1. 1. FUNDAMENTALS OF DAIRY CHEMISTRY THIRD EDITION Editor Noble P. Wong Agricu1tura1 Research Service U.S.Department of Agriculture Associate Editors Robert Jenness formerly Department of Biochemistry University of Minnesota Mark Keeney Department of Chemistry and Biochemistry University of Maryland Elmer H. Marth Department of Food Science University of Wisconsin A Chapman & Hall Food Science Book An Aspen Publication@ Aspen Publishers, Inc. Gaithersburg, Maryland 1999
2. 2. The author has made every effort to ensure the accuracy of the information herein. However, appropri- ate information sources should be consulted, especially for new or unfamiliar procedures. It is the responsibility of every practitioner to evaluate the appropriateness of a particular opinion in in the context of actual clinical situations and with due considerations to new developments. The author, editors, and the publisher cannot be held responsible for any typographical or other errors found in this book. Aspen Publishers, Inc., is not affiliated with the American Society of Parenteral and Enteral Nutrition. Library of Congress Cataloging-in-Publication Data Fundamentals of dairy chemistry. p. cm. An AVI Book (AVI is an imprint of Van Nostrand Reinhold) Originally published : New York : Chapman & Hall, 1988. Includes bibliographical references and index. (Formerly published by Chapman & Hall, ISBN 0-442-20489-2) ISBN 0-8342-1360-5 1. Milk-Composition. 2. Dairy Products- Composition. I. Wong, Noble P. SF251.F78 1988 637 87-21586 CIP Copyright 01988, 1999by Aspen Publishers, Inc. All rights reserved. Aspen Publishers, Inc., grants permission for photocopying for limited personal or internal use. This consent does not extend to other kinds of copying, such as copying for general distribution, for advertising or promotional purposes, for creating new collective works, or for resale. For information, address Aspen Publishers, Inc., Permissions Department, 200 Orchard Ridge Drive, Suite 200, Gaithersburg, Maryland 20878. Orders: (800) 638-8437 Customer Service: (800)234-1660 About Aspen Publishers For more than 35 years, Aspen has been a leading professional publisher in a variety of disciplines. Aspens vast information resources are available in both print and elec- tronic formats. We are committed to providing the highest quality information available in the most appropriate format for our customers. Visit Aspens Internet site for more information resources, directories, articles, and a searchable version of Aspens full catalog, including the most recent publi- cations: http://www.aspenpublishers.com Aspen Publishers, Inc. The hallmark of quality in publishing Member of the worldwide Wolters Kluwer group Editorial Services: Ruth Bloom Library of Congress Catalog Card Number: 87-21586 ISBN: 0-8342-1360-5 Printed in the United States ofAmerica 2 3 4 5
3. 3. To BYRON H. WEBB for his outstandingdedicated service to the dairy in- dustry that spans half a century and whose persist- ence and guidance has led to another edition of Fun- damentals.
