopic investigations of the influence of temperature and feeding conditions on the crystal structure of fat globules in cream. Fett. Wissenscaft. Technol. 90:300-308. 300. Precht, D. 1988. Fat crystal structure iri cream and butter. In N. Garti and K. Sato (eds.), pp. 305-361. Crystallization and Polymorphism of Fats and Fatty Products. Marcel Dekker, New York. 301. Walstra, P. 1974. High melting triglycerides in the fat globule membrane: an artefact? Netherlands Milk Dairy J. 28:3-9. 302. Walstra, P. 1983. Physical chemistry of milkfat globules. In P. F. Fox (ed.), Developments in Dairy chemistry. 2. Lipids, pp. 119-158. Applied Science Publishers, London. 303. Walstra, P. 1987. Fat crystallization. In J. M. V. Blanshard and P. Lillford (eds.), Food Structure and Behaviour, pp. 67-85. Academic Press, New York. 304. Walstra, P., and E. C. H. van Beresteyn. 1975. Crystallization of milkfat in the emulsified state. Netherlands Milk Dairy J. 29:35-65. 305. Walstra, P., and E. C. H. van Beresteyn. 1975. Additional evidence for the presence of mixed crystals in milk fat. Netherlands Milk Dairy J. 29:238-241. 306. Buchheim, W., and P. Dejmek. 1990. Milk and dairy-type emulsions. In K. Larsson and S. Friberg (eds.), Food Emulsions, 2nd edit., pp. 203-246. Marcel Dekker, New York. 307. Bouzas, J., A. R. Kamarei, and M. Karel. 1985. Storage stability of the milkfat globule membrane. J. FoodProc. Pres. 9:11-24. 308. Berger, K. G. 1990. Ice Cream. In K. Larsson and S. Friberg (eds.), Food Emulsions, 2nd edit., pp. 367-444. Marcel Dekker, Inc., New York. 309. Oortwijn, H., and P. Walstra. 1979. The membranes of recombined fat globules. 2. Composition. Netherlands Milk Dairy J. 33:134-154. 310. Walstra, P. 1975. Effect of homogenization on the fat globule size distribution in milk. Netherlands Milk Dairy J. 29:279-294. 311. Keenan, T. W., T. W. Moon, and D. P. Dylewski. 1983. Lipid globules retain globule membrane material after homogenization. / . Dairy ScL 66:196-203. 312. McPherson, A. V., M. C. Dash, and B. J. Kitchen. 1984. Isolation and composition of milk fat globule membrane material. II. From homogenized and ultra heat treated milks. /. Dairy Res. 51:289-297. 313. Oortwijn, H., P. Walstra, and H. Mulder. 1977. The membranes of recombined fat globules. 1. Electron microscopy. Netherlands Milk Dairy J. 31:134-147. 314. Walstra, P., and H. Oortwijn. 1982. The membranes of recombined fat globules. 3. Mode of formation. Netherlands Milk Dairy J. 36:103-113. 315. Oortwijn, H., and P. Walstra. 1982. The membranes of recombined fat globules. 4. Effects on properties of the recombined milks. Netherlands Milk Dairy J. 36:279-290. 316. McPherson, A. V., M. C. Dash, and B. J. Kitchen. 1984. Isolation and composition of milk fat globule membrane material. I. From pasteurized milks and creams. J. Dairy Res. 51:279-287. 317. Darling, D. F., and D. W. Butcher. 1978. Milkfat globule membrane in homogenized cream. J. Dairy Res. 45:197-208. 318. Henstra, S., and D. G. Schmidt. 1970. On the structure of the fat-protein complex in homogenized cow's milk. Netherlands Milk Dairy J. 24:45-51. 319. Dalgleish, D. G., and E. W. Robson. 1985. Centrifugal fractionation of homogenized milks. /. Dairy Res. 52:539-546.

320. Goff, H. D., M. Liboff, W. K. Jordan, and J. E. Kinsella. 1987. The effects of Polysorbate 80 on the fat emulsion in ice cream mix: evidence from transmission electron microscopy studies. Food Microstruct. 6:193-198. 321. Goff, H. D., J. E. Kinsella, and W. K. Jordan. 1989. Influence of various milk protein isolates on ice cream emulsion stability. / . Dairy Sci. 72:385-397. 322. Aguilera, J. M., and H. G. Kessler. 1988. Physico-chemical and Theological properties of milkfat globules with modified membranes. Milchwissenschaft 43:411-415. 323. Walstra, P. 1969. Studies on milkfat dispersion. II. The globule size distribution of cow's milk. Netherlands Milk Dairy J. 23:99-110. 324. Walstra, P. 1969. Studies on milkfat dispersion. HI. The distribution function of globule size in cow's milk and the process of milkfat formation. Netherlands Milk Dairy J. 23:111 -123. 325. Walstra, P., and H. Oortwijn. 1975. Effect of globule size and concentration on creaming in pasteurized milk. Netherlands Milk Dairy J. 29:263-278. 326. Walstra, P., H. Oortwijn, and J. J. de Graaf. 1969. Studies on milkfat dispersion. I. Methods for determining globule size distribution. Netherlands Milk Dairy J. 23:12-36. 327. van Boekel, M. A. J. S., and P. Walstra. 1981. Stability of oil-in-water emulsions with crystals in the disperse phase. Colloids Surfaces 3:109-118. 328. Darling, D. F. 1982. Recent advances in the destabilization of dairy emulsions. J. Dairy Res. 49:695-712. 329. Abrahamson, K., P. Frennborn, P. Dejmek, and W. Buchheim. 1988. Effects of homogenization and heating conditions on physico-chemical properties of coffee cream. Milchwissenschaft 43:762-765. 330. Ogden, L. V., Walstra, P., and H. A. Morris. 1976. Homogenization induced clustering of fat globules in cream and model systems. / . Dairy Sci. 59:1727-1737. 331. Goff, H. D., and W. K. Jordan. 1989. Action of emulsifiers in promoting fat destabilization during the manufacture of ice cream. / . Dairy Sci. 72:18-29. 332. Brooker, B. E., M. Anderson, and A. T. Andrews. 1986. The development of structure in whipped cream. Food Microstruct 5:277-285. 333. Anderson, M., B. E. Brooker, and E. C. Needs. 1987. The role of proteins in the stabilization/ destabilization of dairy foams. In E. Dickinson (ed.), Food Emulsions and Foams, pp. 100-109. Royal Society of Chemistry, London. 334. Brooker, B. E. 1979. Milk and its products. In J. G. Vaughan (ed.), Food Microscopy, pp. 273-311. Academic Press, New York. 335. Schmidt, D. G., and A. C. M. van Hooydonk. 1980. An SEM investigation of the whipping of cream. SEM 111:653-658. 336. Buchheim, W., N. M. Barfod, and N. Krog. 1985. Relation between microstructure, destabilization phenomena and Theological properties of whippable emulsions. Food Microstruct. 4:221-232. 337. Brooker, B. E. 1990. The adsorption of crystalline fat to the air-water interface of whipped cream. Food Struct. 9:223-229. 338. Birkett, R. J. 1983. Role of interfacial processes in the whipping of dairy cream. In J. V. McLoughlin and B. M. McKenna (eds.), Proceedings of the 6th International Congress of Food Science and Technology, Vol. 2, pp. 149-150. Boole Press, Dublin. 339. Brooker, B. E. 1985. Observations on the air serum interface of milk foams. Food Microstruct. 4:289-296.

340. Ruegg, M., and U. Moor. 1988. Effect of temperature between 15 and 25C on the density of milk. Dairy Sci. Abstr. 050-02705. 341. Sherbon, J. W. 1988. Physical properties of milk. In N. P. Wong, R. Jenness, M. Keeney, and E. H. Marth (eds.), Fundamentals of Dairy Chemistry, 3rd edit., pp. 409-460. Van Nostrand Reinhold, New York. 342. USDA. 1965. Marketing Research Report 701. United States Department of Agriculture, Washington, DC. 343. Goff, H. D., and V. J. Davidson, 1992. Flow characteristics and holding time calculations of ice cream mixes in HTST holding tubes. /. Food Prot. 55:34-37. 344. Swindels, J. F., R. Ullman, and H. Mark. 1959. Viscosity. In A. Weissberger (ed.), Technique of Organic Chemistry, 3rd edit., Vol. 1 Part 1. Interscience Publishers, New York. 345. Eilers, H., R. H. J. Saal, and M. van den Waarden. 1947. Chemical and Physical Investigations on Dairy Products. Elsevier, New York. 346. Phipps, L. W. 1969. The inter-relationship of the viscosity, fat content and temperature on cream between 40 and 800C. / . Dairy Res. 36:417-426. 347. Frederick, A. 198S. An improved cryoscopic procedure for lactose determination in milk. Dairy ScL Abstr. 50-02790. 348. Castaneda, R., G. Fernandez, M. CaIo, and A. Pasqualini. 1987. Cryoscopic method for detection and estimation of rennet whey total solids in whole and skim milk powders. Netherlands Milk Dairy J. 41:69-79. 349. Cabezas, L., A. Marcos, M. A. Esteban, J. Fernandez-Salguero, and M. Alcala. 1988. Improved equation for cryoscopic estimation of water activity in cheese. Food Chem. 30:59-66. 350. Harding, F. (ed.) 1983. Measurement of extraneous water by the freezing point test. IDF Bull. 154. 351. Hortvet, J. 1923. The cryoscopic examination of milk. /. Assoc. Off. Agric. Chem. 5:470-474. 352. Shipe, W. F. 1956. The use of thermistors for freezing point determinations. / . Dairy Sci. 39:916. Abstr. 353. Prentice, J. H. 1978. Freezing point data on aqueous solutions of sucrose and sodium chloride and the Hortvet test: a reappraisal. Analyst 103:1269-1273. 354. Association of Official Analytical Chemists. 1970. Methods of Analysis, 13th edit. Washington, D.C. 355. Association of Official Analytical Chemists. 1984. Methods of Analysis, 14th. edit. Washington, D.C. 356. Richardson, G. H., M. S. Mortensen, and R. G. Crockett. 1978. Quantification of added water in milk using vapour pressure osmometry. / . Assoc. Offic. Anal. Chem. 61:1038-1040. 357. Henningson, R. W. 1969. Thermistor cryoscopic determination of the freezing point value of milk produced in North America. J. Assoc. Off. Anal. Chem. 52:142-151. 358. Packard, V., and R. Ginn. 1990. An evaluation of freezing point changes in raw milk analyzed by Dairy Quality Control Inst. Inc. over ten years, 1979-88. Dairy Food Environm. Sanitat. 10:347-351. 359. Packard, V., and R. Ginn. 1990. A 12-month study of freezing point of regional raw milk supplies within the State of Minnesota. Dairy Food Environm. Sanit. 10:597-599. 360. Eisses, J., and B. Zee. 1980. The freezing point of authentic cow's milk and farm tank milk in the Netherlands. Netherlands Milk Dairy J. 34:162-180.

361. Brathen, G. 1983. Milk constituents and freezing point depressing. In F. Harding (ed.), Measurement of Extraneous Water by the Freezing Point Test. International Dairy Federation Doc. 154. 362. Shipe, W. F. 1959. The freezing point of milk. A review. / . Dairy Sci. 42:1745-1762. 363. Buchberger, J. 1986. Stage-by-stage monitoring of addition of water to milk. Molkerei Zeitung Welt Milch 40:497-503. 364. Bradan, A. E. 1987. Milk conductivity and its use for detection of mastitis. Indian Vet. J. 64:733-737. 365. Kozanecki, M., A. Sciubisz, and A. Kasperwicz. 1982. Interrelationships between the somatic cell number and lactose level and conductivity in cow's milk and their diagnostic significance in detection of mastitis. In Proceedings of the 12th World Cong. Diseases of Cattle 2:1054-1058. 366. Fernando, R. S., R. B. Rindsig, and S. L. Spahr. 1982. Electrical conductivity of milk for detection of mastitis. J. Dairy Sci. 65:659-664. 367. Dejmek, P. 1989. Precision conduct-ometry in milk renneting. J. Dairy Res. 56:69-78. 368. Clark, W. M. 1960. Oxidation-Reduction Potentials of Organic Systems. Williams & Wilkins, Baltimore. 369. Dickinson, E., and G. Stainsby. 1982. Colloids in Foods. Applied Science Publishers, Essex, England. 370. Boulet, M., and D. Rose. 1954. Titration curves of whey constituents. J. Dairy Res. 21:229-237. 371. Visser, S. A. 1962. Occurrence of calcium phosphates in the presence of organic substances, especially proteins. J. Dairy Sci. 45:710-716. 372. Pyne, G. T., and T. C. A. McGann. 1960. The colloidal phosphate of milk. II. Influence of citrate. / . Dairy Res. 27:9. 373. Brule, G., and J. Fauquant. 1981. Mineral balance in skim milk and milk retentate: effect of physicochemical characteristics of the aqueous phase. / . Dairy Res. 48:91-97. 374. Hill, A. R., D. H. Bullock, and D. M. Irvine. 1985. Buffer capacity of cheese wheys. / . Food ScL 50:733-738. 375. Hill, A. R., D. H. Bullock, and D. M. Irvine. 1985. Composition of cheese whey: effect of pH and temperature of dipping. Can. Inst. Food Sci. Technol. 18:53-57. 376. Sturtevant, J. M. 1959. Calorimetry. In I. A. Weissberger (ed.), Technique of Organic Chemistry, 3rd edit., Vol. 1. Part 1. Interscience, New York. 377. Phipps, L. W. 1957. A calorimetric study of milk, cream and the fat in cream. / . Dairy Res. 24:51-67. 378. Watson, E. S., M. J. O'Neill, J. Justin, and N. Brenner. 1964. A differential scanning calorimeter for quantitative differential thermal analysis. Anal. Chem. 36:1233-1237. 379. Bertsch, A. J. 1982. Specific heat capacity of whole and skim milk between 50 and 14O0C. Lait 62:265-275. 380. Peeples, M. L. 1962. Forced convection heat transfer characteristics of fluid milk products: a review. / . Dairy Sci. 45:297-302. 381. Woodams, E. E., and J. E. Nowrey. 1968. Literature values of thermal conductivities of foods. Food Technol. 22:150-158. 382. Goulden, J. D. S. 1961. Quantitative analysis of milk and other emulsions by infra-red absorption. Nature 191:905-906.

383. Walstra, P. 1965. Light scattering by milk fat globules. Netherlands Milk Dairy J. 19:93-109. 384. Sjaunja, L.-O., and J. Schaar. 1984. Determination of casein in milk by infra-red spectrophotometry. Milchwissenschaft 39:288-290. 385. Goulden, J. D. S. 1963. Determination of SNF in milk and unsweetened condensed milk from refractive index measurements. / . Dairy Res. 30:411-447.

CHAPTER

2
Analyses
Genevieve L Christen
2.1 Introduction, 85 2.1.1 Purpose of Analysis of Dairy Products, 85 2.1.2 Sources of Additional Information, 86 2.1.3 Types of Analyses, 86 Sampling, 86 2.2.1 General Comments, 86 2.2.2 Sampling of Liquid Products, 87 2.2.3 Sampling of Dry Products, 88 2.2.4 Sampling of Butter, 88 2.2.5 Sampling of Cheese, 88 Tests for Milk Composition, 89 2.3.1 Fat, 89 2.3.1.1 Gravimetric Methods, 89 2.3.1.2 Volumetric Methods, 91 2.3.1.3 Automated Methods, 94 2.3.2 Total Solids, 96 2.3.2.1 Drying Methods, 96 2.3.2.2 Lactometer Method, 96 2.3.2.3 Automated Methods, 97 2.3.3 Protein, 98 2.3.3.1 Kjeldahl Method, 98 2.3.3.2 Dye-Binding Methods, 99 2.3.3.3 Automated Methods, 99 2.3.4 Lactose ,99 2.3.4.1 Polarimetric Method, 99 2.3.4.2 Gravimetric Method, 100 2.3.4.3 Enzymatic Method, 100 2.3.4.4 HPLC Method, 100 2.3.4.5 Automatic Method, 100 2.3.5 Ash, 101 2.3.6 Vitamins, 101 2.3.7 Minerals, 102 Tests for Milk Quality, 102 2.4.1 Titratable Acidity, 102

2.2

2.3

2.4

2.5

2.6

2.4.2 Added Water, 105 2.4.2.1 General Comments, 105 2.4.2.2 Cryoscopic Methods, 105 2.4.2.3 Vapor Pressure Osmometric Method, 106 2.4.3 Sediment, 106 2.4.4 Antibiotics, 107 2.4.4.1 General Information, 107 2.4.4.2 Bacterial Growth Inhibition Methods, 107 2.4.4.3 Competitive Binding Methods, 109 2.4.4.4 Other Methods, 111 2.4.5 Acid Degree Value, 112 2.4.6 Iodine and Hypochlorites, 113 2.4.7 Aflatoxins, 113 2.4.8 Pesticides, 114 Tests for Abnormal Milk, 115 2.5.1 4 'Cow-Side" Tests, 115 2.5.1.1 California Mastitis Test, 115 2.5.1.2 Conductivity Measurement, 116 2.5.2 Wisconsin Mastitis Test, 116 2.5.3 Somatic Cell Count, 117 2.5.3.1 Direct Microscopic Somatic Cell Count, 117 2.5.3.2 Electronic Somatic Cell Counting Methods, 118 2.5.3.3 Membrane Filter-DNA Method, 120 Mien ^biologic al Methods, 120 2.6.1 Aerobic: Plate Count, 121 2.6.1.1 General Introduction, 121 2.6.1.2 Standard Plate Count, 121 2.6.1.3 Spiral-Plating Technique, 122 2.6.1.4 Rehydratable Film Method, 123 2.6.1.5 Impedimetric Methods, 124 2.6.1.6 Hydrophobic Grid-Membrane Filter Method, 124 2.6.1.7 Pectin-Gel Method, 125 2.6.1.8 Reflectance Colorimetry, 125 2.6.2 Coliform Count, 126 2.6.2.1 General Introduction, 126 2.6.2.2 Most Probable Number, 126 2.6.2.3 Violet Red Bile Agar Methods, 127 2.6.2.4 Rehydratable Film Method, 128 2.6.2.5 Pectin-Gel Method, 129 2.6.2.6 Impedimetric Methods, 129 2.6.2.7 Hydrophobic Grid-Membrane Filter Method, 130 2.6.2.8 Fluorogenic Assay Methods, 130 2.6.3 Tests for Specific Spoilage Bacteria, 131 2.6.3.1 Psychrotrophic Bacteria, 131 2.6.3.2 Lipolytic Bacteria, 132

2.6.3.3. Proteolytic Bacteria, 132 2.6.3.4 Yeasts and Molds, 133 2.6.3.5 Spore-Forming Bacteria, 134 2.6.4 Tests for Specific Pathogenic Bacteria, 135 2.6.4.1 Listeria, 135 2.6.4.2 Staphylococcus aureus, 136 2.6.4.3 Salmonella, 137 2.7 Selected Analytical Techniques for Dairy Products, 139 2.7.1 Assurance of Adequate Pasteurization, 139 2.7.2 Total Solids in Butter and Cheese, 141 2.7.3 Salt in Butter and Cheese, 142 2.7.4 Sorbic Acid in Cheese, 144 2.7.5 Overrun in Frozen Dairy Desserts, 145 2.8 Sensory Analysis, 146 2.8.1 Sensory vs. Chemical and Microbiological Methods, 146 2.9 Summary, 148 2.10 Future Developments, 148 2.11 References, 149

2.1.1 Purpose of Analysis of Dairy Products


Dairy products must conform to specifications established by regulatory agencies, processors, and consumers. These specifications are for composition, quality, and shelf life. Analytical techniques have evolved over the last 100 years or so to ensure conformation to specifications. Historically, volume and fat content have been indicators of the value of raw milk. Dr. Babcock introduced a method for determination of fat in milk in 1890. This method, slightly modified, remains as one of the primary techniques of the dairy industry. Other analytical techniques have been developed and applied to dairy products. Many of these, although serving a useful purpose when first developed, have since been replaced by improved methodology. Sources for analytical techniques include university and government laboratories, private testing laboratories, and suppliers of testing materials and equipment. In this chapter, all techniques that have or can be applied to dairy products will not be discussed. Techniques that are widely used or many that have been evaluated in collaborative studies will be presented. Techniques not mentioned are excluded not for lack of validity but for lack of space. This chapter is not intended to be a "how-to" manual for dairy product analyses. Rather it is intended to be a discussion of basic techniques. Section 2.1.2 will direct the reader to sources of step-by-step analytical methodology.

Laboratory safety and quality control cannot be ignored. However, detailed discussion of either is beyond the scope of this chapter. Information on both topics should be gathered before attempting any analytical technique. This information, along with method selection, are the responsibility of the analyst with the assistance of management.

2.1.2 Sources of Additional Information


There are two primary agencies in the United States that serve to approve and adopt analytical techniques for the dairy industry. These are the American Public Health Association (APHA), which published Standard Methods for the Examination of Dairy Products (SMEDP), Compendium of Methods for the Microbiological Examination of Foods and Standard Methods for the Examination of Water and Wastewater, and the Association of Official Analytical Chemists (AOAC), which published Official Methods of Analysis, Journal of the Association of Official Analytical Chemists, and Bacteriological Analytical Manual (BAM). On an international level, the International Dairy Federation (IDF), through its many commissions, adopts analytical techniques and disseminates them through Bulletins. These publications should be consulted for detailed information on specific procedures.

2.1.3 Types of Analyses


Dairy products are analyzed by chemical, physical, microbiological, and sensory methods. Chemical and physical techniques are frequently used to determine milk composition and quality including the presence or absence of adulterants. Microbiological techniques are used when the analyst is interested in milk quality. Sensory techniques are used to determine milk quality as well as the acceptability of products. In this chapter, individual techniques will be discussed under the broad categories of tests for milk composition, milk quality, and abnormal milk; microbiological methods; and sensory analysis. Selected analytical techniques for dairy products will also be discussed. Sampling of dairy products will be covered initially and the chapter will conclude with discussion of some future developments.

2.2 Sampling 2.2.1 General Comments


The results obtained from analysis of any sample are only as good as the quality of the sample. Incorrect conclusions come from improperly collected or handled samples. Milk and its products are heterogeneous. Care must be taken to ensure adequate mixing prior to extraction of the sample. Samples change with temperature fluctuations; care must be taken to ensure that the sample is taken at the appropriate temperature and remains at the appropriate temperature until test results are completed. Sampling is the extraction of a larger approximate quantity of material representative of the whole. The sample usually must be further subdivided by partitioning

into a smaller exact quantity for analysis. The person charged with extraction of the larger sample must carefully record the necessary information on the sample container, including identification of the sample and date taken. Frequently additional information may be required including temperature at sampling time, name of person taking sample, etc. These samples are then transferred to the point of analysis. This may be as simple as carrying the samples to the laboratory, or as complicated as storing the samples in appropriate containers and shipping them under appropriate conditions to a laboratory at some distant point. Sampling of dairy products is done for several different purposes. The sample may be needed for chemical, microbiological, or sensory analysis. The end result determines how the sample is to be taken. Frequently, all three types of tests will be applied to a sample, so the most stringent specifications should be followed. For chemical analysis, the container need only be clean and dry to ensure that the sample is not contaminated with foreign compounds. For microbiological analysis, the container must be clean, dry, and sterile. Frequently, single-use, commercially available containers are used. For sensory analysis, the container must be clean, dry, sanitized, and free from odorous compounds.

2.2.2 Sampling of Liquid Products


Raw milk is particularly difficult to sample, as a cream layer forms when the milk remains quiescent. The milk to be sampled must be thoroughly mixed by pouring milk in small containers from one to another several times; by plunging bulk samples several times, moving a submerged plunger from place to place (this is especially true for cream sampling); or by agitation of bulk samples with a mechanical stirrer. However, care must be taken to avoid mixing too long or too violently as this could lead to churning, homogenization, or foaming which will alter final results. The preferable method of sampling is to use individually wrapped, sterile or presterilized, plastic, single-service sampling tubes which may be dipped directly into the milk. Care must be taken not to handle the portion of the container that will contact the milk.1 Alternatively, sanitized tubes or stainless steel metal dippers may be used to transfer the sample into sanitized or sterile containers. Direct-line sampling is also possible, if the lines are equipped with sanitized gaskets that will permit such sampling. The needle of a disposable, plastic, sterile hypodermic syringe can be inserted through the gasket. Care must always be taken during sampling not to introduce contamination. Samples for microbiological analysis must be kept between 0 and 4.4C and analyzed within 24 h of sampling. Samples for sensory analysis should likewise be kept between 0 and 4.4C and analyzed as soon as possible. If chemical analysis is to be done on the same set of samples the portion needed for microbiological and sensory testing should be removed aseptically first. If only chemical analyses are required on the samples, they may be chemically preserved with a suitable preservative (e.g., potassium dichromate or bronopol). The samples may then be transported at ambient temperature. Preserved samples may not be analyzed for bacteria or for sensory properties.

2.2.3 Sampling of Dry Products


Sampling of dry products presents some challenges, depending on the method of partitioning within the lot. A sample may be desired that is representative of many bags within a shipment, or it may be of one sample container as small as an individual consumer size package or as large as a rail car. Statistically sound sampling plans must be developed to ensure that the sample is representative of the whole. Once the sampling plan has been developed, several points need to be considered in sampling of dry products. The first is that dry dairy products are very hygroscopic; they should be exposed to the atmosphere for a minimal time to avoid moisture adsorption. Generally, a composite sample of several bags or areas within a bulk quantity is preferred. Care should be taken to prevent moisture adsorption during the compositing process. Dry samples are particularly susceptible to oxidation, and should be protected from oxygen and light. When the final sample is extracted for analysis in the laboratory, the composite sample should be mixed by shaking prior to taking the subsample.

2.2.4 Sampling of Butter


Butter may be sampled directly from the churn, from bulk quantities, or from consumer size prints. Source will determine the exact details of sampling, but the general procedure is the same. Butter samples should be cooled to between 0 and 4.4C immediately after sampling. Samples should be placed into sterilized (or sanitized if for chemical or sensory analyses only) containers. Usually butter is sampled with a stainless steel butter trier. The trier can be sterilized between samples by wiping with a clean disposable towel or tissue, dipping in 70% alcohol, and flaming to remove excess alcohol, and tempered by twice inserting into the butter to be sampled. If samples are taken only for chemical or sensory examination, the trier can be cleaned and dried between each sample, without sterilization in alcohol. Sampling should be accomplished from several different points to ensure a representative mixture. The trier should pass diagonally through the butter from top to bottom, through the center to obtain a representative sample.

2.2.5 Sampling of Cheese


The sampling procedure applied to cheese depends on its shape, type, and size. In general, sampling is done with a cheese trier and the sample quantity is >50 g. Following extraction of the sample, care must be taken to close the hole to ensure that mold growth does not occur in the rest of the cheese. The hole may be sealed with a sealing compound such as a mixture of molten paraffin, beeswax, and white petrolatum (1:1:2) or a mixture of white petrolatum and paraffin (hi). 1 Cheese can be resealed in a vacuum-heat-sealable plastic pouch. Cheese sampling in larger than 40-lb blocks is more difficult because cheese cools slowly from outside to inside and the moisture pattern becomes fixed with the highest on the outside, the lowest in the center, and the average somewhere in between.2

Exact details on sampling of barrel cheese may be obtained from the National Cheese Institute (888 16th St., N.W., Washington, D.C. 20006). Subsamples are taken following passage of the cheese through a food chopper three times. Alternatively, the cheese may be cut or shred very finely and mixed. High moisture cheese samples such as cottage cheese should be in original containers if feasible. Coliform bacteria decrease in number in acid environments so tests for these organisms should be performed within 24 h after product manufacture. Samples should always be protected from contamination and stored between 0 and 4.4C. Subsamples of soft cheese which are impossible to grind may be prepared by homogenizing in a blender. Care should be taken to prevent the sample temperature from exceeding 250C.

2.3 Tests for Milk Composition 2.3.1 Fat 2.3. Ll Gravimetric Methods
The Roese-Gottlieb procedure involves determining the weight of fat in a sample following extraction by solvents. This method has been accepted as the international method for fat determination in milk through an agreement between the International Dairy Federation (IDF), the International Organization for Standardization (ISO), and the Association of Official Analytical Chemists (AOAC). It is the final action reference method for fat determination due to the precision of the results. ("Official methods are designated first action or final action, and, in a few cases, procedures. A first action method has undergone collaborative study, has been recommended by the appropriate General Referee and Methods Committee, has been approved interim first action by the chairman of the Official Methods Board, and has been adopted official by the Association members at an annual meeting. A method may be adopted final action a minimum of 2 years after it has been adopted first action, and again, after it has been recommended by the appropriate General Referee and Methods Committee and voted on by the Association members at an annual meeting."3). However, because of the time, expense, and skill required to perform the analysis it is not commonly encountered in most laboratories. The Mojonnier ether extraction method is a slightly modified version of the Roese-Gottlieb method, and has been accepted as a first action procedure by the AOAC. The two procedures differ in the quantity of ammonium hydroxide used to dissolve the casein and in the addition of ethanol in the second extraction step. The second addition of ethanol helps to prevent gelation during extraction. The Mojonnier method is the recommended reference method for the determination of fat content in raw milk for the calibration of infrared milk analyzers.4 The basic procedure for the Mojonnier method is to accurately weigh a sample of product to be tested (weights vary depending on product; exact weights are given in ref. 4) into an empty, dry, preweighed Mojonnier flask (Fig. 2.1). Water may be added to rehydrate or dilute certain products. Ammonium hydroxide is added to

Figure 2.1 Flask, pipette, and drying dish used in the Mojonnier fat determination. dissolve casein, and a few drops of phenolphthalein indicator are added to help visualize the interface between the aqueous and the solvent phase. Ethyl alcohol is added to prevent gel formation when ethers are added. Ethyl ether and petroleum ether are added separately to dissolve fat. Following addition of each reagent, the mixture is shaken for a prescribed time in the Mojonnier flask. The flask and its contents are centrifuged at approximately 600 rpm for at least 30 s to allow phase separation. Alternatively, layers will separate if allowed to stand for sufficient time. Once phases are separated, the upper solvent phase is carefully decanted into a preweighed dish. The analyst must avoid pouring over any of the suspended solids or aqueous phase. While the second extraction is being performed, the dish may be placed on a hot plate at 1000C to evaporate the first ether layer. (CAUTION: ETHER IS EXTREMELY FLAMMABLE. An effective volatile removal system should be used throughout this procedure.) A second extraction is performed to remove additional quantities of fat by adding more ethanol and ethers, and repeating the previous steps. Some high fat products require a third extraction. With this extraction only ethers are added and the previous steps repeated. After the last extraction, the solvents are completely evaporated at <100C to avoid spattering. The dishes containing the fat are dried completely in a vacuum oven at 70 to 75C under <20 inches of vacuum until a constant weight is reached (usually slightly more than 7 min). The dishes may be dried in a forced air oven at 102 2C for a minimum of 30 min. The dry dishes are removed from the oven and placed in a desiccator at room temperature. Once equilbrated to room temperature, the fat is determined by

weight. Fat percentages are calculated by dividing the weight of the fat by the weight of the sample and multiplying by 100. Reagent blanks should be run daily and subtracted from the weight of the fat. Reagent blanks should be from 0 to 0.0020 g and consistent within batches of reagent. If the blank is <0, errors have been made and should be identified. If the blank is >0.0020 g, some reagent contains excessive residue and should be identified and replaced. The same analyst should obtain duplicate results on the same sample within 0.03%. If greater than this, the test should be repeated.

2.3.1.2 Volumetric Methods


The Babcock method for determination of fat in raw milk was accepted by the AOAC as an official final action procedure in 1920 and reclassified as a revised first action procedure in 1989.5 Babcock's original method has been refined over the years to increase its accuracy and improve its efficiency. The refinements include: (1) the use of laboratory grade water of a specified temperature to raise the fat column into the calibrated bottle neck; (2) controlling and standardizing acid addition; (3) use of a mechanical shaker to aid in digestion of the sample; (4) maintaining a constant temperature of the product and acid mixture; and (5) improving the accuracy of the glassware.4 The Babcock fat method is classified as a volumetric method and fat content is expressed in percentage based on volume of fat measured in specially calibrated bottles. The method combines physical and chemical reactions to break the oil-inwater emulsion of the milk and release the fat so that it may be collected and measured in the neck of the bottle. The accuracy of the test is dependent on the accuracy of the transfer pipets used to measure the sample and of the Babcock bottle. There are a variety of Babcock bottles available and selection is dependent on the sample type (Fig. 2.2). Skim milk bottles are calibrated between 0 and 0.5% fat in 0.01% divisions. These bottles are double necked because the sample cannot be forced down the tiny bore of the fat column. Care must be taken when using these bottles that during centrifugation the fat is forced up the small bore rather than the sample introduction neck. Standard Babcock milk bottles are calibrated from 0 to 8% fat in 0.1% increments. The sample is introduced down the same neck in which the fat is collected. Both skim milk bottles and standard milk bottles are calibrated for an 18-g sample. Ice cream, cream, and Paley bottles are calibrated for 9-g samples. Ice cream bottles are designed for samples containing between 5 and 20% fat and are calibrated in 0.2% increments. Cream bottles are designed for samples containing between 10 and 50% fat and are calibrated in 0.5% divisions. Paley bottles are designed for use with solid samples such as cheese. These bottles are calibrated from 0 to 20% fat in 0.2% divisions and have a stoppered opening for introduction of sample. The analyst is responsible for choosing the correct bottle type for the product being analyzed. The quantity of liquid product introduced into the test bottle is determined volumetrically. The temperature of the test mixture will determine the volume transferred as liquids expand with heating. In addition, the temperature of the sample will

Figure 22 Babcock bottles commonly found in daily laboratories. (A) Whole milk test bottle; (B) Skim milk test bottle; (C) Cream test bottle; (D) Paley test bottle.

impact the physical state of the fat. The test mixture should be equilibrated to 38 1C prior to sampling. Specially designed pipettes are used to transfer 17.60 0.05 ml of milk to the bottle with removal of the last drop using a slight air force. Eighteen grams of milk are transferred based on the volume and the specific gravity of milk. Concentrated sulfuric acid is added to break the oil-in-water emulsion and to provide heat to dissolve the fat allowing it to separate by gravity. The specific gravity of the sulfuric acid is important in controlling the acid/milk reaction temperature. Reference 4 should be consulted for specific instructions on how to adjust the specific gravity to obtain the desired reaction temperature. If the acid is too weak, the reaction temperature will be too low and incomplete freeing of the fat will occur, resulting in low test results. If the acid is too strong, burning of the sample will occur from too high a reaction temperature, and charred particles will be present in the fat column that interfere with reading of the results. Other factors that impact the milk/ acid reaction temperature include the amount of acid added, the rate of acid addition and subsequent swirling, and the temperature of the milk and acid. Person-to-person differences in technique exist which make it essential to tailor the quantity of acid added to the technician performing the test. Following the addition of the acid, the sample and acid are carefully swirled until the last traces of curd disappear. The samples are then shaken on a mechanical shaker for at least 1 min after the last bottle is inserted in the shaker. [At this point it is prudent to include a word of warning about the sulfuric acid. Just as the acid dissolves

the milk protein, it will dissolve human skin (as well as lab coats, shoes, and many types of countertop materials). Care must be used in handling of the acid. Protective garments should include rubber gloves, rubber lab apron, and goggles. Care should be taken to point the bottle neck away from yourself and all others in the lab during the swirling process. CAUTION: Acid should always be added to milk in the bottle, not the reverse. Adding aqueous materials to concentrated sulfuric acid will cause violent reactions which are dangerous.] When the last sample has shaken a minimum of 1 min, the bottles are transferred to a heated Babcock centrifuge (600C) where additional heat and centrifugal force bring the fat to the top of the mixture. Sulfuric acid is much denser than the fat (approximately 1.83 specific gravity vs. approximately 0.93 specific gravity). The combined specific gravity of the mixture of sulfuric acid and aqueous components is approximately 1.43, causing physical separation of the phases.6 Centrifugation enhances the physical reaction. When the bottles are placed in the centrifuge they must be counterbalanced. The centrifuge has two rows for bottle placement. Fill the outer row first, placing the bottles opposite one another in the centrifuge. It has been the experience of the author that when only two samples are centrifuged, if they are placed in the inside holders, the necks of the bottles break during centrifugation, leaving a lost test and a mess to clean. The first centrifugation is for 5 min, the centrifuge stopped (carefully) and water (600C) added down the side of the bottle so that it layers underneath the fat, raising it to within 0.6 cm of the base of the bottle neck. Centrifugation is repeated for 2 min and additional hot water is added to raise the fat column into the graduated portion of the bottle. The final centrifugation is for 1 min. The bottles are transferred into a water bath where the fat column is adjusted to 57.5 1C and tempered a minimum of 5 min. The water level in the bath should be slightly above the top of the fat column. The volume of fat is determined using calipers placed at the top and the bottom meniscus of the fat column and carefully transferred so that the lower point rests on the zero mark and the upper point rests somewhere within the calibrations of the column. The fat content is read directly in percentage to the nearest 0.05%. Other fluid products are tested similarly to milk with some modifications. Products such as chocolate milk, ice cream mix, ice cream, and other frozen desserts that are high in sugar must be handled differently due to the tendency to char.7 Ammonium hydroxide and normal butyl alcohol are incorporated into the reaction mixture to improve the results. These reagents are also added to improve fat recovery with cottage cheese samples. Skim milk, low-fat milk, buttermilk, and whey have normal butyl alcohol included in the reagents to aid in obtaining a fat column free of charred material. Roccal solution (a 50% concentrate of benzalkonium chloride, U.S.P.) may serve as a wetting agent to prevent charring of chocolate milk, skim milk, buttermilk, and whey samples.4 Although not as widely applied in the U.S. as the Babcock test, the Gerber fat test method is an official alternative first action method of the AOAC5 as well as being described in SMEDP.4 It is a volumetric test procedure and is applicable to raw, pasteurized, homogenized, and composite milk samples. It is also used for low-

fat milk and skim milk and as an in-plant control test for frozen desserts.4 The Gerber procedure is used on a much wider scale in the international community.

2.3.1.3 Automated Methods


As number of samples and labor costs increased, automated methods were developed for determining fat content. A variety of instruments have been introduced during the last 30 years, so many that the APHA and AOAC approve instrument performance rather than individual instruments.4'5 There are two basic methods for automatically determining fat content: turbidimetric and spectroscopic. The turbidimetric method is suitable only for milkfat, whereas multiple components may be analyzed spectroscopically. Regardless of the method selected, calibration of the instrument is essential. Both SMEDP and AOAC give performance specifications for calibration.4^ Calibration samples may be obtained from state and private laboratories and are useful for those labs not routinely performing ether extraction fat analysis. Once calibrated, performance should be checked daily using six to ten samples of known fat content (by ether extraction) within the range of the samples being tested. Determination of fat content based on turbidity relies on the fact that milkfat scatters light. The quantity of fat may be estimated if the sample is sufficiently dilute to remove the interference by casein. Tetrasodium ethylenediaminetetraacetate (EDTA) disperses colloidal casein particles and the fat is dispersed by homogenization to produce fat globules of uniform size. A beam of light is passed through the prepared sample in a photocell and light scattering is measured. The amount of scattering is proportional to the amount of fat. The percentage of fat is reported directly, often as a printed report generated via computer linkage. This instrumental method is frequently combined with automatic sampling devices where the sample is taken from various points in a milk processing line and transported by pumping to the instrument. The results are obtained and automatically fed to a standardizing computer that makes adjustments as necessary in the flow of milks of various fat content to maintain a constant fat content in the finished product. Laboratories with many samples to test for fat, protein, lactose, and/or total solids find it economical to invest in a spectroscopic instrument able to estimate these components automatically. Such instruments are frequently found in laboratories of cooperatives or Dairy Herd Improvement Associations where the information gained is provided to dairy farmers (Fig. 2.3). Recently, these instruments have become more common in cheese plants where multicomponent information regarding milk has an impact on cheese yields. As the value of milkfat declines and the value of other components increases, we will see these instruments appear in more dairy testing laboratories. The basis of analysis of milk by spectroscopic methodology is that infrared energy (IR) of specific wavelength is absorbed by the chemical constituent in milk. The -CH groups in the fatty acid chains absorb at 3.48 /xm whereas the carbonyl groups in ester linkages of fat molecules absorb at 5.723 |xm. Originally, only ester linkages were measured to quantify fat, but there was a lack of correlation with gravimetric

Figure 2 3 Equipment for the automatic determination of fat and protein in milk at the TN DHIA Services Laboratory, Knoxville, TN.

methods. Triglycerides of differing numbers of carbons each have three ester linkages but have different weights. When carbon groups are measured, the variation in weights is taken into account and the method better correlates with the gravimetric procedure. The specific absorption of other components will be discussed under those sections. Like the instrument for measuring fat by turbidity, calibration is the key to successful results. Energy of the desired wavelength is created by passing an IR beam through an optical filter. The filtered beam passes through the milk sample and unabsorbed energy passes on to the filter. The amount of energy absorbed is proportional to the concentration of the component in the sample. The IR beam is also passed through an optical filter which transmits energy at a wavelength where there is minimal absorption by the component. This beam passes through the milk and on to the detector. The two signals are compared at the detector and the concentration of the component determined. Scattering by components in the milk impact the amount of energy reaching the detector. Degree of homogenization of the sample, either before or during analysis, may impact the results due to variation in scattering by the sample.4 It is essential that the interior of the instrument remain dry because moisture can cause changes in optical zero and a shift in calibration.5 Desiccant should be changed

on a daily basis and 3 to 4 h prior to the next use. Calibration should be performed with the type milk that is to be analyzed; mixtures of milk and cream should not be used. Abnormal milks should also not be used for calibration. As with all procedures, it is the analyst's responsibility to ensure that the instrument is working properly, as malfunctions that affect calibration can cause large errors.5

2.3.2 Total Solids 2.3.2.1 Drying Methods


Total solids represent the components that remain after the complete removal of water. A prescribed amount of sample is weighed into a preweighed, clean, dry sample container. For greatest accuracy, weights are determined to the nearest 0.0001 g. Heat is applied to the sample until a constant weight is attained, the sample is cooled and the weight again determined. Total solids (%) are calculated as the weight of the sample after drying divided by the weight of the sample before drying multiplied by 100. Any variations in procedures are in the method of applying heat. The official final action procedure accepted by the AOAC, ISO, and IDF specifies dehydration under atmospheric pressure.5 The basic procedure is to precisely weigh 2.5 to 3 g of prepared sample into a weighed flat bottom dish 5 cm or greater in diameter. The sample is preheated on a steam bath 10 to 15 min, then transferred to an air oven at 98 to 1000C for 3 h. The dish and sample are cooled in a desiccator, quickly weighed, and results calculated. Specific precautions to apply to all weight determinations, particularly very precise ones, are that fingerprints and air vapor have mass. Once the dishes are predried they should be exposed to the atmosphere for a minimum amount of time and should be handled only with forceps or tongs. All cooling should be done in a clean desiccator. Although, not officially listed among AOAC of SMEDP procedures, there are several procedures that are applicable to milk for rapid screening but do not serve as official test methods. Laboratories equipped with Mojonnier fat testing apparatus also have the capability to determine total solids by the Mojonnier method.6*8 Moisture is removed by predrying the sample on a hot plate at 1800C until the first traces of brown appear. The sample is completely dried in a vacuum oven (not less than 20 inches) for 10 min at 1000C. Moisture can be approximated using a self-contained rapid drying procedure that incorporates an IR lamp (not to be confused with the automated method using IR energy) to dry the sample as it sits on a balance.8 Microwave energy may be used to remove moisture. The use of microwave will be described in Section 2.7.2.

2.3.2.2 Lactometer Method


The lactometer is a special hydrometer designed for determination of specific gravity in milk. Specific gravity is a physical property of matterthe weight of a specific volume of material compared to a standard substance. Water at 16C is the standard for liquids and solids. The average specific gravity of milk is 1.032 but varies de-

pending on the solids-not-fat and the fat content. To determine total solids using a lactometer, fat content must also be determined. The lactometer is a specially designed weighted instrument that is calibrated in specific gravity units. Reference 4 specifies two types of lactometers, large and small; the choice depends on the type of milk being tested. The lactometer is allowed to float freely in the milk and specific gravity is read at the top of the meniscus after the lactometer has come to rest. Readings should be repeated to ensure accuracy by withdrawing the lactometer just enough to wipe the stem then slowly immersing it. The cylinder containing the milk should be of sufficient capacity to allow free movement of the lactometer and allow it to float in the milk. Specific gravity is affected by temperature. Therefore, the sample and the lactometer should be at a constant temperature. Cylinders containing the milk should be equilibrated in a water bath held at 39 1C and deep enough to bring the water level to within 5 cm of the top of the cylinder. Temperature of the milk should be recorded at the time of specific gravity determination. The lactometer should be maintained in the 39C bath (a minimum of 3 min) before immersion, removed just prior to the test, and wiped dry. The lactometer is read in increments of 0.2 or 0.5 units and values fall within the range of 24 to 37. These values are converted to specific gravity by including 1.0 before the lactometer reading. For example, if the lactometer reading were 32.5, the specific gravity would be 1.0325. The lactometer degree value read directly from the lactometer is used along with the percent fat from the Babcock test to calculate total solids using Eq. 2.1 for whole milk or Eq. 2.2 for skim milk. % total solids % total solids where F = % fat and L = lactometer reading in degrees. (2.1) (2.2)

2.3.2.3 Automated Methods


Total solids may be determined using mid-IR spectroscopic analysis indirectly. The instrument can measure fat, protein, and lactose. The other component of total solids is ash, which is relatively constant. As with fat determination, calibration of the instrument is essential. At least eight milk samples should be analyzed for total solids by air drying. Fat, protein and lactose are determined instrumentally. The constant, a, is calculated as the difference between the total solids and the sum of the fat, protein, and lactose. Once a has been calculated it is used in the equation, % total solids = a 4- % fat 4- % protein + % lactose. Complete details on calibration are available in ref. 5. Total solids may also be determined in the near IR range in a similar manner.4

2.3.3 Protein 2.3.3.1 Kjeldahl Method


The Kjeldahl method of protein determination has been the standard method for determining total nitrogen in foods and feeds since 1883.4 It remains the standard method for determination of protein in milk and all other methods must correlate with it. However, because of the time, expense, and skill required to perform Kjeldahl analyses, it is not routinely done in dairy laboratories. It is essential that it be described here, though, due to its importance as a standard method. All proteins contain nitrogen. Most food proteins contain 15.7 to 18% nitrogen with 16% commonly given as the average.9 Analysis of nitrogen content can be converted to give an estimate of percent protein. The Kjeldahl procedure cannot differentiate between protein nitrogen and nonprotein nitrogen; thus, for samples that are extensively proteolyzed, protein content will be overestimated. Nonprotein nitrogen varies from farm to farm and ranges from 2 to 10% of the total nitrogen content.10"12 AOAC has recently adopted a new method for determining true protein in milk that accounts for the nonprotein nitrogen component.13 Traditionally, Kjeldahl nitrogen is determined by digestion of a weighed portion of milk by heat and sulfuric acid. Mercury is used as a catalyst although alternative catalysts posing less of a danger to the environment are under investigation. Carbon and hydrogen in the sample are oxidized; protein nitrogen is reduced and transformed to ammonium sulfate. Concentrated sodium hydroxide is added in the second step: the distillation step. With heat, ammonium is liberated and collected as condensate in a standard acid solution (boric acid). The quantity of nitrogen liberated is determined by back-titration using an acid-base indicator. Complete details for performance of the Kjeldahl method on milk are provided in ref. 4. The Kjeldahl method is the official final action method for protein determination accepted by IDF-ISO-AOAC.5 As mentioned previously, nonprotein nitrogen content is included in traditional protein determinations. True protein content is important to cheesemakers because nonprotein nitrogen is lost in whey. Milks high in nonprotein nitrogen are less valuable to cheesemakers. Although few cheese laboratories perform Kjeldahl nitrogen tests, many now analyze milk for total composition using instrumental methods. Calibration of these instruments is very important. If the instrument is calibrated to include nonprotein nitrogen, all test results will be high. Therefore, use of a procedure that excludes nonprotein nitrogen in standardization of the instrument is appropriate. At the 104th AOAC Annual International Meeting, a method was accepted as official first action in which trichloroacetic acid precipitates protein nitrogen from milk.13 Nitrogen content of such a precipitate will represent the true protein content of milk. At the time of the writing of this chapter, this method was not officially accepted to be used for calibration of instruments, although it appears that it may be in the future. There are many adaptations of the Kjeldahl method available on the market speeding digestion, distillation, or titration. Any technique that enhances speed or im-

proves reliability is acceptable. However, it is the responsibility of the analyst to ensure that the technique conforms with the requirements of the standard method.

2.3.3.2 Dye-Binding Methods


Between the time of the introduction of the Kjeldahl method and the introduction of mid-IR spectroscopic methods, scientists searched for more rapid methods for measuring protein. Two methods remain as official today. Both involve the binding of a dye to protein molecules. The Acid Orange 12 method is an official final action method of the AOAC.5 This dye binds specifically under acid conditions to free amino groups, Iysine, the imidazole group of histidine, and the guanidyl group of arginine. Excess dye is added to milk in a dispenser bottle fitted with a spun-glass paper inside the cap. The mixture is shaken vigorously and the filtrate dropped into the cell of an instrument designed to read absorbance. The quantity of free dye is measured and compared to the total dye available for binding. The less dye in the filtrate, the higher the protein content of the sample. The procedure can be automated with the sample filtrate drawn through a flow-through spectrophotometer cell. Calibration curves must be constructed to relate the absorbance values to protein content. Amido Black 1OB also binds specifically to protein. It has an advantage over Acid Orange 12 in that there is a greater change in optical density per unit of milk protein. The Amido Black 1OB procedure has been accepted as an official first action method for determining protein by the IDF-ISO-AOAC.5 The dye binding capacity of milk protein is not affected by homogenization, condensing or heating to 32C for 15 min.9 Extensive proteolysis will increase dye binding because more amino groups are available. Heating milk to the point of browning will reduce dye binding. The dye-binding test is considered suitable for normal milk, but not for atypical milks such as colostrum, mastitic, or from very late in lactation.

2.3.3.3 Automated Methods


Both dye-binding procedures discussed in the preceding section can be automated and instruments are available for that purpose. Additional automated methods have been developed that rely on the specific absorbance of protein molecules of IR energy. The peptide linkages between amino acids of protein molecules absorb at a wavelength of 6.465 jxm. According to manufacturer's instructions, calibration is done with at least eight samples of known protein content.5

2.3.4 Lactose

2.3.4.1 Polarimetric Method


Lactose is an optically active compound; it will interact with polarized light to cause rotation of the plane. The quantity of lactose impacts the degree of rotation. Deter-

mination of lactose by polarimetry has long been the official final method. 5 Other components of the milk first must be removed via precipitation and filtration. Lactose, in a clear filtrate, is introduced in the polarimeter cell (two different sizes) and degree of rotation is measured. The quantity of lactose is calculated from the rotation of the two quantities by formula.4

2.3.4.2 Gravimetric Method


Lactose is a reducing sugar.14 It will react with oxidizing reagents such as copper sulfate under alkaline conditions. In milk, fat and protein must first be removed by precipitation and filtration. On heating a portion of the filtrate, cuprous oxide precipitates and the quantity is weighed. Lactose equivalent to the quantity of cuprous oxide is determined from tabular values (Munson-Walker tables). 3

2.3.4.3 Enzymatic Method


Lactose is a disaccharide of glucose and p-galactose. The enzyme (3-galactosidase (lactase) hydrolyzes the glucosidic bond to produce free monosaccharides. In an enzymatic procedure,15 lactose is determined based on this principle. In the procedure, p-galactose is oxidized further by NAD to galactonic acid in the presence of P-galactose dehydrogenase. The amount of NADH formed is proportional to the quantity of lactose present initially and can be measured by determining absorbance at 340 nm. Because this is an enzymatic reaction, time and temperature are very important in control of results. Assay kits based on this procedure are available commercially.5

2.3.4.4 HPLC Method


Carbohydrates can be separated and quantitively measured by high-performance liquid chromatography (HPLC). 4 A weighed sample is digested with sulfuric acid and precipitate removed by filtration. The clear, colorless liquid is injected directly into an Econosphere NH 2 cartridge column (Alltech Assoc, Inc., Deerfield, IL; at the present time, this method had been evaluated only for this specific column). Lactose peaks are detected by refractive index and are quantitatively determined based on the area of the peaks. Standards of a- and (3-lactose must be run for quantitation. With the incorporation of an autosampler, this procedure can be semiautomated.

2.3.4.5 Automatic Method


Hydroxyl groups in the lactose molecule interact with IR energy at a wavelength of 9.610 |xm. Lactose can be determined automatically along with fat, protein, and total solids. 5 The linearity of output signals from the instrument is checked using lactose solutions and calibration is with milk of known lactose content. The reference method for lactose standardization is the polarimetric method.

2.3.5 Ash
The ash component of milk is small and is composed primarily of minerals. Ash is the material that remains after the organic material is removed by very high heating. Because the sample is exposed to very high heat (550 0 C) for an extended period (12 to 18 h), choice of ashing crucible is important. AOAC 5 specifies platinum crucibles for ash determination of milk. It is the best choice of materials, but expensive; thus should be handled with care. Platinum dishes can corrode, especially in the presence of dirt-containing organic matter. Corrosion from heavy metals can lead to pitting and hole formation. Platinum crucibles should be touched with platinumtipped tongs and placed, after ashing, on clean porcelain or marble surfaces.9 Care should be taken in cleaning the crucibles and mechanical washing should be avoided. In the ashing procedure, approximately 5 g of sample is weighed into a predried, preweighed platinum dish to the nearest 0.0001 g. The sample is evaporated to dryness on a steam bath. Once free moisture is removed, the crucible containing the sample is transferred to an ashing oven at 550 0 C and ignited until the ash is completely free of carbon. The crucible containing the sample is cooled in a desiccator and weighed. The percent ash is calculated as the weight of the ash divided by the weight of the initial sample multiplied by 100. Total solids determination can be combined with ash determination by heating for 3 h in a drying oven, cooling in a desiccator, and recording the dry weight prior to transferring the sample to the ashing oven. If the two procedures are combined, platinum dishes should be used for total solids determination and a 5-g sample used.5

2.3.6 Vitamins
Milk serves as an important source for vitamins A and D. Although whole milk is naturally adequate in vitamin A, vitamin D content is enhanced through addition at time of processing. Low-fat milks are low in vitamin A and it must be restored to original levels. Because these nutrients are added to milk, processors, regulators, and consumers are concerned that they be present in the specified amount. Recent changes in nutritional labeling laws require that the quantity of these and other nutrients be specified on the label. Therefore, determination of these two vitamins is important to the dairy analyst. The method of choice for determination is HPLC. 4 Equipment costs and ease of analysis are such that HPLC has become almost routine. Vitamin A is extracted from room temperature milk using absolute ethanol and hexane combined with centrifugation. Addition of water to the mixture helps in separation of the aqueous and organic phases. On centrifugation, a hexane top layer forms containing the vitamin A. An aliquot from this layer is injected into a HPLC system equipped with a LiChrosorb Si 60 column. The eluting sample is detected by an absorbance detector with an adjustable wavelength of 313 to 325 nm. Peaks are recorded and quantified compared to a standard of retinyl palmitate in hexane. Vitamins D 2 and D 3 can also be measured by HPLC although the procedure is more complex. 4 The sample first must be saponified and any nonsaponifiable constituents extracted. Then cholesterol is removed from the sample by precipitation.

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Vitamin D is separated in a small chromatographic column on neutral aluminum oxide and dried. The vitamin D is identified and quantified by HPLC on a reversephase stainless steel column, Vydac 201 TP, equipped with a variable wavelength detector. USP reference standards for ergocalciferol (D2) and cholecalciferol (D3) are used for quantification.

2.3.7 Minerals
The ash content of milk serves as an approximation of the total mineral content of milk. For quantitation of individual minerals in milk, atomic absorption spectrophotometry is the method of choice. Calcium, magnesium, iron, zinc, copper, manganese, sodium, and potassium may be determined on the same sample by changing the wavelength and flame conditions.5 The sample is predried at 1000C, ashed at 525C for 3 to 5 h, and cooled. The ash should be white and free of carbon (grey particles indicate the presence of carbon). Wet ashing is not recommended as potassium is lost in this process. The sample is diluted in nitric acid (1AO and analyzed in the atomic absorption spectrophotometer. Calibration curves must be prepared for each mineral. Blanks must be prepared for all reagents and glassware and carried through the entire process as mineral contamination can occur at any point. Special care must be given to the quality of water so as not to cause contamination. All glassware must be cleaned by soaking overnight in 20% nitric acid and rinsed three times with distilled-deionized water. Chloride can be determined by titration with silver nitrate (Mohr method).4 Chloride meters are available commercially that automatically titrate chloride ions with silver ions generated internally. When titration is complete, conductivity of the solution increases which can be sensed by electrodes causing the titration to stop. The instrument uses the elapsed titration time to calculate the chloride content.4

2.4 Tests for Milk Quality 2.4.1 Titratable Acidity


When held at above-refrigeration temperatures, microorganisms in milk begin to grow. Some of these organisms produce lactic acid. Traditionally, titratable acidity has been used as an indicator of milk quality, because there is no lactic acid in fresh milk. Under current methods of handling and distributing milk, temperatures rarely are such that lactic acid is produced. If titratable acidity is used as a test to determine acceptance of milk, temperature, odor, and taste should also be noted. Measurement of acidity is impacted by any condition that causes a change in the distribution of calcium phosphate in the sample. Milks high in protein may also have an apparent high acidity, because charged groups on the protein molecule react with alkali. Normal acidity of fresh milk (apparent acidity) is usually 0.15 to 0.16. If values significantly above normal are obtained, the milk is suspect, but other quality tests (especially taste and odor) should be performed prior to rejection. Several million bacteria

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Vitamin D is separated in a small chromatographic column on neutral aluminum oxide and dried. The vitamin D is identified and quantified by HPLC on a reversephase stainless steel column, Vydac 201 TP, equipped with a variable wavelength detector. USP reference standards for ergocalciferol (D2) and cholecalciferol (D3) are used for quantification.

2.3.7 Minerals
The ash content of milk serves as an approximation of the total mineral content of milk. For quantitation of individual minerals in milk, atomic absorption spectrophotometry is the method of choice. Calcium, magnesium, iron, zinc, copper, manganese, sodium, and potassium may be determined on the same sample by changing the wavelength and flame conditions.5 The sample is predried at 1000C, ashed at 525C for 3 to 5 h, and cooled. The ash should be white and free of carbon (grey particles indicate the presence of carbon). Wet ashing is not recommended as potassium is lost in this process. The sample is diluted in nitric acid (1AO and analyzed in the atomic absorption spectrophotometer. Calibration curves must be prepared for each mineral. Blanks must be prepared for all reagents and glassware and carried through the entire process as mineral contamination can occur at any point. Special care must be given to the quality of water so as not to cause contamination. All glassware must be cleaned by soaking overnight in 20% nitric acid and rinsed three times with distilled-deionized water. Chloride can be determined by titration with silver nitrate (Mohr method).4 Chloride meters are available commercially that automatically titrate chloride ions with silver ions generated internally. When titration is complete, conductivity of the solution increases which can be sensed by electrodes causing the titration to stop. The instrument uses the elapsed titration time to calculate the chloride content.4

2.4 Tests for Milk Quality 2.4.1 Titratable Acidity


When held at above-refrigeration temperatures, microorganisms in milk begin to grow. Some of these organisms produce lactic acid. Traditionally, titratable acidity has been used as an indicator of milk quality, because there is no lactic acid in fresh milk. Under current methods of handling and distributing milk, temperatures rarely are such that lactic acid is produced. If titratable acidity is used as a test to determine acceptance of milk, temperature, odor, and taste should also be noted. Measurement of acidity is impacted by any condition that causes a change in the distribution of calcium phosphate in the sample. Milks high in protein may also have an apparent high acidity, because charged groups on the protein molecule react with alkali. Normal acidity of fresh milk (apparent acidity) is usually 0.15 to 0.16. If values significantly above normal are obtained, the milk is suspect, but other quality tests (especially taste and odor) should be performed prior to rejection. Several million bacteria

per milliliter are necessary to produce detectable developed acidity.6 Common spoilage organisms in today's milk supply, (psychrotrophic bacteria) do not produce lactic acid, so will go undetected by this method. Titratable acidity is useful in cultured product manufacturing, where acid development is encouraged, yet controlled. Specifications exist for titratable acidity values expected during various stages of the cheese-making process. Although lactic acid is not the only acid present in fermented milks, it predominates and is used as the basis of calculation of acidity. Either a 9- or an 18-g sample is pipetted (using pipettes calibrated to contain 9 or 18 g of milk) into a beaker or white-interior titration casserole. Two volumes of water are used to rinse completely the pipette into the container. Dry samples can be analyzed by weighing accurately the prescribed amount of sample and suspending it in water. For most samples, phenolphthalein is added (0.5 ml) and the sample titrated to the first permanent (30 s) color change to pink with 0.1 N sodium hydroxide. The concentration of indicator will impact the results, thus should be a constant amount. Also impacting the results are the amount of dilution of the sample, the speed of titration, the amount of indicator, and the temperature of the sample. Therefore, all samples should be titrated as quickly as possible at room temperature using exact amounts of sample, diluent, and reagents.4 The normality of the sodium hydroxide must be determined exactly (0.1000 A O using standard acid titration. Alternatively, prepared sodium hydroxide is available from chemical supply houses. However, it is good lab practice to confirm the normality of such products occasionally. Some sample may be too dark to accurately observe the phenolphthalein endpoint. Such samples should be titrated to a pH of 8.3 using a standardized pH meter and probe. Formulas are available in ref. 4 for calculation of acidity expressed as percent lactic acid for various products. Each milliliter of 0.1000 N sodium hydroxide used in the titration is equivalent to 0.009 g of lactic acid. Specially designed titrators are available that provide percent titratable acidity directly based on this relationship (Fig. 2.4). Sodium hydroxide tends to adsorb carbon dioxide from the atmosphere. When carbon dioxide is dissolved it produces an acid. Normality may decrease during storage and should be verified periodically. If more exact quantities of citric or lactic acid are desired, AOAC5 provides methods for determination of each. Citric acid is determined by a gravimetric method whereas lactic acid is determined by a colorimetric method. Both methods are quite complicated and not applicable to routine analysis. Titratable acidity is related to the pH of the product. Frequently pH measurements are determined because the method is nondestructive and rapid. The most critical part of pH determination is the condition of the pH-sensing electrode. Electrode tips are made of special hydrogen-sensitive glass. If the tip is scratched or clogged, improper results will be recorded. Standardization of the pH meter is also important. Follow pH meter manufacturer's instructions for proper standardization. Standards covering the range of pH values expected should be used daily to check and standardize the instrument. Preferably, premixed standard buffer solutions within code date are used. Temperature causes changes in pH; therefore measurements should be at

Figure 2.4 Titration unit used for the determination of titratable acidity in milk and milk products. the same temperature as the standardizing buffer. Dairy products contain fat and protein that clog the electrode. Care must be taken to clean carefully the electrode as described by the manufacturer. Electrodes should be stored in potassium chloride solution when not in use, unless it will be several months between uses, in which case the electrode should be stored dry. Reference 4 gives an excellent description of problems of and solutions to electrode maintenance. Liquid samples can be measured directly; dry samples need to be rehydrated. Cheese samples must be uniform, obtained by first blending or grinding. The sample is packed into a small container to ensure good electrode contact. The pH of butter is determined only on the serum (aqueous) phase, obtained by melting the butter and sampling the lower phase.

2.4,2 Added Water

2.4.2.1 General Comments


The intentional addition of water to milk is illegal. Occasionally, water may accidentally be added to milk through inadequate drainage of equipment or carelessness. When water is added, molecules in solution, lactose and salts, are diluted. Colligative properties of a solution are those impacted by number of particles in solution rather than nature of the particles. These properties are freezing point, boiling point, osmotic pressure, and vapor pressure. Each is relatively easy to measure; freezing point and vapor pressure are generally employed as measures of water addition. Other conditions can impact the colligative properties besides intentional water addition, and surveillance of milk for presence of added water using these methods must be approached with caution.4 Season of the year, age and health of cow, feed, ambient temperature, breed, time of milking, access to water, weather, and morning or evening milk have been implicated to have an impact on freezing point. Freezing point is also affected by fermentation, vacuum treatment, sterilization, and prefreezing of the sample prior to measurement. Addition of milk solids impacts freezing point. Milk suspected to have had water added must be confirmed by comparison to an authentic sample. 5 An authentic sample is one from the same herd as the suspect sample collected to be certain to exclude water.

