1.Advanced Dairy Chemistry

2. Advanced Dairy ChemistryVolume 2 LipidsThird EditionEdited byP. F. FOX and P. L. H. McSWEENEYUniversity College Cork, Ireland 3. Library of Congress Control Number: 2005928170ISBN-10: 0-387-26364-0 e-ISBN: 0-387-28813-9ISBN-13: 978-0387-26364-9Printed on acid-free paper.2006 Springer ScienceBusiness Media, Inc.All rights reserved. This work may not be translated or copied in whole or in part without the writtenpermission of the publisher (Springer ScienceBusiness Media, Inc. 233 Spring Street, New York,NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use inconnection with any form of information storage and retrieval, electronic adaptation, computersoftware, or by similar or dissimilar methodology now known or hereafter developed is forbidden.The use in this publication of trade names, trademarks, service marks and similar terms, even ifthey are not identiWed as such, is not to be taken as an expression of opinion as to whether or notthey are subject to proprietary rights.Printed in the United States of America (SPI/SBA)10 9 8 7 6 5 4 3 2 1springer.com 4. ContributorsM.A. AugustinC.H. Fitz-GeraldSchool of Chemistry, School of Land and Food SciencesMonash University, Clayton,University of QueenslandVictoria 3800, Australia Brisbane, Qld 4072, AustraliaDale E. Bauman J. Bruce GermanDepartment of Animal Science Department of Food Science and TechnologyCornell University University of California, DavisIthaca, NY 14853, USACalifornia, CA 95616 USAWolfgang BuchheimH. Douglas GoffLornsenstr. 34, D- 24105 KielDept. of Food Science University of Guelph Guelph, ON N1G 2W1 CanadaRichard ChallisUniversity of NottinghamUniversity Park, NottinghamT.P. GuineeNG7 2RD, UKMoorepark Food Research Centre, Teagasc, Fermoy Co. Cork, IrelandMilena CorredigDepartment of Food ScienceUniversity of Guelph Mingruo GuoGuelph, Ontario N1H 2W1 Canada Department of Nutrition and Food Sciences University of Vermont, Burlington VT 05405, USAH.C. DeethSchool of Land and Food SciencesUniversity of Queensland Gregory HendricksBrisbane, Qld 4072 Department of Cell BiologyAustraliaUniversity of Massachusetts School ofMedicineWorcester, MA 01655, USAEric DufourU.R. Typicite des ProduitsAlimentaires Wolfgang HoffmannENITA Clermont-Ferrand, site Federal Research Centre for Nutrition andde Marmilhat, BP35 Food, Location Kiel63370 LEMPDES, FranceHermann-Weigmann-Str. 1, D- 24103 KielNana Y. Farkye T. HuppertzDairy Products Technology Center Department of Food and NutritionalCalifornia Polytechnic StateSciences,University University College, CorkSan Luis Obispo, CA 93407, USA Ireland v 5. vi ContributorsThomas W. Keenan O.J. McCarthyDepartment of Biochemistry Institute of Food, Nutrition and HumanVirginia Polytechnic Institute and HealthState University Massey UniversityBlacksburg, VA 24061 USA PO Box 11222, Palmerston North New ZealandA.L. KellyDepartment of Food and Nutritional P.L.H. McSweeneySciences,Department of Food and Nutritional SciencesUniversity College,University CollegeCork, IrelandCork, IrelandM.K. Keogh P.A. MorrisseyMoorepark Food Research Centre, Teagasc, Department of Food and Nutritional SciencesFermoy,University College, CorkCo. Cork, IrelandIrelandM. Kiely N.M. OBrienDepartment of Food and Nutritional Department of Food and Nutritional SciencesSciencesUniversity College, Cork University CollegeIrelandCork, IrelandKieran Kilcawley T.P. OConnorMoorepark Food Research Centre,Department of Food and Nutritional Sciences,Teagasc, University CollegeFermoy Cork, IrelandCo. Cork, Ireland Donald L. PalmquistAdam L. Lock Department of Animal SciencesDepartment of Animal Science Ohio Agricultural Research andCornell University Development CenterIthaca, NY 14853, USAThe Ohio State University Wooster, OH 44691, USAAlastair K.H. MacGibbonFonterra Co-Operative Group Ltd.,Peter W. ParodiDairy Farm Road, Human Nutrition and Health ResearchPrivate Bag 11029, Dairy Australia Locked Bag 104,Palmerston North, New Zealand Flinders Lane, Melbourne, Victoria 8009 AustraliaAlejandro G. MarangoniDepartment of Food Science Malcolm PoveyUniversity of Guelph Procter Department of Food ScienceGuelph, OntarioUniversity of LeedsCanada, N1G 2W1Leeds LS2 9JT, UKIan H. MatherMike W. TaylorDepartment of Animal and Institute of Food, Nutrition and HumanAvian Sciences Health,University of Maryland Massey University, PO Box 11222,College Park, MD 20742 USA Palmerston North, New Zealand 6. Contributors viiC. Versteeg Amanda J. WrightFood Science AustraliaDepartment of Human Health and671 Sneydes Road, WerribeeNutritional SciencesVictoria 3030, AustraliaUniversity of GuelphGuelph, OntarioCanada, N1G 2W1Robert E. WardDepartment of Food Science and TechnologyUniversity of California, DavisCalifornia, CA 95616 USA 7. Preface to the Third EditionAdvanced Dairy Chemistry 2: Lipids is the second volume of the third editionof the series on advanced topics in Dairy Chemistry, which started in 1982with the publication of Developments in Dairy Chemistry. The Wrst volume,on milk proteins, of the third edition of Advanced Dairy Chemistry waspublished in 2003. This series of volumes is intended to be a coordinatedand authoritative treatise on Dairy Chemistry. In the decade since thesecond edition of this volume was published (1995), there have been consid-erable advances in the study of milk lipids, which are reflected in changes tothis book.Most topics included in the second edition are retained in the currentedition, which has been updated and considerably expanded from 10 to 22chapters. For various reasons, the authors of many chapters have beenchanged and hence, in effect, are new chapters, at least the topic is viewedfrom a different perspective.The new chapters cover the following subjects: Biosynthesis and nutri-tional significance of conjugated linoleic acid, which has assumed majorsignificance during the past decade; Formation and biological significanceof oxysterols; The milk fat globule membrane as a source of nutritionally andtechnologically significant products; Physical, chemical and enzymatic modi-fication of milk fat; Significance of fat in dairy products: creams, cheese, icecream, milk powders and infant formulae; Analytical methods: chromato-graphic, spectroscopic, ultrasound and physical methods.Like its predecessor, this book is intended for academics, researchers atuniversities and industry, and senior students; each chapter is referencedextensively.We wish to thank sincerely the 37 contributors to the 22 chapters ofthis volume, whose cooperation made our task as editors a pleasure. Thegenerous assistance of Ms. Anne Cahalane is gratefully acknowledged.P. F. FoxP. L. H. McSweeney University College Cork, Irelandviii 8. Preface to the Second EditionAdvanced Dairy Chemistry can be regarded as the second edition of Devel-opments in Dairy Chemistry. The first volume in the series, on Milk Proteins,was published in 1992; this, the second volume, is devoted to Milk Lipids.Considerable progress has been made in several aspects of milk lipids duringthe past 11 years which is reflected in revised versions of seven of the eightchapters included in Developments in Dairy Chemistry 2, most of them bythe same authors. The theme of one chapter has been changed from physicalproperties and modification of milk fat to the crystallization of milk fat. Twonew chapters have been added, i.e. chemistry and technology aspects of low-fat spreads and the significance of fat in consumer perception of foodquality, which reflect the continuing consumer awareness of a healthy diet.Low-fat spreads have become increasingly significant during the past decadeand are now the major type of spread in many countries. However, reducingthe fat content of foods generally results in a concomitant decrease in theorganoleptic quality of the food; consumer attitudes to reduced-fat dairyproducts are discussed in one of the new chapters.Like its predecessor, the book is intended for lecturers, senior studentsand research personnel and each chapter is extensively referenced.I would like to thank all the authors who contributed to this book andwhose cooperation made my task as editor a pleasure. P. F. Foxix 9. Preface to the First EditionMany of the desirable flavour and textural attributes of dairy products aredue to their lipid components; consequently, milk lipids have, traditionally,been highly valued, in fact to the exclusion of other milk components inmany cases. Today, milk is a major source of dietary lipids in western dietsand although consumption of milk fat in the form of butter has declined insome countries, this has been offset in many cases by increasing consump-tion of cheese and fermented liquid dairy products.This text on milk lipids is the second in a series entitled Developments inDairy Chemistry, the first being devoted to milk proteins. The series isproduced as a co-ordinated treatise on dairy chemistry with the objectiveof providing an authoritative reference source for lecturers, researchers andadvanced students. The biosynthesis, chemical, physical and nutritionalproperties of milk lipids have been reviewed in eight chapters by worldexperts. However, space does not permit consideration of the more prod-uct-related aspects of milk lipids which play major functional roles in severaldairy products, especially cheese, dehydrated milks and butter.Arising from the mechanism of fatty acid biosynthesis and export offat globules from the secretory cells, the fat of ruminant milks is particularlycomplex, containing members of all the major lipid classes and as many as400 distinct fatty acids. The composition and structure of the lipids of bovinemilk are described in Chapter 1, with limited comparison with non-bovinemilk fats. Since the fatty acid profile of milk fat, especially in monogastricanimals, may be modified by diet and other environmental factors, thebiosynthesis of milk lipids is reviewed in Chapter 2 with the objective ofindicating means by which the fatty acid profile, and hence the functionalproperties of the lipids, might be modified. Lipids in foods are normallypresent as an emulsion, stabilized by a layer of protein adsorbed at the oil-water interface. The fat in milk and cream exists as an oil-in-water emulsionwith a unique stabilizing lipoprotein membrane, referred to as the milk fatglobule membrane (MFGM). The inner layers of the MFGM are formedwithin the secretory cell and are relatively stable; however, the outer layers,which are acquired as the fat globule is exported