4. 4. Contributors Judith S. Acosta, formerly Dept. of Animal Science and Industry, Kansas State University, Manhattan, Kansas 66506. Richard Bassette, formerly Dept. of Animal Science and Industry, Kansas State University; present address 811 Kiowa Dr. East, Kiowa, Texas 76240. Rodney J. Brown, Dept. of Nutrition and Food Sciences, Utah State University, Logan, Utah 84322. Richard M. Clark, Dept. of Nutritional Sciences, University of Con- necticut, Storrs, Connecticut 06268. Daniel P. Dylewski, Kraft Technical Center, 801 Waukegan Rd., Glen- view, Illinois 60025. C. A. Ernstrom, Dept. of Nutrition and Food Sciences, Utah State Uni- versity, Logan, Utah 84322. Harold M. Farrell, Jr., USDA, Eastern Regional Research Center, 600 E. Mermaid Lane, Philadelphia, Pennsylvania 19118. Joseph F. Frank, Dept. of Animal and Dairy Science, University of Georgia, Athens, Georgia 30602. Virginia H. Holsinger, USDA, Eastern Regional Research Center, 600 E. Mermaid Lane, Philadelphia, Pennsylvania 19118. Robert Jenness, formerly Dept. of Biochemistry, University of Minnesota; present address 1837 Corte del Ranchero, Alamogordo, New Mexico88310. Robert G. Jensen, Dept. of Nutritional Sciences, University of Con- necticut, Storrs, Connecticut 06268. Mark E. Johnson, Dept. of Food Science, University of Wisconsin, Madison, Wisconsin 53706. Thomas W. Keenan, Dept. of Biochemistry and Nutrition, Virginia Po- lytechnic Institute and State University, Blacksburg, Virginia 24061. Mark Keeney, Dept. of Chemistry and Biochemistry, University of Maryland, College Park, Maryland 20742. vii
5. 5. viii CONTRIBUTORS Elmer H. Marth, Dept. of Food Science, University of Wisconsin, Madison, Wisconsin 53706. Ian H. Mather, Dept. of Animal Sciences, University of Maryland, Col- lege Park, Maryland 20742. Lois D. McBean, National Dairy Council, 6300 N. River Road, Rosem- ont, Illinois 60018. Charles V. Morr, Dept. of Food Science, Clemson University, Clemson, South Carolina 29631. Ronald L. Richter, Dept. of Animal Science, Texas A&M University, College Station, Texas 77843. John W. Sherbon, Dept. of Food Science, Cornell University, Ithaca, New York 14853. Elwood W. Speckmann, National Dairy Council, 6300 N. River Road, Rosemont, Illinois 60018. John L. Weihrauch, USDA, Human Nutrition Information Service, Federal Center Building, Hyattsville, Maryland 20782. Robert McL. Whitney (deceased),Food Science Dept., University of Illinois, Urbana, Illinois 61801. Noble P. Wong, USDA, Beltsville Agricultural Research Center, Belts- ville, Maryland 20705.
6. 6. Preface Fundamentals of Dairy Chemistry has always been a reference text which has attempted to provide a complete treatise on the chemistry of milk and the relevant research. The third edition carries on in that format which has proved successful over four previous editions (Fun- damentals of Dairy Science 1928, 1935 and Fundamentals of Dairy Chemistry 1965, 1974). Not only is the material brought up-to-date, indeed several chapters have been completely re-written, but attempts have been made to streamline this edition. In view of the plethora of research related to dairy chemistry, authors were asked to reduce the number of references by eliminating the early, less significant ones. In addition, two chapters have been replaced with subjects which we felt deserved attention: Nutritive Value of Dairy Foods and Chemistry of Processing. Since our society is now more attuned to the quality of the food it consumes and the processes necessary to preserve that quality, the addition of these topics seemed justified. This does not minimize the importance of the information in the deleted chapters, Vitamins of Milk and Frozen Dairy Products. Some of the mate- rial in these previous chapters has been incorporated into the new chapters; furthermore, the information in these chapters is available in the second edition, as a reprint from ADSA (Vitaminsin Milk and Milk Products, November 1965)or in the many texts on ice cream manufac- ture. Originally, Fundamentals of Dairy Science (1928)was prepared by members of the Dairy Research Laboratories, USDA. Over the years, the trend has changed. The present edition draws heavily from the ex- pertise of the faculty and staff of universities. Ten of the 14 chapters are written by authors from state universities, three from ARS, USDA, and one from industry. It seems fitting that this is so. The bulk of future dairy research, if it is to be done, appears destined to be accomplished at our universities. Hopefully the chapter authors have presented appropriate material and in such a way that it serves best the principal users of this book, their students. As universities move away from specific product tech- nology and food technology becomes more sophisticated, a void has ix
7. 7. x PREFACE been created where formerly a dairy curriculum existed. It is hoped that this edition of Fundamentals of Dairy Chemistry which incorpo- rates a good deal of technology with basic chemistry can help fill this void. Preparation of this volume took considerably longer than antici- pated. The exigencies of other commitments took its toll. Originally the literature was supposed to be covered to 1982 but many of the chapters have more recent references. I wish to acknowledge with appreciation the contribution made by the chapter authors and the associate editors. Obviously without their assistance, publication of this edition would not have been possible. Dr. Jenness was responsible for Chapters 1, 3, 8, and 9; Dr. Keeney, Chapters 4, 5, and 10; Dr. Marth, Chapters 2, 13, and 14; and Dr. Wong, Chapters 6, 7, 11 and 12.