2.4.2.2 Cryoscopic Methods


Cryoscopes are instruments that precisely measure the freezing point of a sample. Pure water freezes at 0 0 C. Solids dissolved in water depress the freezing point to temperatures less than zero. When solids become more dilute (as with the addition of water), the freezing point approaches that of pure water. The normal freezing point of milk is usually taken as - 0 . 5 2 2 0 C . Cryoscope methods have been available for many years. In early years, methodology was such that freezing points could not be precisely determined. When more precise instrumentation became available, it was discovered that the freezing points of standard salt solutions were not exactly as previously reported. Most cryoscopes are calibrated in terms of 0 H (degrees Hortvet) but results reported in 0 C. It was originally thought that the two temperatures were equivalent. Now it is known that they are not equivalent but formulas are available to convert one to the other. Results reported prior to the discovery of the difference between 0 H and 0 C are not true freezing points. 4 Methods now use 0 C and results should be reported as such. The official method of analysis accepted by IDF-ISO-AOAC for determination of addition of water to milk is the thermistor method. 5 Thermistor cryoscopes were introduced in 1956. 16 Instead of a large thermometer to record freezing point, a small thermistor is used and changes in voltage or current detected and reported (usually as a digital display). The samples are supercooled in a refrigerated bath, cooling is stopped, and the sample allowed to become isothermal. Crystallization is initiated by agitation via a seeding rod. Latent heat of crystallization is dissipated as the sample temperature increases to the freezing point where the temperature stabilizes

and is recorded. The instrument must be standardized using 7 and 10% (w/v) sucrose solutions which freeze at 0.406 and 0.5980C, respectively. Alternatively, sodium chloride solutions may be prepared. Salt solutions have an advantage over sucrose because they are not as subject to microbial decomposition and are stable for a longer period of time. However, freshly prepared sucrose solutions are the preferred standards. Percent added water can be estimated from the freezing point based on the commonly held relationship that for each 1% of water added to milk, the freezing point increases above the baseline by 0.010C. This presumes that the true baseline temperature is known or can be determined. Generally, if the freezing point of a sample of known origin differs from that of a suspect sample by ^0.0100C, the sample is considered to be water-free. Samples that fail to freeze may have a high solids content caused by high acid, or they may be contaminated with cleaner or sanitizer.4 Samples high in bacteria or somatic cells may continue to supercool.4

2.4.2.3 Vapor Pressure Osmometric Method


The pressure exerted by the vapor over a substance at equilibrium is called its vapor pressure. The value is temperature dependent. As solutes increase, vapor pressure decreases. Conversely as solutes decrease, vapor pressure increases. A vapor pressure reading of 280 mOsmol/kg has been found to be normal for milk.17 An osmometer is an instrument that determines vapor pressure. Instruments are commercially available to determine water added to milk although not as commonly used as the thermistor cryoscope.

2.4.3 Sediment
Sediment is the insoluble portion of foreign material that gets into milk from cows, equipment, or the environment. Because most foreign material is soluble, milk that is found to be free of sediment is not necessarily "clean." Milk that contains a large amount of sediment was most likely collected under unsanitary conditions. The sediment test is usually performed in the laboratory on a known volume of milk. Inline procedures are available for qualitative assessment of sediment. The in-line method is a screening test to be used during pumping of raw milk from farm bulk tanks to raw milk transport tanks and not as a substitute for laboratory analysis on a fixed volume of sample. Reference 5 gives a formula for preparation of coarse standard sediment disks that combines cow manure, garden soil, and charcoal each of specific mesh size and in specified proportions. Standard sediment disks are available from the U.S. Department of Agriculture, Standardization Branch, Dairy Division or commercially. Because sediment is insoluble, it is distributed heterogeneously and most tends to settle to the bottom of the container. Sampling is extremely important to obtain proper results. Choice of sampling method depends on container type. Off-bottom samplers are available for 5- and 10-gal cans. For retail containers, the sample is

extracted after thoroughly mixing the container. One-pint or one-gallon samples are used, again depending on the container size. Care should be observed not to introduce extraneous material in the sampling process. The sample is poured into the sediment testing apparatus containing a cotton sediment pad. Vacuum (limited suction only) is applied below the sediment pad and milk pulled through the pad. Flow rates vary depending on fat content or clumping, previous heat treatment, high acidity, abnormal milk, freezing, and amount of sediment. If the sample does not need to be salvaged, it can be diluted with water to speed filtration. While still wet, the pad is mounted on special-sized paper or stored in individual, transparent, waxed envelopes. Amount of sediment is determined by comparing to standards and character by microscopic examination. Determination of sediment in cheese provides useful information on conditions during production. However, simple preparation of a cheese slurry is not possible because casein prevents passage through the filter pad. 18 Natural cheese samples are ground and dispersed in sodium citrate solution whereas processed cheese samples are dispersed in a solution of pepsin and phosphoric acid. Samples are heated for 1 h at <60C, as heat-coagulated protein will not pass through the filter pad.

2.4.4 Antibiotics

2A A.I General Information


Antibiotics are drugs administered to dairy cattle to control diseases. If proper precautions are adhered to, antibiotics should not enter the milk supply. Unfortunately, through faulty practices or lack of understanding, antibiotics do enter the milk supply and are of concern to processors and consumers. Antibiotic residues may induce allergic reactions in sensitive individuals, slow starter culture growth, and may create an environment favorable to resistant bacteria. To prevent antibiotics from entering the milk supply, each tanker of milk is tested prior to acceptance at the processing plant. Milk that is contaminated with antibiotics must be discarded. In the last several years, the number of tests available to detect penicillin and other common antibiotics has multiplied. Both qualitative and quantitative tests exist; some are applicable for dairy farmer use to prevent antibiotics from entering the milk supply at the source. The following discussion will summarize the current technology in antibiotic testing. It is rapidly changing as monoclonal antibody-based tests, specific for certain residues, are introduced. Drugs used to treat cattle are constantly being refined and improved. As this occurs, methods for detecting residues will also be refined and improved. Reference 19 gives information for 20 different techniques. For more information, that publication should be consulted.

2AA.2 Bacterial Growth Inhibition Methods


The first methods for detection of antibiotic residues in milk were based on the inhibition of growth of susceptible microorganisms. A cylinder plate assay method and a filter paper disc method were described in the early 1940s. 2a ~ 22 Initially, Ba-

cillus subtilis was the organism of choice but in recent years, assays have been developed that rely on Bacillus stearothermophilus inhibition. Growth inhibition can be both qualitative and quantitative. These methods are specific for (J-lactams but most have been collaboratively studied only for penicillin.19 The basis for microbial inhibition procedures is the presence of clear zones on an agar plate medium to which bacterial spores have been seeded. The sample to be assayed is placed on the surface of the agar, either on filter paper disks or in stainless steel cylinders. After incubation for the appropriate time, zones are measured (to the nearest 0.1 mm) with calipers. For quantitative determinations, zones of known amount of penicillin are determined and compared to the sample. Several samples are tested on the same agar plate. The depth of the agar is important to the sensitivity and reproducibility of the method. A thin layer is more sensitive than a thick layer. The plates must be allowed to solidify on a perfectly flat surface so that the agar is the same thickness throughout. Penicillinase ((i-lactamase) is an enzyme that specifically inactivates penicillin. It is added to the sample to confirm the presence of penicillin. If a zone of inhibition is present after the milk is heated to 82C for 2 min and treated with penicillinase, another inhibitor and not penicillin is present. In the qualitative B. stearothermophilus var calidolactis disc assay method a control containing 0.008 IU penicillin/ml is tested on each agar plate, varying the location on the plate. This reference gives a zone of inhibition of 16 to 20 mm. Plates are incubated at 64 2C for about 2.5 h. If the zone of inhibition around the disc containing untreated milk is <12.7 mm, the sample is presumed to be free of inhibitory substances. If the heated milk has a zone of > 12.7 mm but the penicillinasetreated milk has no zone, the milk is positive for penicillin. If the zone sizes are equivalent from all sample treatments, inhibitors other than penicillin are present. If penicillinase treatment reduces the zone of inhibition but does not reduce it to zero, penicillin and other inhibitors are present. If there is no zone around the heat-treated milk but a zone was present initially, a heat-labile inhibitor may be present. This disk assay method has been used to successfully detect minimum penicillin G residues of 0.005-0.008 U/ml, as well as ampicillin, cephapirin, and cloxacillin.5 Agar medium should not be kept more than 30 days at 0 to 4C after sterilization. Once plates are poured, they should be used within 5 days and should be stored at 0 to 4C in the petri dish plastic sleeve so as to prevent desiccation. Quantities of (J-lactam antibiotic residue within 0.003 IU/ml of a reference standard containing 0.016 IU/ml can be quantified in a similar manner.5 The penicillin standard is prepared in inhibitor-free milk and both the sample and the reference are heat treated. It is essential that the standard reference milk sample and the unknown sample be treated identically during heating. Plates must be incubated immediately after introducing samples at 64 2C for exactly 2 h and 45 min. Temperature and time are especially important for quantitative determination of inhibitor. Zones of inhibition between the reference and the sample are compared using a t test. A t value of > 1.860 indicates with 95% confidence that the sample contains more than 0.016 IU/ml of penicillin. If the t value is 1.860 or less, but there is a zone of inhibition around the sample, it is reported to contain 0.016 IU/ml of

(5-lactam or less. If there is no zone of inhibition around the sample, the results are reported as "(3-lactam negative." The fact that B. stearothermophilus var. calidolactis produces acid during growth is utilized in a commercially available procedure (Delvotest; GB Fermentation Industries, Inc., Charlotte, NC).23'24 Bromcresol purple dye changes from purple to yellow in the absence of p-lactam inhibitors. If inhibitors are present, the bacteria do not grow and produce acid; there is no change in the indicator. Test kits are available for individual samples or for multiple sample analyses. In the multiple test kit, one plate contains 96 test wells. A plate can be subdivided by the analyst into six blocks each with 16 cups. Positive (0.008 or 0.010 IU/ml) and negative controls are prepared with inhibitor-free milk. Samples and controls are added to the ampule or block of cups and incubated at 64 2C for exactly 2 h and 45 min. Colors are read through the agar for individual ampules or from the bottom for multitest units. Samples giving a purple color to all or part of the solid medium should be confirmed to contain penicillin by heat-treating and penicillinase treatment. Each new lot of ampules or test kits should be checked prior to use to determine the exact time of incubation. Occasionally some kits require longer than 2.75 h for complete change to yellow in the negative control or complete purple in the positive control. The exact time required for each lot must be determined and used for all tests performed with that lot. The multitest procedure does not work well with chocolate milk because chocolate interferes with color reading. A host of inhibitory substances may be detected with a commercially available test kit called the BR TEST AS. This method combines agar diffusion and color reduction techniques, utilizing B. stearothermophilus var. calidolactis spores.25 Drug residues in excess of the detection limit of the method inhibit metabolism of bacteria during incubation. When inhibitors are present, test color remains blue. During incubation of inhibitor-free milk, oxidation-reduction reactions within the mixture cause a change from blue to yellow. The test is useful for raw or pasteurized fluid milks. The modified Sarcinia lutea cylinder plate method for detection of penicillin in milk requires the creation of a standard curve with varying concentrations of penicillin diluted in inhibitor-free nonfat dry milk.19 The basis for the procedure is otherwise similar to the agar diffusion methods using B. stearothermophilus var. calidolactis. One primary difference is that the samples and controls are introduced to the agar medium by pipetting into stainless steel cylinders resting on the surface of the agar rather than on filter paper disks. This procedure is sensitive to 0.01 IU/ml of penicillin.

2.4.4.3 Competitive Binding Methods


Charm Sciences, Inc. (Maiden, MA) has developed a variety of test procedures to detect inhibitory substances in milk. (This company was originally known as Penicillin Assays, Inc., but has developed other methods so changed their name to represent the family that founded the company.) The original test developed by the company, the Charm Test, has been modified several times over the years to im-

Figure 2.5 Counting unit for the Charm competitive binding technique. Planchets are pictured in front of the unit.

prove its sensitivity, accuracy, and expand its selectivity. It was accepted as a final action procedure for assay of (3-lactams in milk in 1984. 5 The basis for this procedure is that p-lactam residues have a specific, irreversible affinity for enzyme sites on the cell wall of microorganisms. In the test procedure, 14C-labeled penicillin and Bacillus stearothermophilus vegetative cells are combined with the sample. If penicillin is present in the sample, it competes for binding sites on the bacterial cell wall and more 14C-label is free in solution. If no penicillin is present in the sample, the labelled penicillin binds with the cell wall and is removed from solution with centrifugation. The supernatant fluid is decanted and the bacterial cells containing the bound penicillin are resuspended and transferred to a metal planchet. The planchet is dried and radioactivity determined in an isotope counting device (Fig. 2.5). Positive and negative controls are prepared and the results from the sample compared to the controls. Results are available within 15 min and the test is applicable to levels of 0.01 IU penicillin/ml or p-lactam equivalent. Many dairy laboratories have converted to the Charm II procedure. This test has been collaboratively studied and is applicable as a screening procedure for seven families of antimicrobial drugs. 26 Two different microorganisms are used to provide necessary binding sites for the seven drug families. Antimicrobial families detected are p-lactam, tetracyclines, macrolides, streptomycin, novobiocin, sulfonamides, and chloramphenicol. The method detects biologically active drugs in about 8 min for one or two families or 15 min for all seven families. Gentamicin can be detected in

a revised version of this procedure but it had not been collaboratively studied at this time. The Charm II procedure uses a liquid scintillation counting device rather than a dry sample counter to detect the labeled compound. Normal levels of radioactive material stored in a testing lab are below the regulated levels for radioactive substances. Most labs do not need a special license to perform usual numbers of this test. Labeled material may be safely disposed through municipal water treatment systems with copious amounts of water. The analyst is responsible for ensuring that levels maintained and disposed are below applicable local and state regulated levels. A test is also available from Charm Sciences, Inc. that is designed for farm and small plant testing.19 Reagents are in tablet form; single tests can be easily performed. The procedure is sensitive to p-lactam antibiotics and all sulfa drugs in raw milk, milk powder, and pasteurized milk. The equipment is contained within a case for portability and operates on a 12-V battery. Another competitive binding method involves the binding of DD-carboxypeptidase (an enzyme) to (3-lactam antibiotics.27 This test is available in a kit as the Penzyme and Penzyme III procedures (SmithKline Animal Health Products, West Chester, PA). Enzyme and sample are incubated 5 min at 47 1C, then substrate [(R)-D-AIa-D-AIa)] is added. Any unbound enzyme is free to react with this substrate. The substrate is contained in a tablet that produces a yellow color on dissolving. The mixture is incubated for 15 min at 47 1C. Also contained in the tablet are reagents necessary to cause the conversion of free D-alanine to pyruvate and H2O2 and produce a color reaction when H2O2 is oxidized. A pink color indicates a negative test, a yellow color indicates an inhibitor residue is present; an orange/yellow color suggests the possibility of P-lactam residues and the sample should be retested to verify the result. The test detects P-lactam residues at 0.01 IU/ml in raw milk. Each new lot of kits should be checked prior to use with penicillin standards. Positive and negative controls should be run along with all samples.

2.4.4.4 Other Methods


The Spot test (Angenics, Worcester, MA) is an immunological agglutination technique.28 Latex beads coated with specific inhibitory molecules (penicillin-G, cephapirin, or cloxacillin) and antibodies to these inhibitory molecules are mixed with the milk sample. If inhibitors are present in the milk, the antibody and inhibitorcoated latex beads do not agglutinate. If no inhibitor is present in the milk, visible graininess is present in the mixture. The test is performed on a glass slide which is rotated during the reaction. As with all inhibitor tests, positive and negative controls are performed. Sulfamethazine can be detected in milk at 1 to 2 ppb using a HPLC technique.29 Sulfamethazine is extracted from milk with chloroform, the chloroform evaporated, and the residue dissolved in hexane. Sulfamethazine is partitioned into an aqueous potassium phosphate layer which is extracted and injected directly into a HPLC. The eluting sample is detected spectrophotometrically at 265 nm. Sulfamethazine adheres to glassware. Plastic should not be used, and all glassware should be rinsed after washing with approximately 1 Af HCl.

Enzyme-linked immunosorbent assays (ELISA) are rapidly becoming available for detection of specific antibiotics in foods. Although each method is unique, they are similarly antibody-antigen reactions which are visualized by linking with an enzyme reaction that produces a color. Color indicates the presence or absence of antibiotic or drug residues. Methods that are being applied to milk at this time include LacTek screening kit, CITE probe kit, SIGNAL detection test, EZ-SCREEN, and Agri-Screen.19 Each has specific advantages and disadvantages and must be evaluated on an individual basis depending on specific requirements for the analysis.

2.4.5 Acid Degree Value


Acid degree value is a measure of the quantity of free fatty acids present in milk. Milk is composed of a large variety of different fatty acids and contains a high proportion of short-to-medium-chain length fatty acids which are very flavorful. Normal milk contains few free fatty acids. Most fatty acids are incorporated into the milk triglyceride. Under normal circumstances, milk triglyceride undergoes little decomposition because initially it is protected from the action of milk lipase by fat globule membrane, and after pasteurization essentially all milk lipase is inactivated. If milk triglyceride and active lipase combine, hydrolysis results, leading to hydrolytic rancidity (lipolysis). Thomas et al.30 introduced the acid degree value (ADV) procedure and reported that when milk reached a certain value, most people could detect the lipolyzed flavor. ADV is defined as the quantity in milliliters of 1 N alkali required to neutralize the acids in 100 g of fat. Normal raw milk is reported to have an ADV of 0.25 to 0.40. Milk with an ADV of 1.2 or greater has undergone sufficient hydrolysis that flavor may be detected by taste or smell by some people. ADV is determined by first dissolving protein and freeing the fat using detergent. The fat is separated and a weighed quantity dissolved in a fat solvent (petroleum ether and Az-propanol). Fat is titrated, using a microburette, to the phenolphthalein endpoint with dilute alcoholic potassium hydroxide. Blank titrations are performed for the fat solvent. ADV is calculated using Eq. 2.3. (ml KOH for sample - ml KQH for blank) X N X 100 weight of fat Where N = normality of alcoholic KOH solution. Fat may be measured by volume if the temperature of the fat column is maintained at 57 3C for 5 min prior to transfer. Weight is calculated by multiplying the milliliters of fat by the approximate density of the fat at 57C (0.90 g/ml). Weighing the fat provides more precise results. ADV should be reported to the second decimal only. Research has shown that although ADV agrees well with sensory results on laboratory-prepared lipolyzed samples, it does not for farm milk samples.31 Farm samples with an ADV as high as 3.24 were classified as slightly lipolyzed whereas a sample with an ADV of 1.07 was classified as moderately lipolyzed by a trained sensory panel. There was no correlation between ADV and log lipolysis scores (r =

.13; p = .16). However, there was good correlation between ADV and the concentration of the major free fatty acids in the milks (r = .93; p = .0001) indicating that ADV does measure fat hydrolysis. Therefore, ADV is a useful measure of hydrolysis of milkfat but should not be used to predict whether or not the sample will taste lipolyzed. At present, research is underway to develop a method that will better correlate with sensory results. Until such method is found, laboratories should use ADV with caution. Milks exhibiting high ADV have undergone fat hydrolysis and reasons for hydrolysis should be determined. Actual lipolyzed flavor should be determined by sensory evaluation. Another frequently used method for determination of free fatty acids in milk is the copper soap procedure.32'33 Correlation between the copper soap procedure and flavor of laboratory-prepared samples was high (r = .82 to .83). The procedure also compared with ADV with a correlation coefficient of .88 to .90. However, the copper soap procedure is not sensitive to short-chain free fatty acids34 and may suffer from the same limitations as ADV. The advantage of the copper soap procedure is that it is a spectrophotometric method; results are not dependent on the perception of color change by individual analysts.

2.4.6 Iodine and Hypochlorites


Iodine and hypochlorite sanitizers are used throughout the dairy industry to control microorganisms and improve milk quality. Although these chemicals are essential to quality, they should not become a part of the milk. Analysis should be performed periodically to ensure that residual sanitizer is not becoming a part of the finished product. Iodine content of milk may also be increased through feeding. Iodine content can be estimated in raw milk using a selective ion electrode.35 The electrode is sensitive only to iodide ions but total iodine content may be estimated. The electrode must first be calibrated using eight concentrations of standard potassium iodide in solution. Potential (m V) is determined for each standard and the value plotted on semilog paper with concentration of the standard on the log scale. Iodide content of milk is calculated by comparison to standards. Sulfhydryl compounds also give a response with the iodide electrode. Some processing factors may impact sulfhydryl compounds. Previous history of processed milks should be known or the test results are questionable. Hypochlorites may be measured in milk with a colorimetric procedure if the milk contains 2.5 ppm copper or less.5 The process is qualitative and involves four steps. Color is observed and compared to tabular results at each step. Milk that is high in hypochlorite will change color during the first step and the process can be stopped. Lower concentrations of hypochlorite require that successive steps be completed. Differentiation can be made between concentrations that differ by twofold.

2.4.7 Aflatoxins
Aflatoxins are carcinogenic compounds produced by the mold Aspergillusflavus and other species. The first aflatoxin was'discovered in 1960. Aflatoxin B1 is produced

by growing mold. Aflatoxin M1 is a hydroxylated metabolite of B1 secreted by mammals who have consumed mold-contaminated feeds. M1 is the type found in milk and dairy products. This discussion will cover aflatoxins, but other molds produce metabolites equally as toxic. Because it is naturally produced, it is impossible to ensure that aflatoxin is absent. Tolerance levels have been established, above which foods are considered unsafe for consumption. Aflatoxin contamination is particularly a problem in areas where the climate is hot and humid, conditions that promote mold growth. Aflatoxin enters dairy products through the feed of the cow. However, mold contamination on cheese is a potential source. Performance of such analysis should be undertaken with caution due to the hazard of working with a highly potent carcinogen. Aflatoxin analyses are not usually performed routinely in dairy labs. Such analyses are available on a fee basis from independent testing laboratories or in corporate laboratories of larger organizations. Aflatoxins are soluble in organic solvents and through a multistep process are extracted. Samples are concentrated by rotary evaporation under nitrogen and separated by thin-layer chromatography (TLC).36 Once separated, aflatoxins can be detected by long-wavelength ultraviolet light (365 nm). Densitometric analysis of the TLC plates provides quantitative information if standards are used. Aflatoxin M1 can be extracted from milk using a Cl8 Sep-Pak (Waters Assoc, Inc., Milford MA) sample preparation cartridge.37 The extracted aflatoxins are eluted with ether onto a silica column from which it is eluted with a solvent mixture of methylene chloride and alcohol. The eluted material is derivatized with trifluoroacetic acid and separated by liquid chromatography. The eluted material is detected with a fluorescence detector and quantified compared to derivatized aflatoxin standards. Aflatoxins may be detected in milk using an ELISA test at residues below 0.5 ppb in <7 min.38'39 These tests are currently used for rapid screening of a large number of samples and results should be verified using one of the AOAC final action methods. Self-contained test kits are available that are easily used with little prior training.3839

2.4.8 Pesticides
Pesticides are a necessary part of today's production agriculture. Without their judicious use, much of our food supply would be lost to insects, weeds, or rodents. The U.S. Environmental Protection Agency is charged with approval of pesticides for specific applications at specific levels. Approved pesticides are published in the Compendium of Registered Pesticides.40 If the compound leaves a residue on a food, tolerances are established for maximum permissible levels. The Food and Drug Administration is charged with determination of residues in foods. A compilation of the methods for detection is given in the Pesticide Analytical Manual41 and in Methods of Analysis42 Residues of chlorinated hydrocarbon pesticides are more likely in dairy products than are those of organophosphates. The hydrocarbon-based pesticides accumulate in fat and are very slowly metabolized by the bovine. Exposure is cumulative and

residues may persist long after exposure. Organophosphates are metabolized by the bovine and the metabolites appear in milk and excreta. They are not accumulated in the fat of the animal. The metabolites are usually less toxic than the original pesticide. Regulations permit only selected pesticides around dairy animals. Thus, limited contamination occurs and residues usually remain below detection limits of the methods. Few laboratories performing routine dairy analyses are equipped to perform pesticide residue determinations. Laboratories with interest in such information usually contract with specialized analytical laboratories. State and Federal regulatory laboratories are equipped to ensure that amounts remain below action levels. AOAC provides detailed methodology for detection of 60 different pesticides in foods and water.42 Both qualitative and quantitative determinations are described based on chromatographic principles. Multiple residues may be detected simultaneously. Generally, the sample requires extraction and clean-up prior to column chromatography by either gas or liquid techniques. Detection is frequently via mass spectrometry for complete identification of pesticide residues at ppb levels.

2.5 Tests for Abnormal Milk 2.5.1 "Cow-Side" Tests


Mastitis is an infection of the mammary gland, induced by invasion of diseasecausing microorganisms. Somatic cells (principally polymorphonuclear leucocytes) are produced in response to the infection. Somatic cell concentrations in excess of 300,000 per milliliter are commonly considered indicative of mastitis or other abnormality. Cows in very early or very late lactation may have elevated numbers of somatic cells and respond positively to tests for abnormal milk.

2.5.1.1 California Mastitis Test


The California mastitis test (CMT) is an on-farm screening procedure that responds to nucleated somatic cells. It is designed to be performed on all four quarters from one cow simultaneously, but may be applied to bulk-tank and other blended samples.43 CMT reagent consists of a detergent and acid/base indicator. Somatic cells are ruptured by the detergent, releasing nuclear material (DNA). Mixing the DNA and detergent results in precipitation or gel formation that is proportional to the quantity of DNA present. The more the sample gels, the more somatic cells that are present in the milk. This test is not intended for diagnosis of mastitis but as a general screening tool for milk abnormality. Samples that are CMT negative may still be infected with disease-producing microorganisms.44 Two milliliters of milk is placed into each cup of a special CMT paddle. The paddle is white, allowing easy observation of thickening and color reaction. At cowside, the 2 ml is estimated and delivered directly from each quarter. An equal portion of CMT reagent (available from dairy supply sources) is dispensed into the paddle

and the milk and reagent mixed by gently rotating in a circular pattern for 10 s. The reaction must be scored immediately because it changes over time. Between tests, the paddle is rinsed with water and excess moisture shaken off. The test may vary from a slight positive or trace amount when only a very slight precipitate forms and disappears with continued movement of the fluid, to a strong positive when a gel forms creating a mass that tends to adhere to the bottom of the cup. Reactions are associated with somatic cell numbers: trace, 150,000 to 500,000; weak positive, 400,000 to 1,500,000; distinct positive, 800,000 to 5,000,000; and strong positive, >5,000,000. Cell populations in excess of one million are considered abnormal. An acid-base indicator allows for detection of alkaline or acid milk. Alkalinity frequently accompanies inflammation whereas acidity is rare. Some precautions in the performance of CMT determinations include use of fresh milk. Unrefrigerated milk over 12 h old or refrigerated milk in excess of 36 h old gives unreliable test results. During storage, DNase hydrolyzes the DNA, making it unavailable for reaction and leading to false-negative results. Mixing of bulk milk prior to sampling is critical because somatic cells associate with milkfat. Timing is critical in reading of test results. After 15 s, weak reactions fade. No more than four tests should be performed simultaneously because it is impossible to make more than four readings within 5 s. The CMT reagent may vary. Suppliers should be verified and reagents used within the prescribed time. CMT results cannot be read in inadequate light. Weak precipitation is not evident if lighting is not sufficient.

2.5.1.2 Conductivity Measurement


Abnormal milk conducts electrical current more readily than does normal milk. In abnormal milk, lactose (a nonconductor) is depressed whereas milk salts (conductors) increase. Battery-operated conductivity meters are available for mastitis screening.43 High conductivity readings do not correlate with the presence of primary pathogens but do correlate with somatic cell, lactose, and protein content of milk.45'46 Sodium chloride solution is used to standardize the meter, which should be done at least weekly. Milk is injected directly from the udder into the cup of the conductivity meter through a funnel. Conductivity measurements are read from a digital display. Measurements in excess of five usually indicate abnormal milk, but results must be confirmed.

2.5.2 Wisconsin Mastitis Test


The Wisconsin mastitis test (WMT) is based on the same principle as the CMT but is designed for laboratory use. The results are more quantitative than those of CMT.43 Viscosity is created from the reaction of a detergent with cell DNA and has some of the same limitations as CMT. Exact quantities of sample and reagent are dispensed into special test tubes equipped with metal caps with an orifice in the center. These tubes are fitted into a rack that firmly holds the tubes, even when inverted. The tubes are mixed in the rack within 30 s after addition of reagent to the first sample. Tubes are mixed in a nearly horizontal position with tilting. The liquid should move back

and forth through the entire length of the tube, making 10 excursions within 8 to 10 s. Vigorous agitation is to be avoided. Temperature control is important and the samples at the time of inversion should be 24 2C. Within 30 s of initiation of mixing, the tubes are inverted in the rack. Timing is essential; tubes should be held in a horizontal position while one waits for the clock to reach a convenient starting point. The rack should be inverted rapidly but smoothly and held in vertical position for 15 s. When inverted, the mixture will flow through the orifice in the tube cap. After 15 s, the tubes are righted, caps removed, and reagent/milk mixture drained in the tubes for at least 1 min. A measuring device is used to record the length of the column remaining in each tube. Readings of 21 mm or higher are indicative of abnormal milk and such tests should be confirmed. Normal milk does not gel but flows rapidly out of the tube, giving low readings. The size of the orifice will impact the results as will the size of the tubes. Both should be periodically checked and tubes discarded if out of specification.

2.5.3 Somatic Cell Count


Results obtained with the previously described tests must be confirmed for the presence of somatic cells. The tests described in this section are accepted confirmation procedures.

2.5.3.1 Direct Microscopic Somatic Cell Count


The direct microscopic somatic cell count (DMSCC) is the reference method to which automated methods must be correlated. The accuracy and reproducibility of the microscopic method is dependent on the training and skill of the technician.47 The procedure is tedious, as individual somatic cells are counted under microscopic examination. The technique is rapid if only one sample is examined, giving results in 10 to 15 min. Somatic cell enumeration is usually on raw milk samples. A precisely measured 0.01-ml sample is spread onto the surface of a special glass microscope slide in a thin film. The slide is marked with circular areas of exactly 1 cm2. The film is air dried and stained with one of three stains approved for cow's milk. One stain is the Levowitz-Weber modification of the Newman-Lampert stain; tetrachlorethane in 95% ethyl alcohol is the solvent for certified methylene blue chloride dye. A modified stain substitutes the less toxic xylene for tetrachlorethane. Another variation adds basic fiichsin dye to the basic Levowitz-Weber formulation. Stains must be stored to prevent evaporation and formation of precipitate. Following staining, excess stain is removed by resting the edge of the slide in an almost vertical position on absorbent paper. The slide is dried thoroughly and rinsed in three changes of tap water at 35 to 45C. After the final wash, the slide is again drained in an almost vertical position and air dried. The dried, stained film is examined under an oil-immersion objective with one drop of immersion oil on the film. A binocular microscope is preferred. The ocular(s) should be calibrated to provide a microscopic factor of 400,000 to 600,000. The microscopic factor is constant for each microscope for each analyst, but varies between microscopes and between analysts on the same

microscope. The reciprocal of the microscopic factor represent the fraction of 1 ml of milk observed in each field (one viewing of the slide). Reference 47 should be consulted for details on calibrating the microscope. The preferred method for counting the film is the field-wide single strip method, a method using as boundaries a single strip the width of the microscopic field and running across the diameter of the film of milk. Occasionally, when counts exceed 10 per field, they may be estimated by counting fewer fields and using the microscopic factor. If the field-wide single strip method is used, a single strip factor (SSF) must first be calculated.47 The number of cells counted during one pass over the strip is multiplied by the SSF, rounded to two significant figures, and reported as somatic cell count per milliliter. Only those somatic cells with an identifiable stained nucleus should be counted. For polymorphonucleated cells, the cell should have two or more discernible nuclear lobes. Other somatic cells should appear essentially intact. If there is doubt as to whether or not the cell is intact, it should not be counted. Slides may be maintained for future reference if stored to avoid dust accumulation. Immersion oil must first be removed by dipping each slide in xylene for 15 to 20 s and allowing the slide to air dry. Films should be protected from dust and other damage. Care must be taken to prevent insect damage to the film especially during extended storage. With the DMSCC procedure, instruments and glassware must be extremely clean and free of foreign materials. Sterility is not necessary. Exactly 0.01 ml must be transferred to the microscope slide. To achieve precision, the transferring instrument must be checked routinely. Care must be taken to dip the sampling device below any surface film in the sample and the device should be rinsed with sample prior to taking the final portion. The entire contents of the sampling device must be transferred to the slide. Even with exacting technique, replicate estimates of somatic cells by direct microscopic examination may vary. Aside from those mentioned in the previous paragraph, other potential sources of error include faulty preparation and staining of slides; failure of some somatic cells to stain; ratio of amount of milk examined to the total quantity in question; lack of homogeneity of distribution of somatic cells in the films; failure to count sufficient number of fields; poor microscopy (including inadequate or excessive illumination, poor focusing, improper use of filters); films of irregular depth; eye fatigue; and errors in observations and calculation.47 Fatigue is a major factor in compromising accuracy and steps should be taken to prevent it from interfering with the examination, especially when many samples are involved.

2.5.3.2 Electronic Somatic Cell Counting Methods


Somatic cells may be counted electronically with a high degree of correlation with DMSCC.43 The Coulter counter electronic method functions by counting particles, and may be semiautomated or automatic. Fat interferes and must first be removed from the sample. This may be accomplished either by centrifugation or chemically, although chemical removal is more popular. Fat is dissolved using an aqueous so-

Figure 2.6 Electronic somatic cell counting equipment in use at the TN DHIA Services Laboratory in Knoxville, TN.

lution of sodium chloride with 12.5 parts of 95% ethanol mixed with Triton X-IOO (a detergent) and formalin (40% w/v formaldehyde). The mixture is buffered to pH 7.0 with tris-(hydroxymethyl)aminomethane and filtered. The solution is commercially available as Somaton (Coulter Electronics, Inc., Hialeah, FL). The somatic cells are fixed initially with a solution of formalin containing eosine dye as a visible indicator of fixation. Somafix is a commercially available fixing preparation (Coulter Electronics, Inc.). The prepared samples are counted electronically in a calibrated counting device. Calibration spheres are available commercially. In the automated procedure, sample dilution, tempering, timing and mixing are incorporated within the instrument. There are a number of electronic somatic cell counting methods based on a fluorescent dye technique. The DNA in the cell nucleus reacts specifically with a dye (ethidium bromide) that fluoresces when excited. The procedure may be automated, semiautomated, or completely computer-controlled (Fig. 2.6) depending on the equipment capabilities and funds available for equipment purchase. Regardless of the degree of automation, each instrument must be calibrated periodically against the DMSCC. Fresh samples do not give accurate results; therefore, unpreserved milk must be held at 0 to 4.4C for 24 h but no longer than 72 h before examination. Preserved milk must be held at least 8 h but not longer than 7 days prior to exami-

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nation. Ethidium bromide solution is very toxic as well as light sensitive. It should be handled carefully and stored in a light-proof, air-tight bottle for not longer than 60 days. As with the Coulter counter method, fat must be removed using a buffered detergent mixture. Individual cells are isolated within the instrument and excited by light, causing the dye to fluoresce. The energy emitted by each nucleus is measured as an electronic pulse which is then converted to a count representing the number of somatic cells in the sample. Only somatic cell DNA reacts with the dye reagent in sufficient quantity to be counted. Intact bacteria will not absorb dye. Dead and partly degenerated bacteria absorb dye but produce signals of such low intensity as to be included in the background noise of the instrument. Factors that interfere with results are insufficient age of samples, inadequate mixing of samples, pipetting errors, loss of ability of cell to absorb dye, and/or degeneration of somatic cells. Cells lose the ability to absorb dye as they age or are exposed to formalin. Cell degeneration due to bacterial growth, high storage temperatures, excessive agitation, and freezing is the primary cause of error. For all electronic cell counting methods, controls should be run at least every hour during operation and at shutdown to ensure appropriate results. Control samples are available commercially or may be prepared in the laboratory. They should cover the entire range of somatic cell counts anticipated and counts should be verified by DMSCC. Samples that have been previously heated should not be used as controls.43

2.5.3.3 Membrane Filter-DNA Method


Somatic cells may be enumerated rapidly using a combination of membrane filtration and DNA specific dye.48'49 The method can be automated for rapid determination of multiple samples. A fluorescent dye (acridine orange) incorporates into cell DNA which is released by treating the sample with trypsin and triton X-100. The treated sample is filtered through a polycarbonate filter prior to exposure to the dye. Excess dye is removed and the sample on the filter fixed to a microscope slide with one drop of immersion oil. The slide is examined under an ultraviolet microscope and orange, orange/red, and orange/yellow fluorescing cells counted. The counting process may be automated. As with DMSCC, a microscopic factor must be calculated to convert counts to cells per milliliter. The illumination of the microscope must be checked weekly when manual counting is performed. This is done automatically in the automated procedure.

2.6 Microbiological Methods


Microbial growth is the primary cause for loss of acceptability of milk and milk products. Milk is a perishable commodity. If not held at refrigerator temperatures, microorganisms grow. During growth, most microorganisms produce metabolites that cause the milk to be unacceptable to consumers. Pathogenic bacteria may grow without visible signs of spoilage; therefore, milk may not be presumed to be safe unless processed to be so. Milk as it comes from the cow is contaminated with many

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nation. Ethidium bromide solution is very toxic as well as light sensitive. It should be handled carefully and stored in a light-proof, air-tight bottle for not longer than 60 days. As with the Coulter counter method, fat must be removed using a buffered detergent mixture. Individual cells are isolated within the instrument and excited by light, causing the dye to fluoresce. The energy emitted by each nucleus is measured as an electronic pulse which is then converted to a count representing the number of somatic cells in the sample. Only somatic cell DNA reacts with the dye reagent in sufficient quantity to be counted. Intact bacteria will not absorb dye. Dead and partly degenerated bacteria absorb dye but produce signals of such low intensity as to be included in the background noise of the instrument. Factors that interfere with results are insufficient age of samples, inadequate mixing of samples, pipetting errors, loss of ability of cell to absorb dye, and/or degeneration of somatic cells. Cells lose the ability to absorb dye as they age or are exposed to formalin. Cell degeneration due to bacterial growth, high storage temperatures, excessive agitation, and freezing is the primary cause of error. For all electronic cell counting methods, controls should be run at least every hour during operation and at shutdown to ensure appropriate results. Control samples are available commercially or may be prepared in the laboratory. They should cover the entire range of somatic cell counts anticipated and counts should be verified by DMSCC. Samples that have been previously heated should not be used as controls.43

2.5.3.3 Membrane Filter-DNA Method


Somatic cells may be enumerated rapidly using a combination of membrane filtration and DNA specific dye.48'49 The method can be automated for rapid determination of multiple samples. A fluorescent dye (acridine orange) incorporates into cell DNA which is released by treating the sample with trypsin and triton X-100. The treated sample is filtered through a polycarbonate filter prior to exposure to the dye. Excess dye is removed and the sample on the filter fixed to a microscope slide with one drop of immersion oil. The slide is examined under an ultraviolet microscope and orange, orange/red, and orange/yellow fluorescing cells counted. The counting process may be automated. As with DMSCC, a microscopic factor must be calculated to convert counts to cells per milliliter. The illumination of the microscope must be checked weekly when manual counting is performed. This is done automatically in the automated procedure.

2.6 Microbiological Methods


Microbial growth is the primary cause for loss of acceptability of milk and milk products. Milk is a perishable commodity. If not held at refrigerator temperatures, microorganisms grow. During growth, most microorganisms produce metabolites that cause the milk to be unacceptable to consumers. Pathogenic bacteria may grow without visible signs of spoilage; therefore, milk may not be presumed to be safe unless processed to be so. Milk as it comes from the cow is contaminated with many

microorganisms, including pathogenic bacteria. Little raw milk is consumed in the U.S. because of the question of its safety. Even when milk is held at refrigerator temperatures, some microorganisms grow, although relatively slowly and eventually spoilage will occur. Volume II, Chapter 5, presents detailed information regarding the various types of microorganisms (both desirable and undesirable) important to the dairy industry. This section will describe some of the more prominent tests applied in the testing of milk to ensure a long shelf-life and a safe product.

2.6.1 Aerobic Plate Count 2.6.1.1 General Introduction


Micoorganisms have different requirements for level of available oxygen. Because milk is a highly aerated product, most common spoilage organisms are aerobic. If the conditions of storage are converted to anaerobic, aerobic mircroorganisms will not grow, leaving an environment favorable to less common milk-spoilage organisms. Procedures that will be described in this subsection give an estimate of the microorganisms most likely to grow under common milk-handling conditions.

2.6.1.2 Standard Plate Count


The standard plate count (SPC) has long been the method of choice for assessing the quality of milk and its products.50 It is the reference method specified in the Grade A Pasteurized Milk Ordinance51 and the industry standard for detecting sources of contamination and determining quality of products. Even though this method has a long history of use, it is constantly under evaluation. As methods of milk production, handling, and processing change, its validity as the method of choice has come under question. SPC is designed to enumerate aerobic organisms that grow on standard methods agar during a 48 2 h incubation at 32 1C. This may not be the ideal condition for growth of the organisms that cause milk spoilage under today's practices. Preliminary incubation has been promoted in conjunction with SPC to provide more useful information about the bacteriological quality of pasteurized milk. 52 SPC is performed under very precise guidelines regarding equipment, materials, and incubation to increase accuracy and repeatability.50 However, it is expensive and time consuming. SPC as described requires that the laboratory have access to sterilization equipment. Other techniques will be described below that can be performed with materials available commercially, although while initially more expensive, are competitive when labor and laboratory set-up are considered. Because SPC remains the industry standard, all methods introduced to decrease time and expense must correlate with it. Cultured dairy products, or products to which bacteria have been added, are not ordinarily tested by SPC. Throughout the process of performing this and all other microbiological procedures, the analyst must be certain not to introduce contamination or allow microorganisms present to have an opportunity to grow (prior to incubation). Samples are

diluted in phosphated-dilution water to give a final count of between 25 and 250 colonies on individual petri plates. It may be difficult to know actual numbers of organisms in samples; serial dilutions are made to bracket the anticipated number. For most milk samples, dilutions of 1:100 and 1:1000 are prepared. Samples should be held between 0 and 4.4C for no more than 36 h prior to testing. Samples that have been frozen should not be tested because accurate determinations are not possible. SPC is a pour-plate technique; the sample is pipetted onto the bottom of an empty petri dish and tempered medium poured onto it. The medium and sample must be quickly and carefully mixed to obtain even distribution of bacterial cells while minimizing splashing on the edges of the plate. No more than 20 min should elapse between the diluting of the first sample and the pouring of agar on the last plate. Controls should be run to check sterility of dilution water, medium, pipet, and petri dishes for each sterilization lot. Air quality plates should be performed for the laboratory area each morning and afternoon of plating. Reference 50 provides detailed step-by-step procedures and precautions for performing SPC. It also provides details on counting-plates and reporting results.

2.6.1.3 Spiral-Plating Technique


The spiral-plating technique does not significantly differ from SPC yet has the advantage of requiring less time, equipment, and space.50-53*54 It does, however, require a spiral plater (Spiral Systems Instruments, Inc., Cincinnati, OH). Bacteria, in suspension, are deposited in an Archimedes spiral on the surface of an agar plate through a stylus. The tip of the stylus must be exactly parallel to the surface of the agar plate for reliable results. Bacteria may be enumerated in solutions containing from 500 to 500,000 per milliliter without dilution. The stylus automatically moves from the center of the plate to the edge while the plate is rotating. As the stylus moves outward, the volume of mixture dispensed decreases. This change in volume causes a dilution in the bacteria from the center to the outer edges of the plate. After incubation, colonies appear along the line where the liquid was deposited. The instrument must be calibrated against SPC to determine the volume of sample deposited in different parts of the plate. Counting grids are used to divide the plate into sections for ease of counting (Fig. 2.7). The counting grid is a 13.2 cm circle divided into four areas by five concentric circles equidistant along the diameter of the circle. The section nearest the center is marked " 4 " and the section nearest the edge is marked " 1 " . The concentric circles are subdivided further into eight 45 octants that are marked "A" through 44H". The outer ring of two opposite octants are subdivided further into quarters by two perpendicular lines. After the plates have incubated (48 3 h at 32 1C), they are individually centered over the grid. Beginning at the edge of the plate within any octant, the plate is counted toward the center within the octant until a total of 20 colonies are counted. When 20 colonies are counted, the remainder of the segment containing the twentieth colony is counted, and the number of the segment (e.g., "2") is recorded along with the count. The opposite segment is counted similarly and counts added together. Using the volume determined by cal-

B 1 A 2 3 H G

C D 4 E F

Figure 2.7 Schematic of template for counting plates prepared by the spiral plating method.

ibration against SPC, the number counted is converted to bacteria/ml. Counts are reported as Spiral Plate Count per milliliter (SPLPC/ml) or estimated SPLPC/ml. If the count in the first segment exceeds 75 colonies, the total count is reported to be >500,000/ml and is estimated. If the count in an entire wedge is <20, then all colonies on the plate should be counted. If there are fewer than 20 colonies on the entire plate, the count is estimated as <500/ml. Plates with irregular distribution of bacteria should not be counted because this is indicative of a dispensing error. Likewise, if spreading colonies cover the entire plate, it should not be counted. If the spreader covers less than one-half of the plate, only the colonies on the welldistributed, spreader-free area should be counted. The solution to be dispensed on the plate must be free from particles, as these clog the dispensing stylus. If suspended material exists after blending, the solution should settle prior to taking the sample. The depth of the agar plate is important and must be uniform ( 2 mm throughout). The stylus tip touches the surface of the agar as it moves across the plate and it must do so at a constant angle without digging into the agar. The agar plates must be at room temperature when plating begins and the surface free of water droplets but not dried or cracked. As with all techniques, controls must be prepared to ensure the sterility of spiral plater and media.

2.6.1.4 Rehydratable Film Method


The Petrifilm SM aerobic count is a plating method using a cold-water-soluble gelling agent.55'56 The specially prepared plating medium is purchased commercially (Medical-Surgical Division/3M, St. Paul, MN). A bottom film is coated with nutrients of standard methods agar along with a cold-water-soluble gelling agent. A flexible top film is coated with a gelling agent and 2,3,5-triphenyltetrazolium chloride as an indicator. Colonies are stained red with the dye for ease of counting. One milliliter of diluted or undiluted sample is plated in the center of the bottom layer,

the upper film carefully rolled into place, and the sample distributed with pressure from a plastic spreader. The plates should be placed on a flat surface for even distribution of the sample. The sample is spread over approximately 20 cm2. The gelling agent solidifies in minutes and the plates are incubated as with SPC. Plates may be stacked up to 10 high during incubation with the clear side up. The top film helps to eliminate spreading colonies. When bacteria grow, they reduce the tetrazolium indicator with differing abilities, giving various shades of red. The film base has approximately twenty 1-cm squares to aid in counting. Plates are counted and results reported in a manner similar to SPC. This method requires dilution of samples; however, laboratories can purchase prepared dilution blanks, disposable pipettes, and the Petrifilm plates. Minimal space is required and an autoclave is unnecessary. Petrifilm plates take up much less space than do standard petri plates; thus incubator space is less, as is the volume of material to be disposed following the test. The initial cost of the plates may be higher, but if one considers labor savings, the final cost is less per sample.

2.6.1.5 Impedimetric Methods


This method is an instrumental method (Vitek Systems, Inc., Hazelwood, MO) that relies on measurement of changes in capacitance, conductance, or total impedance associated with metabolic activity of bacteria growing on agar surfaces.57'58 The procedure is used to measure SPC in raw milk. It has also been used in conjunction with preliminary incubation to estimate shelf-life of processed milk.60 As bacteria grow, they convert nonelectrolytes such as lactose to electrolytes such as pyruvic acid. The conversion from nonelectrolytes to electrolytes causes a change in the ability of the medium to conduct current. Microbial populations of <10 6 CFU/ml cause minimal change in the impedance signal. Once populations exceed this value, metabolic products such as fatty acids, amino acids, and organic acids are present in sufficient quantity to impact the impedance signal. When this occurs, the instrument registers the change as a detection time (DT). Large initial populations of bacteria have a shorter DT than do small populations. The instrument is calibrated against SPC to determine the relationship between DT and bacterial populations. Initial populations of 104 mesophiles or psychrotrophs in milk can be detected within about 4 or 24 h, respectively.50 The instrument is computer controlled. The financial investment required for initial purchase must be weighed against the saving in time to detect bacteria. Positive and negative controls must be performed routinely to ensure the reliability of the instrument.

2.6. L 6 Hydrophobic Grid-Membrane Filter Method


The hydrophobic grid-membrane filter technique (QA Laboratories, Ltd, Toronto, Ontario) is a most probable number method for estimating bacteria in many foods.54 It has been studied for application to milk and dairy products.60'61 Bacteria are distributed into individual compartments of known and equal size that are created by

hydrophobic lines in a grid pattern. The bacteria are filtered onto the membrane (0.45 jxm pore size) using vacuum. The membrane is transferred onto the surface of agar medium and nutrients diffuse through the membrane, providing energy for bacterial growth. The plates are incubated as with SPC and counted. Samples containing paniculate material must be prefiltered. Raw milk and skim milk may be diluted and filtered directly through the hydrophobic grid membrane. Other fluid products must be pretreated with an enzyme solution to remove colloidal material and allow for filtration. Tryptic soy-fast green agar is dispensed in 18-ml portions into 100 X 15 mm petri dishes. The surface of the medium must be dry when the membrane is applied. The membrane is aseptically applied with a rolling motion to prevent the entrapment of air bubbles between the agar and the filter. Colonies on the surface of tryptic soy-fast green agar will be various shades of green. Counts are made of the number of squares containing one or more colonies rather than actual number of colonies present. Squares containing colonies are considered positive. The number of positive squares can be converted to a most probable number by formula taking into account the dilution factor.50

2.6.1.7 Pectin-Gel Method


The pectin-gel method uses pectin as a gelling agent instead of agar.62 Petri plates are available commercially that are pretreated with pectin-gelling agent (RCR Scientific, Inc., Goshen, IN). Low-methoxy pectin is combined with nutrient ingredients. CaCl2 acts as the gelling agent and is spread on the bottom of the petri dish in a thin agar base. Gelation occurs at room temperature when liquid medium is added followed by addition of diluted or undiluted sample. Gelling time is 30 to 40 min, but sample and nutrient mixture should be mixed immediately after placing on the plate. Sample should not be applied to the plate prior to the addition of the liquid medium because this will lock bacteria onto the surface of the plate and they will not be distributed evenly throughout the medium. Once the plates are prepared, the procedure is the same as for SPC. Medium and gelling agent are available commercially as kits. The advantage of this procedure over SPC is that the medium is at room temperature, preventing heat stress to microorganisms by hot agar.

2.6.1.8 Reflectance Colorimetry


Reflectance colorimetry is an instrumental method (Wescor, Inc., Logan, UT) applicable to estimating initial counts of microorganisms.50'63'64 Bacteria growing in aerobic count medium reduce triphenyltetrazolium chloride, producing a color change in the medium. The instrument responds to color changes occurring in proportion to initial populations. Bacteria populations of approximately 107 are necessary before the instrument can respond. Response is possible in clear, turbid, or opaque medium. Time is recorded for the instrument to respond and is inversely proportional to the initial numbers of microflora in the sample. Milk with low bacterial numbers requires preincubation for precise estimates of populations.

When pasteurized milk is tested for shelf-life, benzalkonium chloride is added to reduce interference from Gram-positive microflora.50 Samples are determined in duplicate by pipetting 200 u.1 into 50 jxl of aerobic count medium in sterile microtest plates (96 wells, flat bottoms). Raw milk samples are preincubated at 7C for 48 h in the aerobic count medium prior to placing in the instrument. Pasteurized milk samples are preincubated in the container at 210C for 18 h. Changes in reflectance following either preincubation is usually within 24 h.

2.6.2 Coliform Count

2.6.2.1 General Introduction


The coliform group of bacteria comprises all aerobic and facultatively anaerobic, Gram-negative, non-spore-forming rods able to ferment lactose with the production of acid and gas at 32 or 35C within 48 h (the temperature of incubation should be specified in reporting of results).65 The acid- and gas-producing ability provides a means of selectively identifying coliform organisms among other contaminants. CoIiforms are heat sensitive. Presence of coliforms in pasteurized milk products is indicative of improper heat treatment or postpasteurization contamination. However, absence of coliforms does not ensure freedom from postpasteurization contamination with pathogenic organisms. Tests on raw milk are to be interpreted differently from tests on pasteurized product, as a few coliforms could be expected in raw product. Coliforms are acid sensitive; tests on cultured products should be within 24 h of processing because counts decrease markedly thereafter. Coliforms may be presumed to be present following one of the several tests described below. If found, presence should be confirmed by checking for gas production in 2% brilliant green bile broth. The test is considered completed when material from positive 2% brilliant green bile broth tubes is streaked on eosin-methylene blue (EMB) agar. On EMB agar, coliform colonies are dark or have dark centers and colorless peripheries and a green-metallic sheen. Pure cultures isolated from this agar should ferment lactose and consist only of Gram-negative, non-spore-forming rods.65 Coliform bacteria may be stressed, but not killed, by heat processing. Given time, such bacteria may regenerate and begin to grow in milk products. Liquid media methods are better able to detect stressed organisms than are solid media methods.66 Liquid media methods sacrifice some precision in estimation of total numbers present.

2.6.2.2 Most Probable Number


Although distribution of microorganisms in contaminated foods may be homogeneous, they are more likely to be on the surface or at isolated points randomly located throughout the food. The most probable number (MPN) technique presumes uniform distribution of microorganisms. Fat prevents homogeneity, leading to decreased accuracy and reproducibility of MPN in milk samples compared to water. MPN is most useful in estimating low numbers of bacteria (< 10 per milliliter or gram). When

microbial density is so low that the sample cannot be diluted, participate matter may interfere with plating.67 MPN allows for direct measurement of large quantities of undiluted paniculate samples. MPN results are less accurate than plating procedures if pure cultures are used. MPN is a multiple tube dilution technique. Subsamples of the product are distributed in three or five tubes, at three consecutive dilutions. Ideally, the tubes with the larger portions will show growth while those with the lowest portion will not. 67 Coliform populations are expected to be low in milk. Therefore, an undiluted sample, a 1:10 dilution, and a 1:100 dilution are commonly used. One-milliliter portions of undiluted or diluted sample are placed in tubes containing lauryl sulfate tryptose (LST) broth. In the bottom of the tubes are inverted durham tubes that will reveal the presence of gas. The ratio of sample volume to medium volume should be maintained at one part sample to ten parts medium. Three tubes for each dilution are used under normal conditions. However, when the average coliform count is 1/ml, the distribution is such that about 37% of 1-ml portions will contain no coliforms. Three tubes at each dilution may not be adequate under these conditions. If five portions of the same sample are distributed, completely negative results may be expected < 1 % of the time. 65 Five tubes are preferred when low numbers are expected. Tubes are incubated for 24 2 h at 35 1C. Turbidity indicates growth; displacement of liquid in the inverted durham tube indicates gas production. Tubes that are positive after 24 h should be recorded and confirmatory tests begun. Negative tubes should be incubated for another 24 2 h and examined. The total number of tubes at each dilution showing growth after 48 3 h incubation at 35 1C are recorded. Presumptive MPN is calculated from tables65 and reports expressed as "presumptive coliform (MPN)*' per gram or milliliter. Coliform bacteria should be confirmed by transferring a loopful from each positive LST broth tube to 2% brilliant green bile broth tubes. Gas production after 48 3 h at 35 1C confirms the presence of coliform bacteria. If required, the test may be completed by streaking on EMB agar to confirm the presence of typical colony morphology and appearance. Gram stain should show negative rods.

2.6.2.3 Violet Red Bile Agar Methods


Coliforms are frequently enumerated in the dairy industry on violet red bile agar. The choice of method depends largely on the number of microorganisms anticipated. Other considerations are materials required, laboratory facilities, and labor available. When five or more coliform colonies appear on single plates, counts are usually more precise than are counts obtained by using five fermentation tubes. 68 One or two plates, each containing 1 ml of undiluted sample generally are sufficient. For increased sensitivity on samples routinely <5/ml, a larger sample may be used. Up to 4 ml of sample may be plated if the VRB agar quantity is increased to 15 to 20 ml. 65 Violet red bile agar should not be sterilized by autoclaving. Media is prepared by heating to boiling for 2 min. Inadequate dispersion of VRB agar may cause the medium to appear grainy, to gel at a higher temperature, or to not completely solidify.

The VRB agar must be tempered to 44 to 46C prior to pouring plates. Hot VRB agar may injure heat-sensitive coliforms. The sample is placed in the center of a sterile petri dish, tempered VRB agar poured, and the sample and agar mixed. No more than 20 min should elapse between diluting the first sample and pouring the last plate. The VRB agar and sample mixture should be allowed to solidify on a flat surface (5 to 10 min). An overlay of 3 to 4 ml of agar is poured on the top of the solidified medium and distributed evenly over the surface. This overlay prevents the growth of colonies on the surface. Plates are incubated in an inverted position for 24 2 h at 32 1C. Dark-red colonies measuring 0.5 mm or more in diameter on uncrowded plates are counted. Preferably only plates containing between 15 and 150 coliforms are counted. If colonies are crowded or noncoliforms are suspected, confirmation of lactose fermentation may be performed as described in the confirmed test under MPN techniques. The test may be completed as described previously, if desired. Frequently, processed dairy foods contain injured coliforms and these have difficulty growing under the inhibitory conditions of VRB agar. A modified procedure may be employed using a pour-plate technique. The sample is first plated in about 10 ml of tryptic soy agar (a noninhibitory medium).65 After solidification, an overlay of double strength VRB agar is applied (10 ml). The plates are incubated and counted as previously described. Results are reported as from the modified VRB procedure.

2.6.2.4 Rehydratable Film Method


Petrifilm coliform count plate is a rehydratable film containing violet red bile nutrient, a cold-water-soluble gelling agent, and 2,3,5-triphenyltetrazolium chloride indicator dye.69-70 One milliliter of diluted or undiluted sample is pipetted into the center of the film and the plastic upper film gently rolled into place. A plastic spreader, flat side down, is used to distribute the sample evenly over the approximately 20-cm2 area. The plates are left undisturbed on a flat surface for 1 min while the gel solidifies. The plates are incubated in stacks of no more than 10 plates at 32 1C for 24 2 h. The upper film traps gas bubbles formed by coliforms, giving an additional indication of their presence. Red colonies with one or more gas bubbles associated with them are counted. Red colonies without gas bubbles are not coliforms. The ability to determine gas formation makes this procedure more discriminatory than the VRB method. Confirmation of ability to ferment lactose with the production of gas is generally unnecessary. Petrifilm coliform plates can be used with most dairy products.65 When used for determination of coliform organisms in cheese, citrate buffer cannot be used as a diluent because it inhibits gas production. Petrifilm plates occupy much less space than do a comparable number of petri dishes. Less incubator space is required and less volume of waste is generated for disposal. This method also eliminates the possibility of heat shocking coliforms during enumeration because the gelling agent is cold water soluble.

2.6.2.5 Pectin-Gel Method


This medium is available commercially as VRB Redigel and pretreated plates that solidify at room temperature.71 Liquid medium containing VRB nutrients and pectin are provided in one tube. Pretreated petri dishes contain a thin layer of CaCl2 in agar. The procedure has advantages over traditional VRB coliform determination in that all media is at room temperature, preventing the possibility of heat-shocking coliforms during the plating process. Liquid medium is transferred into the pretreated plates first and the plates swirled to cover the bottom. Plates must be used within 5 min. Inoculum is added to the liquid medium in the plate; the pipet tip is touched once to a dry spot on the inside wall of the plate, above the liquid level, after dispensing the sample. The plate is immediately rocked and rotated to thoroughly mix the sample with the pectin gel. The sample should not be pipetted into the plate before the liquid medium is added. It would be locked into one spot on the plate and individual colonies could not be enumerated. Once the sample has been mixed, the plates are allowed to solidify on a level surface and overlayed with 3 to 4 ml of liquid medium. Once the overlay has solidified, the plates are incubated at 32 1C for 24 2 h. Colonies that are suspected to be coliforms are pink-to-red in appearance. Five colonies from each plate should be confirmed to be coliforms by transferring to 2% brilliant green bile broth and checking for gas production. As with the Petrifilm coliform method, the Redigel method requires a minimum of laboratory equipment and technical skill. The reagents are available as kits; if undiluted samples are evaluated, the only additional equipment required is sterile, disposable plastic pipettes and an incubator.

2.6.2.6 Impedimetric

Methods

Coliforms may be determined instrumentally with the same equipment used for impedimetric determination of total aerobic bacteria. The results are presumptive for the presence of coliform organisms. The method has been evaluated for raw and pasteurized milk, cream, and ice cream.72 The sample is initially mixed with coliform broth medium73 and preincubated for 3 h at 35C. The mixture is shaken and 1.5 ml transferred into each of two impedance modules. Impedance is monitored during incubation in the instrument for 24 h at 35C. The broth medium is selective for coliform growth. As the organisms grow, they produce metabolites that alter the signal received by the instrument. The greater the number of coliforms present, the more rapidly the instrument responds to a change in signals. Low coliform populations require longer impedance times. Results are reported as impedance coliform count per milliliter or gram. Presence of coliforms should be confirmed. Several hundred samples can be handled by the instrument simultaneously. The instrument provides printed results that can be labelled as unacceptable, borderline, or within specifications. When large numbers of samples are evaluated, the instrument is cost effective; the labor involved with counting plates is eliminated.

2.6.2.7 Hydrophobic Grid-Membrane Filter Method


The hydrophobic grid-membrane filter method for detecting coliforms is similar to that for determining total aerobic bacteria but a different medium is used for incubation. A membrane filter imprinted with hydrophobic material in a grid pattern provides individual compartments of equal and known size. Growing organisms are trapped within the compartments and number of positive squares are counted. The procedure is a most probable number technique and total estimates of bacteria are determined with a formula. This procedure has been modified so that both total coliforms and Escherichia coli may be determined from one membrane. 74 Whole milk, low-fat milk, chocolate milk, evaporated milk, cream, cottage cheese, and ice cream must first be pretreated with a trypsin solution. Without this treatment, the samples will not pass through the membrane filter. Skim milk, cheddar cheese, and butter can be tested without enzyme pretreatment.65 Samples are passed through the membrane filter and the membrane is transferred to the surface of a lactose monensin glucuronate agar plate. No bubbles should be trapped between the filter and the agar. After incubation at 35 I 0 C for 24 2 h, squares that contain one or more blue colonies are counted. Any shade of blue is considered positive. The number of positive squares is converted to an MPN using a formula65 and reported as MPN of total coliform bacteria/ ml or g. If the number of E. coli present in the sample is of interest, the filter can be transferred to the surface of a predried-buffered MUG-agar plate. MUG is a fluorogenic substrate, 4-methylumbelliferyl-p-D-glucuronide. E. coli produce P-glucuronidase, an enzyme capable of degrading MUG. The MUG-agar plate with the membrane filter on top is incubated for an additional 2 h at 35C. If E. coli are present, they will fluoresce blue-white under long-wavelength (366 nm) UV light. Only those colonies that are large and fluoresce blue-white are considered positive. The number of squares containing such colonies is recorded and converted to an MPN by formula.65

2.6.2.8 Fluorogenic Assay Methods


There are several commercially available fluorogenic assay methods. Medium can readily be prepared from individual ingredients in laboratories so equipped. 65 ' 75 ' 76 Each is specific for E. coli rather than the coliform group. Very few organisms produce the enzyme (i-glucuronidase. Even fewer produce a positive reaction in lauryl tryptose broth and produce the enzyme. E. coli is one of the few. When the substrate 4-methyl-umbelliferyl-p-D-glucuronide (MUG) is incorporated into nutrient medium, the activity of the enzyme may be detected by observing fluorescence. If E, coli are present, the enzyme cleaves the MUG substrate, producing a compound that fluoresces under long-wavelength UV light. The test results are read as positive or negative and may be quantified by incorporating fluorogenic substrate into lauryl sulfate tryptose broth tubes in the MPN technique described previously. Tubes that

show gas formation and fluoresce under long-wavelength UV light are considered positive.

2.6.3 Tests for Specific Spoilage Bacteria

2.6.3.1 Psychrotrophic Bacteria


Psychrotrophic bacteria are those that are capable of growth at 7C or less regardless of their optimal growth temperature. These organisms may be capable of growing at from subzero temperatures to temperatures as high as 37 to 45C. 59 Some pathogenic bacteria isolated from milk are psychrotrophic; Listeria monocytogenes is an example. Psychrotrophic bacteria enter milk from equipment, water, and dirt. They grow during storage of milk in farm bulk tanks and processor raw milk silos. They are the most common spoilage microorganisms in today's milk supply. The majority of psychrotrophic bacteria are Gram-negative and these are inactivated by proper pasteurization. However, some Bacillus spp. are psychrotrophic and may survive pasteurization. The latter grow more slowly in milk than do Gram-negative organisms. Although most psychrotrophic bacteria are inactivated by pasteurization, they have been shown to produce heat-resistant enzymes that survive pasteurization and exhibit their effects in products held for longer periods of time such as cheese and ultra-high-temperature pasteurized (UHT) milk. Defects in milk associated with the growth of psychrotrophic bacteria include staleness, bitterness, fruitiness, uncleanliness, and rancidity. The presence of psychrotrophic bacteria in significant numbers following pasteurization is indicative of postpasteurization contamination. The traditional test for psychrotrophic bacteria in milk is similar to the aerobic plate count except that incubation is at 7 1C for 10 days. This is much too long an incubation period to provide useful information, as most products are consumed prior to obtaining results. Faster methods for estimating psychrotrophic populations include incubation at 21C for 25 h or 18C for 45 h 77 ' 78 , using selective medium, or incubating samples at elevated temperature prior to performing an aerobic plate count. The most useful information on shelf-life seems to be provided by preliminary incubation of the samples prior to plating.52 In performance of psychrotrophic bacteria counts, one must take special care that the molten agar medium is cooled to 45 1C before pouring plates. 59 Hot medium can inactive bacteria and prevent their enumeration. Gram-negative bacteria can be estimated using a selective medium containing crystal violet tetrazolium in standard methods agar. Plates are incubated at 21 I 0 C for 48 3 h. Gram-negative bacteria appear as red colonies. 59 Gram-negative bacteria may also be estimated by impedance detection. Dairy Gram-negative agar is available commercially that is combined with the sample in an impedance detection module. The sample must first be preincubated at 18C for 18 h in plate count broth (20 ml each of sample and plate count broth). Impedance detection time is determined and quantity of bacteria in the sample estimated from a standard curve prepared with crystal violet tetrazolium agar as the reference method. 59

Although Listeria monocytogenes is a psychrotrophic bacteria, it will not be detected by the techniques described above. Special procedures must be followed and these will be described in the section on pathogenic bacteria.