through the apical mem-brane of the secretory cells, are unstable. Damage to the MFGM leads tochemical and physical instability of the fat phase in milk and hence the x 10. Preface to the First Editionxistructure of the membrane has been the subject of considerable research, theresults of which are reviewed in Chapter 3.Lipids strongly influence, for good or evil, the flavour and texture offoods, especially high-fat products such as butter. The influence of variouscolloidal features of milk fat on the properties of milk and cream is con-sidered in Chapter 4, while the crystallization of milk fat and how this maybe controlled, modified and measured are reviewed in Chapter 5. Unfortu-nately, lipids are subject to chemical and enzymatic alterations which cancause flavour defects referred to as oxidative and hydrolytic rancidity,respectively. The storage stability of high-fat foods, especially mildly fla-voured foods like milk, cream and butter, is strongly influenced by thesechanges which have been reviewed in Chapters 6 and 7.Dietary lipids play many diverse nutritional roles, some of which areessential. However, dietary lipids, especially saturated lipids of animal ori-gin, have been the subject of much controversy in recent years, particularlyin regard to their possible role in atherosclerosis. Various aspects of thenutritional significance of lipids are discussed in Chapter 8.Finally, I wish to thank sincerely the 14 authors who have contributedto this text and whose co-operation has made my task as editor a pleasure.P. F. Fox 11. Contents1. Composition and Structure of Bovine Milk LipidsA.K.H. MacGibbon and M.W. Taylor1.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .11.2. Fatty Acids . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . .3 1.2.1. Origins of the Fatty Acids . . . . . . . .. . . . . . . . . . . . . . . . . . . .4 1.2.2. Saturated Fatty Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5 1.2.3. Cis-unsaturated Fatty Acids . . . . . . . . . . . . . . . . . . . . . . . . . . .5 1.2.4. Trans-unsaturated Fatty Acids . . . . . . . . . . . . . . . . . . . . . . . . .7 1.2.5. Minor Fatty Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9 1.2.6. Variations in Fatty Acid Composition. . . . . . . . . . . . . . . . . . . . 101.3. Triacylglycerols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 1.3.1. Structure of Triacylglycerols . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 1.3.2. Composition of Triacylglycerols . . . . . . . . . . . . . . . . . . . . . . . . 141.4. Polar Lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 1.4.1. Composition and Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 1.4.2. Ceramides and Gangliosides . . . . . . .. . . . . . . . . . . . . . . . . . . . 25 1.4.3. Health Issues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271.5. Minor Constituents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 1.5.1. Sterols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 1.5.2. Carotenoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28 1.5.3. Fat-soluble Vitamins . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 28 1.5.4. Flavour Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 291.6. Milk Fat From DiVerent Animal Species . .. . . . . . . . . . . . . . . . . . . . 30 1.6.1. Gross Composition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 1.6.2. Fatty Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 1.6.3. Triacylglycerols . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 33 1.6.4. Polar Lipids . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 34Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 352. Milk Fat: Origin of Fatty Acids and Influence ofNutritional Factors ThereonD.L. PalmquistAbstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .432.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 442.2. Origin of the Fatty Acids in Milk Fat . . . . . . . . . . . . . . . . . . . . . . . . .45 xiii 12. xivContents 2.2.1.Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . 45 2.2.2.Fatty Acid Transport . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . 45 2.2.3.Lipoprotein Lipase . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . 47 2.2.4.Transport of Long-Chain Fatty Acids into Mammary Cells. . . . 48 2.2.5.Summary of the Supply of Long-Chain Fatty Acids to the Mammary Gland . . . . . . . . . . . . . . . . . . . . . . . . .....50 2.3. Uptake of Non-Lipid Metabolites by LactatingMammary Glands . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ....51 2.4. Fatty Acid Synthesis in Mammary Glands . . . . . . . . . . . . . . . . . ....522.4.1. Sources of Carbon and Reducing Equivalents for Fatty Acid Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . 522.4.2. Acetyl-CoA Carboxylase . . . . . . . . . . . . . . . . . . . . . . . .. . . . 552.4.3. Fatty Acid Synthase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 582.4.4. Regulation of Acyl Chain Length . . . . . . . . . . . . . . . . . . . . . . 60 2.5. Stearoyl-CoA Desaturase . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . 62 2.6. Triacylglycerol Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . 632.6.1. Fatty Acid EsteriWcation by the Monoacylglycerol Pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66 2.7. Synthesis of Complex Lipids . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . 662.7.1. Synthesis of Phospholipids . . . . . . . . . . . . . . . . . . . . . . .. . . . 672.7.2. Sphingolipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 682.7.3. Cholesterol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68 2.8. Physiological Factors That InXuence Milk Fat Composition . . . . . . . . 692.8.1. Genetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . 692.8.2. Stage of Lactation . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . 69 2.9. EVects of Dietary Fat on the Composition of Milk Fat . . . . . . . . . . . . 712.9.1. EVects of Low-fat Diets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 712.9.2. EVects of SpeciWc Fatty Acids . . . . . . . . . . . . . . . . . . . . . . . . . 712.9.3. Feeding for SpeciWc Milk Fatty Acid ProWles . . . . . . . . . . . . . . 732.9.4. Supplementation with Oilseeds and Commercial Fats . . . . . . . . 742.9.5. Low Milk Fat Syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . . . 782.10. Milk Fat Composition and Quality . . . . . . . . . . . . . . . . . . . . . . . . . . 78Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . 80Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 803. Conjugated Linoleic Acid: Biosynthesis and NutritionalSignificanceD.E. Bauman and A.L. LockAbstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 933.1. Introduction . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 943.2. Dietary Sources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 963.3. Analytical Challenges . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 973.4. Origin of CLA in Milk Fat . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99 13. Contents xv 3.4.1. Lipid Metabolism in the Rumen . . . . . . . . . . . . . . . . . . . . . . . .99 3.4.2. cis-9, trans-11 CLA (Rumenic Acid) . . . . . . . . . . .. . . . . . . . . . 102 3.4.3. trans-7, cis-9 CLA . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . 104 3.4.4. The D9-Desaturase Enzyme System . . . . . . . . . . . . . . . . . . . . . . 105 3.4.5. Other CLA Isomers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1063.5. ModiWcation of CLA Content in Milk Fat . . . . . . . . . . . . . . . . . . . . . 107 3.5.1. Dietary and Nutritional EVects . . . . . . . . . . . . . . .. . . . . . . . . . 108 3.5.2. Physiological Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111 3.5.3. Manufacturing and Product Quality Considerations. . . . . . . . . . 1123.6. Biological EVects of CLA Isomers . . . . . . . . . . . . . . . . . . . . . . . . . . . 114 3.6.1. trans-10, cis-12 CLA and Lipid Metabolism . . . . . . . . . . . . . . . . 114 3.6.2. Rumenic Acid and Human Health . . . . . . . . . . . . . . . . . . . . . . 120Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . 1254. Intracellular Origin of Milk Fat Globules and the Natureof the Milk Fat Globule MembraneT.W. Keenan and I.H. Mather 4.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . 137 4.2. Intracellular Origin and Growth of Milk Fat Globules. . . . . . . . . . . . 138 4.3. Intracellular Transit of Lipid Droplets . . . . . . . . . . . . . . . . . . . . . . . 142 4.4. Secretion of Milk Fat Globules . . . . . . . . . . . . . . . .. . . . . . . . . . . . 143 4.5. Isolation and Gross Composition of MFGM . . . . . . . . . . . . . . . . . . . 150 4.6. Lipid Composition of the MFGM . . . . . . . . . . . . . . . . . . . . . . . . . . 151 4.7. Enzymes Associated with the MFGM . . . . . . . . . . . .. . . . . . . . . . . . 153 4.8. Proteins of the MFGM . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . 155 4.9. Molecular Organization of the MFGM . . . . . . . . . . .. . . . . . . . . . . . 1634.10. Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . 164Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . 164Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . 1655. Physical Chemistry of Milk Fat GlobulesT. Huppertz and A.L. Kelly5.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1735.2. The Nature and Size Distribution of Milk Fat Globules. . . . . . . . . . . . 1735.3. DiVerences in the Composition of Milk Fat Globules . . . . . . . . . . . . . . 1775.4. Fat Crystals in Globules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1775.5. Colloidal Interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1795.6. Physical Instability of Emulsions . . . . . . . . . . . . . . . .. . . . . . . . . . . . 1815.7. Separation of Milk . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1835.8. Cold Agglutination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1845.9. Coalescence and Partial Coalescence . . . . . . . . . . . . . .. . . . . . . . . . . . 188 14. xviContents5.10. Rebodying . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...........1905.11. Factors that AVect the Surface Layers of Fat Globules inMilk and Cream . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1915.12. Disruption of Globules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1925.13. Milk Fat Globules in Homogenized Milk and Cream . . .. . . . . . . . . . 1965.14. Milk Fat Globules in Recombined Milk . . . . . . . . . . . . . . . . . . . . . . 1985.15. Free Fat . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2005.16. InXuence of Fat Globules on Rheological Properties ofMilk and Cream . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2015.16.1. Volume Fraction of the Dispersed Phase . . . . . . . . . . . . . . . . 2025.16.2. Rheology of the Component Phases . . . . . . . . . . . . . . . . . . . . 2025.16.3. Droplet Size . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2035.16.4. Colloidal Interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2035.16.5. Particle Charge . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . 2035.17. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . 204Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2046. Composition, Applications, Fractionation, Technologicaland Nutritional Significance of Milk Fat GlobuleMembrane MaterialR.E. Ward, J.B. German and M. Corredig6.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ........ 2136.2. Nutritional and Physiological SigniWcance of the Milk Fat Globule Membrane . . . . . . . . . . . . . . . . . . . . . . . . ....... 214 6.2.1. Biological SigniWcance of Native Globules . . . . . . . . . ........ 216 6.2.2. MFGM Consumption Studies: Physiological andNutritional EVects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2186.3. Composition and Bioactivity of Individual Components . . . . .. . . . . . . 219 6.3.1. Phospholipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . 219 6.3.2. Ceramide Sphingolipids and Glycosphingolipids . . . . . .. . . . . . . 220 6.3.3. Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223 6.3.4. Butyrophilin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 224 6.3.5. Mucins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 226 6.3.6. Xanthine Oxidoreductase . . . . . . . . . . . . . . . . . . . . . .. . . . . . . 2286.4. Fractionation and Technological SigniWcance of Milk Fat Globule Membrane Material . . . . . . . . . . . . . . . . . . . . . . . . ....... 229 6.4.1. EVect of Processing on the Composition andFunctionality of the MFGM . . . . . . . . . . . . . . . . . . . ........ 230 6.4.2. Isolation of MFGM . . . . . . . . . . . . . . . . . . . . . . . . . ........ 232 6.4.3. Application and Utilization of MFGM as a FunctionalIngredient in Foods . . . . . . . . . . . . . . . . . . . . . . . . . ........ 235 6.4.4. Conclusions and Future Research Directions . . . . . . . . ....... 237Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ....... 238 15. Contentsxvii7. Crystallization and Rheological Properties of Milk FatA.J. Wright and A.G. Marangoni7.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2457.2. Crystallization of Milk Fat . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . 245 7.2.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . 245 7.2.2. Nucleation of Milk Fat . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . 247 7.2.3. Growth of Milk Fat Crystals . . . . . . . . . . . . . . . . . . . . . . . . . . 248 7.2.4. Crystallization, Melting and Mixed Crystal Formation .. . . . . . . 248 7.2.5. Polytypism and Polymorphism . . . . . . . . . . . . . . . . . . . . . . . . . 2507.3. Structure and Rheology of Fat Crystal Networks . . . . . . . . . . . . . . . . . 254 7.3.1. Milk Fat and Butter Crystal Networks . . . . . . . . . . . .. . . . . . . 254 7.3.2. Methods used to Determine the RheologicalProperties of Milk Fat . . . . . . . . . . . . . . . . . . . . . . . ........ 254 7.3.3. Rheology of Milk Fat . . . . . . . . . . . . . . . . . . . . . . . ........ 262 7.3.4. Modeling Fat Crystal Networks and Relating Structureto Rheology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2677.4. Modifying the Crystallization and Rheology of Milk Fat . . . . . . . . . . . 271 7.4.1. Manipulations of Butter Composition . . . . . . . . . . . . . . . . . . . . 271 7.4.2. Manipulations During Processing . . . . . . . . . . . . . . . . . . . . . . . 2747.5. Some Case Studies. Milk Fat Crystallization: Structure and Rheological Properties . . . . . . . . . . . . . . . . . . . . . . . . . ....... 277 7.5.1. EVect of Cooling Rate on Milk Fat Crystallizationand Rheology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ........ 277 7.5.2. EVect of Supplementation with Algae Meal onMilk Fat Crystallization and Rheology . . . . . . . . . . . . ....... 279 7.5.3. EVect of Minor Components on Milk FatCrystallization and Rheology . . . . . . . . . . . . . . . . . . ........ 2807.6. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ....... 281Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ........ 2818. Milk Fat: Physical, Chemical and Enzymatic ModificationM.A. Augustin and C. Versteeg8.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2938.2. Physical ModiWcation of Milk Fat . . . . . . . . . . . . . . . . . . . . . . . . . . . 294 8.2.1. Fractionation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295 8.2.2. Physical Blends of Milk Fat with Other Fats and Oils. . . . . . . . . 309 8.2.3. ModiWcation of Milk Fat Properties by Addition ofMinor Lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . 3118.3. Chemical ModiWcation of Milk Fat . . . . . . . . . . . . . . . . . . . . . . . . . . 313 8.3.1. Hydrogenation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 313 8.3.2. Chemical InteresteriWcation . . . . . . . . . . . . . . . . . . . . . . . . . . . 314 16. xviii Contents8.4. Enzymic ModiWcation of Milk Fat .. . . . . . . . . . . . . . . . . . . . . . . . . . 316 8.4.1. Enzymic InteresteriWcation . .. . . . . . . . . . . . . . . . . . . . . . . . . . 317 8.4.2. Enzymic Hydrolysis . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . 3218.5. Cholesterol Reduction . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . 322 8.5.1. Distillation Processes . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . 322 8.5.2. Supercritical CO2 Extraction .. . . . . . . . . . . . . . . . . . . . . . . . . . 323 8.5.3. Treatment with Adsorbents . . . . . . . . . . . . . . . . . . . . . . . . . . . 323 8.5.4. Treatment with Enzymes . . .. . . . . . . . . . . . . . . . . . . . . . . . . . 3248.6. Future Trends . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . 324Acknowledgements . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . 325Bibliography . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . 3259. Chemistry and Technology of Butter and Milk FatSpreadsM.K. Keogh9.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333 9.1.1. Legislation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333 9.1.2. Emulsion Stability . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . 334 9.1.3. Consumer Pressures for Change . . . . . . . . . . . . . . . . . . . . . . . . 3349.2. Technical Aspects of Butter Manufacture . . . . . . . . . . . . . . .. . . . . . . 336 9.2.1. Chemical and Physical Principles . . . . . . . . . . . . . . . . .. . . . . . . 3369.3. Technical Challenges in the Processing of Fat Spreads . . . . . .. . . . . . . 339 9.3.1. Rates of Microbial Growth . . . . . . . . . . . . . . . . . . . . . . . . . . . 339 9.3.2. Phase Inversion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3409.4. Technology of Spread Manufacture . . . . . . . . . . . . . . . . . . . . . . . . . . 340 9.4.1. Processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . 3409.5. Fundamental Aspects of Emulsions . . . . . . . . . . . . . . . . . . . . . . . . . . 342 9.5.1. Emulsions: Theory, Rheology and Stability to Inversion. . . . . . . 3429.6. EVects of Ingredients on Emulsion Stability . . . . . . . . . . . . .. . . . . . . 348 9.6.1. Fat . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 348 9.6.2. EmulsiWers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . 350 9.6.3. Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . 351 9.6.4. Hydrocolloid Stabilizers . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . 353 9.6.5. Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 354 9.6.6. Disodium Phosphate and Trisodium Citrate . . . . . . . . .. . . . . . . 354 9.6.7. pH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . 354 9.6.8. Interactions of Ingredients . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3549.7. Interactions of Ingredients in Low-Fat Spreads . . . . . . . . . . . . . . . . . . 355Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . 357Further Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 362 17. Contentsxix10. Significance of Milk Fat in Cream ProductsW. Hoffmann and W. Buchheim10.1. Introduction . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36510.2. CoVee Cream . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36610.3. Whipping Cream. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36810.4. Cream Liqueurs .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37210.5. Cultured Cream .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 373Bibliography . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37411. Significance of Milk Fat in CheeseT.P. Guinee and P.L.H. McSweeney11.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .....37711.2. EVect of Fat on Cheese Composition . . . . . . . . . . . . . . . . . . . .....37911.2.1. Fat Content . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ....37911.2.2. EVect of Degree of Fat EmulsiWcation as InXuenced byHomogenization of Milk, Cream and/or Curd . . . . . . . . . ....38211.3. Contribution of Fat to the Microstructure of Cheese . . . . . . . . . ....38511.3.1. Microstructure of Rennet-Curd Cheese . . . . . . . . . . . . . .....38511.3.2. Microstructure of Pasteurized Processed Cheese Products(PCPs) and Analogue Cheese Products (ACPs) . . . . . . . .....39111.3.3. EVect of Fat Level on Microstructure . . . . . . . . . . . . . . .....39211.3.4. EVect of Fat EmulsiWcation on Microstructure . . . . . . . . .....39311.3.5. EVect of Fat on Heat-induced Changes inMicrostructure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . 39511.4. EVect of Fat on Cheese Yield . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . 39711.5. EVect of Fat on Cheese Microbiology . . . . . . . . . . . . . . . . . . . . . . . . 40111.6. EVect of Fat on Proteolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40311.6.1. Primary Proteolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40311.6.2. Secondary Proteolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40611.7. Contribution of Lipolysis and Catabolism of Free FattyAcids (FFA) to Cheese Flavor . . . . . . . . . . . . . . . . . . . . . . . . .....40711.7.1. Lipolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ....40811.7.2. Metabolism of Fatty Acids . . . . . . . . . . . . . . . . . . . . . . ....41011.8. EVect of Fat on the Fracture-Related Properties ofUnheated Cheese . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ....41311.8.1. EVect of Fat Content on Fracture Properties . . . . . . . . . ....41411.8.2. EVect of Solid-to-Liquid Fat Ratio on FractureProperties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .....41411.8.3. EVect of Homogenization of Milk or Cream, and Degreeof Fat EmulsiWcation on Fracture Properties . . . . . . . . . . ....41811.9. EVect of Fat on the Functional Properties of Heated Cheese . . . .....41911.9.1. EVect of Fat Level on Cooking Properties . . . . . . . . . . .....419 18. xx Contents11.9.2. EVect of Milk Homogenization and Degreeof Fat EmulsiWcation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42511.9.3. EVect of Milk Fat Fraction on CookingProperties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 427Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42812. Ice CreamH.D. Goff12.1. Overview of Ice Cream Ingredients and Manufacture .. . . . . . . . . . . . 44112.2. Sources of Fat in Ice Cream . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . 44312.3. Contribution of Fat to the Structure of Ice Cream . . .. . . . . . . . . . . . 44412.4. Contribution of Fat to Ice Cream Texture and Flavor. . . . . . . . . . . . 447Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44813. Significance of Milk Fat in Milk PowderN.Y. Farkye13.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45113.2. Overview of Milk Powder Manufacture . . . . . . . . . . . . . . . . . . .. . . . 45213.3. SigniWcance of Milk Fat during Powder Manufacture . . . . . . . . .. . . . 45513.4. SigniWcance of Milk Fat for the Flavor of Milk Powder . . . . . . .. . . . 45713.4.1. EVect of Pre-heat Treatment on Oxidative Stability . . . . . . . . . 45713.4.2. InXuence of Moisture Content and Water Activity on theOxidation of Fat in Milk Powder . . . . . . . . . . . . . . . . . .....45813.4.3. EVect of Oxygen Content and Packaging the Oxidationof Fat in Milk Powder . . . . . . . . . . . . . . . . . . . . . . . . .. . . . 45813.4.4. Lipid Oxidation Products . . . . . . . . . . . . . . . . . . . . . . . . . . . 46013.5. Role of Fat in the Physical Properties of Milk Powder . . . . . . . .. . . . 461Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46214. Significance of Milk Fat in Infant FormulaeG. Hendricks and M. Guo14.1. The Nutritional Role of Lipids . . . . . . . . . . . . . . . . . . . . . . . . . ....46714.2. Fatty Acid ProWle and Fat-Soluble Vitamins of Human Milkand Infant Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . 46914.3. Biological BeneWts of Milk Fatty Acids . . . . . . . . . . . . . . . . . . . . . . . 47314.4. Formulation of Infant Formula Using Milk Fat as an Ingredient .. . . . 47314.5. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . 476Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 477 19. Contentsxxi15. Lipolytic Enzymes and Hydrolytic RancidityH.C. Deeth and C.H. Fitz-GeraldSummary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48115.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . 48115.2. The Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48315.2.1. Cows Milk Lipase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48315.2.2. Human Milk Lipases . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . 48615.2.3. Milk Lipases of Other Species . . . . . . . . . . . . . . . . . . . . . . . . 48715.2.4. Esterases of Cows Milk . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48915.2.5. Lipases of Psychrotrophic Bacteria . . . . . . . . . . . . .. . . . . . . . 49015.2.6. Phospholipases . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . 49415.2.7. Lipolytic Enzymes in Milk Product Manufacture . . . . . . . . . . . 49515.3. Causes of Hydrolytic Rancidity in Milk and Milk Products .. . . . . . . . 49615.3.1. Induced Lipolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49715.3.2. Spontaneous Lipolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50115.3.3. Mastitis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . 50815.3.4. Microbial Lipolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50915.4. Detrimental EVects of Lipolysis in Milk and Milk Products . . . . . . . . 51115.4.1. Flavor Defects . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . 51115.4.2. Technological Consequences . . . . . . . . . . . . . . . . .. . . . . . . . 51615.5. BeneWcial EVects of Lipolysis in Milk and Milk Products . . . . . . . . . . 51715.5.1. Production of Desirable Flavor . . . . . . . . . . . . . . .. . . . . . . . 51715.5.2. Digestion of Milk Fat . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51815.6. Analytical Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . 51915.6.1. Free Fatty Acids . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . 51915.6.2. Lipase Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52415.7. Prevention of Hydrolytic Rancidity . . . . . . . . . . . . . . . . . .. . . . . . . . 529Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . 53016. Lipid OxidationT.P. OConnor and N.M. OBrien16.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . 55716.2. Mechanism of Lipid Autoxidation . . . . . . . . . . . . . . . . . . . . . . . . . . 55816.3. Oxidation Products and OV-Flavors . . . . . . . . . . . . . . . . . . . . . . . . . 55916.3.1. Spontaneous Oxidation in Milk . . . . . . . . . . . . . . . . . . . . . . . 56116.4. Factors that AVect the Oxidation of Lipids in Milkand Milk Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56316.4.1. Oxygen . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . 56416.4.2. Light . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56816.4.3. Metals . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . 57016.5. Antioxidants . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . 57216.5.1. Ascorbic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 573 20. xxii Contents16.5.2. Tocopherols . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . 57416.5.3. Carotenoids . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . 57516.5.4. Thiols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57616.5.5. Proteins and Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57716.5.6. Products of Browning Reactions . . . . . . . . . . . . . . . . . . . . . . 57916.6. Milk Fat Globule Membrane (MFGM) . . . . . . . . . . . . . . . . . . . . . . . 58016.7. Storage Temperature . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . 58216.8. Water Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58216.9. Measurement of Lipid Oxidation . . . . . . . . . . . . . . . . . . . . . . . . . . . 583Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58517. Nutritional Significance of Milk LipidsP.W. Parodi17.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60117.2. Dietary Fat and Obesity . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . 60217.2.1. Randomised Control Trials . . . . . . . . . . . . . . .. . . . . . . . . . . 60317.2.2. Safety of Low-Fat, High-Carbohydrate Diets . . . . . . . . . . . . . 60317.2.3. Energy Value of Milk Fat . . . . . . . . . . . . . . . . . . . . . . . . . . . 60317.3. Dietary Fat and Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60417.3.1. Colon Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60517.3.2. Breast Cancer . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . 60617.3.3. Prostate Cancer . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . 60717.3.4. Comment . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . 60817.4. Milk Fat and Coronary Heart Disease . . . . . . . . . . . .. . . . . . . . . . . 60817.4.1. Plasma Cholesterol and CHD . . . . . . . . . . . . . . . . . . . . . . . . 60917.4.2. Saturated Fatty Acids and CHD . . . . . . . . . . .. . . . . . . . . . . 61017.4.3. Dietary Cholesterol and CHD . . . . . . . . . . . . .. . . . . . . . . . . 61217.4.4. Intervention Studies for CHD Prevention . . . . .. . . . . . . . . . . 61317.4.5. Comment . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . 61417.5. Trans Fatty Acids and CHD . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . 61517.5.1. Clinical Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61517.5.2. Epidemiological Studies . . . . . . . . . . . . . . . . .. . . . . . . . . . . 61617.5.3. Biological Explanation for the Disparate EVects. . . . . . . . . . . 61617.5.4. RA and Atherosclerosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61717.6. Anti-Cancer Agents in Milk Fat . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61817.6.1. Rumenic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61917.6.2. Sphingolipids and Colon Cancer . . . . . . . . . . . . . . . . . . . . . . 62217.6.3. Butyric Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62517.6.4. 13-Methyltetradecanoic Acid . . . . . . . . . . . . . .. . . . . . . . . . . 62817.6.5. Ether Lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62817.6.6. Cholesterol . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . 62817.6.7. b-Carotene and Vitamin A . . . . . . . . . . . . . . . . . . . . . . . . . . 629 21. Contents xxiii17.6.8. Vitamin D and its Metabolites .. . . . . . . . . . . . . . . . . . . . . . . 63017.6.9. Anti-Cancer Agents from Feed . . . . . . . . . . . . . . . . . . . . . . . 63017.6.10. Milk Fat and Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63117.7. Other Nutritional BeneWts . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . 63117.8. Conclusions . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . 632Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63318. Oxysterols: Formation and Biological FunctionP.A. Morrissey and M. Kiely18.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64118.2. Formation of Oxysterols . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . 64218.2.1. Cholesterol Autoxidation . . . . . . . . . . . . . . . . . . . . . . . . . . . 64318.2.2. Initiation of Cholesterol Oxidation . . . . . . . . . .. . . . . . . . . . . 65218.3. Oxysterols in Food Products . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . 65318.3.1. Oxysterols in Dehydrated Systems . . . . . . . . . . . . . . . . . . . . . 65518.3.2. Oxysterols in High-Fat Products . . . . . . . . . . .. . . . . . . . . . . 65618.3.3. Other Factors Involved in Oxysterol Formation. . . . . . . . . . . 65718.4. Sources of Oxysterols In Vivo . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . 65718.4.1. Absorption of Dietary Oxysterols . . . . . . . . . . . . . . . . . . . . . 65818.4.2. Oxysterols Formed Endogenously byNonenzymatic Oxidation . . . . . . . . . . . . . . . . . . . . . . . . . . . 65918.4.3. Oxysterols Formed Enzymatically . . . . . . . . . .. . . . . . . . . . . 66118.5. Biological EVects of Oxysterols . . . . . . . . . . . . . . . . .. . . . . . . . . . . 66218.5.1. EVects of Oxysterols on Cell Membranes . . . . . . . . . . . . . . . . 66218.5.2. Oxysterols and Apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . 66318.5.3. Oxysterols and Atherosclerosis . . . . . . . . . . . . . . . . . . . . . . . 66518.6. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . 667Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66719. High Performance Liquid Chromatography and GasChromatography Methods for Lipid AnalysisK.N. Kilcawley19.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67519.2. QuantiWcation of FFAs . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . 67619.2.1. Gas Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67619.2.2. High Performance Liquid Chromatography . . . . . . . . . . . . . . 67819.2.3. Isolation of FFA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67919.2.4. GC Analysis . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . 68319.2.5. HPLC Analysis . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . 68519.2.6. Conjugated Linoleic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . 686 22. xxiv Contents19.3. Lipid-Derived Volatile Aroma and Flavor Compounds ............ 68719.3.1. Gas Chromatography Mass Spectrometry . . . . . ........... 68719.3.2. Isolation and Concentration of VolatileLipid-Derived Components . . . . . . . . . . . . . . . ........... 68819.4. Tri-, Di- and Mono-Acylglycerols . . . . . . . . . . . . . . . . ........... 69019.5. Chromatographic Methods for the Analysis ofLipolytic Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . ........... 692Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ........... 69220. Spectroscopic Techniques (NMR, Infrared andFluorescence) for the Determination of Lipid Compositionand Structure in Dairy ProductsE. Dufour20.1. Spectroscopic Techniques Used to Study Fats in Dairy Products. . . . . 69720.1.1. Infrared Spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . 69720.1.2. Fluorescence Spectroscopy . . . . . . . . . . . . . . . . . . . . .. . . . . 69820.1.3. Nuclear Magnetic Resonance . . . . . . . . . . . . . . . . . . . . . . . . 70020.2. Characterization of Dairy Products, Including Cheeses . . . . . . .. . . . . 70120.2.1. Direct Determination of the Concentrations ofDiVerent Compounds . . . . . . . . . . . . . . . . . . . . . . . . . ..... 70120.2.2. Direct Determination of the Structure of Fatsin Dairy Products . . . . . . . . . . . . . . . . . . . . . . . . . . . ...... 70220.3. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..... 705Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..... 70621. Applications of Ultrasound to Analysis/Quantitationof Dairy LipidsM.J.W. Povey and R.E. Challis21.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ............70921.2. Theory of Ultrasound Propagation through Solid andLiquid Lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . .............71121.2.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . ............71121.2.2. Physical Determinants of Attenuation, Phaseand Group Velocity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71221.3. Ultrasound Methods for Characterizing Dairy Lipids . . . . . . . . . . . . . 71621.3.1. Tracking Crystallization . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71621.4. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . 720Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 721Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 721 23. Contents xxv22. Physical Characterization of Milk Fat and Milk Fat-BasedProductsO.J. McCarthy22.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72522.2. Thermal Properties: Phase Change Behavior . . . . . . . . . . . . . . .. . . . 726 22.2.1. Melting and Solidification Points: Introduction . . . . . . . . . . . 726 22.2.2. Melting Points . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . 726 22.2.3. SolidiWcation Point . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 729 22.2.4. Dilatometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 729 22.2.5. Nuclear Magnetic Resonance . . . . . . . . . . . . . . . . . . .. . . . 731 22.2.6. DiVerential Scanning Calorimetry . . . . . . . . . . . . . . . .. . . . 731 22.2.7. X-Ray DiVraction (XRD) . . . . . . . . . . . . . . . . . . . . . .. . . . 740 22.2.8. Combined DSC and XRD . . . . . . . . . . . . . . . . . . . . .. . . . 745 22.2.9. Coupled DSC and XRD . . . . . . . . . . . . . . . . . . . . . . . . . . . 74622.3. Thermal Properties: Critical Temperatures . . . . . . . . . . . . . . . . .. . . . 75022.4. Rheological Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 751 22.4.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . 751 22.4.2. Rheological Behavior and Material ClassiWcation . . . . . . . . . 751 22.4.3. Rheological Characterization of Materials . . . . . . . . . . . . . . 752 22.4.4. Viscometers and the Measurement of Fundamental Viscous Properties . . . . . . . . . . . . . . . . . . . . . . . . . . .....753 22.4.5. Solids Rheometers and the Measurement of Fundamental Elastic Properties . . . . . . . . . . . . . . . . . .. . . . 756 22.4.6. Measurement of Linear Viscoelastic Properties . . . . . . . . . . . 759 22.4.7. Measurement of Nonlinear Viscoelastic Properties . . . . .. . . . 760 22.4.8. Measurement of Extensional Viscosity . . . . . . . . . . . . .. . . . 761 22.4.9. Application of Rheological Techniques to Milk Fat and Milk Fat-Based Dairy Products . . . . . . . . . . . . . . . . . . . . . . 76222.5. Density . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . 76522.6. Electromagnetic Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 766 22.6.1. Refractive Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . 766 22.6.2. Color . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 767 22.6.3. Dielectric Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 768 22.6.4. Electrical Conductivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77022.7. Functional Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . 770 22.7.1. Milk Fat and Butter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 771 22.7.2. Ice Cream . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 771 22.7.3. Chocolate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 771 22.7.4. Whole Milk Powders . . . . . . . . . . . . . . . . . . . . . . . . .. . . . 771Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 772Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 772Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 779 24. 1Composition and Structure ofBovine Milk LipidsA.K.H. MacGibbon and M.W. Taylor1.1. IntroductionThe lipids in bovine milk are present in microscopic globules as an oil-in-water emulsion. The primary purpose of these lipids is to provide a sourceof energy to the newborn calf. Both the fat content of the milk and the fattyacid composition of the lipids can vary considerably as a result of changes infactors like breed of cow, diet and stage of lactation. The fat content canvary from about 3.0 to 6.0%, but typically is in the range 3.5 to 4.7%.Changes in the composition of the fatty acids (e.g., 16:0 and 18:1) can bequite marked and can lead to changes in physical properties of the fat. Thesechanges make comparison diYcult between diVerent samples of milk fat,and ideally comparisons should be made between cows in mid-lactation andfed on similar diets. From a practical viewpoint, milk lipids are very import-ant as they confer distinctive nutritional, textural and organoleptic proper-ties on dairy products, such as cream, butter, whole milk powder and cheese.The composition and structure of bovine milk fat have been reviewedextensively. There are early reviews by Morrison (1970), Christie (1978, 1995),Jensen and Clark (1988), and Jensen and Newberg (1995); recent articles includea comprehensive review of recent research by Jensen (2002) and two bookchapters by Vanhoutte and Huyghebaert (2003), and Zegarska (2003). Bovinemilk lipids are similar to the milk lipids of other species as they are largelycomposed of triacylglycerols; however, there are also minor amounts of diacyl-glycerols, monoacylglycerols, free (unesteriWed) fatty acids, phospholipids andA.K.H. MacGibbon . Fonterra Co-Operative Group Ltd., Dairy Farm Road, Private Bag11029, Palmerston North, New Zealand. M.W. Taylor . Institute of Food Nutrition andHuman Health, Massey University, PO Box 11222, Palmerston North, New Zealand.Advanced Dairy Chemistry, Volume 2: Lipids, 3rd edition.Edited by P.F. Fox and P.L.H. McSweeney, Springer, New York, 2006.1 25. 