8. 8. Contents Preface I ix 1. Composition of Milk, Robert Jenness / 1 2. Composition of Milk Products, Richard Bassette and Judith S. 3. Proteins of Milk, Robert McL. Whitney I 81 4. Lipid Composition and Properties, Robert G. Jensen and 5. Lipids of Milk: Deterioration, John L. Weihrauch I 215 6. Lactose, Virginia H. Holsinger I 279 7. Nutritive Value of Dairy Foods, Lois D. McBean and Elwood W. 8. Physical Properties of Milk, John W. Sherbon / 409 9. Physical Equilibria: Proteins, Harold M. Farrell I 461 Acosta I 39 Richard M. Clark I 171 Speckmann I 343 10. Physical Equilibria: Lipid Phase, Thomas W. Keenan, Ian H. 11. Milk Coagulation and Protein Denaturation, Rodney J. 12. Milk-Clotting Enzymes and Cheese Chemistry, PART I-Milk- Mather, and Daniel F! Dylewski / 511 Brown I 583 Clotting Enzymes, Rodney J. Brown and C. A. Ernstrom I 609 PART II-Cheese ChemistrJZ Mark E. Johnson I 634 13. Fermentations, Joseph F. Frank and Elmer H. Marth / 655 14. Chemistry of Processing, Charles V. Morr and Ronald Index I 767 L. Richter / 739
9. 9. 1 Composition of Milk Robert Jenness Milk is secreted by all species of mammals to supply nutrition and immunological protection to the young. It performs these functions with a large array of distinctive compounds. Interspecies differences in the quantitativecomposition of milk (Jennessand Sloan 1970)prob- ably reflect differences in the metabolic processes of the lactating mother and in the nutritive requirements of the suckling young. Human beings consume large amounts of milk of a few species be- sides their own. The principal ones are cows, water buffaloes, goats, and sheep, which furnish annually about 419, 26, 7.2, and 7.3 million metric tons of milk, respectively, for human consumption (FA0 Pro- duction Yearbook 1979).This chapter, and indeed this entire volume, deals primarily with the milk of western cattle-Bos taurus. References to reviews concerning milk of other important species are: Indian cat- tle-B. indicus (Basuet al. 1962);water buffalo-Bubalus bubalis (Lax- minarayan and Dastur 1968);goat-Capra hircus (Parkash and Jen- ness 1968; Jenness 1980; Ramos and Juarez 1981);sheep-Ouis aries (Ramos and Juarez 1981); and humans-Homo sapiens (Macy et al. 1953; Jenness 1979; Blanc 1981; Gaul1 et al. 1982; Packard 1982). In the United States, milk is defined for commercial purposes as the lacteal secretion, practically free from colostrum, obtained by the com- plete 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% milk fat. Minimal standards in the various states may vary from 8.0 to 8.5% for milk- solids-not-fatand from 3.0 to 3.8% for milk fat (U.S.Dept. Agr. 1980). CONSTITUENTS OF MILK Milk consists of water, lipids, carbohydrates, proteins, salts, and a long list of miscellaneous constituents. It may contain as many as lo5differ- ent kinds of molecules. Refinement of qualitative and quantitative techniques continues to add new molecular species to the list. The con- stituents fall into four categories: 1
10. 10. 2 FUNDAMENTALS OF DAIRY CHEMISTRY 1. Organ and species specific-most proteins and lipids. 2. Organ but not species specific-lactose. 3. Species but not organ specific-some proteins. 4. Neither organ nor species specific-water, salts, vitamins. The following sections summarize the constituents of milk and indi- cate how they are quantitated operationally. Detailed descriptions and properties of lipids, lactose, and proteins will be found in later chap- ters. Water Milks of most species contain more water than any other constituent. Certainly this is true of the milks consumed by humans. The other constituents are dissolved, colloidally dispersed, and emulsified in wa- ter. The dissolved solutes in bovine milk aggregate about 0.3 M and depress the freezing point by about 054C (seeChapter 8).The activ- ity of water in milk, a, which is the ratio of its vapor pressure to that of air saturated with water, is about 0.993. A small amount of the wa- ter of milk is bound ;o tightly by proteins and by the fat globule membrane that it does not function as a solvent for small molecules and ions. Water content is usually determined as loss in weight