2.6.3.2 Lipolytic Bacteria


Many bacteria that cause spoilage of milk and its products produce enzymes that are capable of hydrolyzing milkfat to fatty acids and mono- and diglycerides. Free fatty acids produce a flavor defect known as lipolyzed (or hydrolytic rancidity) (see Section 2.4.5). Some persons are very sensitive to this defect whereas others do not respond to it. Those who respond usually find the defect undesirable. Some lipases are heat resistant and can cause problems in long-term storage products such as cheese and butter. Several methods are available for enumeration of lipolytic microorganisms.59 Spirit blue agar has been recommended in SMEDP because of ease of preparation of the medium and interpretation of the results. Victoria blue butterfat agar has been used, particularly in other countries.79'80 Spirit blue agar is available commercially and should be prepared as instructed by the manufacturer. The medium is sterilized and cooled to 50 to 55C prior to the addition of 3% lipase reagent (available commercially). Lipase reagent consists of tributryrin in an emulsifying agent. Lipase reagent must be thoroughly mixed in the medium. Tributyrin frequently undergoes spontaneous hydrolysis, resulting in total clearing of the medium. Dispersion of lipase reagent by sonification helps eliminate this problem. One to two percent lipase reagent is added and when the medium is sonified sufficiently it changes from translucent blue to nearly opaque bluish-white. The sonifier probe must be sterilized by flaming with alcohol. Ten to twelve milliliters of prepared medium are added to sterile petri plates and allowed to solidify. Sample (0.1 ml) is spread on the surface of the solidified medium. Plates are incubated, inverted, at 32 1C for 48 3 h. To increase recovery of psychrotrophic bacteria, the plates may be incubated at 21 I0C for 72 h. Lipolytic bacteria have a clear zone under and around them. These colonies are counted and reported as lipolytic count per gram or milliliter. Tributyrin is a true fat and the simplist triglyceride occurring in natural fats and oils. Some microorganisms will hydrolyze tributyrin but not other triglycerides leading to overestimation of lipolytic bacteria. It is, however, the substrate of choice for screening lipolytic microorganisms of potential importance in foods.80 Presence of lipolytic microorganisms suggests contamination or mishandling of the product and the source should be identified.

2.6.3.3 Proteolytic Bacteria


Proteolytic bacteria growing in milk degrade milk protein. Frequently this degradation is accompanied by bitter flavor and possibly gelation. At a minimum, breakdown fragments too small to be incorporated into cheese curd result in reduced cheese yields. High content of proteolytic bacteria is indicative of potential quality

problems and unsanitary production practices. As with lipolytic bacteria, although the organism is usually heat-sensitive, the enzymes are heat-resistant and can cause quality problems in long-shelf-life products. Proteolytic bacteria can be detected by ability to hydrolyze caseins. One frequently used method is to incorporate 100 ml of 10% sterile skim milk into 1 L of melted standard methods agar immediately prior to pouring plates.59 The sample is introduced using a pour-plate technique. The plates are incubated for 48 to 72 h at 32 1C. Prior to counting, the plates are flooded for 1 min with a solution of 1% hydrochloric acid or 10% acetic acid. The acid is poured from the plates and colonies surrounded by clear zones counted. The skim milk agar method is not sensitive to weakly proteolytic organisms and frequently produces false-positive reactions due to growth of acid-producing organisms. Standard methods caseinate agar is preferred to prevent these difficulties.59 Standard methods agar serves as a base but it is prepared in 0.015 M citrate solution rather than water. A 2% solution of sodium caseinate is prepared in citrate solution. The two solutions are combined and sterilized. Separately a 1 M CaCl2 solution is prepared and sterilized. Twenty milliliters of the sterile calcium chloride solution is added to 1 L of molten agar and mixed just prior to pouring plates. The medium is dispensed to give a thickness of 2 mm in the petri dishes. Sample (0.1 ml; diluted or undiluted) is spread on the surface with a sterile, bent-glass rod. Plates should be dried for 15 min prior to incubation. Plates may be dried by setting lids slightly ajar but in such a manner as to prevent contamination from the atmosphere. Plates are incubated for 48 to 72 h at 32 I0C or for 72 h at 21 1C. Proteolytic colonies will be surrounded by a white or off-white zone; highly proteolytic colonies will be surrounded by a clear inner zone with a white halo. Results are reported as proteolytic count per gram or milliliter. The type of medium and the incubation time and temperature must be designated in reporting of results. Many organisms are highly proteolytic. Frequently, crowded plates will be almost completely clear and it will be difficult to determine which colonies are proteolytic. Higher dilutions should be used to prevent this. Plates of more than 80 colonies each are difficult to read accurately. For weakly proteolytic organisms, longer incubation times are suggested to improve the sensitivity of the method.

2.6.3.4 Yeasts and Molds


The presence of yeasts and molds in dairy products is indicative of unsanitary conditions. Yeasts and molds frequently contaminate dairy products through airborne routes. Thus, routine sampling of air for yeast and mold content may be helpful to determine exposure of product to these organisms. Mold contamination is especially a problem in cheese manufacturing operations. Proper sanitation of equipment eliminates them as a source of contamination. Protection of open product from exposure and separating areas where yeast and mold are expected (such as corrugated containers) from product packaging areas can help control exposure. Traditionally, acidified agar medium has been the method of choice for enumeration of yeasts and molds in dairy products (acidified potato dextrose agar). However, current literature indicates that medium containing antibiotics to suppress bacteria

is preferable.59 Antibiotic medium allows for improved recovery of injured cells, less interference from bacteria, and less precipitation of food particles that interfere with counting. However, if cells are not stressed, both acidified medium and antibiotic medium yield similar results.81 IDF recommends a medium containing chloramphenicol in a glucose-yeast extract agar. This medium is particularly useful for the recovery of injured yeast.59 Antibiotic plate count agar is prepared by adding 2 ml of antibiotic solution per 100 ml of standard methods agar. Antibiotic solution contains 500 mg each of chlortetracycline-HCl and chloramphenicol in 100 ml of sterile phosphate-buffered solution. The antibiotic solution needs no further sterilization and may be stored for up to 2 months at 5C without loss of inhibitory action.59 Yeast and mold counts are more accurate if surface-plating techniques are used. Pour-plating may be used if yeast alone are of interest or if nonstressed mold cells are being detected.59 With surface-plating techniques, 0.1 or 0.2 ml is spread on the surface of predried plates with a sterile bent-glass rod. One milliliter may be spread on the surface of three plates to increase sensitivity. Plates should not be inverted for incubation. Plates are incubated at 25C for 5 days. Potato dextrose agar is available commercially and is acidified by addition of 10% tartaric acid. Final pH of the medium should be 3.5 0.1. Yeast extract-dextrose-chloramphenicol agar (the medium recommended by IDF) is also available commercially and prepared as directed. Dichloran-rose bengal-chloramphenicol agar is available commercially as well. It has been reported to be useful for enumeration of molds when the sample contains species of Rhizopus and Mucor. These two species tend to overgrow plates with their rapid spreading-type growth. Rose bengal and dichloran restrict the growth of spreading fungi without reducing fungal counts.59

2.6.3.5 Spore-Forming Bacteria


Spore-forming bacteria are a potential problem because spores are more resistant to heat than vegetative cells. Bacillus sp. are the most common spore-formers found in raw milk. Sweet-curdling of pasteurized milk and coagulation of canned evaporated milk may be caused by the outgrowth of Bacillus spores. High numbers of sporeforming bacteria in milk may indicate unsanitary practices. However, initial mesophilic spore count of raw milk has not been found to be a good indicator of the potential shelf-life of pasteurized product.82 Enumeration of spores requires that vegetative cells be inactivated and spores activated. This is accomplished by submerging the sample containers in hot (800C) water. The entire contents must be submerged to inactivate vegetative cells that may survive on the lip of the container during heat treatment. Sample (200 ml) is placed in a sterile screw-cap Erlenmyer flask that is sealed with masking tape to prevent airborne contamination. A flask equipped with a thermometer inserted through a rubber stopper must be heated along with the sample to record temperature changes. Flasks are placed in a water bath at 82C and agitated during exposure. When the thermometer in the control flask registers 79C, the tern-

perature of the water bath is lowered to 80 0 C. When the thermometer registers 80 0 C, the flasks are maintained at that temperature for an additional 12 min. The flasks are cooled immediately in an ice bath. The flasks are aseptically opened to prevent contamination and samples plated on standard methods agar containing 0.1% soluble starch. If mesophilic spore-formers are being enumerated, the plates are incubated at 32 1C for 48 h; for psychrotrophic spore-formers, the plates are incubated at 7 10C for 5 to 7 days. If more than one plate is required for each sample, a separate flask should be heat treated for each. Multiple sampling from the same flask encourages contamination.59

2.6.4 Tests for Specific Pathogenic Bacteria 2.6.4.1 Listeria


Although Listeria monocytogenes has been recognized as a human and animal pathogen for over 60 years, it was not until 1981 that the first confirmed foodborne listeriosis outbreak occurred.83 In 1985, a major outbreak was traced to the consumption of Jalisco-brand Mexican-style cheese in California and caused 47 deaths. 84 Listeriosis affects only a small percentage of the population but causes high mortality in those affected, especially newboms, elderly, and immunocompromised individuals. It can cause spontaneous abortion. The presence of Listeria in dairy products and dairy processing plants has come under increased surveillance since the 1985 outbreak. Its isolation from any source within a processing plant can lead to removal of product from the market. Hence, there has been considerable interest in methods for detection of Listeria in recent years. Although its optimum temperature for growth is 30 to 37C, Listeria is capable of growing at refrigerator temperatures, making it an even more serious threat to dairy products. Detection of Listeria requires preenrichment for sufficient populations. This technique increases the likelihood of contamination of the environment; therefore, Listeria isolation generally should not be performed in a food processing plant. The FDA 8 5 recently revised its procedure for detecting Listeria in foods. 86 A 25-ml or 25-g sample is blended with 225 ml of enrichment broth until thoroughly mixed. The mixture is incubated for 2 days at 30 0 C. After 24 h and again after 2 days, a portion is streaked onto Modified Oxford (MOX) agar and onto lithium chloride-phenylethanol-moxalactam agar. MOX agar is preferred over modified McBride agar because it helps suppress growth of competitive organisms. It also produces Listeria colonies with black pigmentation and a black halo, making identification easier. Competitors may produce weakly brownish-black colonies but this takes longer than 2 days. On lithium chloride-phenylethanol-moxalactam agar, Listeria appear "sparkling" blue or white when examined under oblique-transmitted light. Quantification of populations is impossible; only positive or negative results are reported. Typical colonies are selected and identified by classic biochemical tests. Serotype is determined and pathogenicity may be determined. Not all Listeria are pathogenic; however, their presence in pasteurized dairy products indicates postpasteurization contamination or improper pasteurization.

Several rapid methods are available for presumptive identification of Listeria. An ELISA kit is commercially available.87 The FDA, using a virulence gene, were the first to develop a DNA probe.84 The probe is located with a radioactive tracer. A similar probe is commercially available for Listeria detection. The protocol requires a preliminary enrichment step for 22 to 26 h. Also available commercially is a colorimetric assay using a solid-phase extraction system. This is an enzyme-linked system.88 Hydrophobic grid-membrane filters have been used as a solid support to screen for Listeria monocytogenes.*9

2.6.4.2 Staphylococcus aureus


Staphylococcus aureus is more commonly associated with cheese than any other dairy product. Properly pasteurized cheese milk will not contain staphylococci, and normal acid development and aging for 60 days are effective in destroying these organisms should they be present. It is not the viable organisms that produce illness but the toxins produced during growth. Although the organism is inactivated by proper pasteurization, the toxin is heat resistant. Milk that has been stored under conditions to allow growth of the organism prior to pasteurization may be suspect. Large numbers of S. aureus in pasteurized milk cheese indicate unsanitary production practices. It is difficult to separate toxin-producing from non-toxin-producing strains of S. aureus. Generally, a coagulase reaction of 4 H - indicates the presence of toxigenic S. aureus.90 Detection of S. aureus does not require preenrichment. Actual numbers may be determined. Sample (1 ml) is spread on three plates of Baird-Parker agar medium. After inoculum has dried on the surface of the plates, they are inverted and incubated for 45 to 48 h at 35C. Typical 5. aureus colonies are "circular, smooth, convex, moist, 2-3 mm in diameter on uncrowded plates, gray to jet-black, frequently with light-colored (off-white) margin, surrounded by opaque zone and frequently with an outer clear zone".90 Colonies thought to be S. aureus are transferred into small tubes containing 0.2 to 0.3 ml of brain heart infusion broth. A portion is transferred to a suitable maintenance medium for repeat tests if questionable results are obtained. To the remaining broth, 0.5 ml of reconstituted coagulase plasma is added. The mixture is incubated at 35C and examined periodically for clot formation over a 6-h period. A firm, complete clot that stays in place when the tube is inverted is considered positive for S. aureus. Supportive tests to confirm the presence of S. aureus include several biochemical tests and production of thermostable nuclease. The latter is thought to be as specific as the coagulase test but should be used as confirmation rather than as a substitute. The solid medium method is applicable to products expected to contain 10 or more S. aureus per gram. If numbers are suspected to be less, a MPN technique is preferred.54 This method is also useful in foods suspected to contain large populations of competing species. Fifty grams of sample are blended with 450 ml of phosphate-buffered dilution water and serial dilutions made. One milliliter of each test dilution is transferred to each of three tubes containing trypticase soy broth with 10% NaCl and 1% sodium pyruvate. The tubes are incubated for 48 h at 35C. One

loopful of medium from tubes showing growth is transferred to Baird-Parker agar plates (the surface of the plate should be dry). The plates are streaked for isolation and incubated at 35 to 37C for 48 h. Typical colonies on Baird-Parker agar are as described above. One or more typical colonies should be confirmed by performing the coagulase test described previously. Coagulase-positive cultures are considered to be S. aureus and results are reported as MPN of S. aureus per gram from MPN tables. Although the presence of viable S. aureus cells is indicative of potential public health hazard, it is the presence of enterotoxin that confirms this. Enterotoxin at concentrations of 0.1 to 0.01 jxg per ml may be detected by a microslide gel-double diffusion test.54 Coagulase-positive cells are cultured and harvested to produce culture fluid suspected to contain enterotoxin. This culture fluid is transferred to one of five exactly spaced wells on a microscope slide containing gel diffusion agar. Antisera to the toxin is placed in a well in the center of the slide. Reference enterotoxin is placed in a well adjacent to the well containing the sample. If the analysis is for two staphylococcal enterotoxins simultaneously, one well will contain reference for one enterotoxin and the opposite well will contain the reference for the other enterotoxin. If only one enterotoxin is of interest, then only one well contains the reference enterotoxin and three wells contain sample mixtures. The slides are incubated for 48 to 72 h at room temperature in covered petri dishes containing moist sponge strips to prevent drying and cracking of the agar. Following incubation, the slides are examined for lines of precipitation under light against a dark background. During the test process, the antisera diffuses away from the center well through the agar. The solution in the sample wells and in the reference well also diffuses out. When the antisera contacts enterotoxin for which it is specific, precipitation appears as an arc in the agar. On each slide there should be a positive reaction between the antisera and the reference enterotoxin. The more concentrated the enterotoxin in the sample, the closer the arc appears to the well containing the antisera. Reference 54 should be consulted for exact interpretation of results. Foods suspected to contain enterotoxin may be analyzed following extraction and separation of the toxin from the food material. The procedure is described in detail in ref. 54. There are several other methods for detecting enterotoxins produced by S. aureus. Using some methods, enterotoxin may be detected in foods within a day. Each is a variation of an immunological technique and include reverse passive Latex agglutination, enzyme-linked immunosorbent assay, reverse passive hemagglutination, and radioimmunoassay. The various methods have recently been reviewed.91

2.6.4.3 Salmonella
Salmonella are probably the most serious threat to consumers of milk and its products. Salmonella are commonly associated with raw milk but may also enter the milk supply from human exposure and through contaminated water. Exposure to contamination by other warm-blooded animals, especially rodents and birds, may also serve as a route of entry. The presence of Salmonella, even in low numbers, can lead to illness or even death, especially in the very young or very old. Unlike S. aureus,

Salmonella cells cause illness. The cells are inactivated by pasteurization; their presence is indicative of incomplete pasteurization, mixing of raw and pasteurized milk, or postpasteurization contamination. Like Listeria, Salmonella are difficult to detect and preenrichment procedures must be used for isolation. Preenrichment generally should not be performed in a food processing plant. Salmonella has been the subject of intense study for many years. There is a tremendous body of literature regarding methods of detection. If the reader is interested in more information than is provided here, the IFT Food Microbiology Division presented an excellent symposium on the subject.92 Current methodology for the isolation and identification of Salmonella from foods consists of five basic steps.93 The first step is preenrichment. In this step, the food sample is enriched in a nutritious, nonselective medium to allow for repair of injured cells. For milk samples, preenrichment is usually done in a 1% aqueous solution of brilliant green dye for 24 2 h at 35C. Brilliant green provides some inhibition. Some batches of dye are especially toxic; each batch should be tested prior to use to ensure satisfactory results. The second step is selective enrichment. Portions of the preenriched sample are transferred to selenite cysteine broth and tetrathionate broth and both incubated for 24 2 h at 35C. In this step, the sample is enriched further in a growth-promoting medium containing selectively inhibitory reagents. Salmonella grow under these conditions but growth of other bacteria is restricted. The third step is selective plating on solid medium. Growth of bacteria other than Salmonella is restricted and typical colonies of Salmonella may be identified. A loopful of culture from each selective enrichment broth culture is transferred to each of three selective solid media: xylose-lysine-desoxycholate (XLD) agar, Hektoen enteric agar, and bismuth-sulfite agar. The plates are incubated for 24 2 h at 35C. Salmonella colonies appear differently on each of the selective medium. On XLD agar, colonies are pink with black centers. Many have large, glossy black centers or appear to be almost completely black. On Hektoen enteric agar, colonies are blue-green to blue with or without black centers. On bismuth-sulfite agar, colonies are brown, gray, or black and frequently have a metallic sheen. The latter medium should be examined at 24 and 48 h because it turns from brown to black during incubation. The fourth step is biochemical screening. Commercially available biochemical identification test kits may be used. This step is necessary td eliminate most organisms other than Salmonella and provides tentative generic identification of cultures. Finally, the cultures appearing to be Salmonella through the first four steps are serologically identified to provide specific identification. The final step is an immunological procedure using antisera to specific parts of the Salmonella organism. This entire procedure requires approximately 1 week to complete. Because of the importance of Salmonella detection to the safety of foods, procedures that provide information more rapidly than 1 week have been developed. There are several methods described in ref. 54 for screening samples for the presence of Salmonella. Most still require preenrichment to achieve populations sufficient to detect. The following methods were listed by AOAC54 at the time of this writing. The fluorescent antibody screening method is a microscopic technique which suggests that Salmonella may be present.94 However, other members of the family

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Enterobateriaceae may also react. The method must be followed rigorously, as errors at any point can lead to invalid results. Interpretation of fluorescence of stained cells should be by an analyst with prior training or experience. From selective enrichment medium (incubated for 4 h rather than 24), cells may be stained and examined within one additional hour. Positive samples should be confirmed by biochemical testing and serotyping. The hydrophobic grid-membrane filter screening method95 is similar to methods previously described. Preenrichment is as described earlier; selective enrichment is for 6 to 8 h. The enriched sample is filtered through two hydrophobic grid-membranes. One membrane is placed on the surface of selective-lysine agar while the other is placed on the surface of Hektoen enteric agar. Both are incubated 24 2 h, the former at 43 0.50C and the latter at 35C. Salmonella on selective-lysine agar appear blue-green, blue, or purple, flat but not watery or mucoid. On Hektoen enteric agar, they appear black or green with black centers. Colonies suspected to be Salmonella should be confirmed by biochemical and serological testing. Several ELISA procedures are available for screening foods for the presence of Salmonella?6"100 Some methods use a colorimetric assay to detect the presence of the organism; others use fluorescence. Each is an immunological reaction between Salmonella-specific antibodies in the kit and Salmonella in the sample. Preenrichment is required. The methods are for screening and positive samples must be confirmed. There is some cross-reactivity with non-Salmonella organisms. There are two DNA hybridization screening methods accepted by AOAC54 at the time of this writing. Preenrichment and selective enrichment (but for less time) are still required. In one procedure, bacteria are collected on membrane filters by vacuum filtration.101 The collected bacteria are treated to release DNA; it is denatured and fixed to membrane filters. Filters are incubated with hybridization solution containing radiolabelled Salmonella-specific DNA molecules. If Salmonella are present in the sample, the probe will attach to it; unbound probe is washed away and radioactivity measured with a beta detector. Radioactivity on the filter above threshold levels is indicative of the presence of Salmonella. Because of concern in handling radioisotopes, alternative visualization procedures have been developed. In a similar DNA hybridization method, the probe is labeled with fluorescein isothiocyanate (FITC) and horseradish peroxidase anti-FITC antibodies are used to amplify the bound probe.102 Visualization is by reacting the peroxidase with a chromogen; a blue color is produced that is measured spectrophotometrically. Test kits are commercially available for this reaction (Organon Teknika Corp., Durham, NC).

2.7 Selected Analytical Techniques for Dairy Products 2.7.1 Assurance of Adequate Pasteurization
Temperature recorders should be installed on all pasteurizers to ensure that time and temperature requirements are being met. In the absence of direct access to recorded information or if mixing of raw and pasteurized milk is suspected, the phosphatase

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Enterobateriaceae may also react. The method must be followed rigorously, as errors at any point can lead to invalid results. Interpretation of fluorescence of stained cells should be by an analyst with prior training or experience. From selective enrichment medium (incubated for 4 h rather than 24), cells may be stained and examined within one additional hour. Positive samples should be confirmed by biochemical testing and serotyping. The hydrophobic grid-membrane filter screening method95 is similar to methods previously described. Preenrichment is as described earlier; selective enrichment is for 6 to 8 h. The enriched sample is filtered through two hydrophobic grid-membranes. One membrane is placed on the surface of selective-lysine agar while the other is placed on the surface of Hektoen enteric agar. Both are incubated 24 2 h, the former at 43 0.50C and the latter at 35C. Salmonella on selective-lysine agar appear blue-green, blue, or purple, flat but not watery or mucoid. On Hektoen enteric agar, they appear black or green with black centers. Colonies suspected to be Salmonella should be confirmed by biochemical and serological testing. Several ELISA procedures are available for screening foods for the presence of Salmonella?6"100 Some methods use a colorimetric assay to detect the presence of the organism; others use fluorescence. Each is an immunological reaction between Salmonella-specific antibodies in the kit and Salmonella in the sample. Preenrichment is required. The methods are for screening and positive samples must be confirmed. There is some cross-reactivity with non-Salmonella organisms. There are two DNA hybridization screening methods accepted by AOAC54 at the time of this writing. Preenrichment and selective enrichment (but for less time) are still required. In one procedure, bacteria are collected on membrane filters by vacuum filtration.101 The collected bacteria are treated to release DNA; it is denatured and fixed to membrane filters. Filters are incubated with hybridization solution containing radiolabelled Salmonella-specific DNA molecules. If Salmonella are present in the sample, the probe will attach to it; unbound probe is washed away and radioactivity measured with a beta detector. Radioactivity on the filter above threshold levels is indicative of the presence of Salmonella. Because of concern in handling radioisotopes, alternative visualization procedures have been developed. In a similar DNA hybridization method, the probe is labeled with fluorescein isothiocyanate (FITC) and horseradish peroxidase anti-FITC antibodies are used to amplify the bound probe.102 Visualization is by reacting the peroxidase with a chromogen; a blue color is produced that is measured spectrophotometrically. Test kits are commercially available for this reaction (Organon Teknika Corp., Durham, NC).

2.7 Selected Analytical Techniques for Dairy Products 2.7.1 Assurance of Adequate Pasteurization
Temperature recorders should be installed on all pasteurizers to ensure that time and temperature requirements are being met. In the absence of direct access to recorded information or if mixing of raw and pasteurized milk is suspected, the phosphatase

test is used. The original test for application to dairy products was introduced in 1933.103 It has undergone many subsequent changes for increased rapidity, sensitivity and accuracy. At present, three basic adaptations are popular and these will be presented. Alkaline phosphatase is an enzyme found in milk naturally. The quantity varies with season, breed of cow, stage of lactation, and milk yield.104 The enzyme is inactivated at time and temperature combinations just above those necessary to inactivate non-spore-forming pathogenic microorganisms. The quantity of enzyme present may be easily assayed using one of several colorimetric methods. Properly pasteurized milk will be negative for phosphatase immediately following heat treatment. Reactivation of the enzyme has been observed in products stored at temperatures above 4.4C for extended periods. Certain microorganisms contaminating the product postpasteurization also have been found to produce phosphatase. Previously these were thought to be more heat resistant than milk phosphatase. However, heat resistance of microbial phosphatase is variable depending on the type of microorganisms. Because of this variability, the alkaline phosphatase test has limited application in some cheeses where microbial phosphatase is present. Application of the phosphatase test to cream and butter must be done with caution. Reactivated phosphatase is a particular problem in these products. Phosphatase is inactivated by the acid environment of buttermilk and yogurt; hence, the method has limited usefulness in such products. Phosphatase cleaves ester linkage of phosphate-containing substrates when incubated at proper temperature and pH. Substrates have been found that produce a color on hydrolysis. The amount of color is proportional to enzyme concentration. At present three substrates are popularly used. Disodium phenyl phosphate has been used for the longest period.105"107 When alkaline phosphatase acts on this substrate at pH 9.8 0.2, phenol and sodium phosphate are released. The phenol may be visualized by reacting with 2,6-dichloroquinone chloroimide and copper sulfate. Indophenol is produced; it is blue. This reaction is the basis for the Scharer rapid phosphatase test.108 This test procedure is very sensitive. Trace amounts of phenol from other sources will interfere with the test results. All glassware, stoppers, and reagents must be phenol-free. Stoppers and glass-washing detergent are primary sources of phenolic compounds. Glassware must be thoroughly cleaned and rinsed. It should be protected from contamination during storage. A second substrate is dicyclohexylamine phenolphthalein monophosphate.109 When this substance reacts with alkaline phosphatase at pH 10.15, phenolphthalein is released. At the pH of the reaction, phenolphthalein will be pink. This procedure is commonly known as the Rutgers phosphatase test and is used for screening purposes only. A third substrate is fluorophos R (Advanced Instruments, Inc., Needham, MA). When reacted with alkaline phosphatase at pH 10.0 0.05, fluoroyellow is released.110'111 Fluoroyellow is highly fluorescent and its rate of production may be monitored continuously over a short interval with a fluorometer (excitation at 439

nm; emission at 560 nm). The method is applicable to a wide variety of dairy products but each must be calibrated separately. Positive and negative controls should be performed for all methods. Each may be adapted- for measurement of microbial or reactivated phosphatase. For specific details on methods, ref. 104 should be consulted.

2.7.2 Total Solids in Butter and Cheese


The solids content of butter is approximately 83.5%; of this, 80.5% is fat and the remainder protein, carbohydrate, and ash. Because of the high fat content, spattering during heating may be a problem. If the analyst is concerned only with total solids, sand may be added to the sample to help control this. Determination of fat requires that moisture first be removed. If the two tests are to be done in combination, sand cannot be used and spattering must be controlled by attention to the sample to prevent overheating. A sample of 1.5 to 2.5 g of salted butter or 2 to 6 g of unsalted butter is weighed into a preweighed flat-bottom dish with a diameter of 5 cm or greater. The sample is dried to a constant weight in an oven kept at the temperature of boiling water. This method is the IDF-ISO-AOAC procedure and is applied internationally.5 The boiling point of water varies with location, so it must be determined based on the local altitude. Total solids are determined by weighing the cooled, dried sample and dividing the dried weight by the initial weight and multiplying by 100 to express the value in percentage. Precautions described in determination of total solids of milk by drying methods should be considered. Total solids in cheese may be determined by one of several methods. For most cheeses, drying in a vacuum oven is the reference method. Some cheeses contain large quantities of volatile substances along with water. Blue cheese is an example. Such products will have erroneously low total solids results if determined by drying. A distillation method is described in ref. 5 as the preferred method for such products. Vacuum oven determination of total solids in cheese requires that a 2- to 3-g sample be weighed into a flat-bottom metal dish with a diameter larger than 5 cm and a tight-fitting slip-in cover. The dishes must be predried and stored in a desiccator until used. Weights are determined for the empty dish and lid. Processed cheese and high moisture cheese should be predried on a steam bath prior to placing in the vacuum oven. The vacuum oven is maintained at 100 2C and a pressure of less than or equal 100 mm mercury (13.3 kPa). Samples are dried to a constant weight (4.75 h 15 min). During drying, a slow current of air is admitted to the oven to remove moisture. SMEDP* calls for drying the air by passing through a calcium sulfate moisture trap whereas AOAC 5 recommends sulfuric acid. Flow rate should be about 117 ml/min or approximately two bubbles each minute through the sulfuric acid. When samples are dry, vacuum is slowly released and dry air admitted to the oven. If this process is done too rapidly, lids will pop off the containers and results will be in error. Dishes are removed from the oven using tongs, then cooled in a desiccator for at least 30 min and reweighed. Total solids are calculated as the residual weight divided by the initial weight. The results are multiplied by 100 to express results in percentage.

Total solids in cheese may be determined in a forced-draft oven. For natural cheeses the forced-draft oven is equilibrated at 100 2C. Processed cheeses should not be determined using force-draft methodology. Disposable aluminum moisture dishes are predried for at least 3 h at 1000C. Fiber glass covers are predried for 1 h at 1000C. Both are stored in a desiccator until used. Cheese sample (3.0 0.5 g) is weighed into the predried pan and transferred to a forced-draft oven. Drying time is 16.5 0.5 h. After drying, cooling, and weighing, total solids are calculated as described for the vacuum oven. Microwave energy has been used to remove moisture from cheese. The time to do so is significantly decreased. Results are affected by the time, sample size, position of sample in oven, and energy of the oven. Because microwave ovens vary from unit to unit, each must be evaluated individually. Power setting and time may vary between units and with age of the unit. Most units are equipped with an internal balance, and results are automatically reported. Care must be taken to cover the sample to prevent spattering. Exact power settings and time should be determined with samples of various total solids content and compared to results of the same set of samples determined by vacuum oven drying. Controls should be routinely run to ensure that power drifts or interruptions are not distorting results. Microwave determinations are especially useful for in-process determination of moisture because results may be obtained in minutes rather than hours. Processed cheese samples are uniformly sized using a circular cutter giving a 4 to 4.5 cm circle with a thickness of 1.5 to 2 mm.

2.7.3 Salt in Butter and Cheese


The concentration of salt (sodium chloride) in butter and cheese is important for consumer acceptance and product stability. Salt acts as a preservative. It should be present at a sufficient concentration to be effective. However, current nutrition trends are toward decreasing the consumption of sodium. Therefore, content of salt is being reduced in many products. Amount present must be stated on the label, especially if a reduction from normal is claimed. Analytical techniques are available to ensure that the content of salt is as declared. The chloride ion of sodium chloride reacts stoichiometrically with the silver cation of silver nitrate. In products such as butter and cheese, the chloride must first be freed from the product matrix so that the titration can be performed. Because the molecular weights of sodium chloride and silver nitrate are known, the concentration of chloride may be determined from the quantity of silver nitrate needed to reach an end point. The end point may be determined colorimetrically or potentiometrically. Salt in butter and other high fat spreads may be determined after fat and moisture are removed.4 Once the fat and moisture are removed, the solids are dissolved in hot water. A portion of the solution is titrated with OA N silver nitrate to the first visible pale red-brown color lasting 30 s. Potassium chromate is added as an indicator. The end point is sharper if titration is performed under a yellow light. The percent sodium chloride may be calculated using Eq. 2.4.

_ ml AgNO3 X N AgNQ3 X dilution vol X .0585 C X ml titrated (2.4) where C = initial weight of sample. The value 0.0585 represents the number of equivalents of sodium chloride titrated by each milliliter of a one normal solution of silver nitrate. Alternatively, a 5-g (weighed to the nearest 10 mg) sample may be dissolved in 100 ml of boiling water, cooled to 50 to 55C, and titrated directly.5 The Volhard method5 for determining total chloride was developed in the mid1930s.112'113 In cheese, salt is bound within a matrix and must be released before it can be titrated. The method is a back titration. Excess silver nitrate is added to a weighed 3-g sample. Nitric acid and water are added along with one clean boiling chip and the sample heated to boiling. As the solution boils, 15 ml of 5% potassium permanganate is added in 5-ml portions. Addition of potassium permanganate during the heating process causes the solution to become yellowish and clear. When clear, the solution is cooled and filtered into a 200-ml volumetric flask. All material adhering to the filter paper is transferred by washing with water. The solution is diluted to 200 ml and excess silver nitrate titrated with potassium thiocyanate (0.100 AO. Saturated ferric ammonium sulfate acts as an indicator (the end point is the first pale red-brown color that persists for 30 s). A blank is prepared substituting 2 g of water for the sample. Equation 2.5 is used to determine percent chloride and Eq. 2.6 is used to determine percent sodium chloride (salt). % chloride = [(ml X A f AgNO 3) - (ml X W KSCN)] X 0.0355 X 100 E grams of sample (2.5)

% salt =

[(ml X N AgNO3) - (ml X N KSCN)] X 0.0585 X 100 grams of sample (2.6)

There are several points of caution to be observed during performance of the Volhard procedure. Toxic fumes are generated so all procedures after weighing the sample must be performed under a fume hood. Eye protection should be worn. When potassium permanganate is added to the boiling solution, extreme care must be exercised. The permanganate should be poured down the side of the flask in small portions. If poured into the center of the boiling mixture, hot acid solution might spatter on the analyst. Total chlorides may be determined potentiometrically using a silver chloride electrode.5 This method is recommended by IDF-ISO-AOAC. Two to five grams of cheese are suspended with blending in 30 ml of water at approximately 55C. The salt must be released from the cheese matrix by treating with nitric acid but the sample does not require heating nor any additional solutions. The mixture is titrated with standardized silver nitrate114 to the end point (determined as the inflection point

in the titration curve115). Percent chloride is calculated using Eq. 2.7 and percent sodium chloride (salt) is calculated using Eq. 2.8. ^ ., HiIAgNO3 X WAgNO 3 X 0.0355 X 100 % chloride = ^-J grams of sample
n

(2.7) (2.8)

, ml AgNO3 X N AgNO3 X 0.0585 X 100 % salt = grams of sample


n

Chloride may also be titrated automatically with silver ions generated coulometrically from a silver electrode.4 When a constant direct current voltage is applied across a pair of silver electrodes immersed in a dilute sample, silver ions are released. Chloride ions in the sample precipitate as silver chloride. On titration of all chloride ions, excess silver ions cause the conductivity of the mixture to rise. Electrodes sense the rise in conductivity and stop the titration. Quantity of chloride ions present is directly proportional to the elapsed titration time. As with previous methods, chloride ions must be released from the cheese matrix with nitric acid. Moisture content of the sample must be known because it contributes to the dilution volume. The instrument must be checked daily for accuracy in calibration using a known chloride standard. Lack of reproducibility is most commonly due to dirty electrodes in the instrument or inaccurate pipetting.4

2.7.4 Sorbic Acid in Cheese


Sorbic acid and its potassium and calcium salts are widely used as an antimycotic agent in cheese. When applied properly and in reasonable amounts, their presence does not affect taste or aroma. Permissible level and application method vary throughout the world and between cheese types. The following methods are suggested for determining sorbic acid content in cheese products. The final action procedure described by AOAC is a spectrophotometric method.116 It is applicable to fresh dairy products such as cottage and mozzarella cheeses, sour cream, and yogurt. A portion of cheese is suspended in metaphosphoric acid solution with the aid of a high-speed blender. The mixture is filtered and the filtrate extracted with a mixture of petroleum and anhydrous ether. The aqueous layer is discarded and the ether layer is dried with the addition of anhydrous sodium sulfate. Absorbance of the dried ether layer is determined at 250 nm. Known concentrations of sorbic acid are used to construct a standard curve. Other materials may be present that absorb at 250 nm. Confirmation that absorbance at 250 nm is due to sorbic acid is by adding some potassium permanganate solution. Absence of a peak at 250 nm that was previously present confirms that sorbic acid was present. Sorbic acid may also be determined by a distillation-oxidation procedure.116 Concentration of sorbic acid in the final mixture is determined spectrophotometrically against known standards of sorbic acid. Sulfuric acid and manganese sulfate are added to 1.5 to 2.0 g of sample in a distillation tube. The mixture is distilled and condensate collected. The condensate serves as the sample. It, along with solutions containing known amounts of sorbic acid, are heated with sulfuric acid and potas-

sium dichromate in a boiling water bath. The tubes are cooled and thiobarbituric acid is added. The tubes are returned to the boiling water bath. After 10 min, the tubes are removed, cooled, and absorbance determined at 532 nm. Concentration of sorbic acid is determined from a standard curve.

2.7.5 Overrun in Frozen Dairy Desserts


Overrun is defined as the volume of ice cream obtained in excess of the volume of mix.7 It is usually expressed as a percentage. The increase in volume is due mostly to the incorporation of air during the freezing process. The amount of air incorporated depends on the legal requirements of the market; the type of ice cream being processed; the composition of the mix and the way it is processed; and the desired body, texture, and palatability of the final product.7 Too much air will produce a fluffy product; too little air will produce a heavy, soggy product. There are two basic ways to calculate percentage overrun and each has three variations.7 Choice of method depends on desired end results. Overrun may be calculated by volume. The simplest and perhaps most widely used formula is for plain ice cream (Eq. 2.9) or when an approximation is all that is required for a flavored ice cream. The plant overrun formula (Eq. 2.10) calculates overrun as a percentage of the flavored mix and is more accurate and useful for cost studies. This formula is especially useful for bulky-flavored ice creams. Finally the formula for overrun on plain mix (Eq. 2.11) is used to determine the overrun as a percentage of the plain mix. Flavorings and colorings add little to overrun and are excluded in Eq. 2.11. Overrun may also be calculated by weight. This method relies on determination of the density of the mix and ice cream. The volume does not have to be one gallon as given in the examples. It must be the same for all weights within the formula. Frequently, a smaller, sturdy container is used to contain the mix and later a portion of the frozen product. As with calculation of overrun by volume, there are three variations for calculation of overrun by weight. The first is the simple formula (Eq. 2.12) and is useful for plain ice cream or when only an approximation is desired. The plant overrun formula (Eq. 2.13) calculates the overrun as a percentage of the flavored mix. With this formula, the weight of the flavored mix is taken rather than the weight of the plain mix. This formula is more accurate when the flavoring material alters significantly the density of the mix. The final variation for calculating overrun by weight is a formula that relates the weight of the flavored mix to the volume of plain mix (Eq. 2.14). overrun % = overrun % = volume of ice cream (volume of mix + volume of flavor) ; T^ ; TTt volume of mix + volume of flavor
x 10

volume of ice cream volume of mix X 100 volume of mix

(2.9)

(2.10)

overrun % = volume of ice cream (volume of mix + volume of flavor) volume of plain mix (2.11) overrun % = weight of 1 gal mix weight of 1 gal ice cream ~ X 100 weight of 1 gal ice cream (2.12) overrun % = weight of 1 gal flavored mix weight of 1 gal ice cream weight of 1 gal ice cream (2.13) overrun % = Equation 2.13 X volume of plain mix 4- volume of flavor , X 100 : volume of plain mix (2.14) For quality control purposes, frequent weighing of filled packages is required. The net weight necessary to achieve the desired overrun is determined, usually from Eq. 2.13. Desirable gross weight is determined by including the weight of the packaging container. Comparison to the desired weight is then made frequently during the production run. For regulatory purposes, AOAC describes two procedures for determining overrun in finished product where original mix formulation is not available.5 Ice cream is removed from its container and weighed to the nearest 1 to 2 g. Then the ice cream is entirely submersed in a container filled with kerosene. The container is equipped with an overflow spout through which the displaced kerosene flows into a graduated cylinder. The displaced kerosene is weighed and the net weight of the kerosene is divided by its specific gravity and designated as V. The weight per unit volume of the ice cream is calculated from the weight of the ice cream multiplied by 8.345 divided by V. An alternative method involves the displacement of a measured weight of a solution of polysorbate 80 by a volume of preweighed ice cream in a specially fitted plastic desiccator. The weight per unit volume of the ice cream is determined by comparing the weight of the filled container before and after the ice cream was placed in it to the weight of the piece of ice cream in air.

2.8 Sensory Analysis 2.8.1 Sensory vs. Chemical and Microbiological Methods
Thus far, individual components of milk and dairy products have been discussed as though they existed separately. In reality, they exist together and it is the interactions

among the various components that give us the products we recognize. It is only through sensory analysis that we can evaluate the many interactions among components. Sensory evaluation is the ultimate test for acceptance of milk and its products. Sensory evaluation cannot measure the amount of fat, although we can perceive the richness of high fat milk, and the watery mouth-feel and bluish color of skim milk. Neither can sensory evaluation determine the number of psychrotrophic microorganisms in the milk. We can, however, detect their activity by a bitter taste or fruity odor. Quality may also be impacted by hidden characteristics such as vitamin A content or presence of aflaxtoxin or pesticides. Again sensory evaluation cannot provide us with much information regarding these characteristics. Sensory evaluation is a category of food quality of its own merit. It provides the producer and processor with a guide to the consumer acceptance of the food. Milk may meet regulations in regard to fat content, be free of antibiotics and pesticides, and have low numbers of bacteria, but if the cow has consumed onions prior to milking and that flavor transfers into the milk, it will be unacceptable. At present, we have no better way to detect defects such as this except by sensory evaluation. Evaluation of sensory properties is affected by personal preference. Every individual does not respond to stimuli in the same manner. Complicating the matter even further, every individual does not respond to the same stimuli in the same manner on all occasions. With training and experience, individuals develop skills that help to overcome variations. Sensory evaluation must be done in such a manner that the results are statistically valid. Frequently, in the dairy industry we rely on evaluation by someone who has always done the evaluation with no verification that they are responding to the correct stimuli. Use of reference samples and participation in training sessions with known samples is most useful to be certain that what one person calls rancid, for example, is the result of the same reaction that some one else calls rancid. Problems with sensory evaluation procedures should not prevent the dairy industry from using sensory evaluation frequently at all levels of production, processing, and distribution. It is the best tool we have to measure the final quality of our products. However, we need to use the tool correctly to be certain that the results produced are not misleading. One of the biggest problems with sensory evaluation is bias. In training students for dairy products judging team, the author has experienced that all too often novices cannot perceive a defect until told it is there. This same bias may enter into product evaluation if not careful. When looking for a defect, it can frequently be found; or conversely, if we hope the defect is not there, it probably will not be. For this reason, sample preparation should be so as to avoid associating sample identification with a particular lot, producer, or production run. Chapter 3 specifically addresses sensory evaluation of dairy products. The dairy industry has a long history of product evaluation. Let it not forget that it is ultimately the sensory characteristics that sell its products.

2.9 Summary
This chapter has addressed the many methods of analyzing milk and its products. Every possible method has not been addressed. Methods most commonly found in dairy laboratories have been addressed in some detail. The choice of method depends on the desired results. The analyst, with the aid of management, is charged with the responsibility of selecting the method best suited to their needs. Milk and its products are analyzed for chemical composition, physical characteristics, microbiological quality, and sensory characteristics. Each is a measure of the quality of the product. Results obtained from any analysis are no better than the quality of the sample. Sampling must be done so as to be representative of the whole. Tests for milk composition include those for fat, total solids, protein, lactose, ash, vitamins, and minerals. Traditional and automated procedures have been described. Tests for milk quality include those for titratable acidity, added water, extraneous material, antibiotics, acid degree value, sanitizers, and aflatoxins and pesticides. Tests for abnormal milk include the California and Wisconsin mastitis tests and somatic cell counts. Microbiological quality may be evaluated by many different techniques. Total aerobic plate count gives an indication of total microflora and is the standard of acceptance for raw and pasteurized milks. Coliform bacteria give an indication of sanitary quality of milk. Properly pasteurized milk should have a very low coliform count. A variety of methods exist for determining coliforms in milk. Specific spoilage microorganisms found in milk and its products include psychrotrophic bacteria, those capable of growing in refrigerated milk; lipolytic bacteria, those that degrade milk fat; and proteolytic bacteria, those that degrade milk protein. Yeasts and molds and spore-forming bacteria may also cause spoilage of dairy products. Milk and its products are good vehicles for pathogenic microorganisms. Listeria, S. auerus, and SaImonella are often associated with raw milk. Each bacteria is heat sensitive; if found in pasteurized product an error has occurred in processing. Volume II, Chapter 5, provides additional information on the microbiology of milk and its products. Several selected analytical techniques for dairy products were described. Tests for assurance of adequate pasteurization are especially important given the growing attention to food safety. Methods to quantify total solids and salt in butter and cheese as well as sorbic acid in cheese were described. Standards for overrun in frozen desserts are legally specified. Methods for determining overrun in the plant and on finished product were described. Finally, the relationship between sensory evaluation and chemical and microbiological tests was briefly discussed. Detailed information on sensory evaluation is available in Chapter 3.

2.10 Future Developments


Analytical techniques for dairy products have made significant advancements since the introduction of the Babcock test in 1890. We will see more, although perhaps

not as dramatic, during the next century. As we learn more about the molecular structure of compounds, we will likely see increased use of DNA probes as tools to analyze materials specifically of interest. Computer-integration of processing and analytical results will likely increase. As increasing amounts of data become available, the only way it can be managed is with computers. Robotics have entered the laboratory environment. This will likely continue, especially for repetitive actions. Equipment will likely be down-sized as space becomes more valuable. Laboratories will require more educated employees to handle the sophisticated equipment and masses of computer-based data. Educational institutions need to prepare graduates to meet the challenges of the future by developing logical thinking abilities and computer skills, along with technical knowledge and scientific facts.

2.11 References
1. Grace, V., G. A. Houghtby, S. E. Barnard, and J. Lindamood. 1985. Sampling dairy and related products Chapter 4. In G. H. Richardson, (ed.), Standard Methods for the Examination of Dairy Products, 15th edit., American Public Health Association, Washington, D.C. 2. Blattner, T. M., N. F. Olson, and D. W. Wichem. 1985. Sampling barrel cheese for moisture analysis: comparison of methods. J. Assoc. Off. Anal. Chem. 68:718-721. 3. Helrich, K., ed. 1990. Official Methods of Analysis, 15th edit. Association of Official Analytical Chemists, Arlington, VA. 4. Bradley, R. L., Jr., E. Arnold Jr., D. M. Barbano, R. G. Semerad, D. E. Smith, and B. K. Vines. 1992. Chemical and physical methods. In R. T. Marshall (ed.), Standard Methods for the Examination of Dairy Products, 16th edit., Chapter 15. American Public Health Association, Washington, D.C. 5. Richardson, G. H. 1990. Dairy products. In K. Helrich (ed.), Official Methods of Analysis, 15th edit., Chapter 33. Association of Official Analytical Chemists, Arlington, VA. 6. Campbell, J. R., and R. T. Marshall. 1975. The Science of Providing Milk for Man. McGraw-Hill, St. Louis, MO. 7. Arbuckle, W. S. 1986. Ice Cream, 4th edit. Van Nostrand Reinhold, New York. 8. Atherton, H. V., and J. A. Newlander. 1977. Chemistry and Testing of Dairy Products, 4th edit. AVI, Westport, CT. 9. Pomeranz, Y., and C. E. Meloan. 1987. Food Analysis Theory and Practice, 2nd edit. Van Nostrand Reinhold, New York. 10. Szijarto, L., D. A. Biggs, and D. M. Irvine. 1973. Variability of casein, serum protein and nonprotein nitrogen in plant milk supplies in Ontario. / . Dairy Sci. 56:45-51. 11. Bruhn, J. C , and A. A. Franke. 1979. Regional differences in nitrogen fractions in California herd milks. / . Dairy ScL 62:1326-1328. 12. Franke, A. A., J. C. Bruhn and C. H. Lawrence. 1988. Distribution of protein in California milk in 1983. J. Dairy Sci. 71:2373-2383. 13. Barbano, D. M., J. M. Lynch, and J. R. Fleming. 1991. Direct and indirect determination of true protein content of milk by Kjeldahl analysis: collaborative study. /. Assoc. Off. Anal. Chem. 74:281-288. 14. Aurand, L. W., A. E. Woods, and M. R. Wells. 1987. Food Composition and Analysis. Van Nostrand Reinhold, New York.

15. Kleyn, D. H., and J. R. Trout. 1984. Enzymatic-ultraviolet method for measuring lactose in milk: collaborative study. / . Assoc. Off. Anal. Chem. 67:637-640. 16. Shipe, W. F. 1956. The use of thermistors for freezing point determinations. J. Dairy Sci. 39 (Abstr.): 916. 17. Pensiripun, K., E. C. Campbell, and G. H. Richardson. 1975. A vapor pressure osmometer for determination of water in milk. J. Milk Food Technol. 38:204-207. 18. Spicer, D. W., and W. V. Price. 1938. A test for extraneous matter in cheese. / . Dairy Sci. 21:1-6. 19. Bishop, J. R., G. F. Senyk, and S. E. Duncan. 1992. Detection of antibiotic/drug residues in milk and dairy products. In R. T. Marshall (ed.), Standard Methods for the Examination of Dairy Products, 16th edit., Chapter 12. American Public Health Association, Washington, D.C. 20. Abraham, E. P., E. Chain, C. M. Fletcher, H. W. Florey, A. D. Gardner, N. G. Heatley, and M. A. Jennings. 1941. Further observations on pencillin. Lancet 241:177-189. 21. Loo, Y. H., P. S. Skell, H. H. Thomberry, J. Ehrlich, J. M. McGuire, G. M. Savage, and J. C. Sylvester. 1945. Assay of streptomycin by the paper-disc plate method. / . Bacterioi 50:701-789. 22. Vincent, J. G., and H. W. Vincent. 1944. Filter paper disc modification of the Oxford cup penicillin determination. Proc. Soc. Exp. Biol. Med. 55:162-164. 23. Kelley, W. N. 1982. Qualitative ampule and multitest for beta-lactam residues in fluid milk products: collaborative study. J. Assoc. Off. Anal. Chem. 65:1193-1207. 24. Pater, B. 1977. A collaborative study of the Delvotest-P method to detect low concentrations of penicillin in milk. / . Food Prot. 40:23-24. 25. Muller, F. J. 1988. Sulfonamide residues in milk. Dtsche. Molkerei Zeitung 42:1322-1325. 26. Charm, S. E., and R. F. Chi. 1988. Microbial receptor assay for rapid detection and identification of seven families of antimicrobial drugs in milk: collaborative study. J. Assoc. Off. Anal. Chem. 71:304-316. 27. Knight, A. H., N. Shapton, and G. A. Prentice. 1987. Collaborative trial of the Penzyme assay: a rapid method for the detection of 0-lactam antibiotics in milk. J. Soc. Dairy Technol. 40:30-33. 28. Ryan, J. J., E. E. Wildman, A. H. Duthie, H. V. Atherton, and J. A. Aleong. 1986. Detection of penicillin, cephapirin, and cloxacillin in commingled raw milk by the Spot Test. / Dairy Sci. 69:1510-1517. 29. Weber, J. D., and M. D. Smedley. 1989. Liquid chromatographic determination of sulfa-methazine in milk. /. Assoc. Off. Anal. Chem. 72:445-447. 30. Thomas, E. L., A. J. Nielsen, and J. C. Olson, Jr. 1955. Hydrolytic rancidity in milka simplified method for estimating the extent of its development. / . Am. Milk Rev. 17:50-52, 85. 31. Duncan, S. E., G. L. Christen, and M. P. Penfield. 1991. Rancid flavor of milk: relationship of acid degree value, free fatty acids, and sensory perception. J. Food Sci. 56:394-397. 32. Bandler, D. K., S. E. Barnard, C. W. Hinz, and E. T. Wolff. 1989. Guidelines for Preventing Rancid Flavors in Milk. Northeast Dairy Practices Council Publication No. NDPC 23, Cornell University, Ithaca, NY. 33. Shipe, W. F., G. F. Senyk, and K. B. Fountain. 1980. Modified copper soap solvent extraction method for measuring free fatty acids in milk. / . Dairy Sci. 63:193-198. 34. Shen, N., and G. L. Christen. 1991. Comparison of methods to extract free fatty acids from milk. / . Dairy Sci. 74 (Abstr.): 130. 35. Bruhn, J. C , and A. A. Franke. 1978. An indirect method for the estimation of the iodine content in raw milk. J. Dairy Sci. 61:1557-1560.

36. Scott, P. M. 1990. Natural poisons. In K. Helrich (ed.), Official Methods of Analysis, 15th edit., Chapter 49. Association of Official Analytical Chemists, Arlington, VA. 37. Stubblefield, R. D., and W. F. Kwolek. 1986. Rapid liquid chromatographic determination of aflatoxins M1 and M2 in artificially contaminated fluid milks: collaborative study. J. Assoc. Off. Anal Chem. 69:880-885. 38. Park, D. L., B. M. Miller, S. Neshein, M. W. Trucksess, A. Vekich, B. Bidigare, J. L. McVey, and L. H. Brown. 1989. Visual and semiquantitative spectrophotometric ELISA screening method for aflatoxin B1 in corn and peanut products: followup collaborative study. J. Assoc. Off. Anal. Chem. 72:638-643. 39. AOAC. 1990. Changes in Official Methods of Analysis of the Association of Official Analytical Chemists, First Supplement, 1990, to the 15th edit. Association of Official Analytical Chemists, Arlington, VA. 40. EPA. 1969. EPA Compendium of Registered Pesticides. U.S. Government Printing Office, Washington, D.C. 41. FDA. 1990. Pesticide Analytical Manual. U.S. Dept. of Health and Human Services, Washington, D.C. 42. Sawyer, L. D., B. M. McMahon, W. H. Newsome, and G. A. Parker. 1990. Pesticide and industrial chemical residues. In K. Helrich (ed.), Official Methods of Analysis, 15th edit., Chapter 10. Association of Official Analytical Chemists, Arlington, VA. 43. Hinz, C. W., G. L. Hein, S. Hinckley, J. Althaus, and H. Bengsch. 1992. Methods to detect abnormal milk. In R. T. Marshall (ed.), Standard Methods for the Examination of Dairy Products, 16th edit., Chapter 11. American Public Health Association, Washington, D.C. 44. Marshall, R. T., and J. E. Edmondson. 1962. Value of California mastitis test records to the practioner. JAVMA 140:45-49. 45. Okigbo, L. M., M. A. Shelaih, G. H. Richardson, C. A. Ernstrom, R. J. Brown, and E. L. Tippetts. 1984. Portable conductivity meter for detecting abnormal milk. / . Dairy Sci. 67:1510-1516. 46. Sheldrake, R. F., G. D. McGregor, and R. J. T. Hoare. 1983. Somatic cell count, electrical conductivity, and serum albumin concentration for detecting bovine mastitis. / . Dairy ScL 66:548-555. 47. Packard, V. S., Jr., S. Tatini, R. Fugua, J. Heady, and C. Gilman. 1992. Direct microscopic methods for bacteria or somatic cells. In R. T. Marshall (ed.), Standard Methods for the Examination of Dairy Products, 16th edit., Chapter 10. American Public Health Association, Washington, D.C. 48. Pettipher, G. L., and U. M. Rodrigues. 1980. Rapid membrane filtration epifluorescent microscopic technique for the direct enumeration of somatic cells in fresh and formalin-preserved milk. / . Dairy Res. 48:239-246. 49. Pettipher, G. L., and U. M. Rodrigues. 1983. Semi-automated counting of bacteria and somatic cells in milk using epifluorescence microscopy and television image analysis. Appl. Environ. Microbiol. 53:323-329. 50. Houghtby, G. A., L. J. Maturin, and E. K. Koenig. 1992. Microbiological count methods. In R. T. Marshall, (ed.), Standard Methods for the Examination of Dairy Products, 16th edit., Chapter 6. American Public Health Association, Washington, D.C. 51. U.S. Dept. of Health and Human Services. 1980. Grade A Pasteurized Milk Ordinance, no. 017-001-00419-7. U.S. Government Printing Office, Washington, D.C. 52. Byrne, R. D., Jr., J. R. Bishop, and J. W. Boling. 1989. Estimation of potential shelf-life of pasteurized fluid milk utilizing a selective preliminary incubation. J. Food Prot. 52:805-807.

53. Peeler, J. T., J. E. Gilchrist, C. B. Donnelly, and J. E. Campbell. 1977. A collaborative study of the spiral plate method for examining milk samples. /. Food Prot. 40:462-464. 54. Andrews, W. H., and J. Messer. 1990. Microbiological methods. In K. Helrich (ed.), Official Methods Of Analysis, 15th edit., Chapter 17. Association of Official Analytical Chemists, Arlington, VA. 55. Ginn, R. E., V. S. Packard, and T. L. Fox. 1984. Evaluation of the 3M dry medium culture plate (Petrifilm SM) method for determining numbers of bacteria in raw milk. / . Food Prot. 47:753-755. 56. Ginn, R. E., V. S. Packard, and T. L. Fox. 19S6 Enumeration of total bacteria and conforms in milk by dry rehydratable film methods: collaborative study. /. Assoc. Off. Anal. Chem. 69:527-531. 57. Firstenberg-Eden, R. A., and M. K. Tricarico. 1983. Impedimetric determination of total mesophilic and psychrotrophic counts in raw milk. J. Food Sci. 48:1750-1754. 58. Firstenberg-Eden, R. A. 1984. Collaborative study of the impedance method for examining raw milk samples. / . Food Prot. 47:707-712. 59. Frank. J. F., G. L. Christen, and L. B. Bullerman. 1992. Tests for groups of microorganisms. In R. T. Marshall (ed.), Standard Methods for the Examination of Dairy Products, 16th edit., Chapter 8. American Public Health Association, Washington, D.C. 60. Entis, P. 1986. Hydrophobic grid membrane filter method for aerobic plate count in foods: collaborative study. /. Assoc. Off. Anal. Chem. 69:671-676. 61. Entis, P., and P. Boleszczuk. 1986. Use of Fast Green FCF with tryptic soy agar for aerobic plate count by the hydrophobic grid membrane filter. J. Food Prot. 49:278-279. 62. Roth, J. N. 1988. Temperature-independent pectin gel method for aerobic plate count in dairy and nondairy food products: collaborative study. J. Assoc. Off. Anal. Chem. 71:343-349. 63. Richardson, G. H., R. Grappin, and T. C. Yuan. 1988. A reflectance colorimeter instrument for measurement of microbial and enzymatic activities in milk and dairy products. / . Food Prot. 51:778-785. 64. Zmarticki, S., T. C. Yuan, and G. H. Richardson. 1991. Improved estimations of total and psychrotrophic microflora in raw milk using reflectance colorimetry. / . Food Safety 11:189-196. 65. Christen, G. L., P. M. Davidson, J. S. McAllister, and L. A. Roth. 1992. Coliform and other indicator bacteria. In R. T. Marshall (ed.), Standard Methods for the Examination of Dairy Products, 16th edit., Chapter 7. American Public Health Association: Washington, D.C. 66. Roth, L. A., M. E. Stiles, and L. F. L. Clegg. 1973. Reliability of selective media for the enumeration and estimation of Escherichia coli. Can. Inst. Food Sci. Technol. J. 6:230-234. 67. Mayou, J. 1976. MPNmost probable number. In M. L. Speck (ed.), Compendium of Methods for the Microbiological Examination of Foods, 2nd edit., Chapter 6. American Public Health Association: Washington, D.C. 68. McCrady, M. H. 1915. The numerical interpretations of fermentation-tube results. / . Infect. Dis. 17:183-212. 69. Ginn, R. E., V. S. Packard, and T. L. Fox. 1986. Enumeration of total bacteria and coliforms in milk by dry rehydratable film methods: collaborative study. J. Assoc. Off. Anal. Chem. 69:527-531. 70. Nelson, C. L., T. L. Fox, and F. F. Busta. 1984. Evaluation of dry medium film (Petrifilm VRB) for coliform enumeration. J. Food Prot. 47:520-525. 71. Roth, J. N., and G. L. Bontrager. 1989. Temperature-independent pectin gel method for coliform determination in dairy products: collaborative study. /. Assoc. Off. Anal. Chem. 72:298-302.

72. Firstenberg-Eden, R., M. L. Van Sise, J. Zindulis, and P. Kahn. 1984. Impedimetric estimation of coliforms in dairy products. /. Food ScL 49:1449-1452. 73. Marshall, R. T. 1992. Media. In R. T. Marshall (ed.), Standard Methods for the Examination of Dairy Products, 16th edit., Chapter 4. American Public Health Association, Washington, D.C. 74. Entis, P. 1989. Hydrophobic grid membrane filter/MUG method for total coliform and Escherichia coli enumeration in foods: collaborative study. / . Assoc. Off. Anal. Chem. 72:936-950. 75. Feng, P. C. S., and P. A. Hartman. 1982. Fluorogenic assays for immediate confirmation of Escherichia coli. Appl. Environ. Microbiol. 43:1320-1329. 76. Moberg, L. J. 1985. Fluorogenic assay for rapid detection of Escherichia coli. Appl. Environ. Microbiol. 50:1383-1387. 77. Oehlrich, H. K., and R. C. McKellar. 1983. Evaluation of an 18C/45-hour plate count technique for the enumeration of psychrotrophic bacteria in raw and pasteurized milk. J. Food Prot. 46:528529. 78. Griffiths, M. W., J. D. Phillips, and D. D. Muir. 1980. Rapid plate counting techniques for enumeration of psychrotrophic bacteria in pasteurized double cream. / . Soc. Dairy Technol. 33:8-10. 79. International Dairy Federation. 1967. Standard method for the count of lipolytic organisms. FlLIDF 41: 1966. 80. Smith, J. L., and J. L. Alford. 1984. Lipolytic microorganisms. In M. L. Speck (ed.), Compendium of Methods for the Microbiological Examination of Foods, 2nd edit., Chapter 11. American Public Health Association, Washington, D.C. 81. Henson, O. E., P. A. Hall, R. E. Arends, E. A. Arnold, Jr., R. M. Knecht, C. A. Johnson, D. J. Pusch, and M. G. Johnson. 1982. Comparison of four media for the enumeration of fungi in dairy productsa collaborative study. J. Food ScL 47:930-932. 82. Mikolajcik, E. M., and N. T. Simon. 1978. Heat resistant psychrotrophic bacteria in raw milk and their growth at 7C. / . Food Prot. 41:93-95. 83. Schlech, W. F., Ill, P. M. Lavigne, R. A. Bortolussi, A. C. Allen, E. V. Haldane, A. J. Wort, A. W. Hightower, S. E. Johnson, S. H. King, E. S. Nicholls, and C. V. Broome. 1983. Epidemic listeriosisevidence for transmission by food. N. Engl. J. Med. 308:203-206. 84. Wolcott, M. J. 1991. DNA-based rapid methods for the detection of foodborne pathogens. /. Food Prot. 54:387-401. 85. Lovett, J., and A. D. Hitchins. 1989. Listeria isolation. In R. B. Read, Jr. (ed.), Bacteriological Analytical Manual, 6th edit., Chapter 29. Association of Official Analytical Chemists, Arlington, VA. Supplement, 2nd printing. 86. FDA. 1990. Fed. Regist. 55:38953-38954. 87. Mattingly, J. A., B. T. Butman, M. C. Plank, and R. J. Durham. 1988. Rapid monoclonal antibodybased enzyme-linked immunosorbent assay for detection of Listeria in food products. / . Assoc. Off. Anal. Chem. 71:679-681. 88. King, W., S. Raposa, J. Warshaw, A. Johnson, D. Halbert, and J. D. Klinger. 1989. A new colonmetric nucleic acid hybridization assay for Listeria in foods. Int. J. Food Microbiol. 8:225-232. 89. Peterkin, P. L, E. S. Idziak, and A. N. Sharpe. 1989. Screening DNA probes using the hydrophobic grid-membrane filter. Food Microbiol. 6:281-284. 90. Bennett, R. W. 1984. Staphylococcus aureus. In R. B. Read, Jr. (ed.), Bacteriological Analytical Manual, 6th edit., Chapter 14. Association of Official Analytical Chemists, Arlington, VA.