2 A.K.H. MacGibbon and M.W. TaylorTable 1.1.Main classes of lipids in milkaLipid class Amount (%, w/w)Triacylglycerols98.3Diacylglycerols0.3Monoacylglycerols0.03Free fatty acids 0.1Phospholipids0.8Sterols0.3Carotenoids traceFat-soluble vitaminstraceFlavour compounds traceaWalstra and Jenness (1984)sterols. Trace amounts of fat-soluble vitamins, b-carotene and fat-solubleXavouring compounds are also present in the bovine milk lipids (Table 1.1).Because the triacylglycerols account for about 98% of the total fat,they have a major and direct eVect on the properties of milk fat, for examplehydrophobicity, density and melting characteristics. These triacylglycerolsare a complex mixture, and vary considerably in molecular weight anddegree of unsaturation. After milking, fresh milk contains only smallamounts of diacylglycerols and monoacylglycerols and free fatty acids.The small proportion of diacylglycerols are largely sn-1,2 diacylglycerolsand are, therefore, probably intermediates in the biosynthesis of triacylgly-cerols rather than the products of lipolysis (Lok, 1979). The proWle of freefatty acids in freshly-drawn milk diVers somewhat from the proWle of thefatty acids esteriWed to the triacylglycerols (e.g., there appears to be verylittle free butanoic acid), also indicating that they are unlikely to be the resultof lipase action (Walstra and Jenness, 1984).Phospholipids account for only 0.8% of milk lipids. However, theyplay a major role in milk due to their amphiphilic properties. About 65% ofthem are found in the milk fat globule membrane (MFGM), whereas therest remain in the aqueous phase. Phosphatidyl choline, phosphatidyl etha-nolamine and sphingomyelin are the major phospholipids of milk, whichtogether comprise about 90% of the total. Sterols are also a minor compon-ent, comprising about 0.3% of the fat; cholesterol, being the principal sterol,accounts for over 95% of the total sterols.Milk fat is present in spherical droplets, which range from about 0.2 to15:0 mm in diameter, with the bulk of the fat being in globules 1.0 to 8:0 mmdiameter. The MFGM, which envelopes the fat globule, consists largely ofproteins and lipids. The protein of the membrane has a complex compositionand over 40 polypeptides have been identiWed. Xanthine oxidoreductase, 26. Composition and Structure of Bovine Milk Lipids3 0.4 0.3 Heat flow (W/g) 0.2 0.1 0.0 40 30 20 100 10 20 3040 Temperature ( C)Figure 1.1. Melting proWle of New Zealand milk fat, determined by diVerential scanningcalorimetry (MacGibbon, 1988).butyrophilin, PAS 6 and PAS 7 are found to be the major proteins. The lipidsin the membrane are largely phospholipids and triacylglycerols. In contrast tothe MFGM, the fat globule core almost exclusively consists of triacylglycerols(Keenan and Dylewski, 1995; see Chapter 4, Keenan and Mather).The chemical properties of milk lipids can have a considerable inXuenceon the melting characteristics of milk fat, which in turn can have a markedeVect on the functional properties of a number of dairy products, such ascheese and butter (Chen et al., 2004). Milk fat melts over a wide range, fromabout 358C to 388C (Figure 1.1). There is a small broad peak centred atabout 78C, a major melting peak at about 178C, and a plateau from 228C to368C. It can be seen that a substantial proportion of milk fat melts between108C and 208C. This broad melting range is directly attributable to the largenumber of diVerent types of triacylglycerols present in the milk fat.1.2. Fatty AcidsBovine milk fat is regarded as one of the most complex naturally-occurringfats and oils, because of the large number of fatty acids with a variety ofstructures. Using a combination of chromatographic and spectroscopic 27. 4 A.K.H. MacGibbon and M.W. Taylortechniques, researchers have identiWed approximately 400 fatty acids inmilk fat. A listing of the various types of fatty acids has been compiled byJensen (2002). The vast majority of these acids are present in extremelysmall quantities (0.01% (Table 1.5), while theremainder exist in trace amounts. Most of these fatty acids are of little practicalimportance and, hence, their nature and structure is of academic interest only.Among the minor saturated fatty acids are branched-chain and odd-numbered carbon fatty acids with a range of chain length from C3 to C27 .Examples of odd-numbered fatty acids are 13:0 (0.19%), 17:0 (0.6%) and19:0 (0.15%) (Table 1.5). The monomethyl branched-chain fatty acids arequite signiWcant, accounting for about 2.5% of the total fatty acids. Ex-amples are the C15 branched-chain fatty acids, 13-methyl tetradecanoic acid(the iso conWguration) and 12-methyl tetradecanoic acid (the anteiso con-Wguration), which together make up about 0.8% of milk fat (Table 1.5).There are about 200 minor monoenoic, dienoic and polyenoic fattyacids in milk fat ranging in chain length from C10 to C24 , and consisting ofboth positional and cis/trans isomers. A number have considerable nutritionalsigniWcance; for example, eicosapentaenoic acid (20:5, 0.09%) and docosahex-aenoic acid (22:6, 0.01%) are present in the metabolic pathway of the n-3 fattyacids, while arachidonic acid (20:4, 0.14%) is part of the n-6 pathway.Jensen (2002) has reported that milk fat contains about 60 hydroxyfatty acids. The C4 -hydroxy and C5 -hydroxy acids are of interest as they 33. 10A.K.H. MacGibbon and M.W. Taylor Table 1.5. Minor fatty acids in bovine milk fata Composition (%, w/w, of total fatty acids) Saturated Unsaturated bStraight-chain Branched-chain MonounsaturatedPolyunsaturatedFA %(w/w)FA %(w/w) FA %(w/w)FA %(w/w)11:0 0.20 13:0i 0.03 10:1 0.1520:2 0.0713:0 0.19 14:0a 0.02 12:1 0.0620:3 0.1017:0 0.60 15:0i 0.40 13:1 0.0320:4 0.1419:0 0.15 15:0a 0.44 17:1 0.3620:5 0.0920:0 0.35 16:0i 0.40 19:1 0.1622:2 0.0421:0 0.04 17:0i 0.50 20:1 0.3222:3 0.0722:0 0.20 17:0a 0.52 21:1 0.0422:4 0.0323:0 0.12 18:0i 0.16 22:1 0.0622:5 0.0424:0 0.14 19:0i 0.1022:6 0.0125:0 0.0326:0 0.06aIverson and Sheppard (1986). Minor fatty acids present at levels $ 0.01%.bi iso, a anteisotransform to the respective 4-carbon (g) and 5-carbon (d) lactones, which aremajor contributors to the overall Xavour of the milk fat. Approximately 60keto (oxo) acids have been isolated and identiWed in milk fat (Weihrauch,1974; Brechany and Christie, 1992). When milk fat is heated, b-keto acidsare decarboxylated to form methyl ketones, which contribute to cookedbutter Xavours.1.2.6. Variations In Fatty Acid CompositionThe fatty acid composition of milk fat is not stable and is inXuenced bya number of factors. These include, breed of cow, stage of lactation and type,and quality and quantity of feed (Grummer, 1991; Beaulieu and Palmquist,1995; Hawke and Taylor, 1995; Auldist et al., 1998; Zegarska et al., 2001).These issues are discussed in detail in Chapter 2 (Palmquist).In most countries, there exists a regularly recurring seasonal pattern offatty acid variation in milk fat, which is caused largely by changes to the cowsdiet. This seasonal variation can have an impact on the properties of high-fatdairy products, e.g., the hardness of butter (MacGibbon and McLennan,1987). The seasonal variation for French milk fat is presented in Table 1.6(WolV et al., 1995). It can be seen that 16:0 has a markedly lower value inspring and summer than in winter. The C6 to C14 fatty acids together show asimilar trend, although the magnitude of the change is much smaller. In 34. Composition and Structure of Bovine Milk Lipids 11Table 1.6. Fatty acid composition of French butters collected at diVerent periods of the yeara Composition (%, w/w)Fatty AcidJanuary MarchMayJuneJulyAugust OctNov4:0 4.04.4 3.8 4.24.36:0 2.52.7 2.4 2.52.68:0 1.51.6 1.4 1.51.510:03.53.5 3.1 3.13.312:04.04.0 3.6 3.53.814:0 12.1 11.911.011.2 11.314:11.11.0 0.9 1.10.915:01.21.2 1.2 1.21.116:0 33.3 32.527.128.3 29.316:11.51.6 1.4 1.51.718:09.09.011.010.59.618:1 cis 16.8 17.319.719.2 19.418:1 trans2.42.4 4.3 3.73.218:21.41.5 1.2 1.31.418:2 conj 0.40.5 0.7 0.70.518:30.30.3 0.6 0.50.5Minor 5.04.6 6.6 6.05.6aWolV et al. (1995).contrast, 18:0 and 18:1 (cis and trans) show a reverse trend with lower levels inwinter. Generally, higher values of 16:0 tend to be associated with higherlevels of total lipids and greater hardness of the fat. These variationsare attributed to a change from winter feed of hay and concentrate to a dietof fresh grass in spring. The lipids in fresh grass contain high levels of 18:2and 18:3 which, as a result of biohydrogenation and desaturation reactionsin the cow, lead to increased levels of 18:0 and 18:1 in milk fat. Similar seasonal trends in fatty acid composition have been found inother countries where the pattern of dairy husbandry practices is similar(Hughebaert and Hendrickx, 1971; Muuse et al., 1986; Lindmark-Manssonet al., 2003).1.3. TriacylglycerolsBovine milk fat contains various triacylglycerols, which vary considerably inmolecular weight and degree of unsaturation. This complexity is the directresult of the large number and wide variety of fatty acids which make up thetriacylglycerols. 35. 12A.K.H. MacGibbon and M.W. TaylorTable 1.7. General composition of triacylglycerols of bovine milk fatTriacylglycerol Composition %(w/w)German milk fata New Zealand milk fatbCarbon NumberAverage RangecTypical RangedC26 0.20.20.3 C28 0.60.50.8 0.6 0.40.8C30 1.21.01.9 1.2 0.81.4C32 2.62.13.2 2.5 1.82.9C34 6.04.86.9 5.8 4.46.4C3610.99.212.4 11.0 9.111.8C3812.8 12.113.6 13.311.814.6C4010.19.511.2 10.7 9.712.1C42 7.16.27.9 7.4 6.57.9C44 6.75.47.8 6.7 5.67.3C46 7.46.38.3 7.2 5.67.8C48 9.18.010.78.6 6.99.9C5010.99.712.0 10.6 9.712.8C52 9.57.212.39.4 7.712.6C54 4.62.77.8 4.7 3.77.0C56 0.4 0.40.6aPrecht and Frede (1994)bMacGibbon (unpublished)cRange of values for diVerent regions.dRange of values over a dairying season.As noted earlier, there are some 400 fatty acids in milk fat, whichmeans that theoretically milk fat could contain many thousand triacylgly-cerols. Even if one considers only the 15 or so fatty acids that are present atconcentrations above 1% (Table 1.2), and ignores the placement of thesefatty acids at speciWc positions on the triacylglycerol molecule, there are still680 compositionally diVerent triacylglycerols.The general composition of triacylglycerols can readily be determinedby capillary GC. Typical triacylglycerol compositions of milk fats fromGermany and New Zealand are presented in Table 1.7. The triacylglycerolsshow a wide molecular weight range (from acyl carbon 26 to 56), whicharises from the large diVerences in chain length of the constituent fatty acids(from C4 to C18 ). The triacylglycerol composition is dominated by triacyl-glycerols with 36 to 40 acyl carbons (about 35% of the total) and 46 to 52acyl carbons (about 36% of the total). The range of values for the diVerentcarbon numbers is considerable, indicating that there is signiWcant variationin triacylglycerol composition both throughout the dairying season andbetween diVerent dairying regions. Interestingly, data for the two countriesare remarkably similar. 36. Composition and Structure of Bovine Milk Lipids 13It should be noted that simple capillary GC, while convenient, justseparates triacylglycerols into groups of similar molecular weight, and doesnot provide information on individual triacylglycerols carbon number 38,for example, will consist of several diVerent triacylglycerols (e.g., 4:0, 16:0,18:0; 4:0, 16:0, 18:1; 6:0, 14:0, 18:1, etc.).1.3.1.Structure of TriacylglycerolsTriacylglycerols are synthesised in the mammary gland by enzymicmechanisms that exert some selectivity over the esteriWcation of diVerentfatty acids at each position of the sn-glycerol moiety (Moore and Christie,1979). A triacylglycerol molecule showing the three sn-positions is shown inFigure 1.2.StereospeciWc analytical procedures have been developed that haveenabled the determination of the positional distributions of fatty acids onthe triacylglycerols. The results obtained using these procedures show thatthere is a highly selective stereospeciWc distribution of fatty acids in thetriacylglycerols of bovine milk fat (Table 1.8). For cows fed a normal diet,the fatty acids 4:0 and 6:0 are esteriWed almost entirely at position sn-3. Incontrast, 12:0 and 14:0 are esteriWed preferentially at position sn-2, while16:0 is incorporated preferentially at positions sn-1 and sn-2. 