upon drying under conditions that minimize decomposition of organic con- stituents, e.g., 3 hr at 98-100C (Horwitz 1980). Lipids The lipids of milk, often simply called fat, consist of materials that are extractable by defined methods. Simple extraction with a nonpolar solvent like ether or chloroform is not efficient because the fat is lo- cated in globules protected by a surface membrane. A widely used gravimetric method is the Roese-Gottlieb extraction (Walstra and Mulder 1964) using NH40H, ethanol, diethyl ether, and petroleum ether. Volumetric methods such as that of Babcock and Gerber (Ling 1956; Horwitz 1980)use H&04 to liberate the fat, which is then mea- sured. Rapid determination of the amount of fat in milk can be done by measurement of the absorption of infrared radiation at 3.4 or 5.7 pm (Chapter 8; Goulden 1964; Horwitz 1980). The lipids of milk are composed of about 98% triglycerides, with much smaller amounts of free fatty acids, mono-and diglycerides, phos- pholipids, sterols, and hydrocarbons. Chapter 4 deals in detail with the composition of milk lipids. The fat in milk is almost entirely in the form of globules, ranging
11. 11. COMPOSITON OF MILK 3 from 0.1 to 15 pm in diameter. Size distribution is an inherited charac- teristic that varies among species and among breeds of cattle. Bovine milk contains many very small globules that comprise only a small fraction of the total fat. The total number is about 15 x lo9globules per milliliter of which 75%are smaller than 1 pm in diameter. The fat globules and their protective membrane of phospholipids and proteins are described in Chapter 10. Carbohydrates In bovine milk, and indeed in all milks consumed by humans, the over- whelming carbohydrate is lactose. This disaccharide, 4-O-@-~-galacto- pyranosyl-D-glucopyranose,is a distinctive and unique product of the mammary gland. It has been found in milks of almost all of the species analyzed to date (Jennesset al. 1964)and nowhere else in nature except in low concentration in the fruits of some of the Sapotaceae (Reithel and Venkataraman 1956). Lactose is discussed in detail in Chapter 6. Lactose in milk can be quantitated by oxidation of the aldehyde of the glucose moiety (Hinton and Macara 1927; McDowell 1941; Perry and Doan 1950; Horwitz 1980),by polarimetry of a clarified solution (Grimbleby1956; Horwitz 1980),by colorimetry of the product of reac- tion with phenolic compounds (Marier and Boulet 1959), by infrared absorption at 9.6 pm (Goulden 1964; Horwitz 1980),by enzymatic as- say with @-galactosidaseand galactose dehydrogenase (Kurzand Wal- lenfels 1974), and by chromatography (Reineccius et al. 1970; Beebe and Gilpin 1983; Brons and Olieman 1983).Only the last two of these methods are specific for lactose, but bovine milk contains solittle other material that is oxidizable, that exhibits optical rotation, that reacts with phenolic compounds, or that absorbs at 9.6 pm that the first four give reasonable estimates of lactose. Older analyses were made by oxi- dation or polarimetry. Published values for lactose contents obtained with these methods must be scrutinized carefully because some were calculated on the basis of lactose monohydrate and are thus 5.26%too high (360/342). Carbohydrates other than lactose in milk include monosaccharides, neutral and acid oligosaccharides, and glycosyl groups bound to pro- teins and lipids. Glucose and galactose are detectable by thin layer chromatography (TLC) and gas-liquid chromatography (GLC) of bo- vine milk. Of course, hydrolysis of lactose is an obvious source of these two monosaccharides, but with precautions taken to avoid hydrolysis, concentrations of 100-150 mglliter of each have been found by GLC (Reinecciuset al. 1970).Specific enzymatic methods, however, have in- dicated considerably lower concentrations-about 30 mg glucose and