91. Bergdoll, M. S. 1990. Analytical methods for Staphylococus aureus. Intl. J. Food Microbiol 10:91-100. 92. Symposium of the EFT Food Microbiology Division (1985) 44th Annual Meeting. Recent developments in the detection of Salmonella in foods. Food Technol. 39:75-108. 93. Andrews, W. H., P. L. Poelma, and C. R. Wilson. 1984. Isolation and identification of Salmonella species. In R. B. Read, Jr. (ed.), Bacteriological Analytical Manual, 6th edit., Chapter 7. Association of Official Analytical Chemists, Arlington, VA. 94. Fantasia, L. D., J. P. Schrade, J. F. Yager, and D. Debler. 1975. Fluorescent antibody method for the detection of Salmonella: development, evaluation, and collaborative study. J. Assoc. Off. Anal. Chem. 58:828-844. 95. Entis, P. 1985. Rapid hydrophobic grid membrane filter method for Salmonella detection in selected foods: collaborative study. / . Assoc. Off. Anal. Chem. 68:555-564. 96. Flowers, R. S., K. Eckner, D. A. Gabis, B. J. Robison, J. A. Mattingly, and J. H. Silliker. 1986. Enzyme immunoassay for detection of Salmonella in foods: collaborative study. /. Assoc. Off. Anal Chem. 69:786-798. 97. Flowers, R. S., M. J. Klatt, B. J. Robison, J. A. Mattingly, D. A. Gabis, and J. H. Silliker. 1987. Enzyme immunoassay for detection of Salmonella in low-moisture foods: collaborative study. J. Assoc. Off. Anal. Chem. 70:530-535. 98. Curiale, M. S., M. J. Klatt, B. J. Robison, and L. T. Beck. 1990. Comparison of colorimetric monoclonal enzyme immunoassay screening methods for detection of Salmonella in foods. J. Assoc. Off. Anal. Chem. 73:43-50. 99. Flowers, R. S., M. J. Klatt, and S. L. Keelan. 1988. Visual immunoassay for detection of Salmonella in foods: collaborative study. / . Assoc. Off. Anal. Chem. 71:973-980. 100. Flowers, R. S., M. J. Klatt, S. L. Keelan, B. Swaninathan, W. D. Gehle, and H. E. Chandonnet. 1989. Fluorescent enzyme immunoassay for rapid screening of Salmonella in foods: collaborative study. J. Assoc. Off. Anal. Chem. 72:318-325. 101. Flowers, R. S., M. J. Klatt, M. A. Mozola, M. S. Curiale, D. A. Gabis, and J. H. Silliker. 1987. DNA hybridization assay for detection of Salmonella in foods: collaborative study. J. Assoc. Off. Anal. Chem. 70:521-535. 102. Curiale, M. S., M. J. Klatt, and M. A. Mozola. 1990. Colorimetric deoxyribonucleic acid hybridization assay for rapid screening of Salmonella in foods: collaborative study. J. Assoc. Off. Anal. Chem. 73:248-256. 103. Kay, H. D., and W. R. Graham, Jr. 1933. The effect of heat on milk phosphatase. A simple method for distinguishing raw from pasteurized milk, raw from pasteurized cream, and butter made from raw cream from that made from pasteurized cream. J. Dairy Res. 5:63-74. 104. Murthy, G. K., D. H. Kleyn, and T. Richardson. 1992. Alkaline phosphatase methods. In R. T. Marshall (ed.), Standard Methods for the Examination of Dairy Products, 16th edit., Chapter 14. American Public Health Association, Washington, D.C. 105. Sanders, G. P., and O. S. Sager. 1946. Modification of the phosphatase test as applied to Cheddar cheese and application of the test to fluid milk. / . Dairy Sci. 29:737-749. 106. Sanders, G. P., and O. S. Sager. 1947. Phosphatase test of various dairy products. / . Dairy ScL 30:909-920. 107. Sanders, G. P. 1948. Report on the phosphatase test in pasteurization of dairy products. / . Assoc. Off. Anal. Chem. 31:306-327. 108. Scharer, H. 1953. Scharer modified phosphatase methods. / . Milk Food Technol. 16:86-88.

109. Kleyn, D. H., and S. H. C. Lin. 1968. Collaborative study of a new alkaline phosphatase assay system for milk. /. Assoc. Off. Anal. Chem. 51:802-807. 110. Rocco, R. M. 1990. Fluorometric analysis of alkaline phosphatase in fluid dairy products. /. Food. /Vor. 53:588-591, 630. 111. Rocco, R. M. 1990. Fluorometric determination of alkaline phosphatase in fluid dairy products: collaborative study. J. Assoc. Off. Anal. Chem. 73:842-849. 112. Stone, C. B. 1935. Report on cheese. J. Assoc. Off. Anal. Chem. 18:401-402. 113. Stone, C. B. 1937. Report on cheese. / . Assoc. Off. Anal. Chem. 20:339-341. 114. Poortvliet, L. J., and W. Horwitz. 1982. Determination of chloride concentration in cheese: collaborative study. J. Assoc. Off. Anal. Chem. 65:1350-1356. 115. Horwitz, W. (ed.) Official Methods of Analysis, 13th edit. Association of Official Analytical Chemists, Washington, D.C. 116. Fazio, T. 1990. Food additives: direct. In K. Helrich (ed.), Official Methods of Analysis, 15th edit., Chapter 47. Association of Official Analytical Chemists, Arlington, VA.

CHAPTER
3

Sensory Evaluation of Dairy Products


Lynn V. Ogden
3.1 The Senses, 158 3.1.1 Introduction, 158 3.1.2 Taste, 159 3.1.3 Smell, 162 3.1.4 Sight, 163 3.1.5 Hearing, 165 3.1.6 Touch, 166 3.2 Sensory Evaluation Techniques, 166 3.2.1 Introduction, 166 3.2.2 Affective Testing, 168 3.2.3 Discrimination Testing, 170 3.2.4 Descriptive Analysis, 171 3.3 Application of Sensory Analysis to Dairy Products, 174 3.3.1 The Philosophy of Judging of Dairy Products, 175 3.4 Descriptive Sensory Defects of Dairy Products, 175 3.4.1 Fluid Milk and Cream, 175 3.4.2 Cottage Cheese, 185 3.4.3 Butter, 198 3.4.4 Ice Cream and Related Products, 214 3.4.5 Cheese, 229 3.4.6 Cultured Products, 243 3.4.7 Yogurt, 254 3.4.8 Dry Milk, 267 3.5 References, 274

3.1 The Senses 3.1.1 Introduction


Human senses are classified into five primary modalities: sight, hearing, touch smell, and taste.1'2 These have been further subclassified to include temperature sensation, pain, hunger, thirst, fatigue, balance, loudness, pitch, hue, brightness, and contrast to name a few. A total of 22 subdivisions of the senses are generally recognized.3 Specialized organs on and in the human body respond to stimuli and send messages about the stimuli via the central nervous system to specialized areas of the brain. The retina in the eye with its rods and cones is the visual receptor, the taste buds in the tongue are the taste receptors, and the olfactory tissue at the top of the nasal cavity detects smells. The organ of Corti in the ear is the hearing receptor, and the nerve network that branches into human tissue is responsible for the sense of touch.4 It is by these senses that what we know about our environment has been received into our consciousness.5 The term modality is a more technically precise term for sense. A group of impressions detected by one organ combine to form a sense. The sense of smell, for example, is a modality. Dudel classifies not only the five senses as modalities but also the subsenses temperature, vibration, pain, equilibrium, thirst, hunger, shortness of breath, and visceral sensation within each modality.4 The subsenses are known as qualities. Vision for example has the qualities of hue and brightness, while taste has the qualities of sweet, sour, salty, and bitter. The term stimuli refers to environmental, chemical, or physiological factors that elicit sensory impression of certain qualities.4 A combination of sensory impressions is integrated into a sensation. Interpretation of those sensations with respect to experience is perception. For example, judgment as to the ideality of the intensity of a quality in a particular setting is a perception.5 Two products may have an equal intensity of the quality sweetness but one product, such as bread, will be perceived as too sweet whereas a cake will be perceived as not sweet enough. In analyzing human response, scientists have distinguished between objective and subjective physiology. Responses of the nervous system to a stimulus are objective sensory physiology whereas perceptions and expressions of those perceptions are subjective sensory information.4 Sensory analysis of foods involves the use of statistics to treat data obtained from those subjective judgments. Quantitative relationships have been developed relating objective and subjective responses.4 As the intensity of a stimulus increases various types of threshold values can be detected objectively and subjectively. These have been valuable tools in establishing the relationship between the magnitude of a stimulus and sensations perceived. The amount of stimulus that is required to perceive sensation is the detection threshold, stimulus threshold, or absolute threshold designated as RL.3 In objective measurements, the amount of stimulus needed to achieve this threshold is the reference unit. Stimulus levels for other degrees of sensation are expressed as multiples of that.4 Above the absolute threshold, the difference threshold can be determined. It is the stimulus difference necessary to produce a change in sensation and is often desig-

Taste Pore

Epithet Microvilti Sensory cells Synapse Perigemmal cell


Basal cell

Supporting cell

Neural afferencies id

Figure 3.1 A human taste bud and its structure and innervation. The microvilli of the sensory cells protrude into a fluid-filled space in the taste pore. Only two afferent fibers are drawn, while actually about 50 fibers branch within just one taste bud, which has its cells (about 40 to 70) assembled like the slices of an orange. (Reproduced with permission from ref. 7.)

nated as DL. The minimum amount of stimulus that results in correct recognition of the quality of the stimulus is the recognition threshold. The magnitude of stimulus above which increases in intensity are not detected is the terminal threshold.3 The subjective measurements are the verbal or written information obtained from the taster whereas the objective measurements are obtained by measuring the frequency of action potentials of neurons. The RL is the weakest stimulus intensity that results in a change in frequency of action potentials and the DL is amount of stimulus change that produces a frequency change of the action potentials of a neuron.6 Taste and smell are chemical senses in that the organs that sense taste and smell respond to chemical stimuli. Sight, hearing, and touch are physical senses responding to physical stimulation such as electromagnetic radiation, sound waves, and contact or pressure.

3.1.2 Taste
Taste receptors are flower-bud-shaped groups of 30 to 70 sensory cells at different stages of maturity plus basal and supporting cells (Fig. 3.1) located on moist surfaces in the oral cavity and pharynx. A fluid-filled pore is lined with microvilli that are attached to the ends of the sensory cells. Each of the active sensory cells in the taste bud have microvilli that are exposed to the pore. The cells of the taste bud are

Papillae foliatae Pap. fili formeP s ap. vallataPap. fungifor mis

VallatedRinsing glandsTaste Striated Taste buds (vonEBNERI nerves muscles ditch

Figure 3.2 Taste papillae on the human tongue from surface and sectioned view. (Reproduced with permission from ref. 9.)

innervated with about 50 afferent nerve fibers.7'8 Most of these taste buds are on the tongue, usually on the surface of or in the folds around papillae (nipplelike protrusions) (Fig. 3.2). There are four types of papillae. The filiform papillae are most numerous, with about 1000 on the surface of the tongue. No taste buds are associated with them. About 7 to 14 vallate papillae each about 5 to 7 mm in diameter are located in a V-shaped line between the anterior surface and the base of the tongue (Fig. 3.3). As many as 200 taste buds are located in the vallated ditch around each papillae. About a hundred fungiform papillae, 3 mm high and 0.3 to 2 mm in diameter, are located over the surface of the tongue except for an area in the center. Fungiform papillae may have several taste buds on their surface but half of the fungiform papillae have no taste buds associated with them. Foliate papillae are located on the side edges of the tongue. Each person has 15 to 20 of these papillae with about 10 taste buds each (Table 3.1). 10 A few taste buds not associated with papillae are located on the soft palate, pharynx, and larynx embedded in the mucous membrane.11 The four qualities that can be sensed by the taste receptors are sweet, sour, salt, and bitter.5 Different areas of the tongue vary in sensitivity to these qualities. Bitter is best sensed on the back of the tongue, the sides of the tongue are most sensitive to the sour taste, and sweet and salty are best sensed on the tip of the tongue (Fig.

I N N E R V A T E D BY: N. lingualis (tngeminus. N. Y.. Chorda tympani.N.Ml v.

SWEET SALTY fungiform papillae SOUR filiform papillae BITTER foliate papillae vallate papillae "Tonsilla lingualis" I= bottom or base of the tongue I

N.glossopharyng.( N.IX) N. vagus ( N. X. N. laryng. sup.)


Figure 3 3 Scheme of the tongue surface showing the distribution of the taste papillae, the innervation, and the areas of maximal sensitivity for each taste quality. (Reproduced with permission from ref. 10.)

Table 3.1

HUMAN TONGUE PAPILLAE AND THEIR TASTE BUDS IN ADULTS


Number of Papillae Taste Buds per Papilla 100-200 =-10 0-4 0 Taste Buds in AU Papillae 1000-1500 150-200 300-400 0

Circumvallate

(P. vallatae)
Foliate

8-12 (7-14) 15-20 -100 =-1000

(P.foliatae)
Fungiform

(P.fungiformes) Filiform (P. filiformes)

Reproduced with permission from ref. 10.

THE SENSE OF SMELL

Olfactory nerve

Trigeminal nerve

Trigeminal nerve

Figure 3.4 A representation of the lateral wall of the human nasal cavity showing the nasal turbinates and distributions of olfactory and trigeminal nerves. (Reproduced with permission from ref. 13.)

3.3). Taste receptors are able to sense multiple qualities but they are somewhat specialized in that they respond better to some qualities than others.10 Some individuals are taste-blind to some qualities. Blakesly and Fox demonstrated that approximately 30% of subjects are blind to the bitter taste of phenylthiocarbamide (PTC) and the lack of taste acuity for that quality is an inherited trait.12 They also demonstrated taste-blindness for other substances.

3.1.3 Smell
The sense of smell in man results from stimulation of chemoreceptors on the olfactory and trigeminal nerve systems. The olfactory epithelium is located in the dorsoposterior or upper rear of the nasal cavity (Fig. 3.4) and is yellow in color as opposed to the pink color of the respiratory epithelium. The olfactory epithelium is covered with cilia that extend into the mucous layer. Four types of cells make up the tissue: receptor neurons, microvillar cells, supporting cells, and basal cells (Fig. 3.5). A ciliated protrusion of the receptor neuron at the mucosal surface is called the olfactory knob.15 The microvillar cells also appear to be sensory neurons with microvilli extending into the mucosal layer.14 The basal cells give rise to new receptor cells. Bowman's glands below the olfactory epithelium secrete mucous through ducts to the mucosal layer. The supporting cells also secrete fluid.16 Volatile odorant molecules smaller than 400 MW dissolve in the mucus before reacting with the receptor

Cilia Mlcrovllli Mlcrovillar ceil Olfactory receptor neuron Olfactory knob

Supporting cell
Basal cell Lamina Axon propria Figure 3.5 A representation of the structure of the human olfactory epithelium. (Reproduced with permission from ref. 14.)

cells.17'18 The axons of the olfactory receptor neurons from each nasal cavity travel through the cribriform plate to the olfactory bulb in the brain.19-20 This olfactory system is very sensitive, responding to very low concentrations of some chemicals. A typical threshold for allyl mercaptan is 107 molecules per milliliter. It is also very discriminating. A trained perfumer can distinguish 150 to 200 odor qualities.21 Because the olfactory tissues are out of the mainstream of nasal airflow, odorants reach them by turbulent eddies that are maximized by "sniffing." Odor sensations are not noticed when the breath is held. To enhance the sense of smell, a subject must " s n i f f air that has been in contact with the food. It also helps to move air out through the nose while food is in the mouth.5 The trigeminal nerves respond to chemical irritants such as ammonia, ginger, horseradish, onion, chili peppers, and menthol. Sensations experienced in the mucosa of the mouth and nose include coolness, heat, and pungency. Usually the concentrations required are much higher than those required by the olfactory system, but it is difficult for subjects to separate trigeminal sensations from olfactory and gustatory ones.

3.1.4 Sight
Vision is an extremely important component of sensory perception of foods. Attractive appearance of dairy products enhances acceptability. Colors are almost inseparably associated with flavors. Coloring some flavors atypically makes recognition difficult. The eye is a complex instrument complete with a clear cornea to protect the iris and lens, a clear liquid called the aqueous humor between the cornea and the lens, an adjustable lens that focuses an image on the retina at the back of the orb, an iris

Optic array

. Fovea

Pupil
Cornea Lens Iris

Blind spot Optic nerve Retinal image Retina Rod Retina

Cone

Optic arr^y (distribution of light ^i the eye): the proximal stimulus distribution

Light Connective cells

Figure 3.6 The eye, showing the lens, retina, blind spot, and optic nerve. The fovea is a small region, central in the retina, that is highly sensitive to detail and consists entirely of cones. (Reproduced with permission from ref. 22.) to adjust the amount of light falling on the retina, and a clear liquid medium called the vitreous humor through which the light passes from the lens to the retina (Fig. 3.6). The retina, which covers much of the back of the eye, contains rods which detect 400 to 700 nm light and cones which are sensitive to the wavelength of light enabling us to see color. When the rhodopsin pigment in the rods is exposed to light, it produces a nerve impulse as it is chemically changed. Color vision of the cones is explained by the Young-Von Helholtz theory that three types of receptors are present each of which is sensitive to one of the primary colors. Stimulation of the three receptors at different relative intensities results in color sensation. Impulses from the rods and cones travel through the optic nerve to the brain where the sensation is perceived and interpreted.22"24 Cone vision is trichromic and the color of any light can be matched by mixing red, green, and blue monochromatic primary light in a suitable blend of intensities. 25 There is also an opposing mechanism in which green is opposite red, blue is opposite yellow, and black is opposite white. 26 Modem colorimeters use these three coordinates to define the hue (color), value (lightness), and chroma (saturation) of the light coming into the eye from an object. 27 The eye adapts to the level of light supplied by constriction or dilation of the pupil and adjustment of the sensitivity of the retina.23 It also adapts to the wavelength. When the eye is exposed to bright monochromatic light, sensitivity to that hue is suppressed and it begins to appear more dull. When this occurs, a white surface will appear momentarily to be the opposite hue. For example, after several seconds of exposure to bright blue it will begin to appear more dull. At a glance, white will momentarily appear to be yellow. Appearance of objects will be affected by the extent to which objects transmit, diffuse, or reflect light. Clear materials allow light to pass through them (water).

Auricle Cartilage Mastoid Ceils Malleus Incus


Vestibule

Semicircular Canals

Vestibular N Facial N

Cochlear N

Internal Auditory Canal


External Auditory Canal
Cochlea

Round Window Stapes Drum Membrane Mastoid Tip Cross Section of Eustachian T u b e

Figure 3.7 A semidiagrammatic drawing of the ear. (Reproduced with permission from ref. 29.)

Colored clear materials absorb some wavelengths of light and alter color (colored gelatin dessert). Translucent materials allow the passage of light but diffuse it (fruit juices) and opaque materials reflect diffused light (milk) and may absorb some wavelengths to alter color (cheese). Some light may be reflected to the eyes without diffusion, resulting in highlights and giving a glossy appearance.79

3.1.5 Hearing
A diagram of the human ear is shown in Figure 3.7. Vibrations carried through air or through the bones of the head cause the eardrum or tympanic membrane to vibrate, and the vibrations are transmitted via the small bones in the middle ear to the inner ear where the vibrations are converted to hydraulic motion in the fluid of the cochlea. The spiral-shaped cochlea is divided along its length by the basilar membrane and the vestibular membrane. Numerous hair cells are located along the basilar membrane. The vibrations cause the basilar membrane to move as a traveling wave. That motion stimulates the hair cells, causing them to send impulses to the brain. The impulses travel along the auditory nerve to the brain. In an adult the detectable frequency range is 30 to 15,000 Hz but the most sensitive range is 500 to 4000 Hz. 30 ' 31 When crisp or crunchy foods are consumed, it is expected that the sounds that are generated will be an important factor in texture perception of that food. Loudness

and discontinuity of the sound have been established as the two basic criteria for distinguishing food sounds. "Loud," "snap," and "crackly" were shown to be related to crispness. Loudness was closely associated with crispness but not so closely associated with firmness.32 The sound is helpful but not essential to the perception of crispness. Subjects had no difficulty in judging crispness when a blocking noise was used to mask the sounds and they were able to judge the crispness accurately when listening to a recording of the sounds. Biting a crisp food gives auditory and tactile sensations which can both be used to judge crispness.3334 Few dairy products produce snapping or crunching noises as they are consumed so contributions of hearing to their sensory evaluation are probably minor. Experienced judges can sometimes determine the number and size of eyes in Swiss cheese by tapping the outside of the cheese and the amount of free water in "leaky" butter by the "slushing" sound made as the plug is reinserted into the hole from which it was drawn.5

3.1.6 Touch
A variety of types of nerve endings are responsible for the sensation of touch. Figure 3.8 shows the free nerve endings in the skin, epidermis, dermis, and subcutaneous tissue. They include the tactile discs, Meissner corpuscles, end bulbs of Krause, Ruffini endings, Pacinian corpuscles, and the nerve endings around the hair follicle. These nerve endings are responsible for the "somesthesis" sensations we call touch, pressure, heat, cold, itching, and tickle. These nerves are sensitive in the mouth, lips, and tongue, making detection of small forces and pressures easy during eating. Deep pressure or "kinesthesis" is felt through the nerve fibers in the joints, tendons, and muscles. They sense tension resistance and relaxation. These nerves in the hand, tongue, and jaw are used to sense the pressure and tension used to manipulate, deform, rupture, and masticate food. These nerves combined are very good at distinguishing particle size, crispness, hardness, elasticity, brittleness, fluid viscosity, and temperature and are significant in our sensory perception of foods.30 The trigeminal nerves which have already been covered and are so important to our taste and smell could properly be considered part of our sense of touch.

3.2 Sensory Evaluation Techniques 3.2.1 Introduction


For hundreds of years, the quality of dairy products has been known to be linked to feeding and milk handling practices. A relationship between certain feeds and milk flavor was established early. Turnips for example were known to give an "ill" flavor to butter.36 Product grades and score cards were developed. Attention was drawn to sensory quality of dairy products in 1916 when a collegiate butter judging contest was initiated with nine teams participating. Milk and cheddar cheese were added to the contest the next year. Over the years, vanilla ice cream, cottage cheese, and

Meissner's corpuscle Tactile d i s c s Free n e r v ee n d i n g s S e b a c e o u s gland S m o o t h D e r m i s Epidermis m u s c l e Hair E n d bulbs of K r a u s e

u b c u t a n e o u s Padnian Nerve ending S fat corpuscle wound hair

Duct of Ruffini ItNMt gland ending

Figure 3.8 Composite diagram of the skin in cross-section. Tactile sensations are transmitted from the variety of nerve endings, for example, the free nerve endings and the tactile discs in the epidermis, and the meissner corpuscles, end bulbs of Krause, Ruffini endings, and pacinian corpuscles in the dermis. (Reproduced with permission from ref. 35.)

Swiss style strawberry yogurt were added. With the exception of a few war years, the contest has been held annually. Fifty-nine schools have fielded teams with as many as 33 participating in 1956. 5 ' 37 Several regional collegiate contests are also held each year. At the high school level, the Future Farmers of America conducts an annual state and national dairy foods evaluation contest. These have served to give thousand of students training in the recognition of dairy product defects, their causes, and control. Many other food industries have developed their *'expert" tasters resources. These experts obtained experience through the years and were charged wih the responsibility of determining the material blend or judging the quality of raw materials. They also judge the quality of finished product and identify sources of problems and suggestions for correction when the products are less than perfect. These experts include the perfumers, flavorists, brew masters, wine makers, and coffee and tea tasters. In most of these industries, such as the dairy industry, scorecards and point systems have been developed to help set standards.38

With the growth of the food industry and the expansion of product lines within companies, it has become almost impossible to have dependable expert judges of all products. It has been necessary to develop sensory evaluation systems that are more universally applicable. Sensory evaluation of foods in general with methodology appropriate for either consensus or statistically sound evaluation of foods began to develop in the 1940s and 1950s at the U.S. Army Quartermaster Food and Container Institute in Chicago.39'40 Development began also in the private sector. The Arthur D. Little Company pioneered descriptive analysis by developing a Flavor Profile Method that uses a consensus of a small group of people who are trained to the product in a way that is universally applicable. The single expert was replaced with five or six trained people.41 The University of California at Davis began to offer courses on sensory evaluation in the 1950s. The literature at that time reflects significant development in the application of sensory evaluation. Discrimination tests were developed by Boggs and Hansen,42 Girardot et al.,43 and Peryam et al.39 Ranking and hedonic scales began to be used for consumer acceptance information. Committee E-18 of the American Society for Testing Materials, the Food and Agriculture Section of the American Chemical Society, the European Chemoreception Organization, and the Sensory Evaluation Division of the Institute of Food Technologists got involved by organizing activities focusing attention on sensory evaluation and measurement of flavor and publishing information assisting the food industry in application of the new techniques.40 These methods are all applicable to dairy product evaluation.

3.2.2 Affective Testing


Affective testing is acceptance testing. Its objective is to determine the degree of consumer acceptance or preference for a product. Usually it is determined relative to a product such as an existing product, or an acceptable successful product. The ideality of certain easily understood attributes can be judged by consumers using their concept of ideal as the standard. Hedonic scales are used to rate the degree of liking of products. An example of a nine-point hedonic scale is shown in Figure 3.9. There are a wide variety of hedonic or liking scales that can be and have been used. Recommended scales are balanced with an odd number of choices, with the middle choice being neutral "Neither like nor dislike." Choices above neutral are positive, with the top being "Like extremely" and the choices below neutral being negative and balanced with those above and the bottom being "Dislike extremely." The data can be treated parametrically, yielding means and standard deviations. Liking of products can be compared using the t test or analysis of variance (ANOVA). Parametric treatment assumes that data are distributed normally and that intervals on the scale are equal. There has been considerable discussion about the validity of these assumptions but the practical value of this approach continues to be demonstrated. The data can be converted to preference or ranking and analyzed binomially.40'44'45 Another affective tool is preference testing. Panelists have the opportunity in preference testing to tell which of two samples they prefer (paired comparison) or

Please check a box indicating your feeling about this product.

Like extremely Like very much Like moderately Like slightly Neither like nor dislike Dislike slightly Dislike moderately Dislike very much Dislike extremely Figure 3.9 An example of the nine-point hedonic scale. The subjects indicate to what extent they like or dislike the sample by checking a box by the most correct statement.

Please check a box indicating your feeling about the moistness/dryness of this product Much too moist Slightly too moist Just about right Slightly too dry Much too dry Figure 3.10 An example of a Just-about-right scale. The purpose of the judgment is to establish how close to ideal a product is in an easily understood attribute. The subject checks the box by the statement that best describes his or her feelings about the correctness of the level of that attribute.

to rank more than two samples in order of preference. It is important that each sample is tasted first and last its share of the time to avoid order bias. Analysis of the paired comparison test utilizes binomial statistics. Tables are available giving the number of subjects that must prefer one sample given a certain number of participants for the preference to be significant.46 When ranking is used, tables and formulas are available showing the rank sum difference required for significantly different ranking given the number of samples compared and number of panelists used. 47 An effective tool to determine the ideality of easily understood attributes is the Just-about-right scale. This is the three- or five-point scale with "Just about right" being the middle response with balanced descriptors of the attribute extremes going up and down from ideal (Fig. 3.10). Stone suggests two methods of analyzing the

data to determine if each product deviates significantly from ideal and one method to determine if the samples deviate from one another in ideality.40 One involves using the binomial table of Roessler et al. (p = 0.5, two-tailed) to determine if the number of judgments on one side of ideal is more than can be explained by chance.46 The number of nonideal judgments is n and the number on one side of ideal is found in the column under the appropriate confidence level. The appropriate type of panelist for all affective tests is a "naive" consumer, one who has no knowledge of the objective of the comparison or the technology involved in making the products. The subjects may be screened to be representative of the demographics of a certain target consumer group. Trained panelists who are used in descriptive or discrimination tests should not be used because of their analytical approach which may bias affective judgments.40

3.2.3 Discrimination Testing


Discrimination testing is a very useful sensory evaluation tool that enables one to determine if a perceived difference exists between two products. Often it is preliminary to other types of testing. If no perceived difference exists, it is not necessary to determine which one is preferred or what the difference in the descriptive characteristics are.40 If a development objective is to have no perceived difference, this test can establish that the objective has been met and subsequent sensory testing may not be necessary. There are several methods that may be used to establish whether there is a perceived difference. Methods include paired-comparison, duo-trio, and triangle tests. The paired comparison test is a two-sample test with the task being to determine whether the products are the same or different, or it may be to choose which of the two samples has more of a particular attribute. When the subject is asked if the products are the same or different, it is important that half the panelists receive samples that are the same and half receive samples that are different. In interpreting the data, the number of correct choices are compared with the number of correct selections that can be explained by chance. When the assignment is to indicate which sample has more or less of a certain attribute, it is assumed that the subject recognizes that attribute in the product. It is important that the attributes be simple and easily recognizable. If the number of correct selections if greater than can be explained by chance, one can conclude that the samples are different. Interpretation involves binomial statistics. A table and formula for the significant number of correct judgments is published by Roessler et al.46 The correct table and formula would be those where the probability of being right by chance in one selection is one in two (p = 0.5). It is a one-tailed test. The tail of interest is being correct more frequently than can be explained by chance. The other tail not of interest is being wrong more frequently than can be explained by chance. Protection against a type I error (finding difference when none exists) is selected by selecting the column with the appropriate a. An a of 0.05 would allow for a 5% chance of a type I error.48 The duo-trio test was developed by Peryam and Swartz as a way to minimize the number of comparisons that have to be made.39 The subject is given a reference

sample and two coded samples. One of the coded samples is the same as the reference sample. The subject is asked to indicate which sample is the same as (or different from) the reference. In variations of the test, the reference sample may be removed after it is tasted to force the use of memory for comparison. Reliance on memory decreases the sensitivity of the test. The same sample may be used as the reference through the entire test, or each sample may take its turn as the reference. It is important that the order of tasting the two samples be rotated so that each sample is tasted immediately after the reference with equal frequency. The data are evaluated using the same formula and tables as for paired comparisons.46 The probability of being correct on one decision is one in two (p = 0.5) and interest is in one tail (being right more frequently than can be explained by chance). The most frequently used discrimination test is the triangle test. It was initially developed by a beer company.49 In this test, the panelist is presented three coded samples. Two are the same and one is different. The panelist evaluates all three and determines which one is different or which two are most alike. This test requires more tasting than the others. Three pairs are compared in making the judgment. Again binomial statistics are used to evaluate the results. The probability of being right by chance (p) in one selection is one in three and it is a one-tailed test (the probability of being wrong more frequently than is explained by chance is the tail that is not of interest).40 The table and formula provided by Roessler et al. are used to determine when the frequency of correct selection exceeds chance.46 Subjects for discrimination tests should like the product, be familiar with the test procedure, have frequent practice with the test, have a record of exceeding chance in choosing correctly in previous tests, and have no specific knowledge about the samples.40 The number of panelists used should be no more than 40 and may be as few as 12 to 15. Too many panelists will result in significant differences when the differences are very subtle and of no practical importance. Too few will allow for a large type II error (finding no difference when difference exists).30'48 It is important to guard against unintended differences. For example, it is easy to have slight temperature, serving amount, piece shape or size, or color differences that are not intended. Panelists are playing a game and will look for any clues that will reveal the different sample. If a conclusion is reached, due to inadvertent hints that samples are different when they are not, the results can be misleading and expensive. Further development or costly consumer or descriptive testing may be mandated.

3.2.4 Descriptive Analysis


Descriptive analysis is the process of developing a total sensory description of a product. In its complete form it involves identifying each flavor, aroma, and textural quality detectable in the product and quantifying each. The time sequence of the detection of the qualities can also be included in the profile. Affective judgments as to the desirability of the sensory qualities are generally not a part of descriptive analysis. It is important that the panel members are highly trained to recognize all of the qualities of the product and to use a standardized terminology to describe

them. Developing and proving a descriptive panel requires skill on the part of the leaders, and dedication, time, patience, and attention to detail on the part of panel leaders and panelists. 30 ' 40 Several methods of descriptive analysis have been developed. Three that represent the development of descriptive analysis and slightly different philosophies are the Flavor Profile, Texture Profile, and Quantitative Descriptive Analysis (QDA). The Flavor Profile method was developed by Arthur D. Little, Inc. in the late 1940s. A small panel of four to six trained judges analyze a product's perceived aroma and flavor qualities, and their order of detection, intensity, and aftertaste. They also assess the degree to which various flavor or aroma characteristics fit together and their appropriateness in the product and call this characteristic amplitude.41'50 Prospective panelists are screened for their ability to detect and discriminate tastes and odors. Their interest and availability and ability to work with a group are assessed in a personal interview. Selected panelists are trained with product examples that represent the extremes of the different qualities that may be encountered. Product is made with a variety of ingredients and processes to produce a wide variety of product. In the actual evaluation session, trained panelists first evaluate a product individually while seated together around a table. The results are reported to the panel leader who leads a discussion that results in a consensus profile. More than one sample can be profiled in a session but they are done one at a time without tasting back and forth. Once a panel is trained, profiles can be obtained easily. 10 ' 40 General Foods developed the Texture Profile method to do for texture analysis what the Flavor Profile method had done for flavor and aroma. 51 " 53 It was different from flavor profiling in that the terminology for different texture qualities was standardized (Table 3.2). The anchors used to standardize the scales were also predefined. Odd numbered categorical scales for each quality were developed. Later quality descriptors were added for semisolid foods, beverages, 54 ' 55 and skin-feel products.56 Prospective panelists are screened based on interest, availability, and attitude. They are further selected on the basis of ability to discriminate known textural differences in the product to be tested. They are introduced to the principles involved in the product to be tested. An evaluation of a product after the panel is trained involves independent evaluation by each panelist using one of a number of possible scales, then the generation of a panel verdict. The verdict may be obtained by discussion and group consensus similar to the method for obtaining a flavor profile or by statistical analysis of the data. Quantitative Descriptive Analysis was developed to overcome weaknesses in the descriptive test previously described. It was designed to be responsive to flavor, aroma, and texture simultaneously, to be applicable to a broad range of products, to be quantitative in evaluation of panelists' qualifications and in development of profiles, to use a small number of panelists, and to have flexible panel-generated terminology. Subjects are qualified before participation. They must be available and be users of the product class. They must demonstrate ability to perceive differences within the class of products and to articulate those differences. The terms used to describe qualities may be available from previous work. If so, the panel learns and experiences the definitions of all the qualities. If not, the terms describing the qual-

Table 3,2

RELATIONSHIP BETWEEN TEXTURAL PARAMETERS AND POPULAR NOMENCLATURE


Mechanical Characteristics

Primary Parameters Hardness Cohesiveness

Secondary Parameters

Popular Terms Soft, firm, hard Crumbly, crunchy, brittle Tender, chewy, tough Short, mealy, pasty, gummy Thin, viscous Plastic, elastic Sticky, tacky, gooey

Brittleness Chewiness Gumminess

Viscosity Elasticity Adhesiveness Geometrical characteristics Class Particle size and shape Particle shape and orientation Other Characteristics Primary Parameters Moisture content Fat content Secondary Parameters

Examples Gritty, grainy, coarse, etc. Fibrous, cellular, crystalline, etc.

Popular Terms Dry, moist, wet, watery Oily Greasy

Oiliness Greasiness

Reproduced with permission from ref. 52.

P l e a s e m a r k this line in a position that indicates how w e a k / f i r m you feel this yogurt body to b e .

Extremely weak

Extremely firm

Figure 3.11 An example of a horizontal line scale used by descriptive panelists to indicate the strength of a particular flavor or aroma quality. The subjects marks the position of the line that describes the intensity of the quality.

ities are selected and defined by the panelist as they train. Reference materials that are examples of the qualities are used to aid in definition of qualities. When evaluating actual product, if new qualities are found, the panel reconvenes to define and train on that quality. Scales used are horizontal lines of a consistent length with word descriptors at or near the ends (Fig. 3.11). Intensity always increases from left to right and the subject marks the line at a position that is appropriate for the intensity of the quality. Evaluation during training and on actual product is done individually

Aftertaste Bitterness Aroma

Sweet

Malt Flavor

Crunch (final)

Sour Crunch (initial)

Figure 3.12 Visual display of the sensory characteristics based on the results of a Quantitiative Descriptive Analysis (QDA) test. For each characteristic, the relative intensity increases as it goes further from the center. (Reproduced with permission from ref. 40.)

and usually in isolated sensory booths to ensure independent analysis. Replicate samples are included so that ANOVA can be applied to evaluate the panelists' consistency as well as to statistically compare the intensity of qualities of the different samples. The panelists who are best able to replicate themselves on all the qualities and who agree best with the rest of the panel on each of the qualities are best qualified to evaluate product. Usually between 8 and 12 qualified subjects constitute a panel. The product QDA profile is a listing of the qualities and the means for each of those qualities. Significance of difference between samples in each quality is obtained by ANOVA.40 Multiple-range tests are applied to establish the significance of differences between multiple samples. Profiles of individual samples can be shown in a number of formats. A "spider web" format is shown in Figure 3.12. Each quality is depicted as a "spoke" of a wheel with its length being indicative of the intensity of the quality. With the ends of the "spokes" connected, a shape is formed that is distinct. A change of intensity in one attribute produces a readily distinguishable difference in shape.

3.3 Application of Sensory Analysis to Dairy Products


The system for evaluating dairy products for defects was developed long before the generally applicable tools of affective, difference, and descriptive analysis. These

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newer generally applicable tools are as useful for dairy products as they are for other foods and are essential when sensory information needs to be quantified for research purposes. Any treatment of sensory analysis of dairy products without their mention would be incomplete. The remainder of this chapter, however, will focus on evaluation of dairy products for defects or judging of dairy products. This ability, although not designed for statistical analysis or research, is still very useful to dairy product manufacturers, enabling them to recognize defects, identify causes and take corrective action.

3.3.1 The Philosophy of Judging of Dairy Products


Judging of dairy products is related to descriptive analysis. It is similar in that flavor (including aroma), texture, and appearance can all be evaluated. It is similar too in that the names of the qualities and their definitions are standardized. The quality terms and definitions have evolved over the years with USDA and industry "experts" involved and a committee of collegiate coaches, who serve as the American Dairy Science Committee on Dairy Product Evaluation, periodically modifying the terms and definitions. It is different from descriptive analysis in that normal ideal base qualities of the products are not identified and only the defects are noted. The judges score the products on flavor, texture, and appearance. Score ranges are established for each defect. Defects that are indicative of serious problems have lower score ranges than less serious defects. Higher scores in that range are given if the defect is slight and scores at the lower end of the range are given when defects are pronounced. In the event of multiple defects, the score is based on the defect that would result in the lowest score. In that way, scoring takes into account the magnitude and seriousness of the defects as determined by these "experts." No attempt has been made to tie the scores to consumer acceptance of the products.

3.4 Descriptive Sensory Defects of Dairy Products 3.4.1 Fluid Milk and Cream 3.4.1.1 Introduction
Fluid milk is the material from which all other dairy products are made. Defects in milk will cany over into those products so it is important that these defects be recognized first. Coaches of collegiate judging teams spend a generous amount of time on fluid milk because the defects of milk are closely related to the resulting defects in products, and because "doctoring" milk to simulate the defects is relatively easy. 5 A wide variety of fluid milk and cream products are available. A listing of products is shown in Table 3.3. Complete evaluation of fluid milk can include examination and scoring of a sediment disk, evaluation of the package, storage temperature, and bacteria count.5 Table 3.4 shows flavor defects that can be found in milk and the range of scores that can be assigned. A score card that includes all these important defect descriptors is shown in Figure 3.13. It is based on a possible 25

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newer generally applicable tools are as useful for dairy products as they are for other foods and are essential when sensory information needs to be quantified for research purposes. Any treatment of sensory analysis of dairy products without their mention would be incomplete. The remainder of this chapter, however, will focus on evaluation of dairy products for defects or judging of dairy products. This ability, although not designed for statistical analysis or research, is still very useful to dairy product manufacturers, enabling them to recognize defects, identify causes and take corrective action.

3.3.1 The Philosophy of Judging of Dairy Products


Judging of dairy products is related to descriptive analysis. It is similar in that flavor (including aroma), texture, and appearance can all be evaluated. It is similar too in that the names of the qualities and their definitions are standardized. The quality terms and definitions have evolved over the years with USDA and industry "experts" involved and a committee of collegiate coaches, who serve as the American Dairy Science Committee on Dairy Product Evaluation, periodically modifying the terms and definitions. It is different from descriptive analysis in that normal ideal base qualities of the products are not identified and only the defects are noted. The judges score the products on flavor, texture, and appearance. Score ranges are established for each defect. Defects that are indicative of serious problems have lower score ranges than less serious defects. Higher scores in that range are given if the defect is slight and scores at the lower end of the range are given when defects are pronounced. In the event of multiple defects, the score is based on the defect that would result in the lowest score. In that way, scoring takes into account the magnitude and seriousness of the defects as determined by these "experts." No attempt has been made to tie the scores to consumer acceptance of the products.

3.4 Descriptive Sensory Defects of Dairy Products 3.4.1 Fluid Milk and Cream 3.4.1.1 Introduction
Fluid milk is the material from which all other dairy products are made. Defects in milk will cany over into those products so it is important that these defects be recognized first. Coaches of collegiate judging teams spend a generous amount of time on fluid milk because the defects of milk are closely related to the resulting defects in products, and because "doctoring" milk to simulate the defects is relatively easy. 5 A wide variety of fluid milk and cream products are available. A listing of products is shown in Table 3.3. Complete evaluation of fluid milk can include examination and scoring of a sediment disk, evaluation of the package, storage temperature, and bacteria count.5 Table 3.4 shows flavor defects that can be found in milk and the range of scores that can be assigned. A score card that includes all these important defect descriptors is shown in Figure 3.13. It is based on a possible 25

Table 3.3 A USTING OF FRESH MILK AND CREAM PRODUCTS WITH FAT CONTENT IN PARENTHESES57
Whole milk (2*3.25%) Skim milk (<0.5%) l%Milk(l%) 2% Milk (2%) Half and half (10.5-18%) Light cream (18-30%) Light whipping cream (30-36%) Heavy cream (s*36%)

Table 3.4

THE ADSA SCORING GUIDE FOR OFF-FLAVORS ON MILK AND CREAM


Intensity of Defect

Flavor Criticisms3 Acid Bitter Cooked Feed Fermented/fruity Flat Foreign Garlic/onion Lacks freshness Light induced (oxidized) Malty Metallic (oxidized) Rancid Salty Unclean

Slight 3 5 8 6 5 9 5 5 8 6 5 5 4 8 3

Definite 1 3 8 4 3 8 3 3 7 4 3 3 1 6 1

Pronounced 0b 1 6 1 1 7 1 1 6 1 1 1 0 4 0

Source: American Dairy Science Association, 1990. a "No criticisms'' is assigned a score of 10. Normal range is 1 -10 for salable product. b An assigned score of 0 (zero) is indicative of unsalable product.

points with 10 possible on flavor, three on sediment, five on package, five on bacteria count, and two on temperature. The electronic score card now used in collegiate competition in which only flavor is judged is shown in Figure 3.14. The flavor of milk is usually judged after sediment, closure, and container are judged. This treatment will cover only flavor. For information on how the other factors are judged see Bodyfelt.5 To best judge flavor, the milk or cream should be tempered to 12.8 to 18C. The judge should swirl the bottle and then smell the milk or cream. Swirling serves to mix the sample and to spread a fine film on the inside of the container which gives maximum opportunity for volatiles to fill the headspace. A small amount of sample should be poured into a clean odorless container. Glass is preferred but plastic or paper is acceptable. The judge should then take a sample into his or her mouth, and move it around in the mouth making sure to coat all the surfaces of the

SCORE CARD FOR MILK QUALITY Product: 1 Flavor 10 No criticism 10 Criticism Score Acid Astringent Barny Bitter Cooked Cowy Feed Fermented/fruity Flat Foreign Garlic/onion Lacks freshness Malty Oxidized light induced Oxidized metal induced Rancid Salty Unclean Date: 2 3 SAMPLE NO. 4 6 5 7 8

Unsalable 0 Normal range 1-10

Sediment 3 Package 5

No criticism 5 Unsalable 0 Normal range 1-5

Score Score Container bulging/distorted Dented/defective Dirty inside Dirty outside Leaky Not full Closure defective Coating flaky/cracked Heat seal defective Illegiblejjrinting Labeling/code incorrect Lip chipped Cover not waterproof Unprotected Score Standard plate count Coliform count Keeping quality

Bacteria

Temperature 2 Temperature (0F or 0C) Total score of each sample Desired % Fat content (%) Desired % Solids not fat (%) Under/over filled Titratable acidity Functional and other tests performe:d on samples Signatures of evaluators

Score Score

Figure 3.13 A modified and expanded version of the ADSA milk score card. (Reproduced from ref. 5, with permission of the ADSA, Champaign, IL.)

M A R K I N GI N S T R U C T I O N S M IP R O P E R M A R K S P R O P E R M A R K MILK

E R A S EC H A N G E SC L E A N L YA N D C O M P L E T E L Y O ON O TM A K EA N YS T R A YM A R K S

N C ST m ie O p c t i* M P 3 0 7 3 6 2 9 3 2 1A 2 4 0 0 S A M P L EN U M B E R

N O C R T IC IS I M FEO 1 0
FLAT

WTTER

N O R M A L R A N G E 1 1 0

GARUC/0NI0N MALTY waomo - Mm*, Mouotft SAtTY

BODY AND TEXTURE


NO CRITICISM 5

NORMAL RANGE 1-5

APPEARANCE AND COLOR


NO CRITICISM 5

NORMAL RANGE 1-5

Figure 3.14 Collegiate contest milk score card. (Reproduced from ref. 5, with permission of the ADSA, Champaign, IL.)

mouth from the front to deep in the back down to the throat, noting any off-flavors. While the sample is in the mouth, airis moved up through the nose to enhance odor detection. The sample should then be expectorated and a few moments allowed to observe aftertaste. Aftertaste and aroma sensation are enhanced by exhaling slowly through the nose. Swallowing sample is not advised. According to Bodyfelt,5 the flavor of whole milk should be pleasant and sweet and with neither a foretaste nor an aftertaste other than that imparted by the natural richness. A listing of flavor criticisms with a scoring guide is shown in Table 3.4. A list of these defects, and their verbal descriptions, causes, and methods of preparing training samples follows.

3.4.1.2 Flavor Defects Acid or Sour Milk


Description. Acid or sour is detected by both the senses of taste and smell. The tip of the tongue is sensitive to the "peeling" or "tingling" sensation. A general feeling of "cleanliness" and enhanced ability to taste is part of the sensation. Other flavors such as diacetyl may accompany acid as byproducts of fermentation.5 Cause. Acid or sour milk is a result of bacterial action on lactose converting it to lactic acid. It can be produced by culture organisms such as Lactococcus lactis ssp. lactis, or Lactococcus lactis ssp. cremoris or by any other lactic acid fermenting organism that purposely or accidentally is present in milk and is allowed to grow. Training Sample Preparation. Small amounts of lactic acid can be dissolved in milk until the desired intensity of acid is obtained. Addition of 25 5 to 10 ml of fresh cultured buttermilk can be added to 575 ml of fresh milk. It should be prepared 1 or 2 days before tasting and held refrigerated until use.5 Usually a diacetyl flavor accompanies the acid flavor.

Astringent
Description. This sensory defect is actually a tactile sensation. Other descriptive words used are mouth coating, dry, puckery, chalky, and powdery. It is classified here with flavor because it is sensed when the product is taken into the mouth. It is not a common defect in beverage milk. After expectoration, the lining of the mouth may feel shriveled or puckered. Cause. Not all the causes are known but it is usually associated with high heat treatment of milk that has caused some aggregation of milk proteins. A specific particle size of milk proteins or other milk constituents is thought to be responsible for the sensation. Training. Green persimmon or alum are extreme examples of astringency. They may be used to demonstrate the sensation.

Barny
Description. The flavors ' 'cowy," ' 'barny," and * 'unclean'' seem to be quite alike but differ in intensity and cause. The descriptive term "barny" is quite accurate,

referring to the typical smell of a poorly maintained bam atmosphere. It is noticed immediately after the milk is expectorated.5 Cause. The smells of the barn are thought to be transmitted to the milk through the cow's respiratory system when cows are stabled and milked in a foul smelling barn environment. Training. Trainees could be taken to some milking operations and the atmospheric aroma noted. Milk could be collected from cows that are kept in this type of closed environment, lab pasteurized, and used soon after as training samples.

Bitter
Description. Bitter is a taste sensation with no associated aroma. It is detected at the base of the tongue. The reaction time is fairly slow so it is most strongly sensed after the milk is expectorated. The intensity builds and it is hard to rinse away and refresh the tongue. It seems to be a component of "rancid" and "soapy" flavors.5 Cause. It is generally acknowledged that some protein fragments taste bitter. These fragments can be produced by enzymatic breakdown of milk proteins. Enzyme sources in milk are likely psychrotrophic microorganisms that have grown in the cool milk. Milk that is stored at temperatures at or slightly above 4C for several days will become bitter if these contaminating organisms are present. Under those conditions they will grow to large populations and release proteases. Certain weeds consumed by the cow will also impart bitterness to the milk. Conditions that produce rancidity may be to blame for bitterness that is a component of rancidity. Preparation of Training Samples. Traces of quinine dihydrochloride or quinine sulfate added to milk will give a clean bitter flavor. A 1 % stock milk or water solution can be made and added at the rate of 1 to 2 ml per 600 ml of milk.5

Cooked
Description. Four kinds of heat-induced flavors have been recognized: sulfurous, rich, caramelized, and scorched. All are easily identified.58 They are detected immediately as the sample is placed in the mouth and are usually considered to be pleasant. The sulfurous and rich descriptors are common in milk. The detection of a cooked egg white smell is characteristic of this defect. Cause. The mild sulfurous flavor develops when milk reaches 76C to 78C.59 This is slightly above HTST pasteurization temperatures. Its development is associated with the breaking of disulfide bonds and the development of conditions that discourage oxidation. The more severe flavors of scorched and caramelized develop at higher temperatures and by a different mechanism and are not normal in beverage milk. The heated flavor is what remains after cooked milk is stored cold for a period of time. Caramelized flavor frequently intensifies and becomes more objectionable with age.5

Preparation of Training Samples. Fresh pasteurized/homogenized milk is heated to 800C and held for 1 min and then cooled.5 This can be done on a plate pasteurizer, in a water bath, or in a pan on a stove top with continual stirring. Cowy Description. Usually a "cowy" flavor suggests a cows-breath-like odor and a chemical aftertaste. It seems to be associated with the presence of acetone bodies in milk.5 Cause. Cows that have acetonemia or ketosis will give milk with this off-flavor defect.

Feed
Description. A "feed" flavor is aromatic and sometimes pleasant. After the milk is expectorated a mild aftertaste of "cleanliness" can be present that disappears rather quickly, leaving the mouth free of off flavors. Cowy, barny, and unclean flavors by contrast persist with an accompanying unpleasant or "dirty" aftertaste. Feed flavor varies with the type of feed consumed. The odor is characteristic of the feed.5 Cause. High-volume roughage feeds consumed within 3 h of milking impart flavors and aromas to the milk.5 Silage, some hays, and brewery waste are particularly notable for this. A change of feed from dry hay to fresh green pasture often initiates a strong feed flavor in the milk. If 3 h is allowed to pass between consumption and milking, almost all feed flavors are absent from the milk.5 Preparation of Training Samples. An alfalfa flavor can be simulated by adding and placing 2 to 3 g of alfalfa hay in 100 ml of fresh pasteurized and homogenized milk and holding for 20 min. The milk is then strained through a cheesecloth or paper towel and used as a stock solution. To 575 ml of fresh pasteurized and homogenized milk, add 20 to 35 ml of this stock milk solution. Grass or corn silage can be used to prepare feed flavored milks in the same manner.5

Fermented/Fruity
Description. This defect is detected by its odor which resembles the odor of sauerkraut, vinegar, pineapple, or apple. There will also be an unpleasant flavor that will linger long after the sample has been expectorated. Cause. This flavor is often found in bulk raw milk after lengthy storage. Certain microorganisms such as Pseudomonas fragi and other Pseudomonas species are among those that produce aromatic fermentation products.60 Preparation of Training Samples. Bodyfelt suggests the preparation of a stock solution of 1% ethyl hexanoate. About 1.0 to 1.25 ml of this solution is added to 600 ml of fresh pasteurized and homogenized milk.5

Flat Description. Flat milk gives a watery sensation or a lack of flavor richness. No aroma is associated with flat flavor but the lack of sweet and salty notes becomes apparent immediately as the milk enters the mouth and the subtle thinner mouth feel may also be notable.5 Cause. Flat flavor is generally caused by dilution with water. It can happen at the farm or in the plant by allowing too much rinse water to pass into the milk before it is diverted. Purposeful dilution with water is also possible. Preparation of Training Samples. To prepare slightly flat samples add 75 to 100 ml of good quality tap water to 500 ml of fresh pasteurized and homogenized milk. For definite flat use 110 to 120 ml of water to 485 ml of milk.5

Foreign
Description. The term *'foreign" is used to describe a number of flavors that are imparted by addition of detergents, disinfectants, and sanitizers to milk. The flavor is characteristic of the chemical that has been added. The flavors are atypical of milk and do not develop in milk. In some cases the chemical may be detected by smell but in others it may not be detected until it is tasted. Cause. Adding milk to a vat or running milk through piping that has been washed or sanitized but not rinsed can cause a foreign flavor especially if allowed to comingle with a considerable amount of liquid containing the chemical. Other possible causes include treating the udder with ointments or medication, contamination with insecticides, and drenching the cow with chemical treatments. Preparation of Training Samples. Bodyfelt et al. suggests that a foreign flavor may be created by adding 3 to 4 ml of twofold vanilla extract to 600 ml of milk and that a foreign flavor caused by sanitizer can be produced by adding 1.0 ml of a 5% sodium hyperchloride solution to 600 ml of good quality milk.5 Samples can be made by adding traces of other nontoxic chemical cleaners and sanitizers to milk at low concentrations.

Garlic/Onion (Weedy)
Description. These flavors are identified by their characteristic pungent flavor and aroma and persistent after taste. Cause. Milk is tainted with these flavors during the warm months when cows are feeding in pastures that are infested with onion, garlic, or other weeds that impart these flavors to the milk. They are especially strong when the cows consume these plants shortly before they are milked. Preparation of Training Samples. To produce a definite garlic/onion intensity, add 0.15 g of garlic or onion salt or two drops of extract to 600 ml of good quality pasteurized and homogenized milk. Vary the amounts to get the desired flavor strength.

Lacks Freshness
Description. This flavor lacks descriptive characteristics. It suggests the loss of fine taste qualities typically noted in good milk. It is not as pleasantly sweet and refreshing or as free of an aftertaste as is typically desired in milk. Frequently lowfat milks when compared with whole milk will exhibit this characteristic. Cause. The ' 'lacks freshness" characteristic is often considered to be early stages of the development of oxidized or rancid flavor or it could be the beginning of degradation by psychrotrophic bacteria. Preparation of Training Samples. This characteristic is often present in milk that is approaching its pull date about a week and a half to two weeks after processing. It can also be simulated by addition of 10 to 15 g nonfat dry milk powder to 600 ml of pasteurized and homogenized milk.5 Malty Description. As is suggested by the descriptive term, this flavor is suggestive of malt. Malt, which is grain (barley) softened by steeping and allowed to germinate, has this characteristic flavor. This flavor can be detected by smelling or tasting the milk and is often accompanied by or is the forerunner of an acid taste.5 Cause. This flavor in milk is usually caused by the growth of Streptococcus lactis ssp. lactis var. maltigenes bacteria. They grow well when the temperature is allowed to rise above 18.2C for 2 to 3 h.60 Preparation of Training Samples. This flavor can be easily transferred from malted cereals to milk. A stock solution is made by soaking 15 g of Grape Nuts in 100 g of milk for 30 min. The milk is filtered through cheesecloth or a napkin. Thirteen milliliters of the stock solution is added to 590 ml of pasteurized and homogenized milk to give a malty flavored milk of definite intensity.

Oxidized (Metal-Induced)
Description. This flavor is a result of lipid oxidation that is induced by catalytic action of certain metals. Other synonymous terms are metallic, oily, cappy, cardboardy, stale, tallowy, painty, and fishy. It is characterized by an immediate taste reaction on placing the sample in the mouth and a moderate aftertaste. A puckery mouth feel characterizes high-intensity oxidized flavors. It is similar to the flavor of metal foil, a rusty nail, or an old penny.5 Cause. The presence of this flavor usually means that some corrodible metal has come in contact with the milk. It usually can be traced to a fitting or some piping that is made of "white" metal. For years, dairy plants and equipment have been made entirely of stainless steel to avoid the development of this defect. Oxidation of the phospholipids that were originally in the fat globule membrane is blamed for the majority of the flavor. Two oxidative products, 2-octenal and 2-nonenal, have this characteristic flavor at <1 ppm.61

Preparation of Training Samples. The flavor can be generated by soaking clean pennies in milk until the flavor intensity reaches the desired level. Another method is to prepare a 1% stock solution of CuSO4 and add the following amounts to 600 ml of milk: 0.75 ml for slight, 1.2 ml for definite, and 1.8 ml for pronounced. These samples are held refrigerated for 1 to 2 days before use.5

Oxidized (light-Induced)
Description. Synonymous descriptive terms that have been used for this flavor are burnt, burnt protein, burnt feathers, cabbagey, and medicinal. Some synonymous terms designating cause are light-activated and sunlight flavor. Cause. Two reactions are involved in the development of this flavor which develops when milk is exposed to sunlight or fluorescent lights. One is produced by lipid oxidation as described for metallic oxidized flavor, and the other by amino acid degradation involving riboflavin. It is proposed that methionine is degraded to 3-methylthiopropanal (methional) by a Strecker degradationlike reaction yielding ammonia and carbon dioxide.36'62 Methional has an odor similar to that of lightexposed milk. Without riboflavin methional does not develop.36 Preparation of Training Samples. Milk with the light-induced oxidized flavor can be prepared by exposing milk in clear or translucent containers to bright direct sunlight for 8 to 15 min. The shorter times will produce slight levels of the defect and the longer time will give definite and pronounced levels.5 Similarly the flavor can be produced by exposing milk to bright fluorescent light for 2 to 8 h. Overnight exposure next to a 40-watt fluorescent light will produce pronounced flavor. Less intense samples can be prepared by diluting strongly flavored samples.

Rancid
Description. There are several characteristics of rancid off-flavor. There is a characteristic odor derived from volatile fatty acids that have been hydrolyzed from the fat. Immediately after putting the sample in the mouth, the objectionable flavor may not be apparent but as the sample reaches the back of the mouth, soapy, bitter, and possibly unclean flavors are perceived. The soapy and bitter notes reside long after the sample is expectorated. A high percentage of prospective judges do not detect or have a high threshold for the soapy and bitter notes.5 Cause. Rancid flavor is usually caused by disrupting the milk fat globule while active lipase is present. The lipase enzyme, which catalyzes the deesterification of the fatty acids from the glycerol, is able to get to its substrate when the fat globule membrane is disturbed. This happens when raw milk is held static in a running centrifugal pump, when raw milk is homogenized before it is pasteurized, or when raw milk is inadvertently mixed with homogenized milk. It may also occur when microorganisms, particularly psychrotrophs, produce and release Upases into homogenized milk.5 Preparation of Training Samples. Rancid milk can be prepared by adding equal quantities of raw milk to freshly pasteurized and homogenized milk and holding

several hours cold while the flavor develops. Bodyfelt suggests mixing 100 ml of raw milk with 100 ml of pasteurized and homogenized milk in a Waring blender or a similar mixer for 2 min, then making it up to 600 ml with pasteurized and homogenized milk. He suggests making it up 2 to 3 days ahead and holding cold while the flavor develops. In both cases, it is important to heat the milk to 700C for 5 to 10 min and cool after the flavor has developed.5

Salty
Description. The descriptive term "salty" is commonly known and a good term to describe this flavor. It is perceived quickly on placing the sample in the mouth. No aroma or odor accompanies the salty flavor. It lends a cleansing feeling to the mouth.5 The author perceives the salty sensation as "warm" and lacking refreshing character. Cause. Cows in the advanced stages of lactation and cows that have clinical stages of mastitis often have high salt content in their milk and a salty flavor. Comingled milk seldom has an abnormal salt level nor a salty taste. Preparation of Training Samples. Add a pinch of sodium chloride at a time to pasteurized and homogenized milk while stirring to dissolve the salt until it is at the desired strength.

Unclean
Description. Milk with an "unclean" flavor is readily noted when the sample enters the mouth. The flavor and odor are offensive, suggesting extreme staleness, mustiness, putrid, "dirty sock," or spoiled. The flavor fails to clean up after the milk is expectorated. Cause. This flavor develops in milk when psychotropic bacteria are allowed to grow to high numbers in milk and particularly when held at temperatures above 7.2C. The presence of psychrotrophs is usually due to poor on-farm sanitation. High numbers are generally due to poor bulk tank cooling. Preparation of Training Samples. To find ' 'unclean'' flavored milk, examine several samples of milk that are beyond their pull date. If the flavor is not found, incubate them for 4 to 12 h at room temperature and reexamine them. When an exemplary sample is found, it may be maintained in the refrigerator and used as an inoculum for production of future training samples.5

3.4.2 Cottage Cheese

3.4.2./ Introduction
Cottage cheese is a curd that is formed by the acid coagulation of pasteurized skim milk. The acid may be formed by lactic acid bacteria that are added to the milk which consume lactose and convert it to lactic acid.63 In one successful method, part

of the acid is added to the milk as acid and the rest is added as an acid anhydride which slowly converts to acid and drops the pH of the quiet skim milk to the isoelectric pH where the curd forms.64 The curd is cut into cubes, cooked to expel the whey and firm the curd, washed to cool the curd and remove lactose, then salted and creamed. The cream contains enough fat to bring the final fat content to the desired level which is commonly 2% or 4%. It is sometimes cultured with lactic acid fermenting and flavor producing bacteria to add flavor and extend shelf life. Variations on the process will produce various curd sizes or a curd mass called ''baker's" cheese. These products are held below 40C throughout distribution and consumed within 2 to 3 weeks.5 Good creamed cottage cheese should have a clean, slightly acidic flavor with a slight cultured or "diacetyl" flavor. It should be slightly salty sufficient to give a balanced flavor. The body should have a meaty consistency without being too firm or rubbery. As the product is masticated, the texture should be smooth. The curd particles should appear fairly uniform in size and shape without shattered curd. The cream should adhere to the curd particles and give moderate but not excessive gloss or sheen. A listing of defects that can be found in cottage cheese and the resulting score ranges is shown in Table 3.5. The ADSA contest score card is shown in Figure 3.15 and the Collegiate Contest Scorecard is shown in Figure 3.16.

3.4.2.2 Flavor Defects


Bitter
Description. Bitter is a taste sensation with no associated aroma. It is detected at the base of the tongue. The reaction time is fairly slow so it is most strongly sensed after the cottage cheese is expectorated. The intensity builds and it is hard to rinse away and refresh the tongue. Cause. Cottage cheese that is stored at temperatures at or slightly above 4C for several days will become bitter when psychrotrophic organisms are present. Under those conditions they will grow to large populations and release proteases. Certain weeds consumed by the cow will also impart bitterness to cottage cheese made from the milk. Preparation of Samples for Training. Solutions of 1% quinine sulfate may be added to creamed cottage cheese. Add 2 ml/lb for a slight and 4 for definite.5

Cooked
Description. A sulfurous aroma is detected as the product is smelled and may be sensed soon after the sample is placed in the mouth. The flavor is usually considered to be pleasant. The detection of a cooked egg white smell is characteristic of this defect.

Table 3.5 THE ASDA SCORING GUIDE FOR SENSORY DEFECTS OF CREAMED COTTAGE CHEESE
Intensity of Defect Slight Flavor criticisms8 Acid (high) Bitter Diacetyl Feed Fermented/fruity Flat Foreign High salt Lacks fine flavor Lacks freshness Malty Metallic Musty Oxidized Rancid Unclean Yeasty Body and texture Firm/rubbery Gelatinous Mealy/grainy Overstabilized Pasty Weak/soft Appearance and color Free cream Free whey Lacks cream Matted Shattered curd Slimy Definite Pronounced

9 7 9 9 5 9 7 9 9 9 6 5 5 5 4 6 4 4 3 4 4 3 4 4 4 4 4 4 2

7 5 7 7 3 8 4 8 7 5 4 3 3 3 2 3 1 2 2 2 3 2 3 2 2 3 2 3 0b

5 1 6 5 1 7 1 7 6 1 1 1 1 1 1 1 1 1 1 1 2 1 2 1 1 2 1 2 0

Source: American Dairy Science Association, 1990. a "No criticisms" is assigned a score of 10 for flavor and 5 for body and appearance. Normal range is 1-10 for flavor and 1-5 for body and appearance for salable product. b An assigned score of 0 (zero) is indicative of unsalable product.