18:0 is esteriWedpreferentially at position sn-1, and 18:1 shows a preference for positions sn-1and sn-3. This overall pattern of fatty acid distribution does not changesigniWcantly either throughout the dairying season or between countries(Pitas et al., 1967; Taylor and Hawke, 1975b; Parodi, 1979; Christie andClapperton, 1982).StereospeciWc analysis of milk fat fractions containing triacylglycerolsof diVerent molecular weight have shown that, for fatty acids of chain lengthC4 to C16 , the general pattern of fatty acid distribution in normal milk fat issimilar to the pattern of distribution in the triacylglycerol fractions of diVerent H2C O C R1sn 1 OR2 C OCH sn 2 O H2C O C R3sn 3 OFigure 1.2. Fischer projection diagram of a triacylglycerol showing the stereospeciWc number-ing (sn-) convention. 37. 14A.K.H. MacGibbon and M.W. TaylorTable 1.8.Positional distribution of fatty acids in the triacylglycerols of bovinemilk fata Fatty Acid Composition (mol %)Fatty Acid sn-1sn-2 sn-34:0 0.4 30.66:0 0.7 13.88:0 0.3 3.54.210:01.4 8.17.512:03.5 9.54.514:0 13.125.66.916:0 43.838.99.318:0 17.6 4.66.018:1 19.7 8.4 17.1aCalculated from the data of Parodi (1979).molecular weight. However, the pattern of distribution of 18:0 and 18:1 variesaccording to the molecular weight of the triacylglycerols; these fatty acids tendto be esteriWed preferentially at positions sn-1 and sn-3 in triacylglycerols ofhigh molecular weight and concentrated at position sn-1 in triacylglycerolsof medium- and low-molecular weight (Parodi, 1982).1.3.2. Composition of TriacylglycerolsAs noted earlier, milk fat contains a very complex mixture of triacyl-glycerols. This complexity has made the identiWcation and characterizationof individual triacylglycerols extremely diYcult. Moreover, the fact that notwo batches of milk fat have exactly the same composition adds to thediYculty. As a result, the majority of the earlier studies were aimed atelucidating the general types of triacylglycerols present rather than obtainingquantitive data about individual triacylglycerols.In a series of investigations, milk fat was fractionated into diVerenttriacylglycerol classes on the basis of molecular weight and degree of unsat-uration, using a combination of chromatographic methods, namely normaland argentation TLC, and GC. This approach, in combination with stereo-speciWc analysis, provided detailed information on the diVerent classes oftriacylglycerols present in milk fat (Breckenridge and Kuksis, 1968, 1969;Taylor and Hawke, 1975a; Parodi, 1980).The high molecular weight fractions of diVering degrees of unsaturationwere found to consist largely of triacylglycerols containing combinations offour long-chain fatty acids, namely 14:0, 16:0, 18:0 and 18:1. The most likelyplacement of these fatty acids at the diVerent positions on the triacylglycerol 38. Composition and Structure of Bovine Milk Lipids 15 SaturatedMonoeneDiene TrieneHighAA18:1 18:1molecularBB16:0 18:1weight 18:018:1 18:1 18:1Medium and A 18:1 18:1low molecularBB18:1weight C CC Triacylglycerol skeleton sn1sn2 A = 16:0 or 18:0sn3 B = 14:0 or 16:0 C = 4:0 or 6:0 Figure 1.3. Probable composition of the major triacylglycerols of milk fat.molecules is shown in Figure 1.3. On the other hand, the medium- and low-molecular weight fractions were comprised mainly of triacylglycerols withcombinations of these four long-chain fatty acids at positions sn-1 and sn-2and a short-chain fatty acid (either 4:0 or 6:0) esteriWed at position sn-3.The saturated and monoene triacylglycerol classes were dominant andeach comprised about 35 to 40% of the total milk fat, while the approximateproportions of the high-, medium- and low-molecular weight fractions were40, 20 and 40%, respectively.More recently, the use of more sophisticated chromatographic tech-niques, particularly HPLC and capillary GC, has lead to the identiWcationand quantiWcation of individual, compositionally-diVerent triacylglycerols.In one painstaking study, Gresti et al. (1993) separated milk fat by reversed-phase HPLC into 47 fractions. Each fraction was then analysed for triacyl-glycerol and fatty acid composition by capillary GC. The data obtained wereused to calculate the proportions of some 220 individual molecular species oftriacylglycerols, accounting for 80% of the total triacylglycerols in the sample.The quantitatively important triacylglycerols, each present at >0.5%, areshown in Table 1.9. This list of 40 major triacylglycerols makes up about55% of the total milk fat. An interesting aspect of the data is that sometriacylglycerols are present in high proportions, for example 4:0, 16:0, 18:1(4.2%); 4:0, 16:0, 16:0 (3.2%); 4:0, 14:0, 16:0 (3.1%); 14:0, 16:0, 18:1 (2.8%);4:0, 16:0, 18:0 (2.5%); and 16:0, 18:1, 18:1 (2.5%). Although, this was a longand exhaustive study, it did not deWnitively identify the constituent triacyl-glycerols of milk fat as the placement of the fatty acids at the diVerent sn-positions on the triacylglycerol molecules was not determined. 39. 16A.K.H. MacGibbon and M.W. Taylor Table 1.9. Proportions (mol %) of the major triacylglycerols in a sample of French milk fata,b,cCarbonSaturated Monoene Diene and TrieneNumberTriacylglycerolsTriacylglycerolsTriacylglycerolsC304:0, 10:0, 16:0, 0.6%C324:0, 12:0, 16:00.8%C344:0, 14:0, 16:03.1%C364:0, 14:0, 18:01.3%4:0, 14:0, 18:11.8% 4:0, 16:0, 16:03.2% 6:0, 14:0, 16:01.4%C384:0, 16:0, 18:02.5%4:0, 16:0, 18:14.2% 6:0, 16:0, 16:01.5%6:0, 14:0, 18:10.9% 6:0, 14:0, 18:00.6%C406:0, 16:0, 18:01.1%4:0, 18:0, 18:11.6%4:0, 18:1, 18:1 1.5% 8:0, 16:0, 16:00.7%6:0, 16:0, 18:12.0%10:0, 14:0, 16:00.7%C42 10:0, 16:0, 16:01.0%10:0, 14:0, 18:1 0.6%6:0, 18:1, 18:1 0.6%12:0, 14:0, 16:00.6%C44 14:0, 14:0, 16:00.6%10:0, 16:0, 18:1 1.6%C46 14:0, 16:0, 16:00.9%12:0, 16:0, 18:1 1.2% 10:0, 18:1, 18:1 0.7%14:0, 14:0, 18:1 0.6%C48 14:0, 16:0, 18:00.7%14:0, 16:0, 18:1 2.8% 12:0, 18:1, 18:1 0.6%C50 14:0, 18:0, 18:1 1.4% 14:0, 18:1, 18:1 1.2%16:0, 16:0, 18:1 2.3%C52 16:0, 18:0, 18:1 2.2% 16:0, 18:1, 18:1 2.5%16:0, 18:1, 18:2 0.6%C54 18:0, 18:0, 18:1 0.8% 18:0, 18:1, 18:1 1.2%18:1, 18:1, 18:1 1.0%aGresti et al. (1993).bTriacylglycerols at concentrations >0.5%.cPosition of fatty acid on triacylglycerol molecule not determined.A number of recent investigations have shown that mass spectrometry(MS) is a rapid and eVective method for the identiWcation of triacylglycerolspecies of milk fat that are compositionally diVerent (Myher et al.,1988, 1993; Laakso and Kallio, 1993; Spanos et al., 1995; Laakso andManninen, 1997; Mottram and Evershed, 2001; Kalo et al., 2004). In fact,a range of mass spectral techniques, such as electron ionization, fastatom bombardment, chemical ionization, atmospheric pressure chemicalionization and electrospray MS, have been used to study triacylglycerols.The later three are soft ionizing techniques, which retain substantialamounts of the molecular ion, rather than fragmenting the molecule intoa number of parts. These methods have allowed the determination of 40. Composition and Structure of Bovine Milk Lipids17fatty acids that contribute to the triacylglycerols, especially with the advent of MS/MS where the further fragmentation of a particular molecular ion can be eVected,displaying fragments derived solely from a species or group (Kalo et al., 2004). In these mass spectral studies there are diYculties associated with theaccurate quantiWcation of the diVerent types of triacylglycerols. First, MSshows a diVerence in sensitivity depending on the degree of unsaturation oftriacylglycerols; fully saturated triacylglycerols tend to show a lower molecu-lar ion response than unsaturated triacylglycerols (Byrdwell, 2001). Second,the studies using tandem MS/MS, in which diacylglycerol ions and fatty acidions are formed from triacylglycerol ions, showed that the diacylglycerol ionswere not representative of the expected random distribution of diacylglycer-ols, but rather contained more of the fatty acids at the sn-2 position. In otherwords, the release of fatty acids from the sn-2 position was less than the releasefrom the sn-1 and sn-3 positions (Currie and Kallio, 1993). Despite these concerns, several researchers have used MS to identifymany of the constituent triacylglycerols of milk fat. These studies invariablybegin with extensive fractionation of the triacylglycerols prior to massspectral analysis, to ensure that the number of triacylglycerol species con-tributing to a particular fraction are as small as possible. In one of theearliest investigations, Myher et al. (1988) studied a milk fat fractionwhich was composed largely of low molecular weight triacylglycerols.After an initial separation using argentation TLC, which separated thetriacylglycerols according to their degree of unsaturation, mass spectralanalysis was used to identify more than 100 triacylglycerols. In a further comprehensive investigation by Spanos et al. (1995), milkfat was fractionated initially by HPLC into 58 triacylglycerol fractions.These fractions were characterised using desorption chemical ionisationMS, followed by MS/MS if the peak contained a mixture of triacylglycerols.The fatty acids contributing to the triacylglycerols in each peak were deter-mined and could be related to the acyl carbon number of the triacylglycerols.Thus, the composition of over 180 triacylglycerols were determined. Com-parison of these results with the data of Gresti et al. (1993) showed that thetriacylglycerols identiWed were similar. For example, for the C38 triacylgly-cerols, Gresti et al. (1993) found the following quantitatively-importanttriacylglycerols:- 4:0, 16:0, 18:1 (4.2%); 4:0, 16:0, 18:0 (2.5%); 6:0, 16:0,16:0 (1.5%); 6:0, 14:0, 18:1 (0.9%); and 6:0, 14:0, 18:0 (0.6%). Although,Spanos et al. (1995) did not quantify the triacylglycerol species, only notingthose that were present in greater amounts, they did identify the same groupof C38 triacylglycerols as being present in signiWcant quantities, with theexception of 4:0, 16:0, 18:0. Mottram and Evershed (2001) undertook a similar study in whichthey fractionated milk fat by two diVerent methods, silica TLC and gel 41. 