12. 12. 4 FUNDAMENTALS OF DAIRY CHEMISTRY 90 mg galactose per liter (Faulkner et al. 1981).Free myo-inositol has been found in the milks of several species (in addition to that bound in phosphatidyl inositols; (seeChapter 4). Bovine milk has only 40-50 mg of myo-inositol per liter, but milks of some other species contain much more (Byun and Jenness 1982). The carbohydrates L(-)fucose(Fuc),N-acetylglucosamine (2-acetami- do-2-deoxy-~-glucose),N-acetyl galactosamine (2-acetamido-2-deoxy-~- galactose), and N-acetylneuraminic acid occur in milk almost entirely in the form of oligosaccharides and glycopeptides. Only small concen- trations are present in the free state, although there is one report of 112 mg of N-acetylglucosamineper liter of bovine milk (Hoff 1963).The total (free and combined) content of N-acetylneuraminic acid is 100- 300 mglliter (de Koning and Wijnand 1965). Bovine milk contains 1-2 g of oligosaccharides per liter, human milk 10-25 glliter. Colostrums of both species have higher concentrations. A recent review (Blanc1981)lists more than 60 oligosaccharides which have been detected in human milk. They range from 3 to 20 monosac- charide units per molecule; not all have been characterized structurally. Five oligosaccharides have been detected and characterized in bovine milk or colostrum. All of the oligosaccharides that have been character- ized in either bovine or human milk have a lactose moiety, ~-Gal-P-(l- 4)-~-Glcin the reducing terminal position. (In a few, the terminal resi- due is an N-acetylglucosamine). The simplest are the trisaccharides fucosyllactose [L-FUCa-(1-2)-~-Gal-P-(1-4)-~-Glc]and N-acetylneurami- nyllactose ANA-(2-3)-~-Gal-p-(1-4)-~-Glc].More complex ones have longer chains with various kinds of branching (Ebnerand Schanbacher 1974; Blanc 1981). Various sugar phosphates occurring in milk are listed later under miscellaneous constituents. The glycosyl groups of several of the milk proteins are described in Chapter 3. Proteins The proteins of milk fall into several classes of polypeptide chains. These have been delineated most completely in bovine milk, and a sys- tem of nonmenclature has been developed for them (Chapter 3; Eigel et al. 1984).One group, called caseins, consists of four kinds of poly- peptides: asl-,as2-,and p-,and K- with somegeneticvariants, post transla- tional modifications, and products of proteolysis. Almost all of the ca- seins are associated with calcium and phosphate in micelles 20-300 pm in diameter (see Chapter 9).The other milk proteins, called whey proteins, are a diverse group including p-lactoglobulin,a-lactalbumin, blood serum albumin, and immunoglobulins (Chapter 3). Almost all
13. 13. COMPOSITON OF MILK 5 milk proteins of nonbovine species defined to date appear to be evolu- tionary homologs of those of the bovine and are named accordingly. Classically, milk protein content has been determined by Kjeldahl analysis for nitrogen (N)(Horwitz 1980).This has the advantages that N is a major constituent, comprising about one-sixth of the mass of the protein, and that the N contents of the individual milk proteins are nearly the same. Multiplication by 6.38 has been used commonly to convert the N content to protein. This is based on an old determination of 15.67%N in milk proteins, but a modern weighted average of the N contents of individual milk proteins indicates that the factor should be 6.32 (Walstraand Jenness 1984).Thus older results may be nearly 1% too high. A more serious error is that protein contents have often been calculated as 6.38 x total N. Such crude protein values are 4-870 too high because they include N from nonprotein nitrogenous constitu- ents. Various procedures are used to separate milk proteins into fractions or individual components that can quantitated separately. A classic method of fractionation is by precipitation at pH 4.6, which separates the proteins into two groups-caseins in the precipitate and whey pro- teins in the supernatant. All proteins are precipitated from a second aliquot with trichloroacetic acid at 12% (wlv)concentration (Rowland 1938), and concentrations of casein and whey proteins are calculated as follows: Casein = 6.38 (TN-NCN) Whey protein = 6.38 (NCN-NPN) where TN is total nitrogen, NCN is nitrogen in the pH 4.6 filtrate, and NPN is nitrogen in the trichloroacetic acid filtrate. About 80% of the proteins of bovine milk fall into the category of caseins, but the propor- tions differ greatly among species (see Table 1.10). Numerous other methods have been proposed for routine determina- tion of protein on large numbers of samples. Several are reviewed by Booy et al. (1962).They include colorimetric determination of ammo- nia, colorimetric determination of peptide linkages by the biuret method, analysis for tyrosyl groups, titration of protons released from lysyl groups upon reaction with formaldehyde, binding of anionic dyes to cationic protein groups, turbidimetric procedures (Kuramoto et al. 1959), and absorption of infrared radiation of 6.46 pm (Goulden 1964; Horwitz 1980).Individual milk proteins can be assayed by specific im- munological tests (Larson and Twarog 1961; Larson and Hageman 1963;Babajimopoulos and Mikolajcik 1977;Guidry and Pearson 1979; Devery-Pocius and Larson 1983), by ion-exchange chromatography (Daviesand Law 1977),by gel filtration (Davies 1974),by zone electro-
14. 14. 6 FUNDAMENTALS OF DAIRY CHEMISTRY phoresis (Swaisgood 1975; West and Towers 1976, Bell and Stone 1979),and by high performance liquid chromatography (Diosady et al. 1980; Bican and Blanc 1982). Salts For the purpose of this discussion, milk salts are considered as ionized or ionizable substances of molecular weight 300 or less. Ionizable groups of proteins are not included here, although, of course, they must be taken into account in a complete description of ionic balance and equilibria. Trace elements, some of which are ionized or partially so in milk, are considered in a later section of this chapter. Milk salts include both inorganic and organic substances; thus they are not equivalent to either minerals or ash. The principal cations are Na, K, Ca, and Mg, and the anionic constituents are phosphate, citrate, chloride, carbon- ate, and sulfate. Small amounts of amino cations and organic acid an- ions are also present. General methods for quantitating minerals (especially metals) use absorption or emission of radiation of specific wavelengths (Wenner 1958; Murthy and Rhea 1967).The former is a measure of absorption of the energy required to raise electrons to a higher energy-excited state and the latter is a measure of the energy released when excited electrons revert to their original state. These methods are particularly suitable for Na and K, for neither of which are volumetric or gravimet- ric methods of sufficient sensitivity available. Calcium and magnesium can also be determined by emission or absorption but often are ana- lyzed by specific chemical methods. Dry ashing or wet digestion with H2S04-H202or HN03-HC104are often used to destroy organic mate- rial before analysis for minerals, but in some procedures diluted, un- fractionated samples are injected directly into the flame photometer. Defatted and deproteinized extracts, usually acid, are used to deter- mine the content of organic salts such as citrate; they are sometimes used for analyses of mineral constituents as well. Classically, calcium was determined by precipitation as calcium oxa- late, which was then titrated in H2S04solution with KMn04but this has been largely replaced by titration with the chelating agent ethylen- ediamine tetraacetate (EDTA),using as the indicator a dye (murexide) which changes color when it binds calcium (White and Davies 1962). Another more sensitive method for Ca determination is a colorimetric procedure using glyoxal bis (2-hydroxyanil),whose calcium complex absorbs strongly at 524 nm (Nickerson et al. 1964). Phosphate inter- feres with both methods; it can be removed by treatment with an anion exchanger or by precipitation with potassium meta-stannate. Alterna-