Cause. This flavor can originate from high heat treatment of the skim milk before cottage cheese is made for the creaming mixture that is added to the curd. Preparation of Samples for Training. Wash the cream from cottage cheese curd and replace it with half and half that has been heated sufficiently to 8O0C. Salt curd and cream to taste.

Date:

CONTEST COTTAGE CHEESE SCORE CARD A.D.S.A. Contestant No: SAMPLENO. 1 2 3 4 5 6 Criticisms 10 Contestant Score Score Criticism Acid (high) Bitter Diacetyl Feed Fermented/fruity Flat Foreign High Salt Lacks fine flavor Lacks freshness Malty Metallic Musty Oxidized Rancid Unclean Yeasty Contestant score Score Grade Grade

Flavor

No criticism 10

Normal range 1-10

Body and texture 5

No criticism 5 Normal range 1-5 Appearance and color 5

Criticism Firm/rubbery Gelatinous Mealy/grainy Pasty Weak/soft Contestant score Score Grade

No criticism 5 Normal range 1-5

Criticism Free cream Free whey Lacks cream Matted Shattered curd Slimy Surface discolored Translucent Unnatural color Allowed perfect in contest Total score of each sample Total grade per sample
Final grade Rank

Package Score

Source: American Dairy Science Association (1987)

Figure 3.15 The ADSA contest score card for the sensory evaluation of cottage cheese. (Reproduced from ref. 5, with permission of the ADSA, Champaign, IL.)

M A R K I N GI N S T R U C T I O N S V MN O :T M N C I l <S*tVf P R O P E R M IP R O P E R M A R K M A R K S E R A S EC H A N G E SC L E A N L YA N O C O M P L E T E L Y D ON O TM A K EA N YS T R A YM A R K S C R I T I C I S M S F L A V O R
COOKED NO CRITICISM 10 RAT NORMAL RANGE 1-10 LACKS WN6 ftAVO MAtTY MUTY RANCID YEASTY HtGHACtD FEtO

A U P R C O N T E S T A N TN O D COTTAGE CHEESE

N C ST n r a O p c t i* M P 3 0 7 3 6 3 S 3 2 1A 2 4 0 0 S A M P L EN U M B E R

BODY A N D TEXTURE NO CRITICISM 5


OVERSTABIUZED

OELATWOUS

KWJUgcfoTSl NORMAL RANGE 1-5 APPEARANCE A N D COLOR NO CRITICISM 5 MATTfO FREE WHEY

NORMAL RANGE

1-5

Figure 3.16 Collegiate contest cottage cheese score card. (Reproduced from ref. 5, with permission of the ADSA, Champaign, EL.)

Diacetyl
Description. Diacetyl is a pleasant and desirable ' 'buttery" flavor in cottage cheese and cultured products. This description is for situations where the flavor overpowers acidity and other flavor and aroma notes. Cause. This results when Lactococcus lactis ssp. lactis var. diacetylactis or Leuconostoc sp. has grown better or faster than the lactic culture so that excessive flavor and aroma components have been produced. Preparation of Samples for Training. Slight or definite cottage cheese can be simulated by adding 0.1 or 0.2 ml of food grade diacetyl to 400 g of creamed cottage cheese.5 A stock solution of diacetyl in milk can be made to make measurement easier.

Feed
Description. Feed flavor in cottage cheese is a result of feed flavor in the milk from which it is made or the milk from which the creaming solution was made. A "feed" flavor is aromatic and sometimes pleasant. After the cottage cheese is expectorated a mild aftertaste of "cleanliness" can be present that disappears rather quickly, leaving the mouth free of off-flavors. Feed flavor varies with the type of feed consumed. The odor is characteristic of the feed.5 Cause. High-volume roughage feed within 3 h of milking impart aromas to the milk. Silage, some hays, and brewery waste are particularly notable for this. A change of feed from dry hay to fresh green pasture often initiates a strong feed flavor in the milk. Almost all feed flavors disappear if 3 h is allowed to pass between consumption and milking.5 Preparation of Samples for Training. Half and half can be treated to have a feed flavor as described in the section on milk and cream. The treated cream can be added to creamed cottage cheese or to washed curd. A little salt may be needed to give a typical salt flavor level.1

Fermented/Fruity
Description. A "whiff" of a freshly opened package of cottage cheese with this defect will be suggestive of pineapple, apples, bananas, or strawberries. The taste will confirm those qualities but coming on late may be an unpleasant, lingering aftertaste. Cause. Some psychrotrophic bacteria produce these characteristic aromatic compounds. The package will be near its sell-by date or will have been stored at slightly elevated temperatures. Preparation of Samples for Training. Addition of 1 Vi tsp of banana or pineapple yogurt to 400 g of cottage cheese will simulate fermented/fruity flavor. Addition of 1 to 11A ml of 1% aqueous solution of food grade ethyl hexanoate to 400 g of cheese will give slight to definite levels of this flavor.65

Hat Description. The absence of characteristic flavor and aroma is called "flat." The absence of culture or diacetyl flavor and absence of salt gives that impression. Cause. Insufficient flavor producing culture in the cream and insufficient salt will give this flavor. The early stages of oxidized flavor may tend to give a flat taste and aroma. The delayed flavor perception may give the impression of a metallic flavor.5 Preparation of Samples for Training. The flat flavor may be simulated by washing the curd and replacing the cream with half and half. Salt may be added but less than enough to give the optimum saltiness.

Foreign (Chemical, Medicinal)


Description. A foreign off-flavor is one that is entirely unlike any off-flavor that might be anticipated to develop in cottage cheese. Cause. Most of these atypical flavors are caused by cleaning compounds, chlorine, iodine, or phenol. Any one of many compounds that are inadvertently added to product or whose fumes are absorbed by product may be responsible for the flavor. Preparation of Samples for Training. Foreign flavor caused by sanitizer can be produced by adding 1A ml of a 5% sodium hyperchloride solution to 300 ml of good half and half.5 That cream could be lightly salted to taste and used to cream good quality washed cottage cheese curd. In the same manner, traces of other nontoxic chemical cleaners and sanitizers could be used to taint cream which in turn will taint cottage cheese.

High Acid (Sour)


Description. Acid is a normal component of good cottage cheese flavor. It is a clean and sharp sensation that generally cleans up well and leaves no aftertaste. When it gets high enough that the flavor predominates over other natural components of the flavor and covers the desirable flavors, it should be criticized. Cause. The culture organism can slowly work on remaining lactose in the curd until it is gone. High moisture facilitates this acid production. If the curd is insufficiently washed, then lactose will be present in the curd and this defect will develop. Preparation of Samples for Training. For a slight high acid defect, add 15 to 20 ml of cultured skim milk to 385 g of cottage cheese. For a definite acid flavor intensity use 30 to 40 ml of cultured.skim milk in 365 g of creamed cheese.

High Salt
Description. The "high salt" flavor is characterized by a sharp, biting sensation. The reaction and adaptation time are both short. The initial piercing sensation subsides and it is replaced by copious flow of saliva.

Cause. High salt is a formulation error resulting from addition of excessive salt to the creaming mixture or the curd or both. The proper amount is 0.6% to 1.0%. Preparation of Samples for Training. Salty cottage cheese can be made by adding an additional Vi to 1% additional salt to properly salted, good quality creamed cottage cheese. Stir and allow to dissolve.

Lacks Fine Flavor (Acetaldehyde)


Description. This is a "green" or "green apple" or plain yogurt flavor atypical of the mild diacetyl or buttery flavor that is characteristic of cottage cheese. Cause. An improper lactic culture has been used to make the cream dressing or a contaminating lactic culture has grown up that produced a lot of acetaldehyde. Yogurt has a characteristic acetaldehyde flavor. Preparation of Samples for Training. Cottage cheese that has this defect can be made by adding a tsp. of plain yogurt to 400 g of cottage cheese.5

Lacks Freshness (Stale or Storage)


Description. This is a group of closely related off-flavors. All are related to the age of the product. Stale is a more obvious, more intense version of lacks freshness. Lacks freshness just lacks the refreshing fresh flavor of recently made product. Storage flavor is the changing of character due to absorption of flavors from the products and materials stored around it. Cause. Lacks freshness and stale defects are caused by the occurrence of microbiological and chemical changes resulting in deterioration of typical flavor. This is expected to occur to even the best product near the end of its 2- to 3-week shelf life. Occasionally this will begin prematurely due to contamination of the product or storage at elevated temperatures. The storage flavor is sometimes called absorbed flavor. It is due to absorbed flavors of products that are stored in the same refrigeration unit. Preparation of Samples for Training. Low-fat cottage cheese may demonstrate this defect. One could also obtain cottage cheese samples that are near their pull date and screen them to select samples that demonstrate the lacks freshness or stale defect. Malty Description. This flavor resembles malted milk or the flavor of Grape Nuts cereal. A sourness may accompany the malty flavor. The malting process of steeping barley and allowing it to start to sprout causes this flavor to develop. It generally has a quick reaction time and the aftertaste is not prolonged. Cause. The bacteria Streptococcus lactis spp. lactis var. maltigenes produces that defect in milk if it is able to grow.

Preparation of Samples for Training. Half and half or milk may be soaked in Grape Nuts for 30 min and then filtered through a paper towel. The filtrate is added to and stirred into good flavored cottage cheese. Sufficient is added to give the level of intensity desired.66 Musty Description. This is a serious but seldom encountered defect that resembles the aroma of a damp, poorly ventilated cellar. Cause. This defect is due to the growth of a variety of microbial contaminants including molds. The curd may have become contaminated with Pseudomonas taetrolens which are psychrotrophic bacteria. Poor plant sanitation is responsible for allowing them into the product and marginal cooling temperatures are responsible for allowing their outgrowth.5 Training. No method is suggested in the literature for preparation of samples. Exemplary samples may be found among survey samples that have been held beyond their pull date or held at slightly elevated temperatures.

Oxidized, Metallic Oxidized, Sunlight Oxidized


Description. These three flavors are grouped together because they are thought to be chemically related.' 'Metallic'' has a slight astringent character and a ' 'rusty nail'' like taste. "Oxidized" has a flavor similar to wet cardboard or paper. "Sunlight" flavor is described as burnt, burnt protein, burnt feathers, cabbagey, and medicinal. Cause. AU three flavors are thought to be due to milk fat autoxidation in the cream used to produce the cottage cheese cream. It can be catalyzed by traces of copper or corrodible metal. "Sunlight" flavor also is caused by exposure to sunlight or fluorescent lights which causes an amino acid degradation involving riboflavin. Methional produced in the reaction may cause the flavor.5 Preparation of samples. For "metallic" flavor add 3 to 3 ml of 1% aqueous CuSO4-5H2O solution to 5 ml of milk or half and half which in turn is added to 400 g of creamed cottage cheese. Allow 24 h for the flavor to develop. A sunlight oxidized flavor can be developed by exposing milk or half and half to bright fluorescent light for 6 h and then adding that to the creamed cottage cheese.

Rancid
Description. "Rancidity" in cottage cheese as in milk may be described as an astringent, puckery feeling at the base of the tongue and throat. A bitter and soapy aftertaste may be associated with it. There is a slow response time to this flavor. After expectoration it is difficult to clean the flavor out of the mouth. Cause. This flavor is due to enzymatic action of lipase on milk fat. Ester bonds are broken, leaving free fatty acids and mono- and diglycerides. The shorter free fatty acids, particularly butyric, are flavorful. Mid-length fatty acids taste soapy. Raw

milk contains lipase and some psychrotrophs produce lipase. Conditions are ideal for lipolytic action when these enzymes are present and when the fat is disturbed and new interface is produced. Inadvertent mixing of raw and freshly homogenized milk is such a case. Homogenized milk in which psychrotrophic organisms have grown to great numbers is another. If the cottage cheese cream has been subjected to those conditions, rancidity will probably occur.5 Preparation of Samples for Training. Add rancid milk, prepared according to the directions in the section on milk, to the creamed cottage cheese mixture. Be sure the rancid milk has been laboratory pasteurized. Try 10 to 15 ml in 400 g of creamed cottage cheese.

Unclean
Description. The terms "dirty" and "dirty sock" have been used to describe this flavor. The flavor of limburger cheese has been used to simulate it. An "unclean" flavor is readily noted when the sample enters the mouth. The flavor and odor are offensive suggesting extreme staleness, mustiness, putrid, or spoiled. The flavor fails to clean up after the cottage cheese is expectorated. Cause. This flavor develops in cottage cheese when psychrotrophic bacteria are allowed to grow to high numbers in milk and particularly when held at temperatures above 7.2C. The presence of the psychrotrophs is usually due to poor on-farm sanitation and the high numbers are generally due to poor bulk tank cooling.5 Preparation of Training Samples. To obtain product with this defect, screen several old cottage cheese samples for unclean flavor. If none are unclean, subject them to 4 to 12 h at room temperature, then rescreen the samples looking for this defect.

Yeasty (Vinegarlike)
Description. The "yeasty" and "earthy" flavor and aroma reminiscent of rising bread dough is a good demonstration of the "yeasty" flavor. It is often associated with an acetic acid or "vinegar" flavor. Cause. Growth of yeast is usually responsible for this flavor but it may be due to bacterial fermentation. Certain kinds of psychrotrophic bacteria can be responsible for this objectionable off-flavor. It is due to poor sanitation and lack of temperature control.67-68 Preparation of Samples for Training. High-quality half and half could be purposely inoculated with yeast and sugar and allowed to ferment for a few hours at room temperature until the flavor begins to develop. It could be lightly salted and used to cream cottage cheese to give it this defect. This flavor and aroma can be learned by smelling and tasting yeast leavened bread dough as it is rising.

3.4.2.3 Body and Texture Defects


Description and probable causes for body and texture defects are listed here. Preparation of samples to simulate the defects is extremely difficult. It is recommended

that a number of commercial samples be surveyed. There will likely be product readily available that exemplifies the body and texture defects.

Rrm/Rubbery
Description. Curd that is too firm will be overly resistant to deformation between the roof of the mouth and the tongue. Resistance to mastication will also be noticed. Cause. Firm curd may be due to use of too much rennet, curd cooking temperatures that are too high, cooking the curd for too long, or pH too high at the time of setting or cutting too soon.

Gelatinous
Description. Sticky or ' 'jellylike'' translucent curd is indicative of this defect. The curd may resemble tapioca pudding. A bitter flavor may also be present. Cause. This defect is usually due to growth of psychrotrophic bacteria in the cottage cheese. The product is often unpalatable and unsalable. An attempt to make a cottage cheese product with rennet without sufficient acid will result in gelatinous, translucent curd.5

Mealy/Grainy
Description. This very common defect can be detected by masticating the curd and then pressing the curd against the roof of the mouth with the tongue and noticing the presence of gritty or cornmeallike sensation. Another way to detect this defect is to knead washed curd and smear it between the fingers. The kneaded curd should be silky smooth. A rough gritty mass is indicative of this defect.5 Cause. This defect is caused by overdeveloping the acid during curd formation or too low a moisture level in the curd. It can also be caused by nonuniform cutting of the curd, uneven heating during cooking, cooking too fast, inadequate agitation during cooking, or allowing portions of the curd to come in contact with hot surfaces during cooking.5

Overstabilized (Slick)
Description. coating. Individual curd particles will be surrounded by a thick, pasty, slick

Cause. The use of too much stabilizer in the cottage cheese dressing is the cause of this defect. Processors will often thicken the dressing excessively in an attempt to minimize free cream or free whey. Reduction in the amount of stabilizer in the cream will usually overcome this defect.

Pasty
Description. Other descriptors for this defect are sticky and doughy. This is closely associated with and considered to be the advanced stages of soft and weak curd (discussed next). The curd particles tend to stick together in clumps.5 Cause. See the causes of weak/soft curd(next).

Weak/Soft (Mushy)
Description. Weak and soft curd is high in moisture. The curd offers too little resistance to deformation when pressed between the tongue and the roof of the mouth. Rather than the desired meaty texture, the curd has almost no body and reduces to a liquid on minimal mastication. Cause. Conditions that encourage excessive water to be retained in the curd thereby giving a weak curd are excessively high pasteurization temperatures of the skim milk which denatures the whey protein and predisposes them to bind more water, cutting the curd too late after the curd is excessively firm and the pH is too low thereby hindering expulsion of water during cooking, curd cooking temperatures too low, and overdressing the curd.5

3.4.2.4 Appearance and Color Defects


Free Cream
Description. When creamed cottage cheese is placed on a plate in a mound, as with an ice cream scoop, the cream should cling to the curd with minimal cream running free at the base of the mound of curd. Excessive cream flowing out on the plate is evidence of this defect. Cause. Conditions that can cause the free cream defect are excessively firm curd that does not absorb cream, insufficient washing of the curd after cooking, cutting the curd while the pH is too high thereby producing firm nonadsorbing curd, and too rapid a temperature rise during cooking causing the surface of the curd to resist the adsorption of cream.5 Preparation of Samples for Training. Add half and half or whole milk to creamed cottage cheese until the amount and viscosity of the cream is sufficient to give a zone of free cream around a dollop of cottage cheese.

Free Whey
Description. No clear solution should be evident at the edges of the cream at the base of a dollop of cottage cheese. Presence of a clear solution is evidence of this defect and that destabilization of the cream has occurred. Cause. Free whey can be caused by the following conditions: undercooked curd that is retaining excessive amounts of whey, insufficient washing of the curd such

that excessive whey is still present in the curd, and cutting the curd at too high a pH, making it unable to adsorb liquid.5 Preparation of Samples for Training. Add sufficient whey from wheyed off buttermilk, yogurt, or even tap water to cottage cheese so that clear fluid appears at the edge of the cream when a dollop of the product is placed on a plate.

Lacks Cream
Description. When insufficient cottage cheese cream is added to the curd, it will appear dry and no cream at all will run to the bottom of a mound or scoop of cottage cheese. Cause. This problem is generally caused simply by under-creaming. It is often done purposely for food service customers to avoid any free cream and to facilitate a mound of cottage cheese that does not flow or flatten. Preparation of Samples for Training. This defect may be staged by obtaining some uncreamed curd and blending it with ideal product to give the appropriate appearance. Dry curd may be obtained by rinsing cottage cheese curd free of cream with warm water and then draining off the water.

Matted
Description. Ideally the curd particles in cottage cheese should be individual. Curd exhibiting the matted defect will have curds tightly stuck together into large clumps. Cause. Conditions that will cause matted curd are cutting of the curd at too high a pH so that the curd will be sticky during the initial stages of cooking, insufficient agitation during the first stages of cooking so that curd particles are allowed to matt, or cooking the curd too rapidly so that high moisture curd will become sticky and tend to clump.5 Preparation of Samples for Training. This defect is so common that finding some matted curd will be quite easy. Matted curd can be ' 'planted'' on a dollop of creamed cottage cheese to demonstrate this defect.

Shattered Curd
Description. Ideal cottage cheese will have curd particles of uniform size with no fine particles or "dust." These curd particles can be observed on the creamed surface of the curd. Usually this defect is not called unless at least four or more curd dust particles are present on each curd particle. They can also be seen on the back of a spoon used to sample cottage cheese.5 Cause. Shattering of curd to cause these fine particles can be caused by the following conditions: excessive heat treatment of skim milk causing the curd to be fragile, cutting at too low a pH when the curd mass has set to some extent making it difficult to cut without shattering, low-solids milk producing fragile curd, overly

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severe agitation, excessive amounts of coagulator use, and rough handling of curd during draining, creaming, pumping, and packaging. Preparation of Samples for Training. This defect is also common. Examples of this problem will probably be easy to find in commercial cottage cheese. The more difficult task will be to find a sample that is free of this defect. Such a sample could be made by washing curd free of cream on a sieve that allows the passage of curd dust, then recreaming with half and half.

3.4.3 Butter

3.4.3.1 Introduction
Butter is made by agitating chilled cream to first form granules and then a butter mass. The butter mass is drained of serum (buttermilk) and it may be washed. The mass is worked to reduce the size of the water droplets, and to disperse and dissolve salt. Butter may be made in a churn but most is made in continuous butter makers that take in a steady stream of cream, perform all the operations, and produce a steady stream of butter and buttermilk. As defined in the Code of Federal Regulation, butter contains no less than 80% milk fat and is made from pasteurized cream.20 The majority of the butter marketed in the United States is sweet cream butter made from cream with a titratable acidity of 0.20% or less. If acid has developed in the cream to higher acidities, then the product is sour cream butter. Cultured butter is made by adding lactic cultures that produce aromatic butter flavored compounds to the cream just before churning. Salt is generally added to butter. Lightly salted butter contains about 1.5% salt.5 A number of spreads emulate butter. Margarine, butter-margarine blends, and reduced fat spreads are currently available. Their sensory properties vary widely and, although their defects are not treated here, they should be free of off-flavors and perform as intended. Butter is the standard for these other spreads and the list of possible defects that apply to butter can occur in them. Butter is sampled with a curved bladed double-edged tool known as a trier that is inserted into the block of butter, rotated 180, and removed. It extracts a cylinder of butter for examination. The butter on the trier is passed slowly under the nose while inhaling. The aroma is noted. The color uniformity is next evaluated. The judge then examines the body and texture by pressing the ball of the thumb against the sides of the butter cylinder until it breaks. The smoothness of the break is noted as is the presence or absence of beads of water and the clarity of any water. The judge then breaks off a piece of butter from the end of the plug, usually with a spatula, and places it into the mouth. The sample is chewed while it melts in the mouth. As it is melting, the presence of grit or undissolved salt is noted between the teeth by biting down. It may be observed between the tongue and the roof of the mouth. The melted sample is moved around in the mouth while noting flavors and aromas. The sample is then expectorated. The judge then notices if any aftertaste or off flavor persists. The trier is then cleaned with a soft cloth or paper towel.5

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severe agitation, excessive amounts of coagulator use, and rough handling of curd during draining, creaming, pumping, and packaging. Preparation of Samples for Training. This defect is also common. Examples of this problem will probably be easy to find in commercial cottage cheese. The more difficult task will be to find a sample that is free of this defect. Such a sample could be made by washing curd free of cream on a sieve that allows the passage of curd dust, then recreaming with half and half.

3.4.3 Butter

3.4.3.1 Introduction
Butter is made by agitating chilled cream to first form granules and then a butter mass. The butter mass is drained of serum (buttermilk) and it may be washed. The mass is worked to reduce the size of the water droplets, and to disperse and dissolve salt. Butter may be made in a churn but most is made in continuous butter makers that take in a steady stream of cream, perform all the operations, and produce a steady stream of butter and buttermilk. As defined in the Code of Federal Regulation, butter contains no less than 80% milk fat and is made from pasteurized cream.20 The majority of the butter marketed in the United States is sweet cream butter made from cream with a titratable acidity of 0.20% or less. If acid has developed in the cream to higher acidities, then the product is sour cream butter. Cultured butter is made by adding lactic cultures that produce aromatic butter flavored compounds to the cream just before churning. Salt is generally added to butter. Lightly salted butter contains about 1.5% salt.5 A number of spreads emulate butter. Margarine, butter-margarine blends, and reduced fat spreads are currently available. Their sensory properties vary widely and, although their defects are not treated here, they should be free of off-flavors and perform as intended. Butter is the standard for these other spreads and the list of possible defects that apply to butter can occur in them. Butter is sampled with a curved bladed double-edged tool known as a trier that is inserted into the block of butter, rotated 180, and removed. It extracts a cylinder of butter for examination. The butter on the trier is passed slowly under the nose while inhaling. The aroma is noted. The color uniformity is next evaluated. The judge then examines the body and texture by pressing the ball of the thumb against the sides of the butter cylinder until it breaks. The smoothness of the break is noted as is the presence or absence of beads of water and the clarity of any water. The judge then breaks off a piece of butter from the end of the plug, usually with a spatula, and places it into the mouth. The sample is chewed while it melts in the mouth. As it is melting, the presence of grit or undissolved salt is noted between the teeth by biting down. It may be observed between the tongue and the roof of the mouth. The melted sample is moved around in the mouth while noting flavors and aromas. The sample is then expectorated. The judge then notices if any aftertaste or off flavor persists. The trier is then cleaned with a soft cloth or paper towel.5

Table 3.6

U.S. GRADE CLASSIFICATION OF BUTTER ACCORDING TO FLAVOR CHARACTERISTICS5


Grade Classification by Flavor6

Identified Flavors by Grading3 Feed Cooked Acid Aged Bitter Coarse Flat Smothered Storage Malty Musty Neutralizer Scorched Utensil Weed Whey Old cream

AA Sc D

A D S S S S S S S

B P D D D

D D S S S S S S S D

Source: Code of Federal Regulations (1987). 3 When more than one flavor is discernible the lowest classification b establishes the grade. U.S. Butter Grade as determined by offic cial USDA grading standards. Defect intensity: S = slight; D = definite; P = pronounced.

The USDA grades much of the butter produced in the United States. Before 1977, butter scoring was on a 100-point scale. Now only the letter designation is used. The point system is still occasionally referenced. Grade AA butter scored 93 or more points, A grade required 92 points, and B grade required a minimum of 90 points. Table 3.6 shows flavor grades that are assigned based on flavors present and their intensity. The only flavor defects allowed in Grade AA butter are slight feed and cooked. Any other flavors result in downgrading. Table 3.7 shows how many derating points are assigned for body, color, and salt defects. Derating up to 1A total points does not reduce the grade below the assigned flavor grade. Each 1A derated point beyond that reduces the butter one additional grade.20 The American Dairy Science Association uses a 25-point system with 10 points for flavor, 5 for body and texture, 5 for color and appearance, 3 for salt, and 2 for the package. In collegiate contests only the flavor is judged. The ADSA Flavor scoring guide is shown in Table 3.8 and a body and texture guide is shown in Table 3.9. A sensory scorecard using a 25-point system is shown in Figure 3.17 and the Collegiate Contest Butter Score Card is shown in Figure 3.18.

Table 3.7

CHARACTERISTICS AND DISRATINGS FOR BODY, COLOR, AND SALT FOR U.S. BUTTER GRADES5
Disratingsb

Butter Characteristics Body Crumbly Gummy Leaky Mealy or grainy Short Weak Sticky Ragged boring Color Wavy Mottled Streaked Color specks Salt Sharp Gritty
a b

0.5 0.5 0.5 0.5 0.5 0.5 0.5 1 0.5 1 1 1 0.5 1

1 1 1 1 1 1 1 2 1 2 2 2 1 2

U.S. Butter Grade as determined by official USDA grading standards. Defense intensity: S = slight; D = definite; P = pronounced.

3.4.3.2 Flavor Defects


Acid
Description. Acid and sour are synonymous and refer to a sharp taste on the tip of the tongue as well as an associated "sour" aroma. The sour taste is quickly detected as the sample is placed in the mouth. The acid flavor cleans up quickly after the sample is expectorated, leaving no aftertaste. Cause. Acid tasting butter usually is a result of churning overripe or acid cream. It may be caused by leaving too much ripened buttermilk in the butter after churning. Preparation of Samples for Training. Acid butter or butter with any of the defects may be obtained by surveying product on the market or by asking processors to be on the watch for exemplary product. It can be made by culturing cream with Streptococcus lactis ssp. lactis organisms until it is overly ripe and then churning the cream. Acid butter can be kneaded together with good butter to obtain product with the desired level of acid.

Table 3.8

THE ADSA SCORING GUIDE FOR SENSORY DEFECT OF BUTTER (SUGGESTED FLAVOR SCORES FOR DESIGNATED DEFECT INTENSITIES)5
Intensity of Defect

flavor Criticisms3 Acid (sour) Bitter Cheesy Coarse Feed Flat Garlic/onion Metallic Musty Neutralizer Old cream Oxidized Rancid Scorched Storage Unclean (utensil) Whey Yeasty

Slight 6 6 3 8 9 9 3 4 5 5 6 4 4 7 6 5 6 4

Definite 5 5 2 7 8 8 2 3 4 4 5 3 2 5 5 4 5 3

Pronounced 4 4 1 6 6 7 1 1 2 3 4 2 1 3 4 3 3 2

Source: American Dairy Association, 1990 a "No criticisms" is assigned a score of 10. Normal range is 1-10 for salable product.

Bitter
Description. Bitterness is recognized by the sense of taste alone. Once the butter sample has melted in the mouth, it can be best detected when the sample is moved to the back center of the tongue where the taste buds are most sensitive to bitter. Cause. Bitterness in butter may be caused by action of certain microorganisms or enzymes, consumption of certain feeds or weeds by the cow, impurities in the salt added to the butter, and inappropriate use of some neutralizes.5 Preparation of Samples for Training. Quinine sulfate has strong, clean, bitter flavor in very dilute concentrations. Addition of 1 to 2 ml or 1% quinine sulfate solution to a pint of cream before churning will result in bitter butter. Alternatively, quinine sulfate solution can be kneaded into butter. The level of bitterness can be adjusted by kneading bitter and good butter together in appropriate ratios.

Cheesy
Description. Cheesy butter resembles cheddar cheese in flavor and aroma. The flavor is noticed immediately after the sample is placed in the mouth. It also lingers after the sample is expectorated. Clean up of the flavor is slow.

Table 3.9 A SUGGESTED SCORING GUIDE FOR BODY AND TEXTURE AND COLOR AND APPEARANCE IN BUTTER
Intensity Slight5 Body and texture defect* Crumbly Gummy Leaky Mealy or grainy Ragged boring Short Sticky Weak Color and appearance defect3 Color specks Foreign material Mold Mottled Streaky Surface faded/high Unnatural Wavy Moderate Definite Strong Pronouncedc

4 4 4 4 4 4 4 4

3 3 3 3 3 3 3 3

2 2 2 2 2 2 2 2

1 1 1 1 1 1 1 1

oe
0

3 0 0 3 3 4 0 4

2 0 0 2 2 3 0 3

1 0 0 1 1 2 0 2

0 0 0 0 0 1 0 1

0 0 0 0 0 0 0 0

Reproduced with permission from ref. 5. a b "No criticism" is assigned a numerical score o f ' 5 . " Normal range is 1 to 5 for a salable product. Highest c assignable score for a defect of slight intensity. Highest assignable score for a defect of pronounced intensity. d However, a sample may be assigned a score of " 0 " (zero) (unsalable product). a dash () indicates that the c defect is unlikely to be present at this intensity level. When a product is determined to be unsalable for a given sensory defect, a " 0 " (zero) numerical score is assigned to the sample for the quality attribute(s) in question.

Cause. This flavor results when soured cream is held refrigerated and proteolytic organisms are allowed to grow. When this cream is churned, cheesy butter results. Tendency to develop this flavor is related to the curd content of the butter. Washing the butter well as it is being churned is a precaution against cheesy flavor.5 Preparation of Samples for Training. Cheesy flavored butter can be made by adding cheddar cheese flavor to cream prior to churning or by kneading cheese flavor solution into softened butter. It should be tried with a variety of cheese flavors to ensure that one will have the desired flavor.

Coarse
Description. Coarse butter is one that lacks the pleasing, delicate flavor that is typical of good quality fresh butter. It is really employed when the butter lacks the typical flavor but no other criticism is appropriate. It can be considered the early stages of the "old cream" or "storage" defects but no particular defect has developed, only a flavor that is off ideal.

Cause. Coarse butter generally results when cream that is a little old and perhaps slightly acidic is churned. The defects are not strong enough to be criticized as old cream, acid, or storage. Preparation of Samples for Training. Blending a little slightly aged cream with high quality cream and then churning may result in product that will be criticized as coarse. This defect is quite prevalent among the products on the market. It should not be hard to find product that has this defect. Feed Description. Feed flavors can usually be detected using the sense of smell, then verified by tasting. They are flavors reminiscent of the feeds eaten by the cow. Cause. Feed flavor in product is a result of consumption of feeds within 3 h of milking. Some feeds are particularly potent. Fresh clover and alfalfa are potent in this respect. The spring of the year when the cow goes on pasture is a vulnerable time. Preparation of Samples for Training. An alfalfa flavor can be simulated by adding and holding 2 to 3 g of alfalfa hay to 100 ml of fresh pasteurized and homogenized milk for 20 min. The milk is then strained through a cheesecloth or a paper towel and used as a stock solution. To 575 ml of fresh pasteurized cream add 20 to 35 ml of this stock milk solution. Grass or corn silage can be used to prepare feed flavored milks in the same manner. The cream is then churned into butter which will have a feed flavor corresponding to the material used.5 Flat Description. When the full characteristic buttery flavor is lacking the flavor is considered to be flat. It is noticed very soon after the sample has been placed in the mouth and as the sample melts and is moved around in the mouth. It is not to be confused with a low or unsalted flavor. It is possible for the salt to be absent but the diacetyl and volatile acid flavor notes to be sufficiently present. Lack of salt does suppress the butter flavor though. Cause. Lack of diacetyl and volatile acids are the cause of flat butter. Excessive washing of butter granules can result in flat tasting butter. Consumption of certain feeds has been blamed for milk fat with a low level of volatile flavors. A slight cooked flavor to the cream or culturing the cream are effective ways to give butter some flavor. Preparation of Samples for Training. Flat butter can be prepared by kneading good quality butter in cool clean water to remove some of the water-soluble flavor components.

Garlic/Onion
Description. The distinctive odors and flavors characteristic of garlic or onion are the trademarks of this defect. Both are quite odorous and similar in butter that has

BUTTER SCORE CARD


Product: Date: SAMPLE NO. 1 Flavor 10 No criticism 10 Criticism Acid/sour Bitter Cheesy Coarse Cooked Feed Fishy Flat Foreign Garlic/onion High salt Malty Metallic Musty Neutralizer Old cream Oxidized Rancid Storage (aged) Tallowy Unclean (utensil) Weedy Whey Yeasty Score 2 3 4 5 6 7 8

Unsalable 0 Normal range 1-10

Body and texture 5 No criticism 5 Unsalable 0 Normal range 1-5 Color and appearance 5 No criticism 5 Unsalable 0 Normal range 1-5 Salt 3 No criticism 3 Unsalable 0 Crumbly Greasy Gummy Leaky Mealy/grainy Ragged boring Salvy Sticky Weak

Score

Score Color specks Foreign material Mold discoloration Mottled Streaky Surface faded/high color Unnatural Wavy

Score Excessive (too high) Gritty Uneven distribution

Figure 3.17 Suggested score card for the sensory evaluation of butter. Used with permission.5

BUTTER SCORE CARD (cont.)


Product:
Package and finish 2 No criticism 2 Unsalable O Exposed product Package and liner Careless Damaged Dirty/Unsanitary Not protective Printing defective Unattractive Rough Finish Total score of Score each sample Fat content (%) Weight (Ib) Proteolytic count (per g) Yeast and mold count (per g) Coliform count (per g) Free fatty acid 7-day (21C) keeping quality

Date:
SAMPLENO. Score

Total 25 Laboratory parameters3

Signature of evaluators:
a

The laboratory parameters are not scored; they provide information that helps determine the legal status and company specifications of the product.

Figure 3.17 (Continued)

been warmed to body temperature in the mouth. The aftertaste is persistent and cleanup difficult. Cause. Consumption of onion or garlic by the cow will result in milk with this defect. Butter made from the resulting cream will likewise have the defect. Preparation of Samples for Training. Garlic and onion flavored butter is easy to prepare by kneading a little onion or/and garlic powder or salt into butter.

High Salt/Briny
Description. The acceptable range of saltiness in butter is broad. Calling this defect is appropriate when the salt level is "beyond the range of acceptability." Government graders have a category for salt and call the defect "sharp salt." If salt is the only flavor noticed on tasting a butter sample, then the defect may be called.5 Cause. The problem may be too much salt or poor distribution of the salt. The normal range is extremely broad. Preparation of Samples for Training. Addition of salt above 3 to 4% may be sufficient to produce this defect. The salt should be kneaded into a slightly wanned butter mass until all the salt crystals dissolve.

M A R K I N GI N S T R U C T I O N S U I H O J W N C I L W t V~ M IP R O P E R P R O P E R M A R K M A R K S E R A S EC H A N G E SC L E A N L YA N D C O M P L E T E L Y D ON O TM A K EA N YS T R A YM A R K S C R I T I C I S M S F L A V O R N O mrmm C R T IC IS IM C O A R S E 1 0
F L A T

P R A T F C O N T E S T A N TN O D BUTTER

N C ST r a n s O p c i t* M P 3 0 7 3 S 3 2 3 2 1A 2 4 0 0 S A M P L EN U M B E R

N O R M A L R A N G E 1 1 0

H I G HS A L T M U S T Y O L D C R S A M R A N C I O S T O R A G E W H E Y

B O D YA N D T E X T U R E N O C R T IC IS IM 5 N O R M A L R A N G E 1 5 A P P E A R A N C E A N DC O L O R N O C R T IC IS IM 5 N O R M A L R A N G E 1 5
Figure 3.18 Collegiate contest butter score card. (Reproduced from ref. 5, with permission of the ADSA, Champaign, DL.)

Metallic
Description. This flavor defect, as the name suggests, gives a slight puckery or astringent feeling to the mouth interior similar to putting a nail in the mouth and allowing the saliva to flow and contact it. The flavor is detected right after the butter is placed in the mouth. Strength of the flavor becomes more intense as the sample warms in the mouth. A bitter aftertaste may develop after the sample is expectorated. Cause. This defect is caused by allowing cream to be in direct contact with copper or corrodible metal. Contact need not be for an extended time. One corrodible metal fitting in a system through which the cream is pumped may be sufficient to give the cream and the resulting butter the flavor. Rusty cream cans or cans from which the tin has been abraded can cause the defect. Preparation of Samples for Training. Metallic butter can be made by developing metallic cream and churning it to butter. Clean pennies may be soaked in cream until the flavor intensity reaches the desired level, or 1 ml of a 1% stock solution of CuSO4 can be added to 600 ml of cream. The cream is held refrigerated for 1 to 2 days for the flavor to develop. The cream is then churned into butter.5

Musty
Description. The musty off-flavor of butter resembles the odor of potatoes, a swamp, or a poorly ventilated cellar. Hay that is put up a little damp will develop this smell. It becomes evident after the sample has warmed in the mouth a while and after the sample has been expectorated. The flavor lingers and is difficult to clean out of the mouth. Cause. Morgan attributed musty butter to the growth of Pseudomonas taetrolens and the production of 2-methoxy-3-alkylpyrazine by it. Other causes are storing cream in a poorly ventilated musty environment; consumption of musty feed, slough grass, or stagnant water by the cow; or use of poorly cleaned equipment.60 Preparation of Samples for Training. It is not recommended that any of the above media be added to cream or butter to produce this flavor. Screening a large number of samples to find a musty sample is one possibility. The musty smell can be taught by smelling musty feed or an enclosed musty cellar.

Neutralizer
Description. Different neutralizes have slightly different flavors. It is an alkaline, baking soda, or soda cracker flavor. Bitterness is often part of the profile. It is best detected after the sample has melted in the mouth or after the sample has been expectorated and air is inhaled through the mouth. The aftertaste does not easily clean up. Cause. Neutralization of the acid in cream before churning used to be a common practice and is still practiced to some extent. This defect is the result of adding

excessive quantities or highly concentrated solutions of neutralizes to cream made necessary by high levels of acid. The resulting butter will have this defect. Preparation of Samples for Training. Neutralizer flavored butter can be made by acidifying cream to about pH 6.0, directly with lactic acid or by culture growth, then neutralizing the cream to pH 6.8 with a sodium bicarbonate solution or another neutralizer. Butter made from that cream will have the neutralizer flavor.

Old Cream
Description. ' 4OId cream" is a characteristic flavor of cream that has aged and lost its fresh, sweet, clean flavor. Butter with this flavor has a characteristic staleness that is reminiscent of the background smell in a creamery that does not practice the best sanitation. The flavor of such a sample becomes evident after the sample begins to melt in the mouth. It is noticeable after the sample is expectorated and the flavor lingers in the mouth. Cause. The descriptor "old cream" is indicative of the cause. The effect of age may have been accelerated by poorly cleaned equipment or high storage temperatures. Preparation of Samples for Training. Usually samples of butter can be found that have this defect. Samples can be made by allowing cream to age and making butter from the aging cream. The aging can be accelerated by storage at temperatures above 4C.

Oxidized
Description. This defect could be used to describe a whole family of flavor defects that result from the oxidation of lipids in butter or cream. Other related descriptive terms used are: oily, tallowy, painty, fishy, and storage. Generally fishy, tallowy, metallic, and storage are used separately to describe different versions of these flavors. Oily butter is not seen much so the "oxidized" term here is used to describe a cardboardlike flavor that develops as a result of metal-induced oxidation. It is also used to describe a surface taint that develops on exposed surfaces. Because most tasting is done on samples drawn from deep in the block, the latter is not often seen. Cause. This defect develops on the oxidation of butter fat by free radical degradation that results in the production of short-chain aldehydes, ketones, and acids as the fatty acids break down. Preparation of Samples for Training. These samples are difficult to stage. A large number of samples obtained from various sources could be stored in the refrigerator and evaluated periodically. Some of them should develop exemplary oxidized flavor.

Rancid
Description. Flavor notes that are part of the rancid flavor are soapy, bitter, and butyric acid. It is sensed at the back of the tongue and takes 10 to 20 s to reach full

impact. After the sample is expectorated, the aftertaste remains strong for several minutes and requires rinsing and time to clean up. Cause. Rancidity is the result of the enzymatic action of lipase on fat. Ester linkages connecting fatty acids and glycerol are broken, forming fatty free acids and their salts. These free fatty acids are very flavorful and are responsible for the soapy flavor. Raw milk contains active lipase. Psychrotrophic growth also can produce lipase. The fat surface is available for lipolytic action especially if the globules are disrupted as happens in homogenization or in a centrifugal pump. Preparation of Samples for Training. Adding fatty acids with carbon lengths of 4 to 10 in minute amounts to butter and kneading them into the butter mass will produce this effect. Addition of lipase to butter in very small amounts and allowing time for lipolysis will produce rancid butter. Butter made from raw cream will develop a rancid flavor. After the flavor develops, it should be pasteurized and allowed to resolidify before it is tasted.

Scorched
Description. Scorched cream has a caramelized or burnt flavor. A caramelized or toffee flavor is characteristic of this defect. It is an extreme version of the cooked defect. Cause. Scorched flavor is caused by pasteurization of cream at extremely high temperatures or for too long a time in the presence of developed acidity. The problem is aggravated by burn-on that may occur during the heating.5 Preparation of Samples for Training. Scorched butter can be made by developing some ripened cream with a pH of 6.0 to 6.4 and bringing it to a boil on the stove. The cream is chilled for 12 h and churned into butter.

Storage
Description. Butter with this characteristic lacks the desirable sensory characteristics that are present in and associated with fresh butter. No one particular flavor defect is evident but very low levels of several defects are probably involved. This quality is a difficult one to describe. Cause. Even the best butter, as it ages, will lose the delicate notes that are associated with fresh butter. Low levels of several degradative processes are simultaneously at work slowly and over a long period of time. The best butter will develop this flavor more slowly than lower quality butter. It will develop more quickly at higher storage temperatures than at below 4C. Preparation of Samples for Training. A careful screening of commercial butter samples will probably produce samples with this defect. A number of good quality butter samples could be placed in storage and evaluated periodically. Given enough time, one or more of the samples is likely to develop this characteristic.

Tallowy
Description. This is one of the lipid oxidation related defects. As the name suggests, this flavor resembles the taste and odor of oxidized tallow. Generally it is on the surface of the butter and can be detected by smelling the surface of the butter. The tallowy aroma is quite noticeable in the mouth and nose immediately after the sample has been expectorated. Cause. The extensive degree of oxidative degradation of the unsaturated fatty acids in milk fat is responsible for the tallowy off-flavor. It is developed in butter that is held at high storage temperatures in the presence of light. Contamination with traces of copper or iron will catalyze the development of the defect. Because it is a surface effect, it is most often found in retail butter that is sold in pound or quarter pound units where the surface to volume ratio is high.5 Preparation of Samples for Training. Add 1.0 ml of 1% CuSO4 to 600 ml of cream, then churn it to butter. Place that butter under lights and evaluate periodically for intensity of tallowy flavor. Remove when the desired intensity is achieved.

Unclean/Utensil
Description. Other descriptive terms used to describe this flavor are "dishrag" and *'dirty sock." Its odor is most unpleasant and its strength intensifies as it warms up in the mouth. The flavor remains in the mouth after the sample is expectorated. Characteristics described in milk as ' 'barny'' and' 'cowy'' are placed in this category if found in butter. Cause. Psychrotrophic growth in cream may cause this defect in butter made from that cream. Poor handling and sanitation practices are responsible for their entry into the cream. Elevated storage temperatures facilitate their growth.5 Preparation of Samples for Training. Kneading a little limburger cheese into butter can give a character similar to this. The smell of sour dishcloths or dirty socks is a good way to begin to teach the flavor. Purposeful introduction of psychrotrophs from various sources into good quality cream followed by holding at 5 to 100C until flavors develop will produce a variety of defects in cream. Churning of one of these that has developed this characteristic will produce an exemplary sample of butter.

Whey
Description. Whey flavor is somewhat similar to the combined coarse and acid defects of butter. There is a moderate odor and aftertaste that is typical of cheese whey. Cause. Butter made from cream separated from whey will have this defect. It will be more prevalent if it is poorly washed after it is churned. The flavor will be carried into blends of whey cream butter and fresh cream butter. The amount that can be blended into high quality butter without being detected is limited. The condition of

the whey affects the strength and character of the flavor. Cream taken immediately from fresh whey will not have as pronounced a flavor defect. Preparation of Samples for Training. A sample of whey cream can be obtained and churned in a mixer to give whey cream butter that will exhibit the defect. Alternatively, whey can be added to cream before it is churned. Samples will be stronger if the butter is not washed.

Yeasty
Description. The yeasty flavor and aroma is like the smell of yeast leavened bread dough as it rises. It has a fruity, vinegary, flavor and a fragrant aroma. The flavor is evident when the sample is first taken into the mouth but as the sample warms the flavor becomes more evident.5 Cause. Yeasty butter is caused by byproducts of yeast fermentation that have occurred in abused cream. Preparation of Samples for Training. High quality cream could be purposely inoculated with yeast and sugar and allowed to ferment for a few hours at room temperature, chilled, and churned into butter.

3.4.3.3 Body and Texture Defects


Crumbly
Description. Crumbly butter does not hold together when pressure is exerted on it. The fat particles lack cohesion. Some of the butter will adhere to the trier. It appears dry rather than waxy. It is always difficult to cut into neat patties. Cause. Large fat crystals and a deficiency of liquid fat are associated with crumbly butter. If the cream is held for a long time at the churning temperatures, fat crystals may grow large, tending to give crumbly butter. It is more prevalent in the winter when cottonseed meal and alfalfa is fed and the melting range of the butterfat rises.5

Gummy
Description. Gummy butter tends to stick to the roof of the mouth. It gives a gumlike impression and resists melting as it warms up in the mouth. Cause. Gumminess in butter is considered to be due to an abnormally high percentage of high-melting-point triglycerides causing a firmer than normal butter mass. It is more prevalent where cottonseed products are fed as a protein supplement. It is also more prevalent in the winter months when the melting range of butterfat is naturally higher.5

Leaky
Description. Leaky butter exhibits droplets of moisture on the back of the trier and on the newly cut surface of the butter immediately after the sample is removed.

Cause. Leaky butter is a result of insufficient working of the butter mass after churning and washing. The working is necessary to reduce the size of the water droplets sufficiently to retain them in the butter through cutting, kneading, and printing. Properly worked butter will have a waxy texture.5

Mealy or Grainy
Description. Mealy or grainy butter is detected by compressing a sample of partially melted butter between the tongue and the roof of the mouth. If it has a grainy feel like corn meal mush it has this defect. Cause. Mealy or grainy butter is caused by improperly neutralizing cream before churning or by allowing fat to "oil off" during the butter making process. Oiling off can occur during thawing of frozen cream or during remelting of butter rework into heated cream.

Ragged Boring
Description. Butter that exhibits this defect cannot be easily drawn from a block of butter with a trier. It seems to roll from the trier rather than the trier cutting a distinctly formed plug. It is undesirable because of the anticipated problems that will be encountered in cutting. Cause. Ragged boring is caused by slow cooling after pasteurization, holding temperatures high for a long period of time prior to churning. Any process condition that interferes with the formation of close knit waxy textured butter will contribute to this defect.5

Short
Description. Short bodied butter lacks the desirable characteristics of plasticity and waxiness. When pressure is placed on the plug with the thumb, the butter will tend to break. Cause. Short butter is caused by high-melting-point fats, an extremely low curd content in the butter, manufacturing practices that cause some of the milk fat to be melted during the process, and rapid cooling of recently made butter to extremely low temperatures.

Sticky
Description. Sticky butter adheres to the trier and appears to be quite dry. When a plug is drawn, it appears to be "rough." A cold trier aggravates the problem. It generally goes together with a crumbly defect. Cause. This defect occurs when the fat has a higher than usual melting range. It therefore occurs in the fall and winter. It is a feed-related defect appearing when cows are fed alfalfa. Churn temperatures and churn working conditions affect the occurrence of the sticky defect.

Weak Description. Weak butter has a quicker than usual meltdown and a softness of body. It is difficult to get a good plug of weak butter. When pressure is applied to the butter in the plug, there is no distinct breaking point. Cause. Weak butter can be caused by incomplete fat crystallization. It may either be a result of churning before cream has had a chance to completely crystallize or a higher than usual level of low-melting-point triglycerides.

3.4.3.4 Color and Appearance Defects


Color Specks
Description. The color specks defect is the appearance of black, green, red, white, or yellow specks in the body of the butter. Cause. Specks in butter can be pure solidified butter oil, curd particles, copper salts (green), iron salts (black), and undissolved butter coloring (yellow).5 Foreign Material Description. The presence of any material that is not normally found in butter, seen or unseen, has the foreign material defect. These materials may be discrete particles or added chemicals. Cause. Carelessness in allowing objects or chemicals to enter butter is the general cause. Many routes are possible, for example, dirt or sanitizers from the churn or ammonia from a compressor leak. Mold Description. As the descriptive term states, the mold defect refers to visible mold on the butter. Because mold is aerobic, it can only grow on food surfaces and when exposed to oxygen. Cause. Mold spores are everywhere but growth on a food surface can be prevented by packaging in such a way that air is excluded, using mold inhibitor on the surface, or by killing the mold under the package barrier next to the food.5 Mottled, Streaky, or Wavy Description. Mottled butter has areas of lighter or deeper shades of yellow on the surface of the butter. Streaks on butter are recognizable as an area of light color surrounded by more highly colored butter. Wavy butter has an unevenness of color that appears as waves of different shades of yellow.5 Cause. Mottling, streaking, and waviness in butter are all caused by insufficient working of the butter that may be aggravated by poor mechanical condition of the churn and incomplete incorporation of reworked butter into the butter mass.

Surface Color Faded/High


Description. The acceptable color window is quite broad but this defect can be called if the butter is colored excessively and is a brighter yellow than would ever occur when cows consume grass as the only roughage or if the color is unusually lacking. Cause. Faded color could be caused by bleaching due to storage under lights. High color could result from excessive color addition.

Unnatural Color
Description. Unnatural color is reserved for cases where the color of the butter is not in the characteristic yellow window naturally expected for butter. Shades such as a yellow-green or red would be criticized as unnatural. Cause. Use of colors other than yellow to color butter causes this defect.

3.4.4 Ice Cream and Related Products 3.4.4.1 Introduction


Ice cream is defined in the Code of Federal Regulation, Title 21. 6 9 It is a food produced by freezing, while stirring, a pasteurized mix that consists of one or more of a list of specified milk-derived ingredients plus sweeteners, stabilizers, emulsifiers, flavorings, and color agents. Because it can contain such a variety of ingredients from a broad range of sources, it is susceptible to flavor and texture defects that they can bring to the product. The different sweeteners have a range of sweetening power along with other flavors. They also depress the freezing point based on their molality in the mix. Small molecular weight sugars (monosaccharides) depress the freezing point much more per percent of sweetener than do the large molecular weight (disaccharides) or low dextrose equivalent (DE) corn syrups (polysaccharides). Stabilizers bind water, give meltdown resistance and bite, and hinder the growth of ice crystals to ensure smoothness. Emulsifiers reduce the size of air cells and accelerate the churning of the fat globules so that the product is whipped to maximum dryness and rigidity as it exits from the freezer and enters the package. These agglomerated fat globules are thought to be responsible for the sensation of richness. Emulsifier can also bring undesirable flavors to the ice cream, especially if it has oxidized to some extent before use. The quality and amount of flavoring are extremely important to the quality of the product.5 The ice cream should be tempered or equilibrated to 18 to 15C to facilitate dipping and moderate the numbing coldness of the product while retaining its product characteristics. An ice cream dipper, scoop, or spade should be available to collect the sample from the container. If meltdown is to be observed, a small sample should be placed in a clean petri dish 5 min or so before the product is to be evaluated. Because ice cream changes state so fast, the judge must be ready to evaluate the product rather quickly. A systematic sequence of observations is suggested.5 First

the container is examined. Container type, condition, and defects are observed. The color of the ice cream is then noted. The intensity and hue should be natural and typical of the flavor. A sample of the ice cream is then collected with a dipper. The way the product cuts and feels as the dipper moves through the product is noted. The heaviness or fluffiness is noted. The scoop of product is placed on a small plate. Very little aroma is released from the cold product so smelling the product contributes little. A small sample is placed in the mouth with a spoon. Metal or plastic spoons are preferred because of their neutral taste. A large sample will remove too much heat from the mouth and delay the recovery from the temporary cold-induced numbness. Examinations of the body, texture, and flavor take place simultaneously and rapidly. The judge bites down on the sample and notes the presence and size of ice crystals. A bit of ice cream is pressed against the roof of the mouth, melting the sample quickly while the judge notes the smoothness, coarseness, coldness, and the presence and absence of sandiness. As the mouth warms, flavors will begin to brighten as the numbness leaves. First the fundamental tastes of sweet, salt, and sour will appear, followed by aromas. The sample is then expectorated and the rapidity with which the flavor "cleans up" is observed. The urge to swallow product should be resisted. Perception is soon lost when one starts to consume product. The melting quality should now be noted by observing the melting sample in the petri dish. The judge should notice if the form and original size of the scoop of ice cream has been maintained, and whether melted liquified product appears creamy, curdled, foamy, or watery.5 In competition, vanilla ice cream is judged. Being the mildest flavor, defects can be most easily detected in it. Most of the defects not specific to vanilla will be present but perhaps less detectable in ice cream of other flavors made with the same mix. A complete scorecard based on a 20-point scale with 10 points for flavor; 5 for body and texture; 5 for color, appearance, and package; 3 for melting quality; and 2 for bacterial count, is shown in Figure 3.19. A flavor and body scoring guide is given in Table 3.10, and an appearance scoring guide is given in Table 3.11. The Collegiate Contest Ice Cream Score Card is shown in Figure 3.20. Only flavor, body, and texture are judged in the Collegiate Contest.

3.4.4.2 Flavor Defects


Acid
Description. An acid or sour flavor is characterized by a sharp tingling sensation on the tip or top of the tongue accompanied by a clean refreshing mouth feel. The acid flavor cleans up quickly after the sample has been expectorated. There may be other flavors accompanying the acid that are not so clean.5 Cause. Acid flavor is a result of microbial growth that has converted lactose to lactic acid. It may have developed in one of the ingredients used to make the mix or in the mix after formulation. Preparation of Samples for Training. Acid ice cream can be simulated by adding a cup of cultured skim milk or yogurt to a quart of commercial vanilla flavored ice

Product: Flavor:

ICE CREAM SCORE CARD Date: SAMPLE NO.

Criticism Flavor 10 Score Flavoring system No criticism Lacks fine flavor Lacks flavoring = 10 Too high flavor Unnatural flavor Sweetners Lacks sweetness Unsalable Too sweet = 0 Syrup flavor Processing Cooked Normal range Dairy ingredients = 1-10 Acid Salty Lacks freshness Old ingredient Oxidized Metallic Rancid Whey Others Storage (absorbed) Stabilizer/emulsifiei Neutralizer Foreign 5 Score Body and texture Coarse/icy No Crumbly criticism Fluffy = 10 Gummy Unsalable Sandy = 0 Soggy Normal range Weak = 1-5

10

Figure 3.19 A modified version of the ADSA ice cream score card. (Reproduced from ref. 5, with permission of the ADSA, Champaign, IL.)

cream mix and then freezing it in a batch or home freezer. It may be packaged and hardened, then tempered before use.

Cooked
Description. Cooked ice cream is very common. It has a rich custard or scalded milk flavor. It may have the flavor of condensed milk. More intense versions are scorched, caramel, or burnt. Cause. Cooked flavor can result from any of the milk ingredients having received a high or long heat treatment or from high heat treatment of the mix itself. Double

Product: Flavor:

ICE CREAM SCORE CARD (cont.) Date: SAMPLENO.


Criticisms 1 2 3 4 5 6 7 8 9 10

Color, appearance Score and package No Dull color criticism Non-uniform color = 10 Too high color Unsalable Too j?ale color = 0 Unnatural color Damaged container Normal Defective seal range Ill-shaped container = 1-10 Soiled container (dirt) Soiled container (product) Underfilled Over filled Score Melting quality 3 Curdy No criticism Does not melt Flaky = 3 Unsalable Foamy Watery = 0 Wheyed off Normal range = 1-5 Bacterial content 2 Score Standard plate count Coliform count Total Score of each sample Total solids (%) Fat content (%) Net weight (lbs/gal) Overrun (%) Signatures of evaluator(s): Total 25

Figure 3.19 (Continued)

pasteurization of the dairy ingredients will cause a cooked flavor to develop. Some processors purposely give the mix a high heat treatment for the rich "old fashioned" flavor that it gives. Preparation of Samples for Training. A cooked flavored ice cream can be simulated by reheating a commercial mix to 85C for 30 min on the stove with vigorous stirring. After chilling, it can be frozen in a batch or home freezer.

Lacks Fine Flavor


Description. This term is used to describe ice cream that is good but for some reason just falls short of excellent flavor. The flavor balance may be off, or the

Table 3.10

THE ADSA SCORING GUIDE FOR SENSORY DEFECTS OF ICE CREAM (SUGGESTED FLAVOR AND BODY AND TEXTURE SCORE FOR DESIGNATED DEFECT INTENSITIES)
Intensity of Defect Slight Definite Pronounced 0b 5 4 7 4 7 6 2 2 1 0 5 4 7 7 5 4 1 2 1 1 0 2 1

Flavor criticism Acid (sour) Cooked Flavoring: Lacks flavoring Too high Unnatural Lacks fine flavor Lacks freshness Metallic Old ingredient Oxidized Rancid Salty Storage Sweetener: Lacks Too high Syrup flavor Whey Body and texturec Course/Icy Crumbly (brittle, friable) Fluffy (foamy) Gummy (pasty, sticky) Sandy Soggy (heavy) Weak (watery)

51

4 9 8 9 8 9 8 6 6 6 4 8 7 9 9 9 7 4 4 3 4 2 4 4

2 7 6 8 6 8 7 4 4 4 2 7 6 8 8 7 6 2 3 2 2 1 3 2

Source: American Dairy Science Association, 1990. a "No criticisms" is assigned a score of 10. Normal range is 1-10 for salable product. b An assigned score of 0 (zero) is indicative of unsalable product. c "No criticisms" is assigned a score of 5. Normal range is 1-5 for salable product.

vanilla flavor may be a little off. This is a catch-all last resort descriptor that is used only for minor shortcomings if none of the other terms will describe the problem.5 Cause. The causes are as varied as the reasons for the less-than-ideal flavor possibilities. The most likely cause will be a slightly deficient vanilla blend.5 Preparation of Samples for Training. Screening vanilla flavorings for one that is slightly deficient and using that flavoring at the recommended level is a possible way to stage this flavor. Blending some imitation and high quality flavor in various

Table 3.11 A SUGGESTED SCORING GUIDE FOR COLOR, APPEARANCE, AND PACKAGE OF VANILLA ICE CREAM
Intensity of Defect Defect3 Dull color Nonuniform color Too high color Too pale color Unnatural color Soiled container Product on container Underfill/overfill Damaged container Defective seal Ill-shaped containers Slight5 4 4 4 4 4 3 4 4 3 2 4 Moderate 3 3 3 3 3 2 3 3 2 1 3 Definite 2 2 2 2 2 1 2 2 1 0 2 Strong 1
d d d

Pronounced0
d d d d

1 0 1 1 0 0 1

0 0
d

0 0 0 0

Reproduced with permission from ref. 5. a "No criticism" is assigned a score of 5. Normal range is 1-5 for a salable product. An assigned score of 0 (zero) is indicative of an unsalable product. b Highest assignable score for defect of slight intensity. c Highest assignable score for defect of pronounced intensity. d A dash () indicates that the defect is unlikely to occur at this intensity level.

proportions and freezing mixes with each will give a good range of samples from which to choose. It will also afford a variety of qualities of flavors for demonstration.

Lacks Flavoring
Description. This descriptor is for ice cream that is flat or deficient in the amount of flavoring. The ice cream may be sweet and free from any off-flavors but it lacks the characteristic delicate flavor of an excellent vanilla at the desired intensity.5 Cause. The most probable cause of ice cream that lacks flavoring is inadequate amount of vanilla or flavoring. It could also be caused by inferior vanilla. Preparation of Samples for Training. Vanilla ice cream that lacks flavoring can be made by adding half the recommended amount of top quality flavor to a good commercial mix and freezing in a batch or home freezer.

Lacks Freshness
Description. This is a moderate general off-flavor in ice cream or frozen desserts that can have various characteristics but principally takes the edge off the fresh perfect taste of the product. Cause. This defect can be caused by a little slightly stale powdered milk, slightly stale whey, some slightly old cream, or some milk with the lacks freshness defect. Products that show stronger intensities of these defects are criticized for "old ingredient."5

M A R K I N GI N S T R U C T I O N S
Vmt NO. 3 HNCIt ONLV IMPROPER MARKS PROPER MARK

C O N T F S T A N TM IO D A T E ICE CREAM

ERASE C H A N G E S C L E A N L Y A N D COMPLETELY D O N O T M A K E A N Y STRAY M A R K S

CRITICISMS FLAVOR

N C S T r M O p c it M P 3 O 7 3 5 3 3 3 2 1A 2 4 0 0 S A M P L EN U M B E R

COOKtD NO CRITICISM 10 L A C K S FLAVORJMG

LACKSSVWtTWESS
NORMAL RANGE 1-10 RANCID STORAGE TOOMKiHFLAVCW WNATURALaAVW OLD INGREDIENT

BODY AND TEXTURE


NO CRITICISM 5 GUMMY SOGGY NORMAL RANGE 1-5 CRUMStY

APPEARANCE AND COLOR


NO CRITICISM 5

NORMAL RANGE 1-5

Figure 3.20 Collegiate contest ice cream score card. (Reproduced from ref. 5, with permission of the ADSA, Champaign, IL.)

Preparation of Samples for Training. Dissolve some slightly stale powdered milk at the rate of about 1 to 5% in some good quality commercial ice cream mix. Freeze in a batch or home freezer and harden.

Lacks Sweetness
Description. The name of this defect is very descriptive. It is noted quickly on putting the sample in the mouth as a flat or bland taste. An adequate amount of sweetener would bring out the full flavor. Cause. The cause of this defect is obvious. Either the formula or formulator is in error in making a sweetener deficient mix. When developing formulas, it is important to realize that different sweeteners have different relative sweetness per unit of weight. One of the sweetest nutritive sweeteners is fructose and some of the least sweet are lactose and corn syrup solids. Preparation of Samples for Training. A simple mix can be formulated from scratch as follows: 560 g or 36% fat cream, 1200 g of milk, 125 g of fresh powdered milk, and 115 g of sucrose. The mix should be pasteurized, chilled, and then frozen in a batch or home freezer. As this sample is for flavor only, ingredients to modify the texture are not used. This sample may or may not be homogenized.

Metallic
Description. This flavor is characterized by a puckery mouth feel or a flavor similar to a rusty nail or an old penny. Cause. The mix or one of its dairy ingredients most probably came in contact with copper or iron. These ions catalyzed a free radical lipid oxidation. Dairy plant equipment that comes in contact with dairy products is free of copper and corrodible metal because of the potential for this defect.5 Preparation of Samples for Training. A 2000-ml aliquot of ice cream mix can be treated with 3 ml of a 1% CuSO4 solution and held for 1 to 2 days while the flavor develops before freezing in a batch or home ice cream freezer.

Old Ingredient
Description. Old ingredient flavors are many. Most common are a stale protein flavor or old milk or old cream flavors. Cause. This defect descriptor is reserved for off-flavors brought to the mix from the ingredients. Among the causes of old ingredient flavor are stale milk powder, stale whey powder, or stale or oxidized stabilizer from extended storage; old cream or old milk from age or psychrotrophic growth; and fermented syrup. Preparation of Samples for Training. The stale ingredient flavors can be simulated by dissolving a few percent of a stale milk or whey powder into a good commercial ice cream mix. A stale emulsifier or stabilizer could be dry blended with a little

sugar and added to a commercial mixer at half the recommended level. Old cream also could be added to a finished mix at about 20% of the weight of the mix. Another approach would be to make simple mixes using aged ingredients with the aged or stale flavors that are needed. All these small batches can be frozen in a batch or home freezer.