18 A.K.H. MacGibbon and M.W. Taylorpermeation chromatography. Each set of fractions was analyzed subse-quently by an HPLC-MS system Wtted with an atmospheric pressurechemical ionization source. Some fractions were also analyzed by GC-MS. This comprehensive analysis led to the identiWcation of some 120triacylglycerols.Recently, Kalo et al. (2004) used normal-phase HPLC in combinationwith positive ion tandem MS to obtain quantitative information about theregioisomers of synthetic triacylglycerol mixtures and milk fat fractionscontaining low molecular weight triacylglycerols. In agreement with a pre-vious study (Currie and Kallio, 1993), they found that the diacylglycerolfragment ions, produced by mass spectral analysis from standard triacylgly-cerol mixtures, contained greater amounts of fatty acids at the sn-2 pos-ition than predicted. Furthermore, the ratio of fatty acids at the sn-2position, relative to the fatty acids at the sn-1 and sn-3 positions, variedaccording to the types of fatty acids attached. From the information gainedabout these diacylglycerols, the regioisomers of the synthetic triacylglycerolmixtures could be identiWed. In a similar manner, the regioisomers of thetriacylglycerols in the milk fat fractions were studied, although the fattyacids at the sn-1 and sn-3 positions could not be diVerentiated.Although these later detailed studies have not as yet yielded a methodwhich deWnitively identiWes and quantiWes the constituent triacylglycerols ofmilk fat, the improvement in HPLC and mass spectral analyses have enabledresearchers to develop routine methods that provide detailed informationabout milk fat triacylglycerols. One example is the method developed byRobinson and MacGibbon (1998), in which milk fat triacylglycerols wereseparated into 61 distinct peaks by reversed-phase HPLC (Figure 1.4). Thetriacylglycerols present in each peak were identiWed through initial fraction-ation by argentation TLC, followed by HPLC and MS. This HPLC methodcan be used as a single-injection, routine method, and appears to be sensitiveenough to monitor relatively small changes in peak areas and, hence, minorchanges in the amounts of small groups of triacylglycerols.With the upsurge of interest in CLA, the distribution of CLA inmilk fat triacylglycerols has also become a matter of considerable interest.The distribution of CLA has been determined by a reversed-phase HPLCsystem, in which the eluting peaks were simultaneously detected by bothevaporative light scattering detection (ELSD) and UV absorption at 233 nm(Robinson and MacGibbon, 2000). The UV absorption data clearly showwhich peaks contain esteriWed CLA (the molar extinction coeYcient for CLA1at 233 nm is 23,360 L mol1 cm ). The combined data from the two detec-tion systems show that CLA is found in many diVerent typesof triacylglycerols, which diVer in both molecular weight and degree ofunsaturation. 42. Composition and Structure of Bovine Milk Lipids 19 12 108mV642020 4060 80 100 120 MinutesFigure 1.4. Reversed-phase HPLC chromatogram of milk fat triacylglycerols (from Robinsonand MacGibbon, 1998).1.4. Polar LipidsThe concentration of phospholipids in the milk fat ranges from 0.5 to 1.0% ofthe total (Patton and Jensen, 1976; Table 1.1). About 60 to 65% of thesephospholipids are associated with the intact milk fat globule membrane(MFGM). The remaining 35 to 40% are found in the aqueous phase associ-ated with protein/membrane fragment material in solution, rather than stillattached to the MFGM (Huang and Kuksis, 1967; Patton and Keenan, 1971).The MFGM that surrounds the milk fat droplets is derived from theapical plasma membrane of the secretary cells in the lactating mammaryglands, and is composed of phospholipds and glycolipids, as well as proteins,glycoproteins, enzymes, triacylglycerols and minor components. Estimatesof the proportion of phospholipids in the MFGM vary from 15 to 30%,depending on extraction procedure; however, typical values are at the higherend of the range. For instance, Keenan and Dylewski (1995) reported 26 to31%, and Norris et al. (2003) found 28% of the MFGM as phospholipid (seeKeenan and Mather, Chapter 4). 43. 20A.K.H. MacGibbon and M.W. TaylorH2C O C R1 sn 1OR2 C OCHsn 2OOH2C O P O Polar group sn 3 OWhere the polar group is Ethanolamine (PE) Inositol (PI) Serine (PS) Choline (PC)Figure 1.5. Fischer projection diagram of a glycerophospholipid showing the stereospeciWcnumbering (sn-) convention. While the polar lipids constitute a very small proportion of the totalmilk lipids, they play an important role because of their mixed hydrophilicand hydrophobic nature. This unique characteristic of polar lipids is largelyresponsible for stabilising the suspension of milkfat in the aqueous environ-ment of the milk, allowing the relatively high concentrations of milkfat andprotein to coexist in the same solution (Deeth, 1997). In the above process,the major structural features involved are the large non-polar (hydrophobic)fatty acid chains and the polar (hydrophilic) charged head group residue ofthe phospholipids. The polar lipids contain a variety of polar groups thatcontribute to the charged nature of the molecules. In addition to the chargedhead-group, phospholipids contain a negatively charged phosphate group(Figure 1.5). Dairy phospholipids are important structurally, because they are ableto stabilise emulsions and foams, and to form micelles and membranes(Jensen and Newburg, 1995). Phospholipids also have the potential to bepro-oxidants, because they contain mono-unsaturated and poly-unsaturatedfatty acids and have the ability to attract metal ions. Phosphatidyl ethano-lamine binds copper strongly and is believed to be important in copper-induced oxidation in milk (OConnor and OBrien 1995; Deeth, 1997). Thepolyunsaturated fatty acids and metal ions accelerate lipid oxidation, espe-cially when heat is applied; hence, phospholipids can be degraded during theprocessing of milk. However, in dairy products, the situation is complex andit appears that phospholipids are able to act as either pro-oxidants or anti-oxidants, depending on the pH, ratio of water and phospholipid species(Chen and Nawar, 1991). 44. Composition and Structure of Bovine Milk Lipids21Table 1.10. Approximate phospholipid content of diVerent dairy productsProduct Whole milk Skim milk Cream (40% fat) Butter milkTotal fat (%, w/w) 4 0.06 400.6Phospholipids (%, w/w) 0.035 0.015 0.21 0.13Ratio (g PL/100g total fat)0.925 0.5 22Adapted from Mulder and Walstra (1974)As the milk is processed, the phospholipids are partitioned diVerentlyfrom the neutral lipids (Table 1.10). When the whole milk is separated, thephospholipids tightly bound to the MFGM go into the cream withthe neutral lipids, while the phospholipids associated with the protein/membrane fragments in the aqueous phase are retained in the skim milk.Hence, the ratio of phospholipids to total fat is relatively low in cream andhigh in skim milk. Furthermore, during butter making, a greater proportionof the phospholipids than the neutral lipids from the cream is retained in thebuttermilk, leading to a high ratio of phospholipid to the total fat inbuttermilk (Table 1.10).1.4.1.Composition and StructureThe percentage of phospholipids in milk fat is typically withinthe range 0.51.0 %. Bitman and Wood (1990) found that phospho-lipids in milk tended to decline during lactation, but Kinsella and Houghton(1975) observed little change. While there was a change in thepercentage of total phospholipids, the ratio of the major phospholipidsremained relatively constant, suggesting a constant ratio of phospholipidsin the MFGM.The structures of the major polar lipids found in the milk are shown inFigures 1.5 and 1.6. Glycerophospholipids [phospatidylethanolamine (PE),phosphatidylinositol (PI), phosphatidylserine (PS) and phosphatidylcholine(PC)] have fatty acids at positions sn-1 and sn-2, and a phosphate and apolar head-group on the sn-3 position. Of the minor phospholipids, plasma-logens have a similar structure to phosphatidylcholine and phosphatidy-lethanolamine but with an ether linkage rather than an ester linkage atthe sn-1 position. Lysophospholipids have only one fatty acid in theglycerophospholipid.The sphingophospholipid, sphingomyelin (Sph) consists of a ceramide(a fatty acid linked to a long-chain sphingoloid base through an amidelinkage) with a phosphorylcholine headgroup (Figure 1.6). Sphingomyelin 45. 22 A.K.H. MacGibbon and M.W. Taylor A typical phosphosphingolipid (sphingomyelin) O|| CH3 (CH2)12CH = CHCHOHCHCH2-O-P-O- choline group| NHOCR O A typical glycosphingolipid (glucoceramide) CH3(CH2)12CH=CHCHOHCHCH2-O- glucose NHOCRFigure 1.6. Typical structures of sphingolipids (phosphosphingolipid and glycosphingolipidclasses), based on a d18:1 ceramide (R fatty acid group).is generally included in the phospholipid group as it has similar properties(especially with phosphatidylcholine).Phosphatidylcholine, phosphatidylethanolamine and sphingomyelinare the major polar lipids found in bovine milk and are present in similarproportions in the total phospholipids, about 25 to 35% (Table 1.11).Phosphatidylserine and phosphatidylinositol are present at lower levels,about 3 to 8%. There are also signiWcant amounts of ceramides, as glucocer-amide (GluCer, monohexose) and lactoceramide (LacCer, dihexose), 3 to 6%.Table 1.11 shows Wve sets of analytical data, four of which are relativelyTable 1.11. Proportions of individual phospholipids and ceramides in bovine milk (as percentage of total polar lipids) a (mol %) b %(w/w)c %(w/w)d %(w/w)e %(w/w)Phosphatidylethanolamine 31.834.231.142.036.8Phosphatidylinositol4.7 6.2 5.2 4.8naPhosphatidylserine3.1 2.8 8.5 6.7naPhosphatidylcholine34.525.426.419.232.2Sphingomyelin25.223.628.717.929.6Glucoceramidenaf5.0na 2.7naLactoceramidena 2.9na 6.7naPhospholipids in milk (mg/10 ml)2.282.512.9 2.42Sources and sampling:aJensen and Clark (1988); b Christie et al. (1987), single dairy herd; c Bitman and Wood (1990), 12 cows,42 days of lactation; d Rombault et al. (2005), farm milk; e Fagan and Wijesundra (2004), milk from 21 dairyfarms; f na not available (not determined)Note: not all species identiWed, hence normalisation needs to be carried out prior any to direct comparison 46. Composition and Structure of Bovine Milk Lipids 23similar. The data indicate variations in the proportions of individual phos-pholipids, which may depend on the analytical methods used, the numberand breed of cows in the sample, and the diet and stage of lactation of cows.Interestingly, the reported total phospholipid content of the milk was similar($2.5 mg/10 mL milk