15. 15. COMPOSITON OF MILK 7 tively, the calcium can be precipitated as oxalate before titration with EDTA. Magnesium, formerly determined by precipitation as magnesium ammonium phosphate and determining P in the latter, can be analyzed readily by EDTA titrations. It can be obtained either as the difference between titrations for (Ca and Mg) and Ca alone or by titrating the supernatant after Ca is precipitated as oxalate (White and Davies 1962). Phosphate is determined almost universally by its reaction with mo- lybdate to form phosphomolybdate. The latter can be reduced to a blue compound that absorbs at various wavelengths, of which 640 and 820 nm are often used for colorimetric quantitation (Allen 1940; Sumner 1944; Meun and Smith, 1968). Chloride is analyzed by some form of reaction with silver to form insoluble silver chloride. Direct titration of milk with silver nitrate yields erroneously high and variable results, and pre-ashing cannot be used because chloride is lost by volatilization. Satisfactory procedures involve adding an excess of standardized AgN03directly to milk and back titratingwith potassium thiocyanate (KSCN),using a soluble fer- ric salt as the indicator (Sanderg 1939). Citrate may be oxidized with KMn04and brominated and decarbox- ylated to form the relatively insoluble pentabromacetone; certain methods for detecting citrate in milk, including that of the Association of Official Analytical Chemists (Horwitz 1980),employ this reaction for a gravimetric analysis. It is, however, cumbersome, and pentabro- macetone is somewhat more soluble and volatile than desired in a grav- imetric analysis. In another method, lead citrate is precipitated from a sulfuric acid-alcohol filtrate from milk and titrated with ammonium perchlorato-cerate (Heinemann 1944). A simpler and more sensitive procedure utilizes the Furth-Herrmann reaction, in which a yellow- colored condensation product of citrate with pyridine is formed in the presence of acetic anhydride (Whiteand Davies 1963).Citrate may also be determined enzymatically by cleavage with a bacterial citrate lyase to oxaloacetate; decarboxylation of the latter to pyruvate with oxa- loacetate decarboxylase; and finally, formation of malate and lactate with specific NAD-coupled dehydrogenases (Dagley 1974).The enzy- matic method is the most specific method yet employed. About 10% of the total apparent citrate of milk actually is isocitrate (Faulknerand Clapperton 1981). Inorganic sulfate, So",-,is present in milk in a concentration of about 1mM; it may be determined turbidimetrically after adding barium ion to a deproteinized filtrate (Koops 1965). The total carbonate system (mostly HCO; in equilibrium with Con)
16. 16. 8 FUNDAMENTALS OF DAIRY CHEMISTRY in milk varies with the time after milking and the extent of exposure to heat and vacuum treatments. It is about 2mM in mixed raw milk in equilibrium with air. It can be released by acidification, collected by aspiration into Ba(OH)2,and determined titrimetrically (McDowall 1936), or it can be released and measured in manometric apparatus (Frayer 1940). Table 1.1gives the mean salt composition for 12 bulk milk samples taken from a herd at approximately monthly intervals during a year (White and Davies 1958). The means and ranges of the constituents are similar to those observed by other workers. The sum of Na, K, Ca, Mg, C1, and total phosphate as PO4from these data plus 0.01 gil00 g of inorganic sulfate is 0.73 gil00 g, which is a little short of the re- ported ash content of 0.76 g/lOO g. However, the composition of ash likely differs somewhat from that of the mixture of the components summed because of loss of chloride, conversion of some of the organic S to S024;retention of a little organic C as C0;;and formation of me- tallic oxides during ashing. The extent to which these processes occur depends on the temperature of incineration. The former practice of re- porting the composition of ash in terms of oxides of the metals and of P (asdone in previous editions of this volume) should be regarded only Table 1.1. Salt Composition of Milk. Concentration Mean Range SD Mean Percent Constituent (mgi100 g) (mgi100 g) (mgi100 g) (mM) Diffusible Cutionic Sodium 58 47-77 10 25.2 Potassium 140 113-171 14 35.8 Calcium 118 111-120 2.5 29.5 31 Magnesium 12 11-13 0.6 4.9 65 Amines -1.5 Anionic Phosphorusa 74 61-79 - 23.9 53 Inorganic (PI) 63 52-70 - 20.4 53 Ester 11 8-13 1.7 3.5 Chloride 104 90-127 11.4 29.3 Citrate 176 166-192 9 9.2 90 Carbonate -2.0 Sulfate -1.0 Organic acids -2.0 SOURCE: White and Davies (1958).Twelve samples of herd bulk milk (except for amines, carbonate, sulfate, organic acids; see text). nExcludingcasein P.