Oxidized
Description. Some synonymous terms are cardboard or papery, cappy, tallowy, and painty, representing variations of related flavors that are given the same descriptor. Metallic oxidized is listed separately but is related and considered to be one of this group of related flavors.5 Cause. Exposure of ingredient milk or finished mix to sunlight or fluorescent lights will produce the cardboard flavor. Use of fat products in which the butterfat has undergone autoxidation produces tallowy and painty flavors. Preparation of Samples for Training. Mix or milk from which mix will be made can be placed under fluorescent light for 12 to 24 h prior to freezing. Oxidized butter oil can be made by spiking it with a little rancid soy oil and allowing the flavor to develop for a few days before the oxidized oil is emulsified into a mix.

Rancid
Description. Rancid ice cream, like milk, will have a delayed reaction. It comes on late and is detected on the back of the tongue. It is a very objectionable flavor, tasting soapy and unclean with some bitter notes. It is somewhat masked in ice cream by the sweetness and flavoring but the lingering aftertaste and slow clean up will be evident.5 Cause. Rancidity in any dairy product is caused by the action of lipase on butterfat which releases soapy tasting free fatty acids and their salts. It occurs when lipase, from either raw milk or psychrotrophic growth, comes in contact with a new fat surface. The fat surface is created by homogenization or violent agitation as in a centrifugal pump. Preparation of Samples for Training. Rancid milk can be made by mixing raw and freshly pasteurized milk and allowing lipolysis to proceed for a day or so. That milk is laboratory pasteurized and used to make a small sample of mix that is subsequently frozen.

Salty
Description. The term salty is recognizable as one of the basic tastes. It is detected quickly on the tip of the tongue as the product is placed in the mouth. There is no aroma associated with the salty taste. The sensation is "warm" as opposed to refreshing. It cleans up quickly after the ice cream is expectorated. Cause. Salty ice cream has, as the descriptor implies, too much salt in the mix. It may be coming from salted butter when it is used as the butterfat source; from high

levels of concentrated whey, whey solids, or milk solids; or there could be just too much salt in the formula.5 Preparation of Samples for Training. Salty ice cream can be made easily by adding extra salt to a mix before freezing, or by working a little extra salt into softened ice cream prior to tasting.

Storage
Description. This descriptor is used for a family of defects so it is not a response to one flavor. One description is the lack of fresh, bright, refreshing flavor with no particular defects obvious. In this definition it is quite similar to "lacks freshness" except that this is for more severe cases. Another is the presence of flavors that have been absorbed from the environment such as ammonia, smoke, or chemicals. Cause. The term storage refers to flavors that develop during storage of mix or ice cream. The loss of bright refreshing flavor due to extended storage is one cause. Another is the presence of aromatic materials in the freezer with the ice cream which are absorbed into the ice cream.5 Preparation of Samples for Training. The best source of this flavor would be a good sample of ice cream that has been stored for a year or two. If several samples are put away for the future and evaluated a year or two later, it is likely that some good examples of storage flavor will be among them.

Syrup Flavor
Description. Descriptive synonyms for the syrup are marshmallow, molasseslike, and malty. It has been described as a low level of burnt sugar taste. It is often associated with a gummy body.5 Cause. Sucrose gives a clean sweet taste free of any side flavors. Corn syrups bring with them other flavors. Modest levels of com syrup can be used in normal practice without this defect being evident, but it becomes evident when high levels are used. Preparation of Samples for Training. This flavor can be simulated by dissolving an extra 5% of 36 or 42 DE corn syrup solids into a good commercial mix prior to freezing.

Too High Flavor


Description. When this occurs, this flavor is best recognized when the sample is first placed in the mouth. The flavor is sharp and harsh and the desired balance of vanilla flavors with the other flavors is not achieved.5 Cause. As the descriptor states, this defect is caused by too high a level of vanilla in the mix. Preparation of Samples for Training. Vanilla ice cream with too high a flavor can be produced by adding 25 to 50% more than the recommended level of high quality vanilla to the mix before it is frozen.

Too Sweet
Description. Ice cream that is too sweet is recognized by the candylike taste. The sweetness overpowers the flavor and fails to achieve an ideal blend of flavors. Excessive sweetness detracts from the refreshing quality of a good ice cream.5 Cause. mix. When ice cream is too sweet, excessive sweetener has been added to the

Preparation of Samples for Training. An additional 2 to 5% sucrose can be added to and dissolved into a good quality commercial mix prior to freezing to simulate ice cream that is too sweet.

Unnatural Flavor
Description. There are two types of unnatural flavor. One is a taste that is not in agreement with the label. If it is labeled vanilla but has a hint of butterscotch, unnatural flavor would be the appropriate criticism. The other is presence of flavor notes that are out of balance with the blend of flavors. Sometimes when imitation vanilla is used to boost and extend real vanilla, there will be an initial sharp, piercing, burning sensation on the tongue that is not present with good quality vanilla. Cause. The cause for misflavored ice cream is usually error on the part of the ice cream maker either in adding the wrong flavor or in misjudging the change over point between flavors as it is being frozen and trying to save too much ice cream near the change over. The cause of poorly balanced unnatural flavor is the less than ideal flavor blend that is used to flavor the ice cream. It occurs more frequently in Category 2 and 3 vanilla ice creams. Preparation of Samples for Training. Both types of unnatural flavored ice cream can be simulated. The misbranded type can be achieved by blending some of another flavor into the vanilla while the ice cream is softened. Creating the poorly balanced flavor involves making several batches of vanilla using selected blends of flavors and choosing those that have flavors with examples of the defect.

Whey
Description. The whey flavor in vanilla ice cream is similar to the flavor of graham crackers or stale condensed milk usually accompanied by a slightly salty taste.70 Cause. Federal standards limit the maximum concentration of whey solids in ice cream to 25% of the MSNF.69 That amount may not hurt the quality of product when good quality whey is used, but lesser amounts will be detected when the concentrated or dried whey is of poor quality.5 Preparation of Samples for Training. Dissolve 2 to 4% dried whey into commercial ice cream mix before freezing in a batch or home ice cream freezer.

3.4.4,3 Body and Texture Defects


Coarse/Icy
Description. This defect is evident when the evaluator bites down on the ice cream. The incisors are held slightly apart by the ice crystals until a little more pressure is exerted and the crystals give with a crunchy sound that can be heard through the bones of the head. The feeling is transitory and disappears quickly as the product melts. The product feels unusually cold. Cause. The coarse/icy defect is caused by large ice crystals that form due to slow hardening, high holding temperatures, or cycling temperatures up to near freezing and then back down again. Product that is frozen quickly with stirring and is subsequently hardened at very low temperatures will have very small ice crystals and a smooth texture. When product is held at higher temperatures near freezing, there is a lot of movement of water out of and into crystals and more liquid water. Under these conditions, the small crystals will shrink and the large ones will grow. These large crystals are responsible for the coarse/icy texture.5 Preparation of Samples for Training. This is such a common defect that examples of it will be found by surveying a few commercial samples. The defect can be generated by holding good quality ice cream at around 80C or cycling it between - 8 and -20 0 C. Crumbly Description. The crumbly defect can also be described as brittle and friable. Ice cream with this defect falls apart as it is dipped. It appears dry and open and ice cream particles remain in the scoop.5 Cause. Crumbly ice cream is caused by too low a solids level in the mix, by too high an overrun, or by inadequate stabilization.5 Preparation of Samples for Training. A training sample could be made by diluting a commercial ice cream mix 25% with milk before freezing. The defect could be aggravated by absence of stabilizers. The mix formula suggested for the lacks sweetness defect should be crumbly when frozen. Half the milk powder could be left out to aggravate the defect. Fluffy Description. Fluffy, foamy, or spongy ice cream is light in weight for the volume. It does not offer the usual resistance to dipping of normal ice cream. The texture may be more open than usual and the ice cream is compressible. It melts slowly and yields a small amount of liquid. Cause. This defect is caused by excessively high overrun. It will be evident when the overrun exceeds 100% or when volume of air in the ice cream exceeds the volume of the mix.

Preparation of Samples for Training. A small continuous freezer is the best equipment for making fluffy samples because of overrun control capability. While running regular vanilla, increase the overrun to above 100%, preferably to 150%, or just long enough to collect samples, then return it to normal overrun.

Gummy
Description. Gummy body is sometimes called pasty, sticky, or elastic. It is the opposite of crumbly. The ice cream holds together so well that it resembles taffy. As the scoop is pulled across the surface, the ice cream tends to "curl" behind the scoop. It should be criticized only when the stickiness will interfere with the dipping of the product. If corn syrup is the cause, it will be accompanied by a syrup flavor.5 Cause. This defect is caused by excessive use of stabilizers or corn syrup solids in the ice cream mix. Preparation of Samples for Training. This defect can be caused by the addition of about 5% extra 36 or 42 DE corn syrup solids to a commercial ice cream mix prior to freezing in a batch or home ice cream freezer.

Sandy
Description. Sandy or gritty ice cream has a lack of smoothness and a grittiness that remains on the tongue long after the ice cream has melted and been expectorated. The grittiness feels like fine grains of sand that resist being dissolved. Cause. Crystals of lactose account for the grainy, slow dissolving particles. The form with the lactose content of the mix is high usually due to the high level of use of whey solids in the mix coupled with elevated storage temperatures or cycling storage temperatures that encourage the growth of the lactose crystals.5 Preparation of Samples for Training. Very fine lactose crystals can be blended into softened ice cream to simulate the texture of sandy ice cream.

Soggy
Description. Soggy ice cream has a heavy, doughy, puddinglike body. A given volume of ice cream seems heavier than expected. In the mouth it feels colder than normal.5 Cause. This defect can be caused by too high a solids content in the mix, too much stabilizer, or too low an overrun. Preparation of Samples for Training. Addition of 5% extra nonfat milk solids, 5% corn syrup solids, or reducing the overrun to 20 to 30% will tend to give ice cream with the soggy defect.

Weak
Description. Weak or watery ice cream melts unusually quickly to an uncharacteristically thin fluid. It disappears in the mouth much more quickly than is expected.

It is easily compressed with a spoon or a scoop. It has a tendency to be coarse and icy and crumbly.5 Cause. Low solid mix or unstabilized mix will tend to result in weak ice cream.

Preparation of Samples for Training. Dilution of a commercial ice cream mix with 20 to 30% of its volume of milk or water will tend to result in a weak ice cream after it is frozen in a batch or home ice cream freezer. Formulation of a mix from scratch with a reduced quantity of stabilizer will tend to produce weak and crumbly ice cream.

3.4.4.4 Color and Appearance


Dull Color
Description. Other terms used to describe this defect are dead, soiled white, or gray. If it is obviously caused by ground vanilla beans and is accompanied by little bean flecks, it should not be criticized.5 Cause. Dirty equipment is usually the cause of dull colored ice cream. Lubricant or corrosion that is allowed to come in contact with the ice cream mix will discolor it. Preparation of Samples for Training. Just a trace of black food coloring can be added to commercial mix to cause this defect in finished ice cream.

Nonuniform Color
Description. Nonuniform color is the variation in color shade from one portion of the sample to another. For example, the cream color of vanilla ice cream may change in shade from the bottom to the top of the carton. Cause. This defect is usually associated with product age.5 Bleaching effects can also cause this effect where a portion of the surface is exposed to light. Preparation of Samples for Training. Two ice cream samples with different shades of color can be softened and blended together with very little stirring.

Too High Color


Description. The high color defect refers to uncharacteristically bright color for the flavor. It is objectionable because it gives an "artificial" impression or an impression of cheapness, lack of understanding, and lack of care on the part of the manufacturer.5 Cause. The high color defect is obviously caused by the addition of too much or too bright a color to the ice cream mix. Preparation of Samples for Training. Vivid colored ice cream can be made by adding excessive amounts of color to the mix.

Too Pale Color


Description. This defect refers to an uncharacteristically light color. For vanilla, it refers to a white color that conveys an impression opposite of richness. Cause. The pale color is caused by the absence or deficiency of color in the ice cream mix. Preparation of Samples for Training. A pale colored sample of ice cream can be obtained by pulling a sample of commercial mix before the color is added. An alternate way is to use the formula suggested under lacks sweetness and leave the color out before it is frozen in a batch or home ice cream freezer.

Unnatural Color
Description. Unnatural color is a color that is not characteristic of the flavor of the ice cream.5 For example, purple cherry ice cream or vanilla with a red tint would be unnatural color. Cause. Unnatural colored ice cream is caused by the addition of a color that is not characteristic of the flavor. Preparation of Samples for Training. Addition of tumeric or caramel color to vanilla ice cream prior to freezing would result in unnatural colored vanilla ice cream.

3.4.4.5 Melting Quality


To evaluate the melting quality of ice cream, a small scoop of product is placed on a petri plate or dish and allowed to warm up. Its appearance is noted periodically as it melts. High quality ice cream should melt in 10 to 15 min. Mix should flow from the ice cream as it melts to a smooth uniform and homogeneous liquid.

Curdy
Description. Ice cream that has a curdy melt down will separate into small distinct pieces rather than a smooth uniform white liquid. The surface may appear to have dry, irregular shaped curd particles.5 Cause. Curdy appearing ice cream can be caused by one or more of the following conditions that has destabilized the protein: high acid, high temperature-time, unfavorable salt balance, and certain emulsifiers or stabilizers.5

Does Not Melt


Description. The descriptor is quite adequate in characterizing this defect. A scoop of ice cream put out at room temperature holds its shape or resists slumping and running for longer than 10 to 15 min as it warms to room temperature.5

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Cause. Slow melting ice cream can be caused by certain stabilizers and emulsifiers, high overrun, old ice cream, and several other processing and ingredient interactions that promote gelation of the body of ice cream. Foamy Description. Foamy or frothy meltdown is noticed as a mass of large stable air bubbles when the sample is completely melted. Cause. Foamy ice cream can be caused by high overrun and by emulsifiers that effectively stabilize foam.5 Watery Description. This melting defect is a low resistance to melting with the melted mix being of a thin and watery consistency. Cause. Watery or fast melting ice cream is associated with low solids mixes and coarse, weak bodied ice cream or ice milk. Wheyed Off Description. Ice cream with this defect will develop a ring of clear greenish or bluish fluid collecting around the edges of the scoop of ice cream early in the meltdown test. It may be observable in the mix before freezing.5 Cause. This is a common problem in concentrated mixes and mixes that are stored for long periods before use. It is more common in mixes that have been abused (excessive stirring, aerated, old).

3.4.5 Cheese 3.4.5.1 Introduction


There are at least 400 varieties of cheese with as many as 2000 names. A general definition that applies to all these varieties is a dairy product made by coagulation of milk, with or without its full complement of fat, removing the soluble portion known as whey, and concentrating the insoluble portion into a semisolid cheese mass known as curd. The whey is composed of water, lactose, proteins that are soluble under the conditions of coagulation, and soluble minerals or ash. Some fat usually is present also. The curd is composed primarily of casein and milk fat. It also contains minor amounts of water-soluble materials dissolved in the water portion of the curd. Variations that result in so many different types of cheese include type of milk, method of coagulation (acid or enzyme), culture characteristics, amount of water retained in the curd, method of cutting and handling the curd, fresh consumption versus ripening, and presence or absence of surface ripening organisms. Specific definitions for several types of cheese are given in the Code of Federal Regulations, Title 21 Part 133.71

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Cause. Slow melting ice cream can be caused by certain stabilizers and emulsifiers, high overrun, old ice cream, and several other processing and ingredient interactions that promote gelation of the body of ice cream. Foamy Description. Foamy or frothy meltdown is noticed as a mass of large stable air bubbles when the sample is completely melted. Cause. Foamy ice cream can be caused by high overrun and by emulsifiers that effectively stabilize foam.5 Watery Description. This melting defect is a low resistance to melting with the melted mix being of a thin and watery consistency. Cause. Watery or fast melting ice cream is associated with low solids mixes and coarse, weak bodied ice cream or ice milk. Wheyed Off Description. Ice cream with this defect will develop a ring of clear greenish or bluish fluid collecting around the edges of the scoop of ice cream early in the meltdown test. It may be observable in the mix before freezing.5 Cause. This is a common problem in concentrated mixes and mixes that are stored for long periods before use. It is more common in mixes that have been abused (excessive stirring, aerated, old).

3.4.5 Cheese 3.4.5.1 Introduction


There are at least 400 varieties of cheese with as many as 2000 names. A general definition that applies to all these varieties is a dairy product made by coagulation of milk, with or without its full complement of fat, removing the soluble portion known as whey, and concentrating the insoluble portion into a semisolid cheese mass known as curd. The whey is composed of water, lactose, proteins that are soluble under the conditions of coagulation, and soluble minerals or ash. Some fat usually is present also. The curd is composed primarily of casein and milk fat. It also contains minor amounts of water-soluble materials dissolved in the water portion of the curd. Variations that result in so many different types of cheese include type of milk, method of coagulation (acid or enzyme), culture characteristics, amount of water retained in the curd, method of cutting and handling the curd, fresh consumption versus ripening, and presence or absence of surface ripening organisms. Specific definitions for several types of cheese are given in the Code of Federal Regulations, Title 21 Part 133.71

This treatment will use cheddar and closely related cheese as the standard. Cheddar is the most common type of cheese produced in the United States. It is usually made from pasteurized cow's milk by adjusting the temperature to about 32C and adding a lactic culture (usually Lactococcus lactus spp. lactus or Lactococcus lactus spp. cremorus and chymosin or a related milk coagulant. Annatto color may be added to give deeper orange color. When the milk gel forms, it is cut into small pieces and warmed to drive the water out of the curd. During cooking the acid begins to develop. When some acidity has developed, the whey is drained and the curd is allowed to matt. Matted curd is cut into slabs that are moved and piled as more acid develops. The slabs of curd stretch and flatten under the weight of curd above. The oriented protein fibers give a "chicken breast" texture to the curd. This process is called cheddaring. In an alternative process the curd is not allowed to matt but is stirred continually as the acid develops. That method lends itself to mechanical handling. When sufficient acid has developed the slabs are milled into cubes, salted, placed in hoops, and pressed into blocks. Stirred curd is already in small pieces, so it is salted, hooped, and pressed. Blocks of cheese are sealed in plastic or wax to exclude air and prevent mold growth and aged for a time varying from a few months to a few years depending on the sharpness desired in the cheese. A skilled cheese maker knows how to manipulate the process to achieve the target composition and character. He knows how to avoid phage build up as the culture is developing acid, and he knows the value of and uses good sanitation practices as the cheese is being made. Before examining and grading cheese, the sample is tempered to 10 to 15.5C. Proper tempering is critical for observation of some attributes. The first procedure is visual examination of the cheese and packaging materials. The judge notes whether the sample is neat, attractive, clean, and symmetrical. He looks for evidence of mold growth where air may have had access to the cheese surface. A sample of cheese is removed from the block with a trier similar to the one described in butter judging. The trier is a double-edged curved blade that is pushed into the block, turned 180, and removed bringing with it a tapered cylinder of cheese. The sample is preferably taken from the top and about halfway to the center from the sides. The plug is passed under the nose and the judge notes any aroma. The top 1 to 2 inches is then broken off the pushed into the hole to partially protect the block from mold growth, drying, and cracking. The judge examines the cheese cylinder for clean cut surfaces or featherlike edges as if it were cut with a dull knife. Color is then observed. It should be bright, clear, and uniform, free from faded areas or mottling, dark or light seams. It should be somewhat translucent rather than opaque. The judge then looks for mechanical openings. Their shape and the inside appearance should be noted. A rounded glossy inner surface is indicative of gas while rough irregular inner surfaces indicate poor pressing and curd knitting. The judge then takes the ends of the plug by the tips of the fingers and bends it until it breaks. The plug may show shortness which is resistance to bending followed by an abrupt break, or weakness when it will bend until the ends nearly touch. The judge now takes a piece of cheese from the plug and applies pressure on it between the thumb and forefinger followed by manipulation into a uniform ball. The thumb is then pushed into the ball and re-

moved, noting adherence to the thumb or stickiness. If the ball tends to fall apart under the thumb's pressure, it is crumbly or curdy. The warm molded cheese is now ready for a second pass at aroma detection. The ball should be placed under the nose and smelled. A small unworked portion of the plug is placed in the mouth for tasting. It is chewed, rolling it around in the mouth while observing the taste and aroma sensations, until a semiliquid state is achieved. The judge then expectorates the sample and observes the aftertaste. The trier is cleaned with a soft cloth or a paper towel prior to evaluation of another sample. The judge may freshen his mouth with ambient temperature salt water, grapes, or an apple. He may wish to retaste the best sample in the lot to standardize his tongue on * ideal." Experienced judges learn enough from looking at the sample and evaluating its body and texture that tasting is just reinforcing what they have already learned.5 A scoring guide for flavor and body/texture defects is shown in Table 3.12. The ASDA cheddar cheese score card is shown in Figure 3.21 and the Collegiate Contest Score Card is shown in Figure 3.22. Appearance and color are not judged in the collegiate contest. Much of the cheese sold in the United States is sold and priced based on government grade. Graders employed by the Dairy Grading Branch of the Poultry and Dairy Quality Division, Food Safety and Inspection Service of the USDA assign letter grades based on guidelines summarized in Table 3.13.

3.4.5.2 Flavor Defects


Bitter
Description. The bitterness is a delayed sensation sensed at the base of the tongue. It is somewhat distasteful, resembling quinine or caffeine. It tends to persist long after expectoration. Cause. Bitterness is found most often in aged cheese that has had time to break down. It is associated with excessive acidity, excessive starter, starter with strong proteolytic activity, excessive moisture, and microbial contaminants due to poor sanitation.5 Preparation of Samples for Training. Usually a sample of bitter cheese can be found on the market or at a cheese plant. Bitter processed cheese can be staged by adding 1 to 2 ml of a 1% stock solution of quinine sulfate to 600 g of hot melted cheese. Add 10 g of sodium citrate as an emulsifying salt. Stir to incorporate the salt and quinine and cool.

Fermented/Fruity
Description. The fruity off-flavor resembles the flavor of overripe pineapples or apples. This sweet aromatic flavor intensifies as the cheese gets older and may evolve into an unclean off flavor. The fermented flavor is suggestive of acetic acid or vinegar.5

Table 3.12 THE ADSA SCORING GUIDE FOR SENSORY DEFECTS OF CHEDDAR CHEESE (SUGGESTED FLAVOR AND BODY AND TEXTURE SCORES FOR DESIGNATED DEFECT INTENSITIES)
Intensity of Defect Slight Flavor criticism Bitter Fermented/fruity Flat, lacks flavor Garlic, onion, weedy Heated, cooked High acid, sour Moldy, musty Rancid, lipase, putrid Sulfide, skunky Unclean, dirty Whey taint, sour whey Yeasty Body and texture criticisms5 Corky, dry Crumbly, friable Curdy, rubbery Gassy Mealy, grainy Open Pasty, sticky Short Weak, soft Pasty Weak/soft
3

Definite

Pronounced

9 8 9 6 9 9 7 6 9 8 8 6 4 4 4 3 4 4 4 4 4 3 4

7 6 8 4 8 7 5 4 7 6 7 4 3 3 3 2 3 3 3 3 3 2 3

4 5 7 1 7 5 3 1 4 5 5 1 2 2 2 1 2 2 1 2 2 1 2

Source: American Dairy Science Association, 1990 a "No criticisms" is assigned a score of 10. Normal range is 1-10 for salable product. b "No criticisms" is assigned a score of 5. Normal range is 1-5 for salable product.

Cause. This flavor defect is sometimes but not always associated with high moisture, pasty, weak-bodied cheese. The fruity flavor is thought to be due to the presence of ethanol-forming microorganisms in the cheese milk or in the culture. Esters formed from the ethanol combining with organic acids are responsible for the fruity note and acetic acid generated is responsible for the fermented note.71"73-83 Low acid or low salt also encourage the development of this flavor. Preparation of Samples for Training. Fermented/fruity cheese can usually be found in a survey of a large number of samples. The flavor can be simulated in milk by the addition of a small amount of pineapple juice. A small amount of processed cheese can also be spiked with pineapple juice just before cooling to simulate this

Date:

CONTEST CHEDDAR CHEESE SCORE CARD A.D.S.A. Contestant No: Criticisms Contestant Score Score 1 2 SAMPLE NO. 3 4 6 5 TOTAL GRADES 7 8

Flavor

10

No criticism = 10

Normal range = 1-10

Criticism Acid Bitter Feed Fermented/fruity Flat/lacks flavor Garlic/onion Heated Moldy Rancid Sulfide Unclean Whey taint Yeasty

Grade

Body and texture 5

Contestant score Score Grade

Criticism

No criticism 5 Normal range 1-5

Corky Crumbly Curdy Gassy Mealy Open Pasty Short Weak

Allowed perfect in contest Allowed perfect Finish in contest Total score Total in each sample Total grade per sample Source: American Dairy Science Association (1987) Color

Final grade Rank

Figure 3.21 The ADSA contest cheddar cheese score card for sensory defects. (Reproduced from ref. 5, with permission of the ADSA, Champaign, IL.)

M A R K I N GI N S T R U C T I O N S
X W H O 3 MWCIL 6KlY-

! C O N T E S T A N TN O

D A T E

M IP R O P E R M A R K S

P R O P E R M A R K

CHEDDAR CHEESE

E R A S EC H A N G E SC L E A N L YA N D C O M P L E T E L Y D ON O TM A K EA N YS T R A YM A R K S C R I T I C I S M S F L A V O R
FEEO
NO

N C ST r a n a O p e i t* M P 3 0 7 3 5 3 0 3 2 1A 2 4 0 0 S A M P L EN U M B E R

CRITICISM FLAT/LACKS RAVOR 10 HEATED NORMAL RANGE 1-10 MOtOY SUtFIDC VWiYTAWT

BODY AMD TEXTURE


NO

CRITICISM 5 NORMAL RANGE 1-5 APPEARANCE AND COLOR NO CRITICISM 5 NORMAL RANGE 1-5

Figure 3.22 Collegiate contest cheddar cheese score card. (Reproduced from ref. 5, with permission of the ADSA, Champaign, IL.)

flavor. Addition of 1 to 11A ml of 1 % aqueous solution of food grade ethyl hexanoate to a 400-g batch of processed cheese will produce cheese with this flavor.

Flat/Lacks Flavor
Description. The descriptive term is accurate. Lack of flavor is noticed soon after the sample is placed in the mouth. Odor and flavor are hardly detected either. Cause. It is common for young cheese to have this defect because time is required for cheese flavor to develop. The full flavor of cheese is due to the presence of acid together with products of microbial and enzymatic breakdown products of the fat and protein in the cheese. Some causes of the defect are lack of acid production, use of low fat milk to make cheese, excessively high cooking temperatures that destroy enzymes, curing at too cold a temperature, or too short a curing period.5 Preparation of Samples for Training. Flat cheese has to be found by surveying product that is available on the market or in the plant. A sample can generally be found.

Garlic/Onion
Description. This flavor resembles that of garlic, onions, or leeks and usually has a characteristic odor. The flavor builds up in the mouth and is very hard to wash out after the sample has been expectorated. Cause. Cheese will have the onion/garlic defect when it is made with milk that has the defect. Milk is tainted with these flavors during the warm months when cows are feeding in pastures that are infested with onion, garlic, or other weeds that impart these flavors to the milk. They are especially strong when the cows consume these plants shortly before they are milked.5 Preparation of Samples for Training. Samples with this defect can be produced by making a small experimental batch of cheese from milk with the actual or simulated defect. Alternatively a small batch of processed cheese can be made by melting 800 g of cheese with 16 g of sodium citrate and a small amount of onion or garlic powder.

Heated
Description. The heated or cooked cheese flavor is different from the clean cooked flavor of milk. It resembles the odor of spoiled milk or the odor of melted Bakelite plastic. It is suggestive of the unclean odor and of heated whey.

High Acid
Description. Lactic acid is one of the normal flavor notes in cheddar cheese. Only when the strength of the acid flavor overpowers the other flavor notes of the cheese is it considered a defect. The normal pH range of cheddar cheese is 5.15 to 5.45. At pH values below 5.15 the acid defect may be evident. The acid or sour flavor is

Table 3.13 A SUMMARY OF U.S. GRADES OF CHEDDAR CHEESE


Approximate Score or Score Rangea

Grade AA

General Description of Medium-Cured to Aged Cheddar Flavor: Fine, highly pleasing very slight feed flavor permitted. Body and texture: Firm, solid, smooth, compact, close, translucent, few small mechanical or sweet holes permitted, no gas holes. Color: Uniform, tiny white specks if aged and very slight seaminess permitted. Finish: Sound rind well-protected and smooth, even-shaped. Flavor: Pleasing, may possess limited feed, or acid or bitter flavor (if aged). Body and texture: Reasonably solid, compact, close and translucent, few mechanical holes not large or connected, limited to two sweet holes per plug, no gas holes. Color: Slight white lines or seams. May be very slightly wavy. Finish: Sound firm rind, well protected but may possess to a very slight degree a soiled surface or mold growth; may be slightly lopsided, have high edges or rough, irregular surface. Flavor: May possess certain limited undesirable flavors according to age. Body and texture: Texture may be loose and open and have numerous sweet holes, scattered yeast and other scattered gas holes, pinny gas holes not permitted. Color: May possess about the same defects as Grade A except to a greater degree. Finish: Rind sound, may be slightly weak, but free from soft spots, rind rot, cracks, or openings, bandage may be uneven, wrinkled but sound, surface may be rough, unattractive, but have good protective coating; paraffin may be scaly or blistered; no indication that mold has entered the cheese; may be huffed, lopsided, or have high edges.

93 or above

92

90-91

characterized by a sharp tingling sensation on the tip or top of the tongue accompanied by a clean refreshing mouth feel.5 Cause. The acid flavor can be caused by the development of excessive lactic acid during the making of the cheese, excessive retention of moisture that encourages (Continued) continued bacterial growth, retention of whey and lactose in the curd that provides lactose for the production of lactic acid, the use of excessive starter, and the lack of enough salt in the finished curd.5

Table 3.13
C

(Continued)

Flavor: May possess somewhat objectionable flavors and odors with a certain increase in tolerance according to age and degree of curing. Body and texture: May be loose with large connecting mechanical openings; have various gas holes and body defects with limitations varying with the degree of curing; must be sufficiently compact to permit drawing a full plug. Color: May possess various defects, but not to the extent that the color is unattractive. Finish: Rind may be weak, have soft spots, rind rot, cracks, and openings, with certain limitations varying with degree of curing. Bandage may be uneven, wrinkled, but not torn; may have rough unattractive appearance, paraffin scaly or blistered; mold permitted, but not evidence that mold has entered the cheese; may be huffed, lopsided, and have pronounced high edges.

Reproduced with permission from ref. 5. These are the approximate numerical scores of each U.S. grade if scored by the score-card system. The U.S. grades are reported in letter grades only.
a

Preparation of Samples for Training. High acid cheese is such a common defect that good training samples will generally be found in a screening of products obtained from the market or the plant.

Moldy
Description. A moldy or musty flavor will resemble the smell of a damp potato cellar that is poorly ventilated. The slightly unclean flavor tends to persist after the sample has been expectorated.5 Cause. The most frequent cause is mold growth on the surface of the cheese due to the availability of air at that surface during prolonged storage. Cheese packaging is designed to exclude air at the surface of the cheese but if pinholes in the package allow entry of oxygen, mold growth is inevitable. Preparation of Samples for Training. Moldy cheese is easy to find or make. A block of cheese loosely wrapped in plastic can be placed in the refrigerator for several weeks. After a lawn of mold has grown on the cheese, cut off that mold and a thin layer of cheese under it. The cheese under that is likely to have a moldy flavor.

Rancid
Description. A rancid flavor in cheese is characterized by a short reaction time, a prominent odor that persists after expectoration, and an unpleasant, persistent, soapy, bitter aftertaste. The volatile short-chain fatty acids can usually be smelled.5 A very small amount of this flavor is part of a good cheese flavor, but amounts over and above that are very disagreeable and indicative of problems.

Cause. Rancid cheese is a result of lipase enzyme action on milk fat. The lipase may be from the raw milk or from psychrotrophic organisms growing in the milk. Breaking up of the fat globules exposes new fat surfaces on which the lipase can act. Inadvertent homogenization of raw milk or excessive agitation of raw milk in a pump or tank can cause rancidity in the milk and resultant cheese. Late lactation or mastitic milk can also become rancid.5 Preparation of Samples for Training. A small lot of rancid cheese can be made from milk that was homogenized several hours before pasteurization.

Sulfide
Description. Sulfide, skunky, or spoiled egg flavored cheese has a characteristic flavor similar to water with a high sulfur content. The sulfury odor is noticed in the nasal cavity when the air is slowly exhaled through the nose while working the cheese in the mouth. The flavor is often accompanied by a sticky, pasty body.5 Cause. Release of the sulfur from the proteins in the cheese during aging is normal and is part of the normal cheese flavor profile. Excessive release of sulfur due to unbalanced microbial and enzymatic breakdown is the cause of the sulfide flavor. Preparation of Samples for Training. Incorporation of a trace of sodium bisulfide into a small batch of processed cheese will simulate this flavor. It could also be incorporated into grated or ground cheese and molded into a cold pressed mass. Be careful not to allow asthmatic judges to taste the sample as some asthmatics may have sensitivities to sodium bisulfate.

Unclean
Description. The unclean defect lacks a definitive sensory description. Unclean flavored cheese will have a dirty, lingering, unpleasant aftertaste that persists long after the sample has been expectorated. It is difficult to get the mouth to taste clean again. It may occur in conjunction with high acid, bitter, and whey taint. It has been described as a "dirty sock" taste. Cause. Poor quality or old milk used for cheese manufacture is the cause of unclean cheese. Proteolytic and lipolytic enzymes from the psychrotrophic bacterial growth are responsible for the undesirable fermentations that occur during the aging of the cheese.5 Preparation of Samples for Training. A small lot of cheese can be made from unclean milk to produce cheese that will have this defect. Alternatively a small amount of limburger cheese may be added to a small batch of process cheese or grated cheddar cheese to simulate the defect.

Whey Taint
Description. Whey taint has been described as a slightly sweet, slightly dirty, sour whey flavor with a taste and odor characteristic of fermented whey. Some judges

describe it as an unclean flavor superimposed over a fermented/fruity flavor. The flavor is noticed almost immediately upon placing the sample in the mouth. The body will often have the characteristics of high moisture cheese and it may be accompanied by a white seams.5 Cause. Whey taint cheese is caused by poor whey expulsion from the curd and the entrapment of whey around the blocks as the cheese is cheddared. Preparation of Samples for Training. Whey taint cheese is fairly common and chances are good that a survey of a number of commercial samples will produce some with this defect. Cheese can be made to develop this defect by soaking a small amount of fresh curd in whey, pressing into a block, and aging.

Yeasty
Description. Yeasty flavor is characterized by a sour bread dough, earthy taste, and the aroma of rising bread dough. Yeasty flavor is noticed immediately as the sample is placed in the mouth. It is often accompanied by a gassy body in the cheese. Numerous holes in the cheese with regular spherical shape and shiny inside surfaces are evidence of gas production and yeast.5 Cause. Yeasty flavor cheese is caused by yeast contamination and yeast growth in the cheese. The yeast may have been introduced into the milk after pasteurization or into the cheese curd during manufacture due to uncleanliness of workers or equipment. Preparation of Samples for Training. Exposing trainees to the flavor and aroma of rising bread dough is a good demonstration of the flavors and aromas that will be found in yeasty cheese, A small batch of yeasty cheese can be purposely made by contaminating the milk with yeast prior to the cheesemaking process. A small amount of sucrose may be added to provide food for the yeast.

3.4.5.3 Body and Texture Defects


Corky Description. When cheese is corky, difficulty may be experienced in plugging the cheese with a trier due to resistance to penetration. The extracted plug resists deformation when pressure is applied to the plug by the thumb. When deformed slightly by such pressure there is a tendency to recover the original shape. When a piece of cheese is worked between the thumb and forefinger, the cheese does not work into a smooth paste but instead tends to curl up and be distributed in irregular patches. This defect is associated with dryness, opaque appearance, white seams, or acid cut color.5 Cause. This defect is most often found in cheese that is low in moisture, low in fat, or young. It can also be caused by too little acid production in the curd.

Crumbly
Description. Crumbly or friable cheese tends to fall apart when sliced. Thin slices are very difficult to cut without breaking. A plug of such cheese tends to break easily. It is often accompanied by a mealy texture and acid cut and white seam color defects. Cause. This body tends to develop in high acid and aged cheese. It also occurs in cheese that has been frozen.5

Curdy
Description. Curdy cheese has the properties of fresh cheese curd. It is firm and elastic and, when deformed by finger pressure, tends to spring back into its original shape. It is accompanied by a flat undeveloped flavor. Cause. This is a common body characteristic of *'green" cheese. Normally as proteolysis occurs and the cheese ages, this characteristic will disappear.

Gassy
Description. Gassy cheese will contain regularly distributed holes about the size of a pinhead with shiny internal surfaces. Usually the holes are concentrated near the center of the block of cheese. They are often accompanied by a fruity flavor.5 Cause. Gassy cheese is caused by growth of gas-producing organisms in the cheese. Lactococcus lactis spp. lactis var. diacetylactus or Leuconostoc sp. bacteria will cause gassiness. Coliforms introduced into the cheese due to unsanitary practices may also cause this defect. In some types of cheese this characteristic is encouraged and the cultures are selected to accomplish gas and flavor production.5

Mealy
Description. When mealy cheese is worked between the thumb and forefingers there is a lack of uniform smoothness. The body is interrupted with hard grains of cheese and it spreads in irregular patches over the forefingers. It is also felt in the mouth as the cheese is worked into a paste and rubbed against the roof of the mouth with the tongue. It is generally accompanied by a dry texture with less than the usual elasticity and a sharp flavor. White particles may be visible.5

Open
Description. Open cheese has mechanical openings throughout the body. These openings are irregular in shape and occur at the curd interface. The inside surfaces of these openings are dull in appearance, unlike the shiny inside surface of gassy cheese. This defect is not accompanied by any particular flavor defect.5 Cause. Open cheese is the result of unfavorable pressing conditions that prevent the cheese curds from completely knitting and closing. Press pressures that are too low or curd temperatures that are too cool at pressing will cause this defect.

Pasty
Description. It is difficult to get a full, well rounded plug of pasty cheese. The shape is easily distorted by pressure on the plug by the thumb. There is almost no elasticity. When worked between the thumb and forefingers, it breaks down too easily into a pasty sticky mass that adheres to the fingers. Cause. High acid or high moisture cheese is often pasty. Contamination with atypical microorganisms may also be responsible for the unusually fast and complete breakdown of the cheese body causing the defect. It is often accompanied by a fermented/fruity flavor.5

Short
Description. A plug of cheese with a short texture shows a lack of elasticity. Rather than flexing when it is bent, it breaks easily. It is often accompanied by high acid flavor or a dry or open texture.5 Cause. Shortness can be caused by openness in the body that weakens the curd or dryness that makes the cheese less flexible. It may also be caused by incomplete development and aging of the body.

Weak
Description. Weak-bodied cheese offers little resistance as the cheese plug is cut and drawn. Very little thumb pressure on the plug of cheese will break the curd. It is often accompanied by whey taint, unclean, or fermented/fruity flavor. Cause. Weak cheese is caused by retention of too much moisture or whey in the curd as it was made. The high moisture encourages the growth of unwanted organisms in the cheese, giving the unclean or fermented/fruity flavors.5

3.4.5.4 Color Defects


Acid-Cut
Description. Cheese with acid-cut color defect generally appears bleached, faded, dull, and lifeless. A thinly cut slice of cheese is opaque and lacks translucency. Cause. Cheese having the acid-cut color almost always high acid or sour flavor caused by incomplete removal of whey or moisture from the curd. The residual lactose in the curd is sufficient to allow excessive lactic acid development in the curd.5

Atypical Color Specks


Description. Cheese with this defect has white, black, or red specks or red blotches on the outside surface or the freshly cut inner surface of the cheese. Cause. Carelessness during the manufacture of cheese is responsible for dirt or rust specks being allowed into the milk or cheese curd during manufacture.5 Specks

of undissolved annatto can be present if the color solution has been destabilized. A little residual calcium chloride solution in the container to which annatto is added will cause coagulation of the color that may carry through to the finished cheese.

Color Too High


Description. This defect is characterized by high color intensity such as deep orange hue of the cheese. No particular flavor or textural defects accompany high color.5 Cause. Excessive amounts of color added to the milk are responsible for this defect. A deep color also develops on the surface of precut cheese when it becomes warm. Mottled Description. Cheese with the mottled color defect has irregularly shaped areas of light and dark color with one shade blending into the other. Often the acid-cut defect is evident certain areas with normal color between those areas.5 Cause. This defect may be the result of high moisture whey soaked curd being pressed together with normal curd such that the color defect tends to develop in some areas more than others. It may also be due to unusual microbial growth. When unusual microbial activity is the cause, yeasty, or fermented/fruity flavors are likely to accompany the defect. When nonuniform acid production is the cause, the cheese with the acid-cut defect will have an acid flavor. The defect may be caused by admixing curd with slightly different color intensities.5

Light Seams
Description. Cheese with the light seam or wavy defect is interlaced with lightcolored lines around each original piece of curd. It is most noticeable on a freshly cut surface. This defect may be accompanied by short body or crumbly texture. Cause. This defect is a result of physically altered curd surfaces before hooping. The surface may be covered by free fat due to too warm a temperature or excessive forking. The curds may be dried due to moisture evaporation, or may be unevenly salted due to poor dissolution of salt locally.5

Dark Seams
Description. Unlike the light seamed cheese, cheese with this defect has a darkened band of color between the curd particles. The dark band appears to be wider than the seam itself and is very obvious on freshly cut cheese surfaces.80 Cause. The reason for the dark appearance of the cheese in the seam is not known. Seamy cheese results when milk is warmed to cheesemaking temperatures in the cheese vat. It is avoided by bringing the milk into the vat at cheesemaking temperatures.80

White Specks
Description. Cheese with this characteristic has distinct white specks interspersed throughout the mass of the cheese. The specks vary in size and may be so small that

close examination is necessary to detect them. The larger specks may be detected in the mouth. The presence of these specks is associated with a fully developed flavor.5 Cause. White specks are indicative of mature cheese. They generally contain calcium lactate and tyrosine. The accumulation of tyrosine is indicative of the breakdown to protein associated with the aging process. Colder storage temperatures favor the growth of these particles.5

3.4.5.5 Finish Defects


High or Uneven Edges
Description. Cheese showing this defect has edges that are not square and symmetrical. They tend to curl under onto the end of the cheese, creating a protected area for mold to start growing. These thin long edges are usually quite dry and they do not cure properly.5

Lopsided, Misshapen
Description. Cheese with this defect has nonparallel sides and ends as a result of uneven distribution in the hoops coupled with nonuniform pressure across the hoop. Part of such a block may be undepressed and have a weak body and open texture.5

Uneven Sizes
Description. Cheese blocks should be uniform in size and well within tolerances for that style of cheese. This defect is called when the size variation becomes large. Carelessness in uniformly filling the hoops is the cause of this defect. Blocks of uneven size result in excessive trim loss when the blocks are cut to uniform retail sizes.5

3.4.6 Cultured Products

3.4.6.1 Introduction
Through the ages a natural way to preserve milk was to allow lactose-fermenting organisms to grow in the milk, producing lactic acid and sometimes alcohol. With the pH reduced, spoilage organism growth was discouraged. A variety of products resulted from the various starting materials and treatments applied. In many cultures, these fermented dairy products are preferred over fresh milk. The souring process thickens the body and generated desirable and interesting flavors in addition to offering extended shelf life and improved safety. Consumption of cultured products is growing in progressive countries where fresh fluid milk is preferred, due to health philosopies, trends toward ethnic foods, and changing tastes.5 Products in this class include starter cultures, buttermilk, cultured skim milk, and sour cream. Yogurt is considered a cultured product, but it is so different in character that it is treated separately. Judging of these cultured products is not very well developed. This is partly due to the lack of popularity of the products and the wide

variety of cultured products and opinions about what their characteristics ought to be. The USDA does not grade cultured products and they are not (except for yogurt) judged in competitive situations. Before shaking, the body of cultures to be used for the manufacture of cultured products should be firm and show only a minimal amount of whey. When the container is tilted, the product should break away cleanly from the side and reveal an intact "liverlike" body. When stirred, it should break down to a smooth body. It should have enough body to mound when held in a spoon. When spread thinly, it should be free of lumps. The flavor blend should include acid and diacetyl (buttery) notes and it should clean up nicely after expectoration. No foreign or atypical flavor notes should be present.5 Cultured buttermilk should have the same features as starter culture. Culture organisms used include Streptococcus lactis ssp. lactis, or Streptococcus lactus ssp. cremorous or Lactococcus lactis ssp. lactis var. diacetyllactis with Leuconostoc citrovorum or Leuconstoc dextranicum. Cultured sour cream has similar properties except that it is more viscous due to the 18% or more milk fat content. Milk or cream for either is pasteurized with extra heat treatment to improve water-holding capacity. Cream may be single-stage homogenized warm after pasteurization but before the culture is added. This clusters the fat globules and gives body to the product.5 A suggested score card complete with defect terminology appropriate for the range of cultured products is shown in Figure 3.23. A scoring guide is shown in Table 3.14.

3.4.6.2 Flavor Defects


Astringent
Description. This sensory defect is actually a tactile sensation. Other descriptive words are mouth coating, dry, puckery, chalky, and powdery. It is classified here with flavor because it is sensed when the product is taken into the mouth. It is not a common defect in beverage milk. After expectoration, the lining of the mouth may feel shriveled or puckered.5 Cause. Not all the causes are known but it is usually associated with high heat treatment of milk that has caused some aggregation of milk proteins. A specific particle size of milk proteins or other milk constituents is thought to be responsible for the sensation.5

Bitter
Description. The bitter off-flavor is detected after the sample has been in the mouth for some time and then expectorated. The bitter flavor need not be accompanied by any unusual aroma. Cause. Contaminating organisms are the expected cause of bitterness in cultured products. Breakdown of proteins into bitter peptides by these proteolytic organisms is usually responsible.5

Date: Place:

CULTURED DAIRY PRODUCT SCORE CARD Product: Buttermilk Kefir. Other. Sour cream SAMPLE NO. 1 2 3 4 5 6 10 Criticisms Score Astringent Bitter Chalky Cheesy Coarse (harsh) Cooked Fermented Foreign Green (Acetaldehyde) High acid (sour) Lacks acid (flat) Lacks culture flavor Lacks freshness Metallic/oxidized Rancid Salty (too high) Sauerkraut-like Stabilizer/emulsifier Unclean Vinegar-like Yeasty

Flavor

No criticism 10

Normal range 1-10

Body and texture No criticism 5 Normal range 1-5

Score Curdy Gassy Grainy/gritty Lumpy Too firm (Over-stabilized) Too thin (weak)

Figure 3,23 A suggested score card for the sensory evaluation of cultured milk products. (Reproduced from ref. 5, with permission). (Continued)

Cheesy
Description. Cheese cultures lack the typical cultured flavor and generally have a proteolytic flavor note and a slightly bitter aftertaste.5 The flavor and aroma are similar to that of Cheddar cheese. Cause. This flavor also is a result of contamination of the culture with proteolytic microorganisms and a breakdown of the protein and fat into components that give the cheese flavor.

Appearance No criticism 5 Normal range 1-5

5 Score Churned fat Dull (Lacks gloss) Lacks uniformity Unnatural color Wheyed-off (Syneresis)

Product Acidity

Score % Titratable acidity PH Score Short-fill Over-fill Soiled Dusty

Container and 3 Closure

Total Score Evaluators:

25

Score per Sample

Figure 3.24 (Continued)

Coarse
Description. This descriptor is one that has a broad meaning and multiple causes. It refers to a general lack of delicate appeal, flavor balance, or bouquet that constitutes a well balanced cultured flavor. It may have excessive acid or just be lacking in some of the volatile compounds that are needed for good balance.5 Cause. Culture strains that lack the ability to produce some of the important flavor compounds may be the cause of coarse flavor. That may be due to improper culture selection inappropriate propagation methods. It may also be due to overripening and be accompanied by high titratable acidity.5

Fermented
Description. The fermented flavor describes a culture or cultured product that has an acetic acid or vinegar flavor note. Cause. Organisms that produce acetic acid in considerable quantities are responsible for the fermented flavor of cultures or cultured products.5

Foreign
Description. The term foreign is used to describe a number of flavors that are imparted by addition of detergents, disinfectants, and sanitizers to milk or products made from milk. The flavor is characteristic of the chemical that has been added. The flavors are atypical of dairy products and do not develop in them. In some cases the chemical may be detected by smell but in others it may not be detected until it is tasted.5 Cause. Adding milk or milk product to a vat or running milk through piping that has been washed or sanitized but not rinsed can cause a foreign flavor especially if allowed to comingle with a considerable amount of liquid containing the chemical. Other possible causes include treating the udder with ointments or medication, contamination with insecticides, and drenching the cow with chemical treatments.5

Green
Description. Green or acetaldehyde flavored cultures or cultured products have the flavor of green apples. Acetaldehyde is a normal product of cultures and a normal note in the flavor profile of cultures and cultured products, especially yogurt. When the flavor is unbalanced because of the intensity of the green apple flavor it is said to be green.5 Cause. Green flavor is caused by inappropriate cultures or cultures that have produced excessive amounts of acetaldehyde.

High Acid
Description. A sharp acid taste in cultured products is common and expected in cultures and cultured products. It is detected by a sharp tingling sensation on the tip of the tongue almost immediately after the product is placed in the mouth. It can be accompanied by a lack or an excess of cultured flavor. Only if it is out of balance and excessive for the product is it considered a defect. Cause. The acid flavor is caused by the conversion of lactose to lactic acid by the culture organisms. It becomes excessive by allowing the culture or product to overripen before it is cooled. In a product, if the acid is excessive relative to the other flavors, citrate may be necessary to assist in production of other flavors, the inoculation rate of the lactic acid fermenter may be excessive, or the incubation time too long.5

Lacks Acid (Hat)


Description. This defect refers to a lack of the normal amount of acidic flavor in cultures or cultured products. Without sufficient acid the culture or cultured product tastes flat. The flavor is accompanied by a higher pH or lower titratable acidity than normal. Cause. The low acid flavor is caused by the production of too little acid. That may be the result of too short an incubation time, inactivity of the culture, or incubation

Table 3.14 A SUGGESTED SCORING GUIDE FOR THE SENSORY DEFECTS OF CULTURED MILK PRODUCTS WITH ASSIGNED SCORES FOR DESIGNATION DEFECT INTENSITIES
Intensity of Defect Slight5 Flavor defects3 Astringent Bitter Chalky Coarse (harsh) Cooked Fermented (vinegary) Foreign41 Green (acetaldehyde) High acid (sour) Lacks acid (flat) Lacks freshness Metallic/oxidized Rancid Salty (too high) Sauerkraut-like Stabilizer/emulsifier Unclean Yeasty Body and texture defectsf Curdy Gassy Grainy/gritty Lumpy Too firm (overstabilized) Too thin (weak) Wheyed-off (syneresis) Definite Pronounced0

7 8 8 8 9 7 6 8 9 9 8 6 4 9 7 8 4 5 4 4 4 4 4 4 4

5 5 5 6 8 5 3 7 8 8 7 4 2 8 6 7 2 3 3 3 3 3 3 3 3

3 2 2 4 6 2 (f 6 7 7 6 2 0 6 5 5 0 0 2 2 2 2 2 2 2 (Continued)

temperatures either too high or too low for the specific culture involved.5 Inactive culture can be due to sanitizers that have residual activity such as quarternary ammonia, phage specific to the culture, or a culture with poor viability. It can also be due to lack of nutrients in the media to support growth.

Lacks Culture Flavor


Description. This defect is characterized by the absence of cultured aroma and flavor. Often the flavor is a sharp high acid taste instead of the balanced cultured flavor. Cultured products that have this defect have little or no flavor appeal. Cause. This defect could be due to inappropriate culture strains, improper culture handling, or presence of general microbial inhibitors or inhibitors specific to the

Table 3.14 (Continued)


Appearancef Churned fat Dull (lacks gloss) Lacks uniformity Surface growth Unnatural color Wheyed-off (syneresis) Product acidity8 PH % Titratable acidity Container and closureh 4 4 4 1 4 4 3 3 3 0 3 3 2 2 2 0 2 2

Reproduced with permission from ref. 5. a "No criticism" for flavor is assigned a score of 10. Normal range is 1-10 for a salable prodb c uct. Highest assignable score for defect of slight intensity. Highest assignable score for defect of pronounced intensity. A score of 0 (zero) may be assigned if the defect renders the product d unsalable. Due to the variety of possible foreign off-flavors, suggesting a fixed scoring guide is not appropriate. Some foreign flavor defects warrant a 0 (zero) score even when their intensity is slight c (e.g., gasoline, pesticides, lubricating oil). An assigned score of zero (0) indicates an unsalable f product. "No criticism" for body and texture and appearance categories is assigned a score of 5. g Normal range for either category is 1-5 for a salable product. "No criticism" for product acidity is assigned a score of 2; penalty point deductions for pH or % T.A. would have to be devised for each h cultured product evaluated by this scoring system. Normal range is 1 - 2 for a salable product. "No criticism" for container and closure is assigned a score of 3; penalty point deductions would have to be devised for any assessed defects or criticisms. Normal range is 1-3 for a salable product.

flavor producing strains. If acid is being produced then the problem is with the flavor producing organisms.5

Metallic/Oxidized
Description. The first impression when tasting metallic or oxidized culture or cultured product may be that the sample is flat but as the sample is held in the mouth, a sort of puckery cardboard, papery off-flavor may become evident.5 The flavor is similar to that of a copper coin. It tends to remain in the mouth after the sample has been expectorated. Cause. This flavor is due to autoxidation in the milk used to produce culture or cultured products. It can be catalyzed by traces of copper or corrodible metal that has come in contact with the product.

Rancid
Description. There are several characteristics of rancid off-flavor. There is a characteristic odor derived from volatile fatty acids that have deesterified from the fat. Immediately after putting the sample in the mouth, the objectionable flavor may not be apparent but as the sample reaches the back of the mouth, soapy, bitter, and possibly unclean flavors are perceived. The soapy and bitter notes reside long after the sample is expectorated. A high percentage of prospective judges do not detect or have a high threshold for the soapy and bitter notes.5

Cause. Rancid flavor is usually caused by disrupting the milk fat globule while active lipase is present. The lipase enzyme, which catalyzes the deesterification of the fatty acids from the glycerol, is able to get to its substrate when the fat globule membrane is disturbed. This happens when raw milk is held static in a running centrifugal pump, when raw milk is homogenized before it is pasteurized, or when raw milk is inadvertently mixed with homogenized milk. It may also occur when microorganisms, particularly psychrotrophs, produce and release Upases into milk or cultured products from which it is made.5

Salty
Description. The descriptive term "salty" is commonly known and is a good term to describe this flavor. It is perceived quickly on placing the sample in the mouth. No aroma or odor necessarily accompanies the salty flavor. It lends a cleansing feeling to the mouth.5 Cause. Salty flavor can come from the milk but in cultured product is most often due to excessive salt added to the product.

Unclean
Description. The unclean defect lacks a definitive sensory description. Unclean flavored culture or cultured product will have a dirty, lingering, unpleasant aftertaste that persists long after the sample has been expectorated. It is difficult to get the mouth to taste clean again. It may occur in conjunction with bitter flavor. It has been described as a "dirty sock" taste. Cause. Poor quality or old milk used for cultured product manufacture is the cause of unclean cultured product. Proteolytic and lipolytic enzymes from the psychrotropic bacterial growth are responsible for the undesirable fermentations that occur during culturing or storage.

Yeasty (Cultured)
Description. The "yeasty" and "earthy" flavor and aroma reminiscent of rising bread dough is a good demonstration of the "yeasty" flavor. It is often associated with an acetic acid or "vinegar" flavor. Cause. Growth of yeast is usually responsible for this flavor but it may be due to bacterial fermentation by certain kinds of psychrotrophic bacteria. It is due to poor sanitation and lack of temperature control.67

3.4.6.3 Body and Texture Defects


Curdy
Description. Curdy buttermilk or sour cream appears to have a rough nonhomogeneous body. Curds can be seen on the lip of the container after pouring out some

of the sample and are also obvious when a small portion of the product is diluted with water. Visible curd particles settle to the bottom of the container. The curdy defect is associated with a thin body.5 Cause. Curdy texture is a result of low level of milk solids in the product base, movement or agitation of the coagulum during incubation, or inappropriate cultures for the product. Gassy Description. When cultures and cultured products that should be free of gas have this defect, there are excessive bubbles in the broken curd or streaks in unbroken coagulum where gas is escaping. If whey has separated, it will collect under or in the middle of the buoyant curd. The product will develop carbonation flavor. These characteristics are normal in buttermilk. It is a defect when it is found in sour cream and most other cultured products.5 Cause. Some cultures are gas producers. Those in buttermilk that produce flavor components are also gas producers, so gassiness is normal in buttermilk. It is not desirable in most cultures and cultured products. When it is out of place, it is usually due to contamination with gas-producing contaminant organisms such as coliforms or yeast. Unclean, fermented/fruity, yeasty, or earthy flavor defects will usually accompany microbial contamination. Gassiness can also be due to the selection of inappropriate starters.5

Grainy/Gritty
Description. Gritty and grainy sour cream is detected in the mouth. Small particles in the body of sour cream are detected by pressing the top of the tongue against the roof of the mouth and noting a mealy feel. Cause. A grainy defect in cultures or cultured products is often due to incompletely dissolved dry ingredients in the product base.5 Lumpy Description. Lumpy cultures or cultured products have large lumps of firm curd interspersed throughout the product. The body may otherwise be normal. Cause. Lumpiness is an aggravated case of the curdy defect caused by premature agitation of the product as it is being incubated. Low solids in the product mix favors the curdy and lumpy defect.5

Ropy
Description. Ropy cultures and cultured product tends to string out as the product is poured or spooned. When product is poured, a continuous string of the product stretches from the container to the product below like thin syrup or mucus. It does

not plop and break. When a spoon full is lifted from the surface as it is poured the same effect is observed. Cause. Ropy defect is usually due to polysaccharide producing bacteria in the culture. In some cultured products this internal stabilization system is desirable. Dutch yogurt is famous for its ropy characteristics and some types of domestic yogurt utilize this type of culture. It is considered a defect when it is excessive or unwanted and is likely due to contamination with inappropriate gum-producing organisms.5 It can also be due to partially broken down stabilizers. Some types of starch, for example, are stringy or can be made to be so by excessive shear.

Too Finn
Description. Product is too firm when it has excessive viscosity and resists pouring. In the case of sour cream it refers to the inability to stir it with a brittle, lightweight plastic spoon without breaking the spoon. Another way to judge sour cream in the original container is to insert a spoon or small spatula near the edge of the product and twist it. If the entire contents of the container turns with the spoon, it is too firm. The product may appear dull and lack the usual sheen.5 Cause. Product that is too firm is generally due to excessive use cf stabilizers or excessive solids levels in the product mix.

Too Thin
Description. Weak-bodied cultures and buttermilk are observed by tilting the intact coagulum to a 45 angle. If the product breaks and flows, it is too weak. The broken agitated curd will break and flow too readily when it has lower than typical viscosity. Low culture titratable acidity ^0.75 often accompanies this defect. Weak buttermilk drips and splashes similar to water and exhibits a dull appearance. It is quite subject to wheying off. Weak sour cream is too thin to spoon onto a potato and once there will not mound and stay in place. It is too thin to have a generous amount cling to a chip when used as a dip. Cause. Thin cultured products can be due to understabilization or too low a solids level in the product mix. It can also be caused by heat treatment of the mix insufficient to denature the whey proteins and potentiate their natural stabilizing ability. Other possible causes are impaired culture activity or excessive proteolytic activity.5

3.4.6A Appearance Defects Dull


Description. This defect occurs in sour cream and is observed as a dull matt appearance to the surface of the product. Sour cream should have a glossy sheen or silky appearance. Cause. A dull appearance often accompanies a thick, overstabilized body.5

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Lacks Uniformity
Description. appearance. This defect is called when the product lacks a homogeneous uniform

Cause. Nonuniform appearance is generally caused by incomplete mixing of the ingredients or absence of or inadequate homogenization of the product mix.

Surface Growth
Description. This defect is easily observed. When colonies or microbial growth are observed on the undisturbed surface of the coagulum or in the headspace of a cultured product, this defect is called. It is found only in product that has been stored refrigerated for weeks. Cause. Product with this defect has been contaminated due to unsanitary practices after processing and prior to or at the filler. The surface of the product serves as the growth medium and air in the headspace supports the growth of the aerobic yeasts and molds. Consumer product can develop this defect when product is opened and partially used, then recovered and placed back in the refrigerator and forgotten.

Unnatural Color
Description. This is a rare defect. It may be present in sour cream as a snowy white appearance lacking the usual cream color. It may be present in buttermilk-containing butter granules that are too light in color to have the necessary contrast. Any time the color of the product is not within the normal range, it is appropriate to assign this defect. Cause. White sour cream and pale butter granules are caused by lack of carotene in the butterfat. The carotene content is low in the winter when the cows are in the feed lot. Other cases of off color may be due to inadvertent inappropriate coloring of the product.5

Wheyed-Off
Description. This defect is observed in unbroken coagulum as a shrunken coagulum with free whey around the edges or pooled on areas on the top of the curd. In buttermilk the whey may be found under the floating curd. Cause. This defect is caused either by understabilization or by inability of the milk proteins to hold the water. Selection of appropriate stabilizers and stabilizer levels will help discourage syneresis. Proteins can be made to hold a maximum amount of water by increasing the pasteurization temperatures or holding times to denature the whey proteins.5

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3.4.7 Yogurt

3.4.7.1 Introduction
Yogurt is the oldest known fermented milk product dating back to at least 5000 B.C. It has been a staple for people in eastern Mediterranean countries and has been known by at least 13 different names. It grew in popularity in Europe in the early 1900s after claims that it would prolong life were circulated. It was successfully introduced into the United States in 1939 on the east coast.5 Flavored, fruited, and sweetened varieties have grown in popularity throughout the United States. Two bacteria, Lactobacillus delveccii ssp. bulgaricus and Streptococcus salaverious ssp. thermophilous, work together in warm (29 to 45C) milk to produce this acidic "green apple" flavored product. Body varies from thin and drinkable to thick and custardlike.5-81'82 Traditionally the milk used had been boiled to increase the solids, add body, and prevent syneresis. The same effects are now achieved by fortification with powder, condensation by vacuum or membrane, then applying a heat treatment to the mix to denature the whey proteins to improve water binding and prevent syneresis.5'63'74 The federal definition of yogurt specifies the cultures, sets the minimum fat, solids, and acidity levels, and allows pasteurization after culturing.75 It also lists allowable sweeteners. Recently, aspartame was approved for use in yogurt.76 Yogurt is a comparatively new product in the United States, so procedures for its evaluation are new and not yet as uniform as for the other products. The USDA does not grade yogurt and collegiate judging of strawberry prestirred yogurt began in 1977. As a rule, 6- or 8-oz. single serving units are evaluated. The cartons are covered or placed inside another carton to conceal their identity. Temperature of evaluation is between 1.7 and 100C to standardize the effect of temperature on body. Before the sample is disturbed, the top of the sample is examined for surface growth, shrinkage away from the sides of the container, and wheying off. Color and overall appearance are observed in the undisturbed sample. Some product is then spooned onto a plate. With the spoon full of sample, it is held up to eye level and examined. A moderate mounding is desired. The spoonful of product is examined for several color, appearance, body, and texture qualities. The aroma is observed and the sample is placed in the mouth. In the mouth, the yogurt is manipulated while observing several body, texture, and flavor qualities. Initial, midpoint, and delayed taste sensations are noted. The basic tastes are observed first, then the aromatic flavors emerge as the product warms. The sample is expectorated and aftertaste and' * clean up" are noted.5 The ADSA score card for Swiss-style flavored yogurt is shown as Figure 3.24, the scoring guide is shown in Table 3.15, and the Collegiate Contest Yogurt Score Card is shown as Figure 3.25.

3.4.7.2 Flavor Defects Acetaldehyde


Description. Synonymous descriptive terms for acetaldehyde flavored yogurt are coarse, green, and green apple flavor. Acetaldehyde is a normal component of yogurt

CONTEST SWISS STYLE YOGURT SCORE CARD


Flavor: Date: Costumer No. A.D.S.A. Criticisms Contestant Score Score Grade Criticisms Acetaldehyde (coarse) Bitter Cooked Foreign High acid Lacks fine flavor Lacks flavoring Lacks freshness Lacks sweetness Low acid Old ingredient Oxidized Rancid Too high flavoring Too sweet Unnatural flavoring Unclean Contestant score Score Grade No criticism 5 Normal range 1-5 Appearance 5 Gel-like Grainy Ropy Too firm Weak Criticisms 1 2 3 SAMPLE NO. 4 5 6 7 8 Total Grades

Flavor

10

No criticism 10

Normal range 1-10

Body and texture 5

Contestant score Score Criticisms Atypical color Color leaching Excess fruit Free whey Lacks fruit Lumpy Shrunken Surface growth Grade

No criticism 5 Normal range 1-5

Total

Total score of each sample Total grade per sample Source: American Dairy Science Association (1987)

Final grade Rank

Figure 3.24 The ADSA contest score card for swiss-style yogurt. (Reproduced from ref. 5, with permission of the ADSA, Champaign, IL.)

Table 3.15

THE ADSA SCORING GUIDE FOR SENSORY DEFECTS OF SWISS-STYLE YOGURT (SUGGESTED FLAVOR, BODY AND TEXTURE, AND COLOR AND APPEARANCE SCORES FOR DESIGNATED DEFECT INTENSITIES) Intensity of Defect Slight Definite Pronounced

Flavor criticisms Acetaldehyde (green) Acid (too high) Acid (too low) Bitter Cooked Foreign Lacks fine flavor Lacks flavoring Lacks freshness Lacks sweetness Old ingredient Oxidized/metallic Rancid Too high flavoring Too sweet Unclean Unnatural flavoring Yeasty Body and texture criticisms0 Gellike Grainy/gritty Ropy Too firm Weak, too thin Appearance criticisms0 Atypical color Color leaching Excess fruit Free whey Lacks fruit Lumpy Shrunken

9 9 9 9 9 8 9 9 8 9 7 6 4 9 9 6 8 6 4 4 3 4 4 4 4 4 4 4 4 4

7 7 8 7 8 7 7 8 7 8 5 4 2 8 8 4 6 4 3 3 2 3 3 3 3 3 3 3 3 3

5 5 6 5 6 6 5 7 6 7 3 1 0b 7 7 1 4 2 2 2 1 2 2 2 2 2 2 2 2 2

Source: American Dairy Science Association, 1990 a "No criticisms" is assigned a score of 10. Normal range is 1-10 for salable product. b An assigned score of 0 (zero) is indicative of unsalable product. c "No criticisms" is assigned a score of 5. Normal range is 1-5 for salable product.

M A R K I N GI N S T R U C T I O N S unwosHNca^y P R O P E R M IP R O P E R M A R K M A R K S E R A S EC H A N G E SC L E A N L YA N D C O M P L E T E L Y D ON O TM A K EA N YS T R A YM A R K S C R I T I C I S M S F L A V O R
R(TTEB

A T E P R C O M T E S T A N TN O D SWISS STYLE YOGURT

N C ST r a n a O p c t i* M P 3 0 7 3 S 2 S 3 2 1 A 2 4 0 0 S A M P L EN U M B E R

NO CRITICISM FOREKSN 10 NORMAL RANGE 1-10


LQWACID

LACKS FWE FLAVOR


LACKS <SMftSS

OXlOiXCO TOO HKSsK $UWORlNG MNWAfUWAt n>VQ8*6

ViASTV

BODY A N D TEXTURE NO CRITICISM 5


TOOFWM GRAtNY

NORMAL RANGE 1-5 APPEARANCE A N D COLOR NO CRITICISM 5


EXCtSSFRUtT COLOR LEACHiMO

NORMAL RANGE 1-5

L A C K S FRUIT SHRUNKEN

Figure 3.25 Collegiate contest swiss style yogurt score card. (Reproduced from ref. 5, with permission of the ADSA, Champaign, IL.)

flavor but when it overpowers the other flavor note, it is a defect. The characterizing flavor should predominate with acid, acetaldehyde, and sweetness filling in. Cause. Acetaldehyde is produced by the Lactobacillus delveccii ssp. bulgaricus culture organisms at incubation temperatures above 37.8C. In plain yogurt, typical acetaldehyde levels are 5 to 40 ppm. The threshold is about 12 ppm. Overripening at higher incubation temperatures may produce excessive amounts of acetaldehyde and produce this defect. Lack of sweetener will also allow the acetaldehyde flavor to come through too strongly.5 Preparation of Samples for Training. Demonstration samples can be prepared by adding acetaldehyde at about 30 to 60 ppm into strawberry-prestirred yogurt. A little stock solution can be made in milk which is added to the yogurt dropwise while stirring until the flavor is at a desired level.

Bitter
Description. Bitter is one of the basic taste sensations that does not necessarily have an associated aroma. It is detected at the base of the tongue. The reaction time is fairly slow so it is most strongly sensed after the yogurt is expectorated. The intensity builds and it is hard to rinse away and refresh the tongue. Cause. Bitter yogurt results from contaminated yogurt cultures, poor quality milk ingredients that have been contaminated with psychrotrophic microorganisms, or extremely poor quality fruit or flavoring. Preparation of Samples for Training. Solutions of 1% quinine sulfate may be added to yogurt. Add 2 ml for a slight and 4 for definite.5

Cooked
Description. A mild sulfur-like, slightly nutty, cooked egg white flavor may occasionally be encountered in yogurt. Yogurt and fruit flavors are effective masking media so the cooked defect has to be strong to be noticed. It is objectionable only when definite or pronounced. It may detract from the intended refreshing characteristic of the yogurt. It is most easily detected when the yogurt lacks acid or lacks flavoring. Cause. Yogurt mix is usually given a severe heat treatment to denature the whey proteins and give syneresis resistance and body to the product. This treatment develops a cooked flavor. The strength of the competing and covering flavors determines whether or not the cooked flavor will be noticed. Preparation of Samples for Training. Yogurt mix can be heated to 900C and held for 30 min prior to cooling and culturing. It can be broken and cooled at pH 4.5, lightly flavored, and lightly sweetened prior to tasting.

Foreign
Description. An off-flavor that is entirely unlike any off flavor that might be anticipated to develop in yogurt. This descriptor is reserved for the flavors introduced

by inadvertently added chemical. The expected characteristics are as varied as the chemicals that might be causing the flavors. Cause. Most of these atypical flavors are caused by cleaning compounds, chlorine, iodine, or phenol.5 Any one of many compounds that are inadvertently added to product or whose fumes are absorbed by product may be responsible for the flavor. Preparation of Samples for Training. A version of foreign flavor caused by sanitizer can be produced by adding 1A ml of a 5% sodium hyperchloride solution to 300 ml of good yogurt. In the same manner, traces of other nontoxic chemical cleaners and sanitizers could be used to taint cream which in turn will taint cottage cheese.5

High Acid
Description. Yogurt is a tart product normally. This defect refers to cases where the tartness is overpowering the other flavors in the system. The high acid defect is often confused with the '' green apple" o r ' ' acetaldehyde'' defect. High acid flavored yogurt is usually below pH of 3.8. It usually dissipates quickly and leaves a refreshed feeling in the mouth. Cause. High acid yogurt is caused by an extended incubation period, high incubation temperatures, or insufficient cooling at the end of the incubation and flavoring process. Other acids in the fruit flavoring can contribute to the excessive acid defect.5 Preparation of Samples for Training. High acid is a common defect in yogurt. Food training samples are probably available on the retail shelf. If samples need to be generated, it can be done by making a small batch of yogurt and incubating until the pH drops well below 3.8. It can also be made by adding citric, malic, or lactic acid to a sample of good quality yogurt sufficient to reduce the pH into that range.

Lacks Fine Flavor


Description. This defect refers to a flavor system that is out of balance. Adjustments in the flavoring system, the quantity of flavoring, the acid level, or the acetaldehyde level are probably all that would be necessary to balance the flavor and achieve a pervect score. It is not far enough off in any one of those factors to be called high or low acid, acetaldehyde, or lacks flavor. Cause. Slight deficiencies or excesses in the acid, acetaldehyde, or flavor level are to blame for this defect.5 Preparation of Samples for Training. A survey of samples available on the market is the best way to find samples with this defect. It is a fairly common defect. One might also blend excellent product one at a time with products that have acetaldehyde, high acid, low acid, or lacks flavoring defects.

Lacks Flavoring
Description. This defect is characterized by a weak characterizing flavor impact. Sweetness, acid, and acetaldehyde flavor overpower the characterizing flavor.5

Cause. The cause of this defect is obviousthe use of too little flavor either purposefully or accidentally. Because good fruit is expensive, the temptation is to minimize the amount that is added. Carrying this too far will result in yogurt that lacks flavoring. Preparation of Samples for Training. This defect can be simulated by blending a good quality yogurt with plain yogurt and sweetening to the appropriate level. The proportions blended will depend on the flavor intensity of the good yogurt and the degree of this defect desired.

Lacks Freshness
Description. Yogurt with this defect is characterized by a stale aftertaste. The flavor of aged milk powder or whey powder is evident in the finished product. This flavor comes on late after the product has been in the mouth a while. The stale aftertaste remains in the mouth after the sample has been expectorated. When the flavor becomes so strong that it is no longer a background flavor, the term old ingredient is used. Cause. The cause of the lacks freshness defect is usually the use of old stale powdered milk or condensed milk to build the solids of the yogurt mix. It can also be due to the use of old stale fruit in the flavoring system.5 Preparation of Samples for Training. If a sample demonstrating this defect cannot be found on the market, one can be made by formulating a small batch of yogurt at home or in the laboratory and purposely using stale powdered milk to build the body to about 12 to 14% milk solids. Another quicker method is to incorporate stale ingredients into finished yogurt. Stirring a small amount of powder into good flavored yogurt may make a reasonably representative sample. The amount of stale powder used is adjusted to achieve the desired intensity of flavor. The powder is worked to a paste in a small portion of the yogurt and then mixed into the sample.

Lacks Sweetness
Description. There is a broad range of sweetness that is acceptable in flavored yogurt. Usually 4% to 12% sucrose is needed to balance the acid and the intensity of the characterizing flavor. When the amount of sweetener is insufficient, this defect is called. It is one of the more confusing yogurt flavors because experts and consumers are not necessarily in agreement as to what is an appropriate level of sweetener. Cause. The lack sweetness defect is caused by insufficient sweetener in the flavored yogurt to balance and enhance the other flavors of yogurt.5 Preparation of Samples for Training. This defect can be simulated by blending an ideal yogurt with plain unsweetened yogurt and then adding flavors sufficient to give a good characterizing flavor and not adding additional sweetener.

Low Acid
Description, Yogurt that has a low acid defect lacks the tart, refreshing character of normal yogurt. It tends to taste more like a neutral pudding than yogurt. The pH of low acid product will be above 4.5. Cause. Low acid yogurt may be caused by imbalanced yogurt culture with a scarcity of acid-loving Lactobacillus delveccii ssp. bulgaricus organisms, underactive cultures due to an inhibitor, insufficient heat treatment of the base, or excessive sweetener level.5 Preparation of Samples for Training. Demonstration product can be made by formulating a small laboratory or home batch and purposefully cutting short the incubation by breaking, flavoring, and cooling as soon as the coagulum is formed at around pH 4.6. It can be accentuated by adding a little excess sweetener.

Old Ingredient
Description. Old ingredient is an extension of the lacks freshness defect. When the stale flavor becomes very obvious and predominates over the acid, acetaldehyde, and characterizing flavors and the sweetness, this descriptor is applied. The stale flavor masks the expected refreshing flavor of the yogurt. Cause. Use of stale milk powder, whey powder, or condensed milk in the yogurt mix is probably the cause of old ingredient defects. It is occasionally caused by stale stabilizer or emulsifier.5 Preparation of Samples for Training. A sample demonstrating this defect can be made by making a small batch or yogurt at home or in the laboratory and purposely using stale powdered milk to build the body to about 12 to 14% milk solids. Another quicker method is to incorporate stale ingredients into finished yogurt. Stirring 2 to 4% stale powder into good flavored yogurt may make a reasonably representative sample. The powder is worked to a paste in a small portion of the yogurt and then mixed into the sample.

Oxidized
Description. Oxidized yogurt has cardboard, tallow, or metallic flavors. It is very uncommonly noticed because of the masking effect of the strong yogurt flavor. Cause. Oxidized yogurt is caused by the use of ingredients in the mix that have developed the defect. The metallic oxidized defect is caused by the presence of corrodible metal in the lines or tanks that come in contact with the mix. These metal ions catalyze lipid oxidation. The sunlight oxidized flavor is caused by exposure of milk to sunlight or fluorescent lights, causing a reaction that involves the riboflavin and causing the cardboard or burnt feathers flavor. That flavor is common in retail milk. Preparation of Samples for Training. Oxidized yogurt can be made by generating oxidized milk either by exposure to sunlight or exposure to copper or CuSO4

followed by a period for development of the flavor, and then using that milk to make a small batch of yogurt. An alternate method would be to add about 10 to 20% of intensely oxidized milk to good quality yogurt.

Rancid
Description. There are several characteristics of rancid off-flavor. There is a characteristic odor derived from volatile fatty acids that have deesterified from the fat. Immediately after putting the sample in the mouth, the objectionable flavor may not be apparent but as the sample reaches the back of the mouth, soapy, bitter, and possibly unclean flavors are perceived. The soapy and bitter notes reside long after the sample is expectorated. A high percentage of prospective judges do not detect or have a high threshold for the soapy and bitter notes.5 Cause. Rancid flavor is usually caused by disrupting the milk fat globule while active lipase is present. The lipase enzyme, which catalyzes the deesterification of the fatty acids from the glycerol, is able to get to its substrate when the fat globule membrane is disturbed. This happens when raw milk or product mix is held static in a running centrifugal pump, when raw milk is homogenized before it is pasteurized, or when raw milk is inadvertently mixed with homogenized milk which is subsequently used as an ingredient for yogurt. In making yogurt it is quite possible that raw and homogenized ingredients are mixed inadvertently. It may also occur when microorganisms, particularly psychotrophs, produce and release lipases into dairy ingredients used to make yogurt.5 Preparation of Training Samples. Rancid milk can be prepared by adding equal quantities of raw milk to freshly pastuerized and homogenized milk and holding several hours cold while the flavor develops.5 The rancid milk can either be used as an ingredient to make a small batch of yogurt normally or pasteurized rancid milk can be blended into good quality finished yogurt at the rate of 10 to 20%.

Too High Flavoring


Description. Yogurt with this defect has too intense a characterizing flavor. It may be excessively aromatic and quite out of balance. What are supposed to be delicate flavor notes seem harsh. Cause. Miscalculation or lack of control in blending flavor materials or in adding those flavors to yogurt are responsible for excessive flavor. Preparation of Samples for Training. Yogurt fruits generally come complete with the flavorings included. Excessive fruit/flavor blend can be added to the yogurt to simulate this flavor. In this case both the show of fruit and the flavor would be excessive. One could obtain just the flavor from the fruit supplier or a flavor house and add an extra dose of flavor to ideally flavored and fruited yogurt.

Too Sweet
Description. When the sweetness of the yogurt is so strong that it overpowers the flavor system and the acid, it is criticized for this defect. The sweetness should be

just enough to complement the berry or fruit flavors and balance the acid but not strong enough to cover it. Cause. This is a common defect of yogurt in the United States. Our sweet tooth encourages many yogurt makers to add excessive sweetener, thinking that the consumer finds it more acceptable. It may also be caused by formulation error. Preparation of Samples for Training. Ideal yogurt can be made to exemplify this defect by adding an additional 5% sucrose and stirring until it is dissolved.

Unnatural Flavoring
Description. Yogurt is unnaturally flavored when the character of the flavor does not agree with the flavor on the label.5 For example, when the strawberry yogurt tastes like cherry or when banana flavor has found its way into the vanilla yogurt it is unnaturally flavored. The flavor may not be identifiable but also not characteristic of the labeled flavor. Cause. Unnatural flavoring can be caused by inadequacies in the flavor source or use of uncharacteristic flavor enhancers or other natural flavors (WONF) that are intended to extend the flavor but succeed only in changing its character. Another cause is lack of control or changes in the process will alter the flavor and cause this defect. Preparation of Samples for Training. There are a lot of possible variations of this defect. A good judge would need to be able to recognize departures from normal flavor. One could obtain a good set of characterizing flavor systems without fruit from flavor houses and add traces to good quality yogurt to give uncharacteristic flavor notes. With a little more effort, fruit and color systems without flavor could be made or obtained. Addition of a fruit and color system of one character and a flavor system of another would result in good training samples.

Unclean
Description. Unclean flavored yogurt is noticeable, unpleasant, and serious. The judge will note a dirty flavor or unpleasant aftertaste that lingers after the sample has been expectorated. It discourages a consumer or a judge from taking a second taste. A "dirty sock" or limburger flavor would be classified as unclean. Cause. The unclean flavor is due to the proteolysis of proteins producing volatile products. Some of the amines such as putricine or cadaverine produced are particularly offensive.5 Preparation of Samples for Training. Working a little limburger cheese into a thin paste and blending it into the yogurt will give product with an unclean character.

Yeasty
Description. The "yeasty" and "earthy" flavor and aroma reminiscent of rising bread dough is a good demonstration of the "yeasty" flavor. It is often associated with an acetic acid or "vinegar" flavor.5

Cause. Growth of yeast is usually responsible for this flavor but it may be due to bacterial fermentation. Certain kinds of psychotrophic bacteria can be responsible for this objectional off flavor. It is due to poor sanitation and lack of temperature control.67-68 Preparation of Samples for Training. Having students smell and taste rising bread dough will acquaint them with the flavor and aroma of products that have this character.

5.4.7.3 Body and Texture Defects


Gellike
Description. When yogurt has this gellike defect, a spoonful of yogurt viewed at eye level will have a high ridge with sharp edges and tend to jiggle like a gelatin dessert when it is moved. Product released from an inverted cup will retain the shape of the cup. It may have a higher degree of gloss than normal and "slick'' gelatinlike mouth feel. As the product is eaten, it offers some resistance and breaks apart into small chunks. That texture dulls some of the refreshing characteristics expected in yogurt.5 Cause. The gellike defect is caused by excessive use of gelatin or other gel-forming stabilizers. It is often done purposely to give the product stability and resistance to syneresis through distribution. Some manufacturers believe the consumer actually prefers that type of yogurt.

Grainy
Description. The grainy defect is best detected in the mouth. Small hard grains will be evident in the body of the yogurt as the tongue is pressed and rubbed against the roof of the mouth. Cause. Grainy yogurt can be caused by incomplete hydration of dry ingredients into the mix, acid development that is too rapid or excessive, incubation temperature too high, homogenization at too high a temperature, excessive amounts of culture, inappropriate stabilization system, or improperly blended yogurt base with fruit.5

Ropy
Description. Ropy yogurt tends to string out as the product is poured or spooned. When product is poured, a continuous string stretches from the container to the product below like thin syrup or mucus. It does not plop and break. When a spoon is immersed and lifted 5 to 8 cm above the yogurt surface, the yogurt strings and stretches like taffy or glue. Cause. Ropy defect is usually due to polysaccharide producing Lactobacillus delveccii ssp. bulgaricus strains in the culture.5 In some yogurt products this internal stabilization system is desirable. Dutch yogurt is famous for its ropy characteristics

and some types of domestic yogurt utilize this type of culture for stabilization. It is considered a defect when it is excessive or unwanted and is likely due to contamination with inappropriate gum-producing organisms. It can also be due to partially broken down stabilizers. Some types of starch, for example, are stringy or can be made to be so by excessive shear.

Too Firm
Description. When yogurt exceeds the consistency of a light custard it is too firm. A spoonful of yogurt viewed from the side at eye level will appear rounded and mounded high. In the mouth it gives the impression of heavy pudding and does not give the refreshing feeling of the ligher bodied product. Cause. Yogurt that is too firm is generally due to excessive use of stabilizers or excessive solids levels in the product mix.5

Weak
Description. Weak yogurt has a thin consistency. When a spoonful is viewed from a side profile, the product is not mounded in the spoon and the surface is flat. Some of the spoon's contents may spill over the edge of the spoon. When spooned or poured out into a dish or on a plate it flattens and does not mound at all. Cause. Causes of weak yogurt are understabilization, low levels of milk solids in the mix, under-incubation such that the product has not fully ripened, or too low a pasteurization temperature to convert the protein system into a good water binder.5

3.4.7.4 Appearance and Color Defects


Atypical Color
Description. When the hue or intensity of the yogurt color does not match the labeled flavor, it has the atypical color defect. This defect is also called when the color is dull or has a gray appearance or when the product is under-colored. The acceptable range is very broad. Strawberry yogurt exhibits this defect if it is almost white, bright pinkish red, or too blue. Cause. Atypical color for strawberry Swiss-style yogurt is caused by color addition that makes the yogurt too light, too vivid, too blue, or too pink. The use of recommended levels of high quality flavorings and colorings at an appropriate pH, following the supplier's recommendations, will usually give an appropriate color.

Color Leaching
Description. The color-leaching defect applies to flavors that have piece integrity when the color in the pieces migrates to the yogurt. It becomes obvious when the yogurt is spooned and the color from the pieces streaks across the cut surface of the yogurt.

Cause. Leaching color is difficult to prevent and is most obvious when the fruit is highly colored. It is aggravated by large fruit and berry pieces, colors that are not acid stable in the pH range of 3.8 to 4.3, and incomplete blending of the fruit with the yogurt base before the filling operation.5 Excess Fruit Description. Since the fruit portion of yogurt is very expensive, this defect is not encountered often. When it does occur, the yogurt will have an excessive show of fruit. The cut surface of the yogurt will have more than the typical number of fruit pieces and it will likely be accompanied by a higher level of color. The body may be somewhat weak due to the dilution of the yogurt with excessive fruit.5 Cause. The cause for excess fruit is usually operator error. Rarely does a manufacturer purposely load the product down with excessive fruit. It may also occur due to incomplete blending of fruit and yogurt so that the fruit is concentrated in portions of the yogurt. Free Whey Description. The free whey defect refers to the expulsion of a clear fluid (whey) from the curd. An undisturbed cup of yogurt exhibiting this defect will have clear fluid around the edges or/and a puddle of whey on the top of the gel. When the cup is tipped, it will run to one side and be more easily seen. In disturbed product it will puddle in the depressions where the product has been spooned out. In the collegiate contest, this judgment is made in an undisturbed cup. Cause. Tendency for yogurt to syneresis or whey off is aggravated by excess or insufficient acid development, disruption of the yogurt by shaking or inverting the carton, low milk solids, or insufficient heat and holding time during pasteurization to give needed water binding character to the protein system.5 Lacks Fruit Description. In berry or fruit flavored yogurt, there is expected to be a certain show of fruit. When the product is spooned or cut, a number of pieces are exposed on the surface to give the impression that a reasonable amount of fruit was used in the flavoring. A scarcity of fruit or berry pieces on that cut surface is indicative of this defect. It is common to have a good flavor impact but very little fruit. Cause. This defect is generally caused by economizing on fruit and using too little flavoring material or too few pieces in the flavoring material. It may also be caused by incomplete blending of the flavoring material with the ripened yogurt mix before the filling process began yielding portions of yogurt that were not sufficiently fruited.5

Lumpy
Description. Lumpy yogurt is characterized by its resistance to stirring to a smooth texture. Instead, it forms individual lumps resist breaking up and smoothing out. It is usually accompanied by a gellike body. Cause. Lumpy appearance in yogurt and gelled body often go together and have similar causes. They are caused by excessive use of gelatin or other gel-forming stabilizers. It is often done purposely to give the product stability and resistance to syneresis through distribution.5

Shrunken
Description. This defect is characterized by the pulling away of the coagulum from the sides of the cup due to the contracting of the coagulum mass. It looks like the product has been reduced in volume. It is usually accompanied by the collection of free whey in the space that is created. Cause. Tendency for yogurt to shrink is caused by some of the factors that cause free whey to develop. It is aggravated by excess acid development and by low milk solids in the mix.5

3.4.8 Dry Milk

3.4.8.10 Introduction
Removal of water is an effective way of preserving dairy products. The low water activity arrests the growth of spoilage organisms. Drying minimizes the weight and volume making shipping and storage more efficient. Drying also makes possible addition of dairy products to formulated dry or concentrated mixes. Baking mixes, baby formulas, and drink mixes are examples of products that contain dried dairy products and only dried products would do. Several drying processes are available. Drum drying, spray drying, and freeze drying are examples. By far the most commonly practiced is spray drying. A concentrated (vacuum evaporated) mixture is pumped through an atomizer which finely divides the liquid into droplets that are ejected into a down draft of hot dry air. With the large surface area per unit volume and high temperatures, the water evaporates in seconds. Before the droplet hits the wall of the dryer, it is dry enough that it will not stick to surfaces. The dry particles are separated from the air by gravity and by centrifugal force in cyclones. The residual is removed by porus bags through which the air flows and in which the powder collects. Temperatures of air-powder mixtures in the dryer are reduced by evaporative cooling so that the final dry powder need not be very hot in a well balanced system. Some dried products may tend to ball up when water is added, making it difficult to rehydrate. Powders may be instantized to overcome this difficulty.77'78 The commonly used method involves exposing cascading powder to steam or a fine water mist. The particles are partially rewetted and as they fall to the bottom of the

Table 3.16 U.S. GRADE CLASSIFICATION OF NONFAT DRY MILK (RELIQUIHED BASIS) BASED ON FLAVOR AND ODOR
Flavor Classification Flavor Characteristics Bitter Chalky Cooked (spray and instant) Feed Flat Oxidized Scorched Roller Spray and instant Stale Storage Utensil U.S. Extra Grade U.S. Standard Grade3 Slight Definite Definite Definite Definite Slight Definite Slight Slight Slight Slight

Slight Slight Slight Slight

Slight

Reproduced with permission from refs. 5 and 28. a Applies only to spray and roller process. Only one grade. "U.S. Extra." is recognized for instant nonfat dry milk.

chamber, they stick to one another and pile up in a loosely packed porus layer. This material is redried and ground. The more open structure and the crystalline lactose facilitates controlled, complete, and rapid rehydration. Some commonly dried milk products are skim milk, milk with varying fat contents, buttermilk, whey, yogurt, and cheese. Standards of Identity for dry whole milk and nonfat dry milk are found in The Code of Federal Regulations Title 21. 5 7 Grading standards for dried milk, cream, and whey are found in Title 7.28 Two grades are established, Extra and Standard. Beverage nonfat dried milk must meet the standards for Extra Grade. The flavor and appearance criteria for the two grades of nonfat dried milk are shown in Table 3.16 and Table 3.17 respectively. In addition to the flavor and appearance criteria, there are compositional and microbiological criteria. These criteria vary only slightly for dried whole milk. A suggested dry milk products score card is shown in Figure 3.26 and a scoring guide is shown in Table 3.18. The flavor and appearance defects not already covered in other products are described below.

3.4.8.2 Flavor Defects


Scorched
Description. The flavor of scorched milk is more intensive than cooked. It is a flavor that is associated with burnt protein. A characteristic burnt aftertaste is also part of the flavor profile. Cause. Scorched flavor is likely to occur in dried products that were dried under excessive heat or stayed in the drying chamber or remained on the contact surfaces

Table 3.17 U.S. GRADE CLASSIFICATION OF NONFAT DRY MILK BASED ON PHYSICAL APPEARANCE CHARACTERISTICS
Classification Physical Appearance Characteristics3 Dry Product Lumpy Unnatural color Visible dark particles Spray Roller Instant Reliquified Grainy Spray Roller Instant U.S. Extra Grade Very slight0 U.S. Standard Grade6

Slight Slight Slight Definite

Very slight Slight

Slight

Slight Slight

Reproduced with permission from refs. 5 and 28. a In general, the dry product shall be white or light cream in color and shall not exceed the intensities of other characteristics as indicated. b Applies only to spray and roller process. Only one Grade, "U.S. Extra" is recognized for instant nonfat dry milk. c Instant product must be reasonably free-flowing (i.e., pours in a fairly constant, uniform stream from the open end of a tilted container or scoop).

too long. It is often accompanied by the presence of scorched particles and darkening of the color. It is more commonly associated with roller dried powder than spray dried powder.5

Stale
Description. Stale powders are the source of stale flavors in so many other dairy products in which milk powders are used. Other descriptors used are lacks freshness, glue like, storage. It is a very distinctive flavor that gets meaning and definition at the first taste of reconstituted stale milk powder. A darkening of the powder follows the development of stale flavor. The stale flavor will be noted before any darkening occurs. Cause. Oxidation of the milk proteins and the milk fat in powders is difficult to prevent because of the vast surface area and the intimate contact with oxygen in the air. This flavor develops even in nitrogen-packed powders because of the presence of some oxygen. If the solution to this problem was found, the acceptability of dried products would take a giant leap forward.5

Chalky
Description. Chalky milk powder refers to powder that, when rehydrated, has the feel of fine insoluble chalk particles. It is as much an objectionable mouth feel

SENSORY EVALUATION OF CONCENTRATED AND DRY MILK Product: Date: SAMPLE NO. 1 Flavor 10 Score Criticism Acid Astringent Bitter Chalky Cooked Feed Fermented Flat Foreign Gluey^ Metallic Neutralizer Oxidized/tallowy Rancid (lipolysis) Salty Scorched Stale Storage Unclean/utensil Weedy 2 3 4 5 6 7 8

No criticism 10

Unsalable 0

Normal range 1-5

Physical appearance

No criticism 5 Unsalable 0 Normal range 1-5

Score Dry product: Caked Dark particles Lumpy Unnatural color Reconstituted Product: Churned particles Dark particles Grainy Undispersed lumps

Package

5 Ruptured vapor Barrier Soiled Unsealed

Score

No criticism 5 Unsalable 0 Normal range 1-5

Figure 3.26 A suggested dry milk products score card. (Reproduced from ref. 5, with permission of the ADSA, Champaign, IL.)

SENSORY EVALUATION OF CONCENTRATED AND DRY MILK (cont.)


Product: 1 Laboratory tests 5 No criticism 5 Score Fat (%) Moisture (%) Titratable acidity (% Lactic acid) Solubility index (ml) Bacterial estimate (per gram) Coliform (per gram) Direct microscopic Clump count (per g) Scorched particles (mg) Dispersibility (modified Moats-dabbah method, %] Phosphatase test Micrograms phenol/ml Undenatured whey protein Nitrogen (mg/g) Oxygen content {%) Copper (ppm) Iron (ppm) Vitamin A (i.u.) Vitamin D (i.u.) Alkalinity of ash (ml/lOOg) Protein content (%) Mesh (screen %) Ash, phosphorus fixed (%) Lead (ppm) Yeast and mold (per 0.1 g) Thermophiles (per g) Reducing sugars (as lactose %) Staphylococcus (coagulase positive) Salmonella (in 100 g) Date: 2 3 SAMPLE NO. 4 5 6 7 8

Unsalable 0

Signatures:

Figure 3.26 (Continued)

Table 3.18 A SUGGESTED SCORING GUIDE FOR THE FLAVOR OF DRY MILK (RELIQUIHED BASIS)
Scores for a Given Intensity3 Defect Acid Astringent Bitter Chalky Cooked Feed Fermented Rat Foreign*1 Gluey Metallic Neutralize/ Oxidized/tallowy8 Rancid (lipolysis) Salty Scorched Stale Storage Unclean/utensil Weedy Slight5 2 8 6 8 9 8 6 9 2 2 4 0 4 5 7 4 4 7 5 3 Moderate 1 7 5 7 8 7 5 8 1 1 3 0 3 4 6 3 3 6 4 2 Definite 0 6 4 6 7 6 4 7 0 0 2 0 2 3 5 2 2 5 3 1 Strong 0 5 3 5 6 5 3 6 0 0 1 0 1 2 4 1 1 4 2 0 Pronounced0 0

0-4 0-2 0-4 5 0-4 0-2


e

0 0 0 0 0 0-1 0-3 0 0 0-3 0-1 0

Reproduced with permission from ref. 5. "No criticism" is assigned a score of 10. Normal range is 1-10 for a salable product. b Highest assignable score for a defect of slight intensity. c Highest assignable score for a defect of pronounced intensity. However, a sample may be assigned a score of 0 (zero) if the defect makes the product unsalable. d Due to the variety of foreign off-flavors, suggesting a fixed scoring range is not appropriate. Some foreign off-flavors warrant a score of 0 (zero) even if their intensity is slight (i.e., gasoline, pesticides, lubricating oil). e The defect is unlikely to be present at this intensity level. f The use of neutralizes is not authorized except in whey. However, dry, sweet-type whey must have an alkalinity of ash not to exceed 225 ml of 0.1 N HCl per 100 g. g When an oxidized off-flavor has progressed to the tallowy stage, the assigned flavor score should be 0 (zero).
a

sensation as it is an off-flavor. The sensation is particularly noticeable after the product has been expectorated. Cause. Not all the causes are known but it is usually associated with high heat treatment of milk that has caused some aggregation and loss of solubility of milk proteins. A specific particle size of milk proteins or other milk constituents are thought to be responsible for the sensation.5

Neutralizer
Description. Different neutralizes have slightly different flavors. It is an alkaline, baking soda, or soda cracker flavor. Bitterness is often part of the profile. It is best detected after the sample has been in the mouth a while or after the sample has been

expectorated and air is inhaled through the mouth. The aftertaste does not easily clean up. Cause. Neutralizes are legal to add to whey to bring the pH to neutral before drying. It is not legal to add to other dairy products prior to drying. These alkaline neutralizes have a characteristic flavor that is detectable in the finished product.

3.4.8.3 Appearance Defects


Caked
Description. When the powder sets into a hard rock it is considered caked. The caked mass breaks off into small hard units but not into powder again. Grinding or sifting is necessary to restore it to a powder.5 Cause. Caking happens in extended storage where a sealed container goes through temperature cycles. These temperature cycles cause relative humidity cycling to occur. When the storage temperature is cold, the relative humidity may get high enough to condense a thin layer of water around each particle. That water dissolves a small amount of material. Then when the temperature warms, the relative humidity drops in the bag, the water dries, and the solutes solidify. After several of these cycles the powder particles are welded together by the solidified solute material. Lumpy Description. Lumpy powder lacks homogeneity in appearance. Small hard lumps the size of wheat grains are present in powder that may be normal otherwise. Cause. The lumps are caused by insufficient drying, a dripping spray nozzle, or powder exposed to moisture-laden air. The resulting clumps are subsequently dried to hard oversized particles.5 It should not be confused with the caking of the powder into large chunks.

Unnatural Color, Browned, or Darkened


Description. This defect refers to the darkening or browning of the product as it ages. It first turns off white to cream and then to gradually darkening brown color. It is associated with a distinctive old or stale off flavor. Cause. Nonenzymatic browning or lactose caramelization are the expected causes of browned or darkened milk powder. The little bit of moisture that is present in milk powder works very slowly at ambient temperatures to develop this color while generating flavors that render the milk powder inedible.

Visible Dark Particles


Description. This defect is characterized by the presence of small dark particles throughout the milk powder. Cause. Usually dark particles are the result of burned-on product that has sloughed off and carried into the finished product. It is most common in drum dried powder

where the traces of product might ride around the drum several cycles then be scraped into the product.5 In other drying processes, any hot surface could collect and darken product which later falls into the final product. Dark particles could also be introduced in other ways.

3.5 References
1. Brown, E. L., and K. Deffenbacher. 1979. Perception and the Senses, p. 57. Oxford University Press. New York. 2. Coren, S., C. Porac, and L. M. Ward. 1978. Sensation and Perception, p. 112. Academic Press, New York. 3. Amerine, M. A., R. M. Pangbom, and E. B. Roessler. 1965. Principles of Sensory Evaluation of Food. Academic Press. New York, 602 pp. 4. Dudel, J. 1981. General sensory physiology, psychophysics. In R. F. Schmidt (ed.), Fundamentals of Sensory Physiology, pp. 1-30. Springer- Verlag, New York. 5. Bodyfelt, F. W., J. Tobias, and G. M. Trout. 1988. The Sensory Evaluation of Dairy Products, pp. 11-478. Van Nostrand Reinhold, New York. 6. Zimmerman, M. 1981. Neurophysiology of sensory systems. In R. F. Schmidt (ed.), Fundamentals of Sensory Physiology, pp. 31-80. Springer-Verlag. New York. 7. Altner, H. 1981. Physiology of taste. In R. F. Schmidt (ed.), Fundamentals of Sensory Physiology, pp. 220-227. Springer-Verlag, New York. 8. Murray, R. G., and Murray, A. 1967. The fine structure of the taste buds of rhesus and cynomolgus monkeys. Anat. Rec. 19:327-353. 9. Rohen, J. W. 1978. Funkionelle Anatomie des Nervensystems, p. 380. F. H. Schattauer, Stuttgart. 10. Plattig, K. H. 1988. The sense of taste. In J. R. Piggott (ed.), Sensory Analysis of Foods pp. 2 - 3 , 11-15. Elsevier, New York. 11. Fabman, S. I. 1967. Structure of chemoreceptors. In H. W. Schultz, E. A. Day, and L. M. Libbey (eds.), Chemistry and Physiology of Flavors, pp. 25-51. AVI, Westport, CT. 12. Blakeslee, A. F., and A. L. Fox. 1932. Our different taste worlds. / . Hered. 23:96-110. 13. Maruniak, J. A. 1988. The sense of smell. In J. R. Piggott (ed.), Sensory Analysis of Foods, pp. 2 - 3 , 11-15. Elsevier, New York. 14. Moran, D. T., J. C. Rowley, and B. W. Jafec. 1982b. Electron microscopy of human olfactory epithelium reveals a new cell type: the microvillar cell. Brain Res. 253:39-46. 15. Graziadei, P. P. C. 1971. The olfactory mucosa of vertebrates. In L. M. Beidler (ed.), Handbook of Sensory Physiology, Chemical Senses I, pp. 27-58. Springer-Verlag, New York. 16. Getchell, M. L., B. Zielinski, J. L. DeSimone, and T. V. Getchell. 1987. Odorant stimulation of secretory and neural processes in salamander olfactory mucosa, / . Comp. PhysioL A 160:155-168. 17. Moncrieff, R. W. 1967. The Chemical Senses. Chemical Rubber Co. Press, Cleveland, OH. 18. Getchell, M. L., G. L. Heck, J. A. DeSimone, and S. Price. 1980. The location of olfactory receptor sites: inferences from latency measurements. Biophys. J. 29:397-412. 19. Allison, A. C, and R. T. Warwick. 1949. Quantitative observations on the olfactory system of the rabbit. Brain 72:186-197. 20. Code of Federal Regulations. 1991. Title 7, Part 58, Subpart P, Paragraphs 58.2621-582635. U.S. Standards for Grades of Butter. U.S. Government Printing Office. Washington, D.C. 21. Harper, R. 1972. Human Senses in Action, pp. 238, 250, and 255. Churchill-Livingstone, London. 22. Hochberger, J. E. 1964. Perception. Prentice-Hall, Englewood Cliffs, NJ. 23. McNamara, B. P. 1968. Vision. In Basic Principles of Sensory Evaluation, ASTM STP 433, pp. 19-23. American Society for Testing Materials. 24. Brown, J. L. 1965. The structure of the visual system. In C. H. Graham (ed.), Vision and Visual Perception. J. Wiley & Sons, New York. 25. Stiles, W. S. 1978. Mechanism of Colour Vision. Academic Press, London.

26. Hurvich, L. M. 1981. Color Vision. Sinaver Associates, Sunderland Massachusetts. 27. Christie, J. S. 1977. On-machine measurement of chromatic aspects of appearance. J. Tech. Assoc. Pulp Paper lndust. 60:119-121. 28. Code of Federal Regulations. 1990. Title 7 Part 58. Dried Dairy Products. Grading Standards. U.S. Government Printing Office, Washington, D.C. 29. Kling, J. W., and L. A. Riggs. (eds.). 1971. Woolworth & Schlosberg's Experimental Psychology, 3rd edit. Holt, Rinehart and Winston, New York. 30. Meilgaard, M., G. V. Civille, and B. T. Carr. 1987. Sensory Evaluation Techniques, VoI I. Chemical Rubber Company Press, Boca Raton, FL. 31. Silbiger H. R. 1968. Hearing. Basic Principles of Sensory Evaluation, ASTM STP 433, pp. 24-29. American Society for Testing Materials. 32. Christensen, C. M., and A. M. Vickers. 1981. / . Food ScL 46:574. 33. Vickers, Z. M. 1984. /. Texture Stud. 15:49;157. 34. Vickers, Z. M. 1985. J. Texture Stud. 16:85. 35. Geldard, F. A. 1972. The Human Senses, 2nd edit. John Wiley & Sons, New York. 36. Demick, P. S. 1982. Photochemical effects on flavor and nutrients of fluid milk. Can. Inst. Food Sci. Technol. J. 15:247-256. 37. Weigold, G. 1981. Building Dairy Careers in the Collegiate Dairy Products Evaluation Contest. Brochure published by the Dairy and Food Industries Supply Association, Rockville, MD. 38. Hinreiner, E. H. 1956. Organoleptic evaluation by industry panelsthe cutting bee. Food Technol. 31:62-67. 39. Peryam, D. R., F. J. Pilgrim, and M. S. Peterson, (eds.). 1954. Food Acceptance Testing Methodology. National Academy of Sciences-National Research Council, Washington, D.C. 40. Stone, H., and J. L. Sidel. 1985. Sensory Evaluation Practices. Academic Press, New York. 41. Caul, J. F. 1957. The profile method of flavor analysis. Adv. Food Res. 7:11 -40. 42. Boggs, M., and H. L. Hansen. 1949. Analysis of foods by sensory difference tests. Adv. Food Res. 2, 219-258. 43. Girardot, N. E., D. R. Peryam, and R. Shapiro. 1952. Selection of sensory testing panels. Food Technol. 6:140-143. 44. Jones, L. V., D.R. Peryam, and L. L. Thurstone. 1955. Development of a scale for measuring soldiers food preferences. Food Res. 20:512-520. 45. Peryam, C. R., and Pilgrim, F. J. 1957. Hedonic scale method of measuring food preferences. Food Technol. 11:9-14. 46. Roessler, E. B., R. M. Pangborn, J. L. Sidel, and H. Stone. 1978. Expanded statistical tables for estimating significance in paired-preference, paired-difference, duo-trio and triangle tests. J Food Sci. 43:940-947. 47. Basker, D. 1988. Critical values of difference among rank sums for multiple comparisons. Food Technol. 42:79. 48. Huntsberger, D. V. 1961. Elements of Statistical Inference, p. 143. Allyn and Bacon, Boston. 49. Helm, E., and B. Trolle. 1946. Selection of a taste panel. Wallerstein Lab. Commun. 9:181-194. 50. Cairncross, S. E., and L. B. Sjostrom. 1950. Flavor profilesa new approach to flavor problems. Food Technol. 4:308. 51. Brandt, F. L, and M. E. Terry, 1963. Texture profile method. / . Food Sci. 28:404-410. 52. Szczesniak, A. S., M. A. Brandt, and H. H. Friedman. 1963. Development of standard rating scales for mechanical parameters of texture and correlation between the objective and the sensory methods of texture evaluation. / . Food Sci. 28, 397-403. 53. Szczesniak, A. S. 1963. Classification of textural characteristics. / . Food Sci 28:385-389. 54. Civille, G. V., and I. H. Liska. 1975. Modifications and applications to foods of the General Foods sensory texture profile technique, J. Texture Stud. 6:19. 55. Civille, G. V., and A. S. Szczisniak. 1973 Guidelines to training a texture profile panel. J. Texture Stud. 4:204. 56. Schwartz, N. 1975. Method to skin care produts. J. Texture Stud. 6, 33. 57. Code of Federal Regulations. 1990. Title,21. Part 131. Milk and Cream Products. U.S. Government Printing Office. Washington, D.C.

58. Shipe, W. F., R. Bassette, D. D. Deane, W. L. Dunkley, E. G. Hammond, W. J. Harper, D. H. Kleyn, M. E. Morgan, J. H. Nelson, and R. A. Scanlan. 1978. Off-flavors in milk: nomenclature, standards, and bibliography. / . Dairy Sci. 61:855. 59. Gould, I. A., and H. H. Sommer. 1939. Effect of heat on milk with special reference to the cooked flavor. Mich. Agr. Exp.Sta. Tech. BuL 164. 60. Morgan, M. E. 1976. The chemistry of some microbiologically induced flavor defects in milk and dairy foods. Biotechnol. Bioeng. 18:953. 61. Jenness R., and S. Patton. 1959. Principles of Dairy Chemistry, p. 337. John Wiley & Sons, New York. pp. 337. 62. Hoskin, J. C. 1979. Sensory Evaluation and Riboflavin Analysis of Milk Held in Light-Exposed OneGallon Containers. M.Sc. Thesis. The Pennsylvania State University, University Park, PA. 63. Kosikowski, F. W. 1977. Cheese and Fermented Milk Foods, 709 pp. Edwards Brothers, Ann Arbor, Michigan. 64. Loter, I., H. G. Dissly, and R. E. Schafer. 1973. Improved Cheese Manufacture Process, U.S. Patent No. 1,400,927. 65. Morgan, M. E. 1970b. Microbial flavor defects in dairy products and methods for their simulation. II. Fruity flavor. / . Dairy Sci. 53:273. 66. Morgan, M. E. 1970a. Microbial flavor defects in dairy products and methods for their simulation. I. Malty flavor. /. Dairy Sci. 53:270. 67. Bodyfelt, F. W. 1981a. Sensory and shelf-life characteristics of cottage cheese treated with sorbic acid. In Proc. Biennial Marschall Intl. Cheese Conf Madison, WI. 68. Bodyfelt, F. W. 198 Ib. Temperature control monitoring for cottage cheese plants. Dairy Rec. 82:84. 69. Code of Federal Regulations. 1990. Title 21-Food and Drugs, Part 135Ice Cream and Frozen Desserts. U.S. Government Printing Office. Washington, D.C. 70. Bodyfelt, F. W. 1979. Ice cream qualitywho should be the judge? Natl. Ice Cr. Retail. Assn. Production Tips. March. 71. Code of Federal Regulations. 1990. Title 21. Part 133. Cheeses and Related Cheese Products. U.S. Government Printing Office, Washington, D.C. 72. Bills, D. D., M. E. Morgan, L. M. Libbey, and E. A. Day. 1965. Identification of compounds responsible for fruit flavor defect of experimental cheddar cheeses. J. Dairy Sci. 48:1168. 73. Bodyfelt, F. W. 1967. Lactic Streptococci and the Fruity Flavor Defect of Cheddar Cheese. M.S. Thesis, Oregon State University, Corvallis, OR, 118 pp. 74. Tamime, A. Y., and R. K. Robinson. 1985. Yogurt Science and Technology. Pergamon Press, New York. 431 pp. 75. Code of Federal Regulations. 1990. Title 21. Part 131.200 Yogurt. U.S. Government Printing Office. Washington, D.C. 76. Code of Federal Regulations. 1990. Title 21. Part 172.804 Food Additives. U.S. Government Printing Office. Washington, D.C. 77. Graham, D. M., J. T. Hutton, and J. M. Mclntire. 1981. Concentrated and dry milks and wheys in the third quarter of the 20th century. J. Dairy Sci. 64:1055. 78. Hall, C. W., and T. I. Hedrick. 1971. Drying Milk and Milk Products, 2nd ed. AVI, Westport, CT. 338 pp. 79. Zapsalis, C , and R. A. Beck. 1985. Food Chemistry and Nutritional Biochemistry, pp. 574-576. John Wiley & Sons, New York. 80. Williams, L. H., and L. V. Ogden. 1990. Effect of warming cold milk in the vat on occurrence of a dark seam defect in cheddar cheese. / . Dairy Sci. 71:8-10. 81. Connolly, E. J., C. H. White, E. W. Custer, and E. R. Vedamuthu. 1984. Cultured Dairy Foods Quality Improvement Manual. American Cultured Dairy Products Institute. Washington, D.C, 40 pp. 82. Duthie, A. H., K. M. Nilson, H. V. Atherton, and L. D. Garrett. 1977. Proposed score card for yogurt. Cultured Dairy Prod. J. 12:10. 83. Vedamuthu, E. R., W. E. Sandine, and P. R. Elliker. 1966. Flavor and texture in Cheddar cheese. II Carbonyl compounds produced by mixed-strain lactic starter cultures. / . Dairy Sci. 49:151.

CHAPTER
4

Functional Properties of Milk Proteins


Olivier Robin, Sylvie Turgeon, and Paul Paquin
4.1 Introduction, 278 4.2 Composition and Principal Physicochemical Properties of Major Milk Proteins, 280 4.2.1 Major Protein Components in Milk, 280 4.2.2 Principal Physicochemical Properties of Milk Proteins, 281 4.3 Major Functional Properties of Milk Proteins, 282 4.3.1 Water-Protein Interactions, 282 4.3.1.1 Hydration/Rehydration Properties, 284 4.3.1.2 Solubility, 289 4.3.2 Protein-Protein Interactions, 292 4.3.2.1 Rheological Behavior of Protein Dispersions, 292 4.3.2.2 Gelling Properties of Globular Proteins, 297 4.3.3 Protein-Surface Interactions, 302 4.3.3.1 Interfacial Properties of Milk Proteins, 303 4.3.3.2 Dispersed Systems: Emulsions and Foams, 309 4.3.3.3 Flavor Binding, 324 4.4 Some Selected Processing Effects on the Functional Properties of Major Milk Proteins, 325 4.4.1 Effects of Heat Treatments, 325 4.4.1.1 Effects on Caseins, 325 4.4.1.2 Effects on Whey Proteins, 328 4.4.2 Membrane Separation Processes, 329 4.4.2.1 Reverse Osmosis (RO), 330 4.4.2.2 Nanofiltration (NF), 330 4.4.2.3 Ultrafiltration (UF), 331 4.5 Conclusion, 332 4.6 Acknowledgments, 333 4.7 References, 334

4.1 Introduction
Dairy quotas, prospects for dairy product prices, enormous stockpiles of skim milk powder and butter, the new eating habits of Occidental consumers, and ever stricter environmental laws have resulted in an increasing demand for versatile ingredients, principally proteins, possessing appropriate functional properties.1'2 Proteins, and specifically milk proteins, are important, not only because they possess a wide range of dynamic functional properties, but also because they are easily isolated from raw milk, provide essential amino acids, show versatility during processing, and possess the capacity to form network structures and stabilize emulsions and foams.3-4 A better understanding of the functional properties of milk proteins has led to or contributed to the development of new prospects for meeting consumer expectations (light products), and those of health (body, pharmacological, infant nutrition, enteral, or parenteral products) and beauty professionals (cosmetic products), or simply for a better management of the raw product.1'5"7 The use of milk proteins to give food desirable organoleptic or textural properties is strongly influenced by their functional properties. Functionality is defined as "any property of a food, or a food ingredient, except its nutritional ones, that affect its utilization."8 Cheftel et al.,9 and Lorient10 propose a more accurate definition by classifying functional properties of proteins into three major groups: 1. Properties depending on the behavior of proteins in water: water-protein interactions which include water adsorption, hydration, wetting, solubility, and viscosity. 2. Properties depending on interactions between macromolecules: protein-protein interactions, which include structural properties, and covalent or ionic intermolecular associations. 3. Properties depending on interactions with amphiphilic molecules or with a gas phase: protein-surface interactions which include emulsifying and foaming properties, and flavor binding. If this classification has the merit of being succinct and reasonably complete, these three categories are not, however, mutually independent: gelation involves not only protein-protein interactions but also protein-water interactions.3 Because of the diversified nature of milk proteins (amino acid composition, tridimensional structure), the study of the functional properties of proteins cannot be dissociated from the study of the physicochemical properties of proteins and of the relationships between structure and functional properties in food systems, that is, taking into consideration the importance of various inter-/intramolecular forces/ interactions (e.g. covalent, electrostatic, hydrogen, hydrophobic) which ultimately determine the functional properties of proteins as they do for any other molecule.11'13 Furthermore, these inter-/intra-molecular forces/interactions, and consequently functional properties, are closely related to the environmental conditions (e.g., pH, temperature, ionic strength, salt composition and species, presence of other solutes, etc.) and the modifications due to processing that are involved in obtaining and utilizing a protein ingredient (thermal, physical, chemical, and biological treatments).14

Table 4.1 FUNCTIONAL ROLES OF MILK PROTEINS IN VARIOUS FOOD SYSTEMS


Food System Group Analogue of dairy products Examples of Related Product Low fat spreads UHT coffee cream Whipped topping Cake Cookies Chocolate drinks Red wine (Cabernet) Marshmallow Meringue Ice-cream Fruit yoghurt Processed cheese Ham products Sausage Examples of Milk Protein Ingredient Used Sodium caseinate Sodium caseinate Sodium caseinate Whey Lactic casein Whey Casein Casein Whey Sodium caseinate Sodium casemate Casein Whey Casein

Functional Properties Emulsification, stabilization, resistance to feathering Water/fat holding, emulsification, foaming, texture, appearance Solubility at different pH, heat stability, viscosity, stabilization, removal of phenolic compounds Emulsification, dispersibility, stabilization Emulsification, foaming, viscosity, gelification, coagulation, fat/flavor binding Emulsification, gelation, cohesiveness, water/fat holding

Bakery

Beverages

Confectionery Dairy products

Meat products

Adapted from Refs. 49, 50, and 51.

Therefore, depending on the protein itself and various environmental and processing factors, the functional properties of proteins can induce a wide variety of physical states on the foods in which they are contained (e.g., liquid, solid, gelled, emulsified, dispersed, etc.), as well as confering characteristic organoleptic properties and determining shelf-life. The functional behavior of caseinate and whey concentrates and relationships between functionality and structure in aqueous solutions or in model systems are generally well known and have been the subject of many comprehensive and detailed reviews: Kinsella,15 de Wit,16 Cheftel and Lorient,17 Fox and Mulvihill,18'19 de Wit,20 Kinsella,14 Cheftel et al.,9 Kinsella and Fox,21 Modler,22-23 Morr,24 Leman and Kinsella,25 Vuillemard et al.,26 Paquin and Dickinson,27 Tornberg et a/.,28 Lorient et al.29 Various symposia and books have also been devoted more specifically to the functional properties of macromolecules and proteins: Pour-El,30 Cherry,31'32 Dickinson and Stainsby,33'34 Mitchell and Ledward,35 Brash and Horbett,36 Fox,37 Lorient et a/.,38 Kinsella and Soucie,39 Parris and Barford,40 Harris,41 Larsson and Frieberg.42 The abundance of references in this field is explained in great part by the fact that the primary and tridimensional structures of the six major milk proteins (a s l - as2-, /3-, and K-caseins, /3-lactoglobulin, and a-lactalbumin) are known.43"48 Although there is great difficulty in determining the relationship between simple solution functionality and complex food system functionality, many different protein ingredients are used in a wide range of foods. Table 4.1 shows a number of food

applications where milk protein ingredients are used.49'51 It is well known that in all these food systems (e.g., cheese, cream, ice cream, etc.) interactions will occur between the milk proteins and other components naturally present in the formulation. To be able to develop and to increase the utilization of milk proteins, a better knowledge of molecular behavior in mixtures is necessary. Such a basic understanding would not only lead to the development of better processing techniques so as to retain or improve the functional properties of proteins but would also lead to the development of the functional properties of underutilized food proteins. In this chapter, we emphasize a nonmathematical, molecular description of milk protein functionality. Some equations have nevertheless been used. Although they describe approximate phenomena, they have been used to illustrate the chapter and to clarify some concepts. A more basic detailed, and necessarily mathematical account of the underlying basic concepts, is given by Franks,52-53 Dickinson and Stainsby,33 Becher,54-55 and Bird et al.56 After a succinct review of the various physicochemical properties of milk proteins, basic principles underlying their functional properties, in relation to their environment, will be evoked on the basis of the numerous studies previously mentioned, and on recent work discussing interactions between proteins and nonprotein molecules. Due to the interests of the authors, particular attention will be paid to protein-surface interactions. Then, in a third section, the effect of some physical processes on the functional properties of milk proteins will be briefly discussed. Finally, some present and future trends in the field of research and development involving the functional properties of milk proteins will be presented. It is not intended that this chapter should be an exhaustive review of the extensive literature on the functional properties of milk proteins. There has consequently been a conscious attempt at selection, although it is hoped that no important aspects of this large subject have been ignored.

4.2 Composition and Principal Physicochemical Properties of Major Milk Proteins


A more detailed review of the composition and physicochemical properties of milk proteins can be found in the first chapter of this book.

4.2.1 Major Protein Components in Milk


Normal bovine milk contains 30 to 35 g of protein/Liter.9 The two principal types of milk proteins are caseins and whey proteins. Caseins constitute 76 to 86% of the total milk protein. They are generally found in milk in the form of spherical and macromolecular complexes containing inorganic material, principally calcium phosphate, called micelles.57"59 Caseins comprise four primary proteins, a sl -casein, as2-casein, /3-casein, and K-casein (Table 4.2), with different genetic variants of each, and several minor proteins originating from postsecretion proteolysis of the primary caseins.44'45

Table 4.2 CONTENT OF MAJOR PROTEIN COMPONENT IN MILK


Content of Protein in Milk Weight Contribution (g/L) 24-28 12-15

Protein Type Casein

Protein or Polypeptide

asl-Casein as2-Casein /3-Casein K-Casein y-Casein Whey protein /3-Lactoglobulin a-Lactalbumin Bovine serum albumin Immunoglobulins Proteoses peptones Adaptedfromref. 9.

3-4 9-11 3-4 1-2 5-7 2-4 1-1.5 0.1-0.4 0.6-1.0 0.6-1.8

Whey proteins represent 14 to 24% of milk proteins and are in solution in the serum phase of the milk, normally in monomer or dimer form. In milk, the ratio of whey proteins to casein micelles is about 1500:1.16 The major whey proteins (Table 4.2) are jS-lactoglobulin (/3-Ig), a-lactalbumin (a-la), bovine serum albumin (BSA), immunoglobulins (Ig-G, Ig-A, Ig-M), and proteose peptones (PP-3, PP-5, PP-8 fast, PP-8 slow).45'60 There are several minor proteins including lactotransferrin, lactoperoxidase, lysozyme, glycoprotein, and serum transferrin, as well as casein degradation products.44'45

4.2.2 Principal Physicochemical Properties of MUk Proteins


The caseins and whey proteins can be distinguished on the basis of their physicochemical properties (Table 4.3).51 Caseins, micellar or not, are very sensitive to pH (insoluble at pi 4.6), and to the presence of di- or polyvalent salts and are heat stable, whereas whey proteins are soluble in acid solutions and can be denatured by heat.3 Casein molecules have a particular amphiphilic nature arising from a separation between hydrophobic clusters and negatively charged regions along the peptide chain. Caseins have a relatively small number of cysteine residues so the occurrence of disulfide cross-linkages is infrequent. Consequently, all casein molecules are disordered with little secondary structure.3 This lack of disulfide bridge stabilization renders a sl - and j8-caseins very dependent on pH and on the presence of divalent cations; in neutral or basic media, their voluminosity increases considerably.19 This gives them exceptional viscous and interfacial properties.61'28 Heat has little effect on casein molecules as they are already in an open and extended form.3

Table 4.3 PRINCIPAL PHYSICOCHEM1CAL PROPERTIES OF MAJOR PROTEIN COMPONENTS IN MILK


Protein Type Casein Properties Contains strongly hydrophobic regions Contains little cysteine Random coil structure Heat stable Unstable in acidic conditions Balance of hydrophilic and hydrophobic residues Contains cysteine and cystine Globular structure, much helical content Easily heat denatured Stable in mildly acidic conditions

Whey proteins

FromRef. 51.

Whey proteins are a much more diverse group than the caseins. They are much more structured than caseins due to a more uniform distribution of amino acid types along their peptide chains and the presence of disulfide bridges (higher quantities of cysteine), and are not greatly affected by pH and salts. Their compact structure gives to them the ability to form thick and sticky interfacial films (especially at pi 5.2 for /3-lg) even if their ability to adsorb to interfaces is lower than that of caseins;25 this results in good emulsifying and foaming properties at all pH values.29 As do most globular proteins, whey proteins, and particularly /3-lg, gel easily with heat due to a modification of the spatial structure (hydrophobic interactions, disulphide bridge exchange).62"64

4.3 Major Functional Properties of Milk Proteins


The functional behavior of milk proteins (Table 4.4) is principally a function of14'29: 1. Their behavior in water in relation to their spatial structure and their physicochemical properties (voluminosity, surface hydrophobicity, amphipolarity), and 2. Their flexibility in relation to spatial structure and water content. The following sections present a summary of the functional properties of milk proteins (caseins and whey proteins) taken mainly from previously cited references.

4.3.1 Water-Protein Interactions


Various aspects of the physical chemistry of water-protein interactions with special reference to foods have been thoroughly reviewed by Lumry,65 Kuntz and Kauf-

Table 4.4 FUNCTIONAL PROPERTIES OF MAIN MILK PROTEINS


Properties Hydration Caseins Very high water binding with glue formation at high concentration. Minimum at pi Insoluble at pi Very viscous solutions at neutral and basic pH. Lowest viscosity at pi No thermal gelation except in presence of calcium. Micelle gelation by chymosin Excellent emulsifying properties especially at neutral and basic pH Good overrun but low foam stability: K > P > sl Good flavor binding Whey Proteins Water binding increasing with protein denaturation Very soluble at every pH. Insoluble at pH 5 if thermodenaturated Not very viscous solutions except if thermodenaturated Thermal gelation from 700C: influence of pH and salts Good emulsifying properties except at pH 4-5 if thermodenaturated Good overrun and excellent foam stability /3-lg > a-Ia Retention very variable with the denaturation

Solubility Viscosity Gelation

Emulsifying properties Foaming properties Flavor binding From Ref. 29.

man,66 Chou and Morr,67 Rockland and Stewart,68 Damodaran and Kinsella,69 Kinsella,14 Simatos and Multon,70 Kinsella and Fox,21 Franks,71 Hardman,72 Morr,73 and Kneifel et al?A Water, the major constituent of milk (87%), is not only a solvent but also plays a key role in determining the three-dimensional structure of proteins as well as determining many of the functional properties of proteins in foods. These properties come into play during processing (rehydration of protein ingredients normally preserved dry, emulsification, foaming, cheese processes, etc.) and when the food product is consumed.15'75 The dominant role played by water is principally due to its many unique properties, related to its structure (two areas of positive charge and an equal one of negative charge in a tetrahedral arrangement). Compared to other molecules of similar molecular weight, water has larger values of heat capacity, melting and boiling point, surface tension, heats of fusion, vaporization, and sublimation than would be expected from its components.71 The higher values are related to the extra energy needed to break the intermolecular hydrogen bonds between water molecules.76 Under the general term hydration, Kinsella,15 includes some other practical functions performed by milk proteins such as wettability, water adsorption, voluminosity, swelling, dispersibility, and solubility. Water-protein interactions also affect other functional properties of proteins such as rheological behavior, thickening, gelling, emulsifying, and foaming properties, dough formation, etc.21 Therefore, depending on the affinity of a protein for water, a polymer will either be readily soluble or form a viscous solution, a colloidal suspension, a precipitate, a coagulum, or a gel. Finally,

Table 4.5 APPROXIMATE aw LEVELS OF SOME DAIRY PRODUCTS AT 25C


Food Product Dried milk products Butter, unsalted salted Sweetened condensed milk Cheese, hard soft fresh Cream Frozen desserts Condensed milk products Fermented milk products Milk and whey From Ref. 80. aw 0.1-0.3 >0.99 0.91-0.93 0.77-0.85 0.86-0.87 0.96-0.98 0.98-0.99 >0.99 0.97-0.98 0.98-0.99 0.97-0.99 0.996

because the number and the type of polar or ionized groups and conformational factors necessarily affect water-protein interactions, any environmental factor that will affect either polarity or conformation may also affect water-protein interactions.77

4.3.1.1 Hydration/Rehydration Properties


Definitions and General Considerations
Many terms have been used to describe the uptake of water by proteins, including water hydration capacity, water-holding capacity, water absorption, water-imbibing, and water binding;50*14 this diversity is related to the variety of methods used to measure water-protein interactions.21'74 However, all of them describe the amount of water that can be bound or retained by a protein matrix under defined conditions and this is generally expressed as g of H2O/g of dry protein.19*78 Availability of water in a given system can be evaluated using a thermodynamic quantity, the water activity (a w ), defined as the reactivity of water in solution at constant temperature and pressure.79 This quantity is usually measured as the partial pressure of water above the sample (P), over the vapor pressure of the pure water (P0) at the same temperature and may vary between zero (in a water-free system) and 1 (in bulk water). (4.1) Approximate values of aw of some dairy products are given in Table 4.5. The addition of water to a protein can be represented by a sorption isotherm (Fig. 4.1). Sorption

WATER CONTENT (%)

in!