17. 17. COMPOSITON OF MILK 9 as a mode of expression and not as an indication that these oxides are actually present in ash. Not all of the salt constituents are found in the dissolved state in milk. Calcium, magnesium, phosphate, and citrate are partitioned be- tween the solution phase and the colloidal casein micelles (seeChapter 9 for the composition and structure of these micelles). For analytical purposes, partition of the salt constituents can be achieved by equilib- rium dialysis or by pressure ultrafiltration. In the latter technique, pressures must be limited to about 1atmosphere to avoid the so-called sieving effect (pushing water through the filter faster than the dis- solved components (Davies and White 1960). Table 1.1shows the proportion of the several constituents found in the dissolved, diffusible state. Actually, phosphate is present in five classes of compounds: inorganic dissolved, inorganic colloidal, water- soluble esters, ester-bound in caseins, and lipid. These can be deter- mined by making the following analyses: I. Total P in the dry-or wet-ashed sample. 11. Lipid P in digested Roese-Gottlieb extract. 111. Dissolved P in digested ultrafiltrate. IV. Inorganic dissolved P in undigested ultrafiltrate. V. Acid-soluble P in undigested 12.5% trichloroacetic acid filtrate. Then: Inorganic dissolved P = IV Inorganic colloidal P = V - IV Water soluble ester P = I11 - IV Casein P = I - (I1 + V) Lipid P = I1 The total inorganic phosphate (Pi)is, of course, V. The salt constituentsin the dissolved state (ultrafilterableor diffusi- ble)interact with each other to form various complexes. The concentra- tions of each of these constituents can be calculated (withsuitable com- puter programs) from a knowledge of their several interaction or association constants. Results of such a calculation (Holt et al. 1981) for an ultrafiltrate similar to that of Table 1.1are given in Table 1.2. Na, K, and C1 are primarily present as free ions but Ca, Mg, phos- phate, and citrate are distributed throughout many complexes; those in the highest concentration are CaCit-, Mg Cit-, H2P04-HP0;-and CaHP04.The calculation yields Ca2+and Mg2+concentrations of 2.0
18. 18. 10 FUNDAMENTALS OF DAIRY CHEMISTRY Table 1.2. Concentrations of Ions and Complexes in Typical Milk Diffusate. Complex with Cation (mmoliliter) Free Ion Anion (mmoliliter) Ca2+ Mg2+ Na + K + H,Cit - HCit2 Cit3- H,PO; HP0:- Po: - Glc-1-P2 HZCO, HC0,- so: - RCOOH RCOO Free ion +" 0.04 0.26 7.50 2.65 + 1.59 0.11 0.32 0.96 0.02 2.98 + 0.01 6.96 0.07 0.59 0.01 0.17 0.01 0.07 0.03 2.00 - h - + + 2.02 0.04 0.34 + 0.07 + 0.03 0.02 0.81 - + + 0.03 0.10 0.39 + 0.10 + 0.04 0.02 20.92 - + + 0.04 0.18 0.52 + 0.14 + 0.10 0.04 36.29 - ~~~ SOURCE: Holt et al. (19811 '+ = 80% in micelles >80% in micelles years 1311 8 days -100 days In solution 21 hours -100 days In solution,331 137Cs 30 years -30 days In solution I4OBa 13 days