Il

WATER ACTIVITY (a w )
Figure 4.1 Generalized water sorption showing water uptake of a protein as a function of equilibrium relative humidity or water activity (aw). Region I is a region of adsorption and highly bound water, region II contains adsorbed and some multilayer water, and region III contains these plus physically entrapped bulk water. (From Ref. 21.) isotherms of many products are described in the literature, but there are often important differences between isotherms proposed for the same product. This results essentially from an insufficient standardization of the methods used, and from a great sensibility to the preparation of the tested products (pH, salts, product structure, etc.). In sorption isotherms three zones are generally distinguished (Fig. 4.1)21: (1) in region I, water fixes to the most hydrophilic groups of proteins; (2) the second one corresponds to the hydration of uncharged polar groups; and (3) in region III where interaction forces between water and dry materials are lower than in the previous two, water is essentially retained by capillary forces. At low water activity (0 < aw < 0.3), 0.04 to 0.09 g of K2OJg of protein are adsorbed. In the case of caseinates and /3-lg, approximately 0.06 and 0.07 g of H2OZg of protein respectively are adsorbed.21 At higher water activity (aw = 0.92), globular proteins bind approximately 0.5 g of H2O/g of protein. Sodium caseinate and /3-lg follow this trend with 0.4 and 0.3 g of H2OZg of protein respectively, whereas casein micelles bind larger amounts of water, that is, 2 to 4 g of H2OZg of protein.21'73 This larger amount of water is due to the mechanical entrapment of water in the micellar matrix (via calcium phosphate) and to the binding of water by the hydrophilic part that protrudes from the surface of the micelle.73 A large number of equations (more than 75 according to van der Berg and Bruin79) have been proposed to describe water activity and its estimation in food systems: While each model has its advantages for particular systems, none provides accurate

predictions of moisture sorption data over the complete range of #w. The Guggenheim, Anderson, de Boer (GAB) equation has been suggested as the best describing the region II moisture sorption isotherm for most foods.79-81 This equation is considered as the most satisfactory by many authors.82 In addition, this term correlates well with the rate of many degradative reactions and is used as an indicator of food perishability.9 Complementary information on the immobilization states of water with respect to proteins has been provided by Chou and Morr67 who divided the three previous regions into six states. Definitions of these various forms of water-protein associations with the corresponding a w are given in Table 4.6.21 The first is structural water in which hydrogen bonding to the protein stabilizes the native three-dimensional conformation. This water is not available for chemical reactions. The second type of water is monolayer water which fills the first adsorbed layer around the protein. Monolayer water is attached to specific water-binding sites through hydrogen bonds or electrostatic interactions. This water is also not available as a solvent, but may be available for certain reactions. The third type of bound water is unfreezable water which represents the total water clustered around each polar group. This water may include both structural and monolayer water. The remaining three types of water associated with proteins are not as well defined as the first three. The fourth type of water is that which is associated with proteins via hydrophobic hydration. This type of water has been described as clathrate-type or ice-like structured water, but the real nature of this water is not entirely clear.71 A fifth type is capillary water which is held physically or by surface forces and which acts as a solvent; this water is available for chemical reactions. It is the main type of water found in cheese curd.75 The sixth type of water is called hydrodynamic water. This water, which is transported along with protein molecules, has the physical properties of normal water. Trying to define and categorize the types of water associated with proteins is necessary but difficult because it implies a sharp demarcation between different states of *'bound" water associated with the protein which have unusual physical properties and "normal" water loosely associated with the protein. Furthermore, this problem of definition is also a methodological one, because the numerous methods used to study the interactions of water with proteins often describe different types of bound water. One should not loose sight of the fact that water associated with proteins is a continual transition from highly-structured monolayer water molecules bound to specific groups to the unordered liquid water at the periphery of the multilayers, and that it is difficult to know where one type of water ends and another begins.21-71'75 Consequently, at the present time, there is no uniform set of definitions to describe these states: water molecules interact with each other and with proteins in many ways.83 Other researchers76'84 define only three types of water: constitutional, interfacial, and bulk phase water. Constitutional water corresponds to structural water; interfacial water is made up of vicinal water (the first one or two molecules adjacent to proteins), and multilayer water (the next few layers of water molecules). Interfacial water would correspond to monolayer, unfreezable, and hydrophobic hydration water.75 Bulk phase water, which is the remaining water as-

Table 4.6 CLASSIFICATION OF WATER ASSOCIATED WITH PROTEINS AT INCREASING WATER ACTIVITIES (aj
Structural water:

(flw < 0.05) Monolayer water: (0.05 < aw < 0.2) Unfreezable water: (0 < aw < 0.9) Hydrophobic hydration water: (0.1 < aw < 0.25) Capillary water: (0.5 < aw < 0.95) Hydrodynamic hydration water: (flw > 0.99)

Water hydrogen bonded to specific groups and which participates in stabilization of structure (10 to 20 H2O molecules/protein); unavailable for chemical reactions The first adsorbed water monolayer hydrogen bonded to protein groups; unavailable as solvent, may be available for chemical reactions (4 to 9 mg of H2O/g of protein) This includes all water that does not freeze at normal temperature (0.3 to 0.5 g of H2O/g of protein); amounts varies with polar amino acid content and includes some water available for chemical reactions Structured cage-like water surrounding apolar residues; involved in stabilization of protein structure Water mechanically trapped by surface forces in the protein molecule; similar to bulk water in physical properties Water "loosely" surrounding the protein and that is transported with the protein during diffusion (centrifugation); properties of normal water

From Refs. 3, 21, and 67.

sociated with proteins, constitutes the major type of water. It may be physically free as in diluted protein dispersions or entrapped as in gels. More recently, Kneifel et al.14 proposed dividing the water held in a protein into two main types: (1) that bound to the molecule and no longer available as a solvent, and (2) that entrapped in the protein matrix or in a corresponding comatrix (fat, polysaccharide). The first type can be regarded as adsorbed water and the second as retained water.

Environmental Effects on Hydration/Rehydration Properties


De Moor and Huyghebaert85 reported that the amount of water bound by whey powders and demineralized whey powders is generally low. However, the protein component of these powders has a high water-holding capacity. The evaluation of the amount of water bound by a protein depends not only on the protein itself (composition of amino acid residues, conformation) but also on environmental conditions (pH, ionic species and composition, temperature). Amino acid residue composition necessarily affects the hydration properties of proteins because some amino acids bind more water than others. Proteins that contain large amounts of polar or ionized groups (carboxylic, hydroxyl, and thiol side chains) will tend to bind a large amount of water. The number of polar or ionized group will affect the rate and the extent of water binding to proteins. In contrast, apolar

amino acid residues (aliphatic and aromatic side chains) which show a low affinity for water molecules are preferentially buried in the interior of the protein molecule and are not available for interactions with the solvent. 3 ' 86 The amount of water bound by ionized or polar groups is affected by the steric availability of hydration sites. The unfolding of a protein molecule from a globular conformation to a random coil results in an increase of the net area surface and thus in an increase of availability of extra hydration sites due to the exposure of more ionized or polar amino acid residues and peptide bonds.3 Practically, protein unfolding has relatively little effect on the amount of water bound by a protein. Usually, there is an increase of 0.02 to 0.1 g of H2CVg of protein.21 Depending on the extent of unfolding, it may also result in a decrease of hydration capacity because of increasing protein-protein interactions.21 Another important parameter that affects the amount of water associated with a protein is the net charge on the protein molecule. These charges that give rise to electrostatic repulsions (the concept of electrical diffuse double layers) may provide a driving force to stabilize particles in solution or in colloidal dispersion (see ProteinSurface Interactions). pH is a factor that affects the net charge on a protein. At the isoelectric point (pi), the number of positive and negative charges is equal; that is, the net surface charge equals zero, and therefore the hydration capacity is lower. Moreover, this decrease in repulsive forces and in the hydration shell favors attractive forces leading to protein-protein interactions. The nature and the concentration of salts also affect the hydration properties of proteins by their effects on electrostatic interactions. At low electrolyte concentrations, the amount of water bound to proteins increases with increasing electrolyte concentration. For high electrolyte concentrations, the amount of water bound decreases because of the suppression of the electrical double layer surrounding the protein molecule; this is directly related to the hydration of the ion and hence to its ability to separate water molecules from the protein molecules: ions with smaller unhydrated radii (larger charge density) have larger hydrated radii and thereby produce a greater degree of dehydration of the protein (Hofmeister or lyotropic series). 21 ' 69 Temperature has a major effect on hydration properties because, from a thermodynamic standpoint, in nearly all dairy products water absorption is an exothermal process; that is, the partial molal enthalpy of mixing has a negative sign. 79 ' 87 Therefore, a decrease in temperature causes an increase in equilibrium water content and thereby in hydration properties.80 So, as expected, heating of proteins in most studies decreases hydration.88'89 Bech, 90 however, reported enhanced water-holding capacity by whey proteins after severe heat treatment. Preheating of the base milk prior to the manufacture of sodium caseinate leads to a concomitant adsorption of whey proteins onto caseins, increasing the water-holding capacity of the product. This effect was thought to be due to the thermal denaturation of the whey proteins, creating a spongelike surface on the casein, which retains more water than a caseinate powder produced from unheated milk. However, skim milk powder subjected to varied heat treatments did not show different a water-holding capacity.78

Measurement of Hydration/Rehydration Properties


Numerous techniques have been used to study water-protein interactions. These methods have been reported and listed by Bull and Breese,91 Labuza,92 Franks,71 Chou and Morr,67 Patel and Fry,93 Schnepf,75 and more recently by Kneifel et al?A Chou and Morr67 have grouped the methods into four categories depending on the main properties of the protein-water interactions. The first group includes methods related to the thermodynamic properties of water (e.g., enthalpy, entropy, free energy, <zw, freezing and boiling points), and uses methods such as sorption isotherms. The second group of techniques measures kinetic properties; that is, the mobility of water as it associates with a protein, and changes in proteins that are effected by protein-water interactions. Specific techniques are used to evaluate kinetic properties such as nuclear magnetic resonance (NMR), dielectric dispersion, laser light scattering, and intrinsic viscosity. The third group of methods are spectroscopic techniques which measure the nature and strength of hydrogen bonds. These methods include infrared and Raman spectroscopy, and NMR. The fourth group provides information on the average position and orientation of water molecules and includes light and X-ray scattering, as well as neutron diffraction. However, many of the above techniques are only of academic interest for most food technologists. Kneifel et al.74 have proposed differentiating methods according to whether they measure water adsorption (Baumann apparatus, viscosimetry, farinographic techniques, rehydration tests, sorptions isotherms) or whether they measure water retention (centrifugation test, differential scanning calorimetry, filtration procedure). Table 4.7 (Kneifel et al.14) lists the most important methods used and the underlying principles. However, more work should be done to standardize methodologies for evaluating hydration properties. Two types of procedures for water absorption and retention properties are presently under investigation by the committee on the functional properties of milk proteins of the International Dairy Federation.

4.3A.2 Solubility Definition and General Considerations


Solubility in aqueous systems is a valuable predictor of other functional properties of dairy protein-containing ingredients. Solubility is related to the dispersibility of protein ingredients in water, and to environmental conditions. Solubility can also be used to provide information on protein denaturation (e.g., at pH 4.6 for whey proteins) caused by processing and storage and thereby to predict the usefulness of the powder in food applications (beverages, yogurt, emulsions, foams, etc.).14'50 Solubility itself, however, is no guarantee of useful functionality. Indeed, foaming may be best exhibited by proteins at their isoelectric point, where they are also least likely to remain in solution.93 Practically, solubility (g of dry protein/100 g of H2O) is the amount of protein in a sample that goes into solution or into colloidal dispersion under specified conditions and is not sedimented by low centrifugal forces.3

Table 4.7 PRINCIPAL METHODS USED FOR PROTEIN HYDRATION MEASUREMENTS


Method Bauman apparatus Farinographic techniques Principle of the Method Based on the measure of the uptake of water by a sample at equilibrium Based on the constant dough weight method which allows the calculation of several farinographic characteristics Based on the spectrometrical measurements of the change in transmission density of the dispersed protein as function of time Based on the measure of the weight uptake after exposure of the dry protein sample to an atmosphere of defined relative humidity Based on the increases of viscosity with the water uptake Based on the weight of liquid released after centrifugation Based on the record the difference of enthalpy change that occurs during heat treatment, between a sample and a reference Based on the volume of released water after equilibration of the powder with excess water Examples of Tested Products Whey protein concentrate Milk powder, casemates, coprecipitates Reference 94

89

Rehydration test

Milk proteins

95

Sorption isotherms

Casein

96

Viscosimetry Centrifugation test

Milk protein concentrate Caseinate, whey protein concentrates Whey protein concentrate

97 98

Differential scanning calorimetry

88

Filtration procedure

Sodium caseinates Casein micelles

78

NMR From Ref. 74.

99

Generally proteins are soluble in water when electrostatic or hydration repulsion between proteins is greater than the driving forces for hydrophobic interactions. Thus, the polar and ionizable groups of proteins largely confer water solubility.

Environmental Effects on Solubility


As previously noted, the solubility of proteins depends on many factors including the inherent properties of the protein itself as well as on environmental factors (ionic strength and ion species, pH, temperature, presence of other solutes). The effect of salts on the solubility of proteins has been extensively studied and also exploited as a mean of protein isolation.77 If salt is added progressively, protein

solubility increases (salting-in) and subsequently, after passing through a maximum, starts to decrease {salting-out). The salting-in process is usually considered as a net increase of protein solubility on the basis of nonspecific electrostatic interactions between a.charged molecule and its environment.3'50 However, Arakawa and Timasheff 10 revealed that the salting-in effect of yS-lg by NaCl is strongly related to a preferential interaction of this salt with the protein. On the other hand, the effectiveness of various salts in inducing salting-out depends very strongly on the type of salt used, as indicated by the so-called Hofmeister series.101 Sodium caseinate can be salted out at ionic strengths above 0.2 M NaCl,3 but the solubility of whey proteins, because of their low tendency toward association, is not that drastically dependent on ionic strength or pH.102 pH, by affecting the charges borne by proteins, affects their solubility. Caseinates are often completely soluble at pHs above 5.519 and a solution containing 15 to 25% protein can be readily prepared at pH 6 to 8. The isoelectric point (pi), which is generally related to the pH of minimum solubility of proteins, is usually observed between pH 4.5 and 5.5. The solubility of sodium caseinate increases to 90% above pH 4.5. Whey protein concentrates are less soluble at the pi although solubility always exceeds 60%. 102 Solubility is minimal at the pi unless the net charge on the proteins is controlled in part by highly hydrated cations such as Mg2 + , Ca2 + , and Na + . In the latter situation, coagulation occurs when the H + concentration is great enough to replace the hydrated cations. The more Ca2 + present, the greater the H + concentration required to cause coagulation.3 Increasing the temperature progressively disorders both protein and solvent by disruption of hydrogen and ionic bonds which destabilizes protein structures and causes unfolding (reversible or irreversible). Generally, in the case of an irreversible unfolding, protein-protein interactions are enhanced, resulting in aggregation and precipitation.3'50 Dairy protein fractions are susceptible to heat denaturation in different ways. Caseins show an unusual heat stability. A 3% caseinate solution at pH 7 can be heated at 1400C for at least 60 minutes without significant aggregation of the proteins occurring.19 Other factors such as surfactants, organic solvents (especially water miscible), pressure, and other substances such as enzymes, polysaccharides, etc., by modifying protein structure, affect the balance of intermolecular forces that control protein solubility.3 Back et al.103 have shown that various polyols and sugars can stabilize proteins against heat denaturation by strengthening water structure which indirectly strengthens the hydrophobic interactions that stabilize protein conformations.

Solubility Measurements
The various techniques used to determine the solubility of milk proteins and milk protein ingredients have been summarized by Fox and Mulvihill.18 However, because solubility is an important functional property in the evaluation of proteins, standard methods are needed. Patel and Fry93 reported that two standard methods exist for determining solubility, namely the Nitrogen Solubility Index (NSI)104 and the Protein Dispersibility Index (PDI).105 The International Dairy Federation (IDF)

has accepted a procedure for the determination of the NSI for use as an international standardized technique for all milk protein ingredients. In fact, both methods give an indication of protein dispersibility rather than true solubility.93 The PDI method involves high-shear blending for mixing proteins with water whereas the NSI procedure employs more gentle mixing. In both techniques dispersion is followed by low-speed centrifugation to remove insoluble solids. In the NSI and PDI methods, the protein content of the sample, and of the "soluble" fraction, is estimated by determining the Kjeldahl nitrogen content, and the solubility is expressed as a simple percentage.

4.3.2 Protein-Protein Interactions


Proteins play an important role in controlling the texture of many food products. Consequently, the rheological and gelling behaviors of proteins are important determinants of their functional properties.

4.3.2.1 Rheological Behavior of Protein Dispersions


Definitions and General Considerations
Studies of the rheological properties of proteins are important because106'50 (1) they allow investigation of the conformation (shape) and interactions (hydration, aggregation) of proteins in solution, (2) they make it possible to reduce functional properties to physicochemical characteristics, and (3) they provide a tool for process monitoring and control. If proteins display a varied and complex rheological behavior, this can be interpreted as fundamentally the result of the combined contribution of the hydrodynamic volume, both size and shape, and interactions of the protein particles as schematically represented in Fig. 4.2 (Rha and Pradipasena106). The viscosity of a protein solution is concentration dependent.107"109 Menjivar and Rha108 proposed a model that described the rheological behavior of globular proteins in solution over a wide range of concentrations (Fig. 4.3). In this model each protein is composed of a molecular core (hydrated volume) and an interactive volume whose viscoelasticity is represented by Voigt models; Voigt models consist of a spring and a dashop in parallel.56 The molecular core is the volume of a swollen quarternary structure. The interactive volume includes the effect of hydrodynamic and electrostatic interactions. In this model, three separate relationships between protein concentration and viscosity are distinguished, (1) in very dilute, (2) in semiconcentrated, and (3) in concentrated protein dispersions. 1. The viscosity of very dilute and undenaturated protein (globular or random coil) dispersions, without interactions between protein molecules, is governed by the shape and size of the molecules according to Einstein's equation.110411: (4.2) where r]s and rj0 are viscosities of the suspension and of the continuous phase respectively, /3 is a shape factor (representing the axial ratio of the equivalent ellipsoid

SIZE Molecular weight Hydration HYDRODYNAMIC VOLUME

SHAPE Conformation PACKING

RHEOLOGY OF PROTEINS

ASSOCIATION DISSOCIATION DEFORMATION

INTERACTIONS Disulfide linkage Electrostatic force Van der Waals force Hydrogen bond Hydrophobic interaction Steric hindrance

Figure 4.2 Influence of various factors on the rheology of proteins. (From Ref. 106.)

INTRINSIC VISCOSITY REGIME


Tl r = 1 + [ T l ] C

DILUTE REGIME W ( C [ T l ] )

CONCENTRATED REGIME V V C [ T l ] , C/[CJ)

Cch

'Or
MINIMUM "Interactive volume"

MAXIMUM "Interactive volume"

[T|] Z hydrodynamic volume

log zero-shear viscosity

log zero-shear viscosity

Reduced viscosity

Concentration (C)

Concentration (C)

Concentration (C)

Figure 4.3 Descriptive model of rheological behaviour of proteins. (From Ref. 108.)

or rod, = 2.5 for a spherical uncharged particle), and (f> is the volume fraction of the protein in solution. The intrinsic viscosity [rf], is defined as the viscosity that exists when the molecules are completely isolated, that is, when the concentration of the protein approaches zero. (4.3) and is an indication of the hydrodynamic shape and size of the protein in solution.106 Consequently intrinsic viscosities of isolated proteins have been widely studied as a means of establishing their molecular dimensions. Mulvihill and Fox77 collated some values of [rf] for individual milk proteins under various environmental conditions. 2. When protein concentration increases, deviations from Einstein's equation are observed: they translate the change from dilute to semiconcentrated systems, and are due to the presence of hydrodynamic interactions between protein molecules. These deviations occur above a particular protein concentration, defined as the charateristic concentration (cch) which is estimated from: (4.4) For /3-lg, on the basis of 1/[Ty], Pradipasena and Rha112 have estimated that the semiconcentrated region began above 10%. Above cch, the zero shear rate viscosity no longer increases linearly but increases exponentially with protein concentration (Fig. 4.3). The relative viscosity r)T of spherical particles in the semiconcentrated region may be represented by a series expansion: (4.5) where It1 is the second virial coefficient. 3. The viscosity of concentrated protein solutions is governed by the excluded volume and by interactions between suspended particles. This region starts at a socalled critical concentration (ccr), at which the zero shear rate viscosity approaches infinity (Le., a yield stress is observed). The value of c cr can be estimated from a modified Mooney equation113 using concentration instead of volume concentration and replacing the shape factor by the intrinsic viscosity. (4.6) The critical concentration is equivalent to # m , the maximum packing fraction in the Mooney equation.108 For /3-lg, Pradipasena and Rha112 have estimated that the concentrated region corresponds to levels above 30%. The advantage of using the parameters cch and cCT is that they provide information about the nature of the protein and the extent of the intermolecular interactions in solutions over a wide range of concentrations.50-106

At concentrations > ~ 1 5 % casemates form highly viscous solutions, and at concentrations >~~20%, even at high temperature, the viscosity of solutions is so high that it is difficult to process them.77 In contrast, undenaturated whey proteins, due to their compact globular shapes, form much less viscous solutions.50-77 For protein dispersions (caseinate and whey) containing < 12%, Hermansson,114 and Towler115 have shown that their behavior is Newtonian, that is, that a linear relationship is observed between the shear stress (T) and the rate of shear strain (s): (4.7) At higher concentrations, above 12% and 18 to 20% for caseinates and whey proteins, respectively, dispersions show a more pseudoplastic Theological behavior. Flow properties are better described by a power law, (4.8) where K is the consistency index and n is the behavior index (0 < n < 1). The consistency index increases strongly with concentration.

Environmental Effects on the Rheological Behavior of Protein Dispersions


The viscosity of protein dispersions is a function of various parameters related to production (especially pH) and to processing conditions (concentration, pH, salt nature, thermal treatments, etc.). 116117 Furthermore, chemical modifications allow modifications of physicochemical properties such as flow properties and consequently functional properties.3 The variation of protein solution viscosity as a function of pH is highly complex,117 and is strongly related to the nature of the cation used. In the presence of sodium hydroxide, Hayes and Muller118 have observed a quick increase of the viscosity when the pH increases from 6.9 to 9.5, then a quick decrease for pHs higher than 9.5. These observations were confirmed by Purri et al.119 and Hermansson114 who place the maximum degree of viscosity near pH 10.8-11 and 9.8-10 respectively. Purri et al.119 explained this increase in viscosity by the gradual neutralization of acidic groups, which leads to the formation of growing amounts of strongly hydrated caseinates. The decrease in viscosity after complete neutralization would be due to a loss of the casein structure. In the presence of ammonium hydroxide,118 a plateau of viscosity is obtained at pHs between 6.5 and 9. Purri et al.119 have compared different caseinates and have observed that the degree of viscosity follows the series NH 4 + , Na + , Ca2 + , with the lowest degree of viscosity occurring in the presence of Ca 2 + . Purri et al.119 attributed the higher values in the presence of ammonium ions to the formation of hydrogen bonds between particular functional groups of casein, such as phenol and e-amino groups. The addition of calcium to sodium caseinate increases viscosity, especially at pHs > 7, but at pHs < 7 the addition of calcium at a concentration of > 8 mg/g causes a decrease in viscosity, presumably due to micelle formation.77 At lower pHs, Korolczuk97 has reported an increase in viscosity when the pH decreases. This effect is explained on the basis of electrostatic repul-

sions; in caseins containing more carboxylic and phosphate groups than amine groups, the net positive charge in acidic solution is lower than the net negative charge in basic solution. Consequently, the probability of aggregate formation is higher in acidic solution, thus resulting in a higher viscosity of these solutions. Moreover, casemate viscosity increases with ionic strength.117 The addition of sodium chloride leads to a strong increase in viscosity, but only for sodium caseinate concentrations > 10%.114 Colas117 reported the existence of a patent120 using soluble aluminum salts to increase viscosity. Indeed, the addition of 1.5% hydrated aluminum sulfate multiplies the viscosity of a 12% sodium caseinate solution by 100; this trivalent cation probably allows a larger reticulation of the protein by the formation of ionic bridges.117 The viscosity of casein and caseinate solutions decreases as the temperature increases.116 However, the magnitude of this classic phenomenon is strongly affected by the pH and by the presence of Ca 2 + ions. In the case of sodium caseinate, at pH 7, the Andrade relation is confirmed: a linear relationship exists between log viscosity and the reciprocal of absolute temperature.115-118121'122 The decrease in viscosity correlates with a decrease in hydration capacity.123 This relation is not, however, confirmed when calcium is added to the solution:121 viscosity decreases quickly when the temperature increases from 30 to 38C, then stays constant to 570C where a gel forms. At acidic pH (2.4 to 2.9), the situation is slightly different:123 the viscosity of solutions decreases when the temperature increases from 25 to 600C, and the hydration capacity remains practically unchanged; between 60 and 800C, viscosity and hydration capacity increase. As previously mentioned, this increase can be explained by a decrease in the net charge of proteins at pH values lower than the pi; moreover, hydrophobic interactions increase with temperature. These two phenomena contribute to the formation of aggregates, and an increase in viscosity.117 The viscosity of whey concentrates in the range from 25% to 40% depends strongly on the composition and preheat treatment of the whey.50 This appears to be caused mainly by the rate of crystallization of the lactose in the concentrates.124 Limited proteolysis by proteinases such as plasmin 125126 or treatment with disulfide reducing or sulfydryl blocking agents127 can also markedly reduce the viscosity of caseinates.

Measurement of the Rheological Behavior of Protein Dispersions


Due to the extensive nature of the subject, the following section is far from exhaustive. The main rheometer classes can be classified as: 56 ' 128 1. Rheometers operating under stationary conditions. They work essentially as viscosimeters to determine viscosities and r-s rheograms of liquid substances. In these rheometers, the sample is submitted to laminar shear forces, independent of time. The following types of rheometers fall into this category: Couette-type rheometers in which the sample is sheared between two solid surfaces, one at rest, and the other mobile.

Poiseuille-type rheometers in which the shearing movement is due to the application, at the ends of a cylindrical tube containing the sample, of a pressure differential. In most cases, this pressure differential is given by the action of gravity. Viscosimeters with falling or rolling spheres whose applications are limited (they allow only the study of rigorously Newtonian liquids), but the are very well known and relatively widely used. 2. Rheometers that work under transitory conditions which allow the study of the viscoelastic properties of material. They are essentially two types of transitory rheometers: Creep compliance rheometers. In creep compliance testing, a small constant stress (r) is applied to the sample, and the resulting strain (s) is followed with time. Stress relaxation rheometers. In a stress relaxation experiment, a small constant strain (e) is applied to a given sample, and the resulting stress (T) is followed over time. 3. Dynamic rheometers. In dynamic testing, conditions are used that will not alter the structure of a material and will satisfy the requirements of a linear viscoelasticity theory based on infinitesimal strains and strain rates where the ratio of stress to strain is a function of time (frequency) alone, and not of stress magnitude.116 From the curves obtained for a given sample, the elastic and viscous components known as the dynamic shear storage modulus (G', which describes the elastic nature of the material) and the dynamic shear loss modulus (G", which describes the viscous nature of the material) can be determined. Two other parameters can also be evaluated, the loss tangent expressed as tang S = G "/G' and the dynamic viscosity tf = GVw, where co is the oscillatory frequency. Generally, two types of devices are used according to whether or not the movement is conserved: Rheometers with forced oscillations which can function over a large range of frequencies or at a particular frequency. Rheometers with free oscillations which allow the measurement of low viscosities by studying the breaking point. These latter two sets of rheometers (2 and 3) are also particularly well adapted to studying the rheological behavior of viscoelastic foods such as gels. In addition, some rheometers can operate under stationary, transitory, or dynamic conditions, if optional equipment is also used.

4.3.2.2 Gelling Properties of Globular Proteins


Definitions and General Considerations
There are many processed foods (cheese, firm yogurt, etc.) in which proteins act as gelling agents and provide a desirable texture.12 Although they hold large amounts of water, one of their characteristic features is to behave as solids while retaining many of the properties of their liquid component.129 On quickly applying and removing a very small stress, a gel reversibly loses its form, in the manner of an elastic

body with a low modulus of elasticity. Flow may occur, but only above a finite yield stress.33 The gel structure usually takes one of the following two forms:33'77 1. Polymer network. The gel structure is provided by a well-ordered, three dimensional cross-linked macromolecular network, or matrix made up of cagelike unit structures of uncoiled polypeptide segments that interact at specific points and are able to retain large amounts of water due to specific intermolecular forces (covalent, hydrogen, and hydrophobic). Gels of this type are formed by globular proteins, and polysaccharides. 2. Aggregated dispersion. The gel structure consists of a highly aggregated dispersion of colloidal particles. Intuitively, it is reasonable to think that aggregation tends to occur more frequently when concentrations are high and pH is in the isoelectric range. Clotting of milk is an example of this type of gelation. A clear distinction between these two types of gels is rather difficult however, especially when hydrocolloids are implicated in the gelation process.33 To define more clearly the gelation process, de Gennes130 has proposed distinguishing two types of Theological behavior: (1) strong gelation, and (2) weak gelation. If crosslinks, once formed, remain intact for a finite time under stress, gelation is considered to be strong. In contrast, when crosslinks are not strong enough to resist a small stress, the system is said to exhibit weak gelation. The gelation process of globular proteins can be caused by heat denaturation or by any other process (changes of pH, addition of salts, action of enzymes, etc.) that converts proteins to a state that favors intermolecular protein interactions. The next section is restricted to heat induced gelation of globular whey proteins.62'63'131"134 From statistical theories of gelation129'135'136 the process of gelation is described as the formation of an infinite network of trifunctional and bifunctional units (Fig. 4.4). If initially there are f reactive sites per molecule, when a critical fraction ac of these have reacted the weight-average molecular weight diverges to infinity: this is the gel point. If the aggregation process is random, theory predicts that f and ac are related by the equation:129 (4.9) At this stage, a gel fraction and a sol fraction coexist. However, the sol fraction decreases as the gel fraction increases beyond a c . Moreover, the sol fraction can disappear, and the gel rigidity can eventually increase with time if crosslinking proceeds far enough.62 Heat induced gelation of globular proteins is a two-step process involving:137 (1) an activation (or denaturation) step, and (2) an association step
heat heat and/or cooling

A B Linear Chain B A A Branching point

B
B .A AB BA A B AB BA A

BA

AB

BB

BB

BA

Gel matrix
Figure 4.4 Formation of an infinite gel matrix. (Adapted from Ref. 129.)

where x is the number of protein molecules, P N is the native protein, and P D is the denatured protein. The initial stage corresponds to a conformational alteration or unfolding of the secondary and tertiary structures due to a decrease in intermolecular forces. This unfolding is induced by an increase in temperature. The denaturation temperature or gelling point (T d ) is the point at which the extent of the reaction is equal to 0.5 and [PNI = [PD]- 1 3 8 Th e magnitude of this unfolding is a function of temperature as well as of environmental factors. The unfolding increases, in general, the exposition of

hydrophobic (favoring aggregation) and thiol residues, and favors formation or exchange of disulfide bridges (irreversible gels). The subsequent polymerization process is affected by the capacity of the protein surface for intermolecular interactions and requires a balance between proteinprotein interactions, protein-solvent interactions, and attractive/repulsive forces between adjacent polypeptidic chains.133 Hydrophobic interactions (favored at high temperature), bridges with Ca 2 + or other divalent salts, hydrogen bonds (favored during cooling) and disulfide bridges represent attractive forces. Electrostatic repulsions, principally at pHs higher than pi, and protein-water interactions, act to keep polypeptide chains separated.9 If protein-protein interactions are too weak (repulsive forces predominate), viscosity will increase, but the fluid can always flow: a gel, strictly speaking, cannot form. In contrast, if protein-protein interactions are too strong (attractive forces predominate), the network will collapse and water will be expelled from the structure.139 The functional properties of gelling proteins such as gelation time and the rigidity modulus are related to several factors:133'140 (1) the nature of the protein, the lowest concentration of protein required to form a gel and desired gel texture, (2) the conditions required for gelation (temperature pH, ions), and (3) matrix geometry, flexibility of the polymer, and strength of the junctions (chemical nature and extent of protein-protein interactions). Experimentally, protein concentration determines both the likelihood of gel formation and also the characteristics of the gel that forms. If the protein solution is heated below a concentration sometimes designated as the critical concentration,62 and which varies according to the protein utilized, gelation will not occur. Indeed, when the protein concentration is too low, a protein network is difficult to establish: protein-protein interactions tend to be intramolecular rather than intermolecular and a gel network cannot be established. As protein content increases, the likelihood of intermolecular interactions increases, and at the critical concentration proteins form a coagulum or at the temperature that initiates the gelation process (generally between 70 and 85C), or during cooling.63-137139 The firmness of gels and the gelation speed increase with the protein content and the heating temperature up to i20C102'141-142 due to a higher probability of proteinprotein interactions. The temperature at which gelation begins decreases as the protein concentration increases; solutions containing 3 or 9% of j3-lg (pH 6.6, 1% of NaCl) begin to gel at 82 and 75C, respectively. Solutions containing 1 or 5% of BSA (pH 6.6, 1% of NaCl) gel at 82 and 77C, respectively.143 Under the same conditions of pH and ionic strength, a-la does not form a gel, even at a concentration of 20%. 143

Influence of Environmental Factors


Gelation of globular proteins are largely influenced by media conditions (e.g., pH, salt nature, presence of other solutes, etc.). An increase of pH of /3-lg solutions from 8.7 to 9.0 decreases the temperature at which gelation begins,16-94144 decreases the rate of gelation,141 and beyond pH 7.5 to 8.0 decreases gel firmness.145 A decrease

of pH, in the acid zone, from 5.0 to 4.0 leads to a decrease in the temperature at which gelation begins, from 700C to 500C. However, the observed increase in viscosity may be due to aggregation rather than real gelation.16'94'146 An increase in NaCl concentration to 0.5 M leads to an increase in viscosity,147 or in gel firmness (10% whey proteins, pH 7.0, 80C/30 min or 100C/15 min respectively),145 but generally leads to a decrease in water-holding capacity as soon as the NaCl concentration goes above 0.3 M.148 The presence of 1 or 2 M NaCl increases the temperature at which gelation begins (10% whey proteins, pH 7.0, 85C/30 min).147 After heating of /3-lg solutions (9%, pH 2.5, 90C/30 min), in the presence of NaCl 0.2 M, Harwalkar and Kalab149 obtained gels with a regular matrix and particles of small size. At higher ionic strength (0.4 M NaCl), the presence of voluminous aggregates (0.5 to 2 mm) can be detected by electronic microscopy.149 The addition of CaCl2 leads to an increase in gel firmness (10% whey proteins, pH 7.0, 100C/15 min) up to a concentration of 0.04 M (with a maximum at 0.011 M), but is associated with a decrease in the elasticity and water-holding capacity of the gel.150 When the protein concentrate is dialysed, the gels obtained are firmer, more elastic, and more translucid than those prepared with the nondialyzed protein concentrate.148 The presence of reducing agents inhibits gelation.151 With a series of whey protein concentrates, a positive correlation has been established between the gelifying power on the one hand and the sulfhydric group content and protein solubility at pH 4.5 on the other.151 The addition of cysteine to a concentration of 40 mM decreases gel quality (10% proteins, pH 7.0, 100C/15 min):150 gel firmness is maximum at a cysteine concentration of 9.7 mM, but the cohesiveness, the elasticity, and the waterholding capacity of the gel decrease. The addition of sucrose delays gelation and increases the temperature at which gelation begins; however, gels with a smooth texture, similar to a baked custard, can be obtained in the presence of 30% sucrose (5% ultrafiltered and diafiltered whey proteins, 115C/5 min). 152153 The presence of other milk proteins can modify the gel quality obtained; for example, gels prepared from whey protein concentrates (10%, pH 7.0, 85C/10 min) are more opaque and less elastic than those obtained from purified /3-lg.63 Under the effect of heat, /3-lg can form complexes with other milk proteins. Doi et al.154 have obtained a gel by heating a solution containing 5% /3-lg and 5% /c-casein (pH 7.1, 70 mM KCl, 90C/10 min), but separate solutions of /3-lg and of a-casein do not gel under the same conditions. Similarly, these authors155 have obtained a gel from a solution containing 2.5% a-la and 2.5% /c-casein (pH 7.6,0.4 NaCl, 90C/30 min), but a solution of a-la alone, under the same conditions, does not gel.

Measurement of Gelling Properties


The Theological and textural properties of gels are important properties for determining acceptance. They are measured when the food is exposed to a certain stress and shear strain rate and therefore Theological measurements are considered relevant for characterizing texture on a nonsensorial level. A large number of instru-

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merits and techniques have been developed for measuring Theological and textural properties.56-156'157 Furthermore, texture profile measurements as defined by Szczesniak158-159 have so far served as bridge between fundamental rheological principles and popular nomenclature. Various techniques can in general be separated in the analyses using small, nondestructive strains (i.e., sample deformation) and destructive techniques (i.e., sample rupture).133 In the first case, rheological characteristics using small, nondestructive strains allows a dynamic measurement of rheological transitions. Changes in rigidity or shear modulus (stress/strain), storage modulus (G') and loss modulus (G") can be measured as a function of time or temperature. These rheological characteristics of a viscoelastic body are independent of size and shape, and can be determined by a large variety of methods.133-160

4.3,3 Protein-Surface Interactions


The boundary between two homogeneous phases is not to be regarded as a simple geometrical plane, on either side of which extend the homogeneous phases, but rather as a lamina or film with a characteristic thickness; the material in this "surface phase" has properties differing from those of the materials in the contiguous homogeneous phases.161 Here we are especially concerned with the interfaces between two immiscible or partly miscible liquids (emulsions), and between a liquid and a

gas (foams).
It is a matter of everyday experience that two immiscible liquids rapidly separate into two distinct phases and consequently the adage, "like oil and water." The reason why oil and water alone, after being mixed by shaking, separate so quickly is that this intense agitation, by inducing the dispersion of one phase (dispersed phase) under droplet form in the other (continuous phase), also induces an extensive increase of the interfacial surface and therefore of the free energy of the system. In following symbols, if y is the force per unit of length tending to contract such a surface or interfacial tension, and if S, T, P, V, A, //,, and n refer respectively to entropy, absolute temperature, pressure, volume, surface area, chemical potential, and number of moles in the system, then (4.10) where G represents the total Gibbs free energy of the system. At constant temperature and pressure and for a given number of moles in the system, this reduces to (4.11) As interfaces between phases necessarily have a positive free energy, the variation of the free energy of the system is reduced by coalescence (4.11). Coalescence is an irreversible phenomenon because the surface area of the new droplets is less than the sum of the surface areas of the two colliding droplets. The effects of Brownian motion, arising from the distribution of thermal energy between the molecules of the system, combined in real systems, to the movements caused by density differences (sedimentation or creaming), which put droplets into contact, and to van

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merits and techniques have been developed for measuring Theological and textural properties.56-156'157 Furthermore, texture profile measurements as defined by Szczesniak158-159 have so far served as bridge between fundamental rheological principles and popular nomenclature. Various techniques can in general be separated in the analyses using small, nondestructive strains (i.e., sample deformation) and destructive techniques (i.e., sample rupture).133 In the first case, rheological characteristics using small, nondestructive strains allows a dynamic measurement of rheological transitions. Changes in rigidity or shear modulus (stress/strain), storage modulus (G') and loss modulus (G") can be measured as a function of time or temperature. These rheological characteristics of a viscoelastic body are independent of size and shape, and can be determined by a large variety of methods.133-160

4.3,3 Protein-Surface Interactions


The boundary between two homogeneous phases is not to be regarded as a simple geometrical plane, on either side of which extend the homogeneous phases, but rather as a lamina or film with a characteristic thickness; the material in this "surface phase" has properties differing from those of the materials in the contiguous homogeneous phases.161 Here we are especially concerned with the interfaces between two immiscible or partly miscible liquids (emulsions), and between a liquid and a

gas (foams).
It is a matter of everyday experience that two immiscible liquids rapidly separate into two distinct phases and consequently the adage, "like oil and water." The reason why oil and water alone, after being mixed by shaking, separate so quickly is that this intense agitation, by inducing the dispersion of one phase (dispersed phase) under droplet form in the other (continuous phase), also induces an extensive increase of the interfacial surface and therefore of the free energy of the system. In following symbols, if y is the force per unit of length tending to contract such a surface or interfacial tension, and if S, T, P, V, A, //,, and n refer respectively to entropy, absolute temperature, pressure, volume, surface area, chemical potential, and number of moles in the system, then (4.10) where G represents the total Gibbs free energy of the system. At constant temperature and pressure and for a given number of moles in the system, this reduces to (4.11) As interfaces between phases necessarily have a positive free energy, the variation of the free energy of the system is reduced by coalescence (4.11). Coalescence is an irreversible phenomenon because the surface area of the new droplets is less than the sum of the surface areas of the two colliding droplets. The effects of Brownian motion, arising from the distribution of thermal energy between the molecules of the system, combined in real systems, to the movements caused by density differences (sedimentation or creaming), which put droplets into contact, and to van

der Waals forces of attraction which pull droplets together, causing them to
coalescence.162

To prevent rapid breakdown of an oil-in-water dispersion, as with, any other emulsion type or foam, it is necessary to add a third componentemulsifier or surfactantwhich, from its amphiphilic nature, spontaneously adsorbs at the interface and prevents or at least reduces contacts leading to coalescence by introducing intermolecular forces of a repulsive nature.163 The adsorption of emulsifiers including proteins is spontaneous because it is thermodynamically favorable. Indeed, emulsifiers minimize their contribution to the free energy of the system by decreasing interfacial tension (4.11), that is, they adopt an orientation such that the hydrophobic groups (usually hydrocarbon chains) tend whenever possible, to adopt a position in the oil phase, whereas hydrophilic groups (such as -OH, -CO2H) are in a lower energy state in the aqueous phase.33

4.3.3.1 Interfacial Properties of Milk Proteins


Definitions and General Considerations
Proteins and, a fortiori, milk proteins are, from their chemical nature, surface active compounds. However, the adsorption of proteins differs from that of low molecular weight emulsifiers. In the first place, there are many possible regions of interaction with an interface along a protein chain so that the energy of adsorption is large even if the energy of adsorption for each individual region is small. Second, if absorbed macromolecules are flexible, they can adopt a large number of configurations at the interface. However, entanglements with neighboring molecules generally prevent the state of lowest energy being attained.164 Fig. 4.5 165 shows the configuration of a protein chain at an oil/water (O/W) interface. Only a fraction of the molecule is in direct contact with the surface in the form of trains. The remainder protrudes into the two contiguous homogeneous phases, as the three dimensional loops and tails, to form an interfacial region that is much thicker than the width of the chain. The proportion between the train and loop regions determines, in part, the interfacial efficiency of proteins.166 Consequently, one can predict that not very structured, flexible proteins such as /3-casein will be more surface active than a rigid globular protein such as /3-lg or a-la. During adsorption, contacts between surface and solvent, and between protein segments forming future trains and solvent are decreased and replaced by direct contact between surface and protein segments.164 The enthalpy of adsorption being positive, and the free energy of adsorption being negative (spontaneous process), the process of adsorption for proteins and other polymers is dominated by the change of entropy. This includes the change in the configurational entropy of the proteins and the entropic change resulting from the solvent released from the surface and the protein.33 Experimentally, the kinetics of protein adsorption can be monitored by following the time dependence of the surface concentration (F) or the surface pressure (/T). The pressure surface is defined as (4.12)

NATIVE

DENATURED

Train

Tail

OIL WATER

Loops Figure 4.5 Orientation of proteins at an interface. Schematic representation of nonpolar (O), polar ( ) , and neutral (@) residues of protein. (From Ref. 165.)

where yo and y are the interfacial tensions in the absence and in the presence of emulsifier.167 Fig. 4.6 168 gives typical results and shows the changes in Hand Ffor /3-casein and &gg white lysozyme (which is almost homologous with a-la) at the air/ water (A/ W) interface, at room temperature. For the disordered and flexible /3-casein, changes in TI and Tare closely coupled (Fig. 4.6a). However, with lysozyme which is a very rigid globular protein, the J7-t curve shows an initial period of "induction", and moreover /7 is still increasing significantly when F has attained its steady state value (Fig. 4.6b). The presence of the induction period in the /T-t curve has been observed by others 169 in dilute solutions, and is not entirely clear in terms of molecular behavior. 170 One explanation assumes that this induction time corresponds to an accumulation of protein segments near the interfacial region before the adsorption occurs. 171 According to Cumper and Alexander, 172 and MacRitchie, 173 the increase of TI with time can be attributed to three molecular processes: (1) the diffusion of protein

r(mgnr 2 )

(a)

t(h)

(a)
r(mgm" 2 ) n(mNm" 1 )
(b)

t(h)
(b)

Figure 4.6 Adsorption of ^-casein and lysozyme at the A/W interface. The surface concentration F (O) and the surface pressure II () are plotted against the time (t) for protein adsorption at 200C, pH 7, and ionic strength = 0.1 mol dm"3, (a) (3-casein, initial concentration = 7.3 X 10"4 kg m~3. (b) lysozyme, initial concentration = 7.6 X 10~4 kg m~3. (From Ref. 168.)

molecules to the surface, (2) the spreading or unfolding of adsorbed protein molecules, and (3) the conformational rearrangement of adsorbed protein molecules. At the outset, protein chains must arrive at an interface by simple molecular diffusion. De Feijter and Benjamins171 have shown that this statement is true only for the very early stages of the process, that is, when JT ^ 1 mNm" 1 . They also drew the conclusion that this diffusion-controlled period coincides with the induction period. Benjamins et al.174 reported diffusion coefficients ranging from 3.3 to 0.7 X 10" 1 0 m 2 ^ " 1 for several proteins, with 0-casein being adsorbed much more rapidly than /c-casein or BSA. For adsorption to occur, only a small section of the macromolecule needs to enter the interfacial region. In addition, the area of contact that is needed is very small (1.0 to 1.75 nm2) and is not related to the total size or molecular conformation.175

n(mNnr 1 )

(a)
1

(b)

(C)

Figure 4.7 Schematic representation of the protein adsorption at liquid interfaces: (a) P-casein at ATW interface: (1) T < 1 mg m~ 2 , (2) Tsat > T > 1 mg m" 2 , (3) T = T8^, (4) T > T5^. (b) p-casein at O/W interface: (1) - (4) as in (a), (c) lysozyme at AAV interface: (1) T < 2 mg m" 2 , (2) T > 3 mg m" 2 , (3) T = Tsat, (4) T > Tsat. Tsat = surface concentration at primary layer saturation. (From Ref. 178.)

Although protein adsorption is a thermodynamically favorable process, the attainment of an equilibrium state, that is, of an equilibrium interfacial conformation, can take time; Kim and Kinsella 176 who studied the ability of BSA to lower the superficial tension, reported that the equilibrium surface pressure was attained only after 24 h. Castle et a!.177 reported that very slow but continuous structural changes, as indicated by surface rheological parameters, take place in adsorbed protein films over a period of several days. If flexible proteins arrive at their equilibrium conformation quickly, it is not generally the case for globular proteins. Films, and principally concentrated ones, can contain protein chains with different degrees of unfolding (Fig. 4.7). 178 Consequently, films are not in an equilibrium state and rearrangements of proteins with individual trains desorbing and others adsorbing occur to obtain the lowest energy state. 179 Moreover, adsorbing proteins are affected by the already adsorbed proteins. The latter exert an energy barrier made up of a physical barrier due to dynamic rearrangements of loops and tails on the aqueous

side and an electrical barrier, unless the system is very close to the pi. 164 This effect is probably related to multilayer formation. However, further "adsorption" may occur, but solely through protein-protein interactions, as shown schematically in Fig. 4.7d.178 An issue that has been under much debate is whether protein adsorption is irreversible at the interface.170 Cohen Stuart et a/.180"182 have proposed a theoretical model; MacRitchie183 has shown experimentally the reversibility of protein adsorption. Norde et al.1S4 reported BSA, adsorbed on various adsorbents, could be removed, totally or in part, by adjusting the pH or ionic strength, or by adding a displacer. For rigid proteins, where the conformational changes on adsorption are small, this is a matter to consider during the experiments. However, for more unfolded proteins with many attachments at the interface, the energy requirements for desorption are very unfavorable. Thus, within the time limits of most experiments, protein adsorption can be regarded as irreversible.2833

Environmental Effects on Interfacial Properties


Beginning with Jackson and Pallansch,185 surface and interfacial properties of individual milk proteins have been extensively characterized (see reviews of Kinsella,14 Leman and Kinsella,25 and Tornberg et <z/.28). Jackson and Pallansch185 found that the interfacial activity of different milk proteins, at a butter-oil/water (O/W) interface at 400C, decreases in the order /3-casein > monodispersed casein micelles > BSA > a-la > a-casein > /S-Ig. These results are consistent with the previous discussion, indicating that caseins may have better interfacial properties than native whey proteins, but thermal unfolding may improve the emulsifying properties of whey proteins.3*29 Mitchell et al.186 continued these studies in more detail by following the surface pressure developed by the six major milk proteins, at the airphosphate buffer (A/W) interface (pH 7,1 = 0.1, T = 25C) as a function of area (J7-A isotherms), subphase concentration (il-C isotherms), and time (/7-t isotherms). With the exception of BSA, the /7-A relationships were independent of the structure of the proteins and were not affected by heating or urea treatment, whereas the /T-C and JFT-t isotherms are strongly dependent on these conditions. This suggested that the protein molecules, with the exception of BSA, unfold to some extent on adsorption at the interface. The TT-C isotherms for open and flexible K structures such as /3-, a sl -, and /c-caseins superimposed exactly. The /7-t isotherms strongly reflected protein structure and showed that caseins, especially /3- and a sl -caseins, are more rapid in lowering surface tension than whey proteins and give rise to a large surface pressure (JI). The order of effectiveness, expressed in y after 50 minutes at a protein concentration of 10" 3 wt%, was as follows: /3-casein (22 mN m " J ) > a sl -casein (16 mN m~ l ) > K-casein (15 mN m" 2 ) > j3-lg (13 mN m~ 2 ) > a sl -la (12 mN m ~ J ) > BSA (8 mN m " ! ) . This order of effectiveness does not completely fit with that observed by Jackson and Pallansch185 at the O/W interface. More precisely, /3-lg is more surface active than BSA at the A/W interface. The latest result was also reported by Tornberg and co-workers.187188 Tornberg et al.2* reported some results on the interfacial activity of /3-, a-, and /c-caseins at the air-water interface (protein concentration of 10" 3 wt%, pH 7, I = 0.2 M NaCl,

T = 4C). The a- and /c-caseins are similar in activity, whereas /3-casein gives rise to a quicker and larger surface tension. Britten et aL189 have also shown similar properties of interfacial measurements of casein micelles and their fractions. The surface Theological properties of adsorbed films of milk proteins are sensitive to pH. Dickinson,27 studying time-dependent surface viscosities for casemate films adsorbed at the O/W interface from 10 ~ 3 wt% buffered protein solutions at pH 3 and 7, reported that the surface viscosity under acidic conditions is an order of magnitude higher than that measured at neutral pH. These results, consistent with bulk Theological measurements,97190 showed that, at similar concentrations, acidic casein solutions are much more viscous than neutral sodium casemate solutions. In practice, however, it is rare that only one type of protein is involved in real systems. One can imagine that a competition for the various adsorption sites exists between the various sources of food macromolecules. Musselwithe191 reported the preferential adsorption of caseins at the O/W interface, at 44C, from an aqueous solution containing, in the same proportion, two disordered macromolecules: gelatin and casein; the surface pressure isotherm being close to that of the casein alone. Recently, Dickinson et al.192 have confirmed Musselwithe's work. Murray,193 by studying the behavior of 50:50 mixtures containing /3-lg and another milk protein (/3-, K-casein or a-la) has reported that the isotherms for the mixed films cannot be simply related to the isotherms of the individual proteins. With the /3-lg H- /3-casein mixture, it was suggested that /3-casein prevents the unfolding of /3-lg at the interface. Dickinson et al.,192 studying the adsorption of a sl , /3-caseins and various casemates onto polystyrene lattices, have shown that the more hydrophobic /3-casein is more surface active than asl-casein and that caseinates have surface properties intermediate between these two. This suggests that these components adsorb independently and not competitively. Recently, Dalgleish and coworkers 194195 have provided information on possible conformations of milk proteins (/3-casein and /3-lg) when adsorbed onto polystyrene lattices. If many studies have been carried out with milk proteins, alone or in mixture, relatively less work has been done with proteins and low molecular weight lipophilic emulsifiers. Paquin et al.,196 and Laliberte et al}91 have investigated the behavior of mixed films of monoglycerides (GSM)/sodium caseinates and GMS/casein at the A/W interface. Results exhibited a high surface pressure region dominated by GMS, and in areas where there was only a small contribution from proteins. This contribution arises from the hydrophobic portion of the casein molecules which stick into the lipophilic (GMS) matrix at high surface pressure. This model is consistent with the interpretation of results obtained by Courthaudon et al. (1991, personal communication) on a model emulsion system containing casemate + C12E2 at the n-tetradecane/W interface. The alternative to competition is cooperation. Larichev et a/.198 found that complexes of BSA with dextran sulfate produced more stable decane/W emulsions than BSA alone.

Measurements of Interfacial Properties


Various techniques can be used to study interfacial properties (see general textbooks on the physical chemistry of surfaces).

The ring of du Noiiy and the capillary rise methods seem unsatisfactory for timedependent solutions. 187 For studying the adsorption of proteins at interfaces, the Wilhelmy plate technique is the most commonly used method 1 6 8 ' 1 7 1 1 7 6 - 1 7 8 1 8 6 1 8 9 ' 1 9 9 and, operates on the following principle. A very thin plate is attached to an arm of a balance and the additional pull on the plate, when it becomes partly immersed, is equal to the product of the perimeter and the surface tension. 161 Compared to the pendant drop and the drop volume method, one of the advantages is that continuous measurements can be performed as a function of time. 28 The pendant drop and the drop volume method, respectively based on the shape of the drop and on the volume (or weight) of a liquid drop that detaches itself from the tip of a vertical tube are less often used. 188 ' 200 ' 201 Tornberg187 has adjusted the drop volume technique to be able to follow the time dependence of the lowering of surface tension by proteins. Another set of methods mainly represented by the Langmuir film balance are also widely used to study adsorption from solutions or the spreading of monolayers. This technique involves measuring the film pressure surface directly, rather than calculating it from surface tension differences (4.12). The Langmuir balance is composed of a trough of inert material whose surface is swept by barriers to clean the surface and to compress monolayers. By means of this arrangement, it is possible to vary the area of a spread monolayer and directly measure the corresponding film pressure. However, althouth the Langmuir is quite a simple device, obtaining unambiguous results is far from simple. Anyone interested in considering this type of experiment should consult the more detailed description given by Games. 202

4.3.3.2 Dispersed Systems: Emulsions and Foams


Emulsions and foams are, by definition (4.11), unstable systems. However, the addition of emulsifiers (low molecular weight emulsifiers and/or macromolecules) allows one to control the kinetics of the instability processes that lead to the breakdown of emulsions and foams by modifying surfaces forces. The latter, as do all static forces, act between particles and depend on particle separation (h). Such forces are affected by the properties of both the particles and the separating medium. 203 Since emulsions and foams are colloidal systems, their behavior is governed by the general aspects of colloidal science, as well as specific factors relating to the presence of proteins at the interfaces. There are three main mechanisms or forces which are generally used in considering the stability of colloidal systems; two are based on the interactions between charged droplets, and the third depends on steric considerations. Detailed reviews on interparticle forces can be found in Mahanty and Ninham, 204 Dickinson and Stainsby,33 Israelachvili,11 de Gennes, 205 Fisher and Parker,163 and Bergenstahl and Claessom. 203 Forces between molecules and particles caused by interactions between permanent and induced dipoles and other multipoles (Keesom, Debye, and London interaction forces) are collectively known as van der Waals forces. The classic omnipresent London interactions (induced dipole-induced dipole) are dominant attractives forces over the distances that are important when considering dispersed system stability. 162 De Boer 206 and Hamaker,207 by integrating the van der Waals forces acting

Primary maximum

POTENTIAL ENERGY (kT)

ELECTROSTATIC REPULSION

Primary minimum

Secondary minimum VANDERWAALS ATTRACTION

DISTANCE OF SEPARATION (nm) Figure 4.8 Potential energy versus distance separation curve for a pair of electrostatically stabilized droplets; also shows the separate contributions of the electrostatic and van der Waals components. (From Ref. 162.)

between the individual atoms making up the particles, calculated the attractive potential VA between two spheres of equal radius (a): (4.13) if h < < a where AH is the Hamaker constant which depends on the density and the polarisability of the material making up the particles.208 In principle, this constant can be calculated, but in practice the estimation of this constraint is fraught with considerable uncertainty, especially as the structures of the particles become more complex.204'209*210 Although not exact, this equation indicates the character of the attractive force which increases more and more rapidly as the droplets approach one another (Fig. 4.8).162 A more accurate theory was developed 20 years later by Lifshitz and coworkers211'212 which described the van der Waals forces as originating from spontaneous electromagnetic fluctuations. This theory, in contrast to the Hamaker and de Boer approach, takes into account many body effects, temperature dependence, and effects due to the finite speed of light, and to continuous medium. It turns out that the van der Waals forces between identical particles are always attractive, whereas such forces, according the latter theory, may be repulsive between particles having different chemical compositions.163 In practice however, this theory

remains quite difficult to apply. The range of van der Waals forces of attraction in an oil-in-water (O/W) emulsions is of the order of 20 nm; at greater distances, the effects of van der Waals potential would be roughly countered by Brownian motion of the particles.163 To impart stability to colloidal systems, it is necessary to have repulsive forces between dispersed particles as strong as, and comparable in range to, the everpresent van der Waals forces. In dispersed systems, this may be achieved by acquiring an electric layer through the ionization of characteristic groups of adsorbed proteins (e.g., -CO 2 " and -NH 4 + groups) or through the adsorption or dissolution of small ions.33 Electroneutrality of the whole system requires that the net charge on the dispersed particles be balanced by oppositively charged ions (counterions) whose concentration decreases as one moves away from the charged surface; ions of the same charge (coions) are repelled near the surface. The region of unequal counterand coions surrounding the charged surface is called the electrical double layer. This double layer can be regarded as consisting of two regions (Stern theory): an inner region of strongly adsorbing ions, and an outer region where charges are diffusely distributed according to a balance between electrical forces and random thermal motions.11-33 Since all proteins carry some net charge, it is certain that adsorption of proteins to an interface will lead to the formation of double layers around the emulsion or foam droplets. It is the interaction of these double layers, as two such particles approach, which leads to a mutual repulsion. This mutual repulsion can also be understood as an osmotic pressure effect: the excess concentration of counterions in the space between the double layers produces a local osmotic pressure difference between the interacting layers and the bulk solution.213 The range of the electrostatic forces is of the order of the ' 'thickness" of the electrical double layer which is usually characterized by the Debye-Hiickel length (1/K): (4.14) where s r and e o are the permitivities of the vacuum and the continuous phase, respectively. K the Boltzmann constant; T is the absolute temperature; e is the electronic charge; and c and Z are the concentration and charge number of the ions in the continuous phases, respectively.167 Calculations of the energy of the electrostatic repulsions require numerous assumptions about the conditions at the surface of the particles when they interact, but Derjaguin and Landau,214 and Verwey and Overbeek213 have demonstrated that it is possible to roughly estimate solutions for the energy of electrostatic interactions between two charged particles by considering either particles which are large and have thin double layers (that is, where Ka > > 1), or which are small and have large double layers (that is, KQ. 1). In the case of protein-stabilized emulsions, it is clear that the first of these cases is applicable,215 and so one of the best known solutions, only valid for low surface potentials (if/ < 25 mV), derived by Derjaguin and Landau214 is given by: (4.15)

where is ip the surface potential. Verwey and Overbeek213 have published tables of VR using more exact solutions valid for higher surface potentials. This equation, however, indicates that the electrostatic repulsion falls off exponentially with distance (Fig. 4.8). The range is very sensitive to the ionic strength of the continuous phase since K is proportional to the square root of the electrolyte concentration. Typically, l//c would fall from 100 nm at 10~ 5 M to 1 nm at 10" ] M for a univalent electrolyte. The total interaction energy VT between colloidal particles can be calculated by adding the van der Waals attractive forces and the double-layer repulsive potentials (4.13 and 4.15). This theory, independently derived by Derjaguin and Landau,214 and by Verwey and Overbeek,213 and known as the DLVO Theory, is undoubtedly one of the greatest steps forward in understanding the stability of colloidal system. Schematic results such as those in Fig. 4.8 162 show how the forms of the functions for VA and VR combine to give a maximum repulsive potential so that particles are prevented from coalescing. From equations (4.13) to (4.15), it is clear that the stability of electrostatically stabilized droplets depends on the height of the primary maximum (Fig. 4.8) which in turn depends on the surface potential (#), the range of the double layer (K), and the Hamaker constant (AH). As a rough rule, if the primary maximum exceeds approximately 15 kT (kT is the average energy expected from local thermal fluctuations), a dispersion is "absolutely" stable with respect to coagulation into the primary minimum.33 Furthermore, if the secondary minimum (Fig. 4.8) is sufficiently deep, 2 kT or more, then when the droplets come together they will form aggregates with a lifetime dependent on the depth.162 These aggregates of droplets are generally easily dispersed by agitation.179 If the DLVO Theory has the merit to be entirely quantitative, it unfortunately can rarely explain emulsion stability in many food emulsions because double-layer forces are not very important.216 Typical food emulsions stabilized by proteins or hydrocolloids have small surface charge densities corresponding to low zeta potentials, normally between 1 and 20 mV.203 Also, in many food emulsions the electrolyte concentration is rather high, which reduces the Debye-Hiickel length and consequently the electrostatic repulsion.179 The third major mechanism by which the stability of colloidal systems can be influenced is due to the presence of flexible polymers {i.e. disordered or denatured proteins) on particle surfaces or in solutions which affect forces acting between these particles. These steric forces can be strong enough to provide a metastable thermodynamic equilibrium and prevent droplets from approaching closely enough for the attractive van der Waals interactions to be sufficiently powerful to permit coagulation.215 Models describing the interaction between irreversibly adsorbed flexible polymers have been described by Flory,217 de Gennes,205'218 and by Scheutjens and Fleer.219"221 The interactions between polymer/polymer segments of adsorbed macromolecules may generate a repulsive effect due to an entropic contribution to the free energy, rather than being a true repulsive potential, for two reasons. Firstly, the approach of two interacting droplets may compress the surface layers of adsorbed macromolecules. This compression, by diminishing the volume available to the macromolecule, produces a loss of configurational entropy {i.e. macromolecules are

constrained to be effectively less flexible). Secondly, the adsorbed macromolecules of two approaching particles can interpenetrate, and in is case the entropy of intermixed macromolecular chains is not favoured by a close approach.205'215 Therefore, adsorbed flexible macromolecules tend to promote stability of colloidal systems. Relatively recent studies by Pargesian, Rand and co-workers,222"225 by Pashley,226 and by Marra and Israelachvili227"228 have experimentally shown that a further strong repulsive force can be generated between two surfaces covered with hydrated macromolecular groups when they are brought close together in an aqueous environment. This interpenetration of the adsorbed layers may disrupt the binding water of the macromolecules, and will contribute unfavourably to the overall free energy of aggregation. These short-range forces become measurable at about 3 nm, and decrease exponentially with distance according to Parsegian and coworkers. 222 " 225 The forces described by Marra and Israelachvili227'228 have a more complex functional form. Despite these differences, explained mainly on the basis of different experimental conditions, the hydration forces are strong enough to prevent adhesion of lecithin bilayers.227'228 Two further mechanisms are also associated with the presence of adsorbed and nonadsorbed macromolecules, namely (1) polymer bridging, and (2) depletion flocculation. 1. Polymer bridging occurs when the polymer concentration is low or when the time of adsorption is short, for instance, during homogenization processes.33'229 Consequently, bridging gives arise to an attraction force at relatively large separation distances, that is, comparable to the length of the adsorbed polymer chain that protrudes into the solvent. As the surface concentration increases the effect of bridging polymers becomes less important, whereas adsorbed polymer/polymer interactions become more important. 2. Depletion flocculation arises when the particle surfaces are sufficiently close together that the nonadsorbed dissolved macromolecules cannot fit between them, and the concentration of macromolecules between the surfaces is therefore lower than the bulk concentration. This produces an osmotic attractive force that tends to drive particles together.230 At sufficiently high nonadsorbed dissolved macromolecule concentrations, theory also predicts that forces induced by free macromolecules can actually change from attractive to repulsive, leading to what has been called depletion stabilization.231"233 Although, in practice, it is extremely difficult to quantitatively estimate colloidal interaction between dispersed particles, some trends can often be infered. Table 4.8 234 summarizes the primary factors (particle size, pH, ionic strength, etc.) involved. Furthermore, modem developments in the theory of stability of colloidal systems include additional factors, especially the effects the steric forces, and a more precise definition of the interaction forces.11163'205

Emulsifying Properties
Definition and Formation of Emulsions. Emulsions as well as foams are dispersed systems; they contain two distinct phases. According to the traditional definition,235

Table 4.8 VARIABLES AFFECTING THE THREE MAIN TYPES OF COLLOIDAL INTERACTIONS BETWEEN SPHERICAL PARTICLES IN AN AQUEOUS MEDIUM. A STAR DENOTES THAT THE VARIABLE IS IMPORTANT. ALL VARIABLES, EXCEPT PARTICLE SIZE, MAY IN TURN AFFECT THE COMPOSITION OF THE SURFACE LAYER
Variable Particle size Particle material Surface layer pH Ionic strength Solvent quality From Ref. 234. van der Waals Attraction * * (*) Electrostatic Repulsion * * * * Steric Repulsion (*) (*) *

emulsions are colloidal dispersions of liquid droplets in a second immiscible liquid phase. If the continuous phase is water, they are termed oil-in-water (O/W) emulsions (e.g., milk, cream, mayonnaise, etc.); the opposite arrangement is called a water-in-oil (W/0) emulsion (e.g., margarine, butter, etc.). However, this classic definition is too narrow to include most food emulsions; many of which are in fact considerably more complex: the dispersed phase can be partially solidified as in dairy products and the continuous phase may also contain crystalline material, as in ice cream, or it may be a gel as in many desserts. In addition, air bubbles may have been incorporated as in whipped creams. Also, a