HtOTEXNS OF THE HOMOGENIZED MILK FATj GLOBULE MEMBRANE Robert Howard Jackson AN ABSTRACT Submitted to the School for Advanced Graduate Studies of Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHTLOSGPHT Department of Dairy 1959 Approved. ABSTRACT ROBERT HOWARD JACKSON The fat-globule membranes of milk are materially altered as a result of homogenization- In the literature* one can find analytical, ultracentrifugal, and electrophoretic evidence showing the difference in membrane material before and after homogenization- The principal objective of this research was to isolate, characterize, and identify the proteins of the homogenized milk-fat globule. Also, the effect of heat was studied on the total amount of protein associated with the milk fat after homogenization. For the isolation of homogenized fat-globule membrane proteins, a scheme developed for the isolation of nonhomogenized fat-globule mem­ brane proteins was modified and extended- Sucrose was used to increase the specific gravity differential of the fat and serum of the homogenized milk. In so doing, greater yields of membrane materials were obtained. The total amount of protein on the homogenized fat-globule membrane was found to be inversely dependent upon the amount of heat applied to the milk prior to homogenization- At 60°, 70°, and 80° C., the amount of protein associated with 100 grams of homogenized milk fat was 2.27, 1 .60 , and 1.31 grams, respectively. The 60° C. preparation had a total protein value 5 to 6 times greater per 100 grams of fat than the average protein value reported for the nonhomogenized fat-globule membrane, based on grams of protein per 100 grams of fat- When one considers that homogenization increases the fat surface of milk by a factor of 5 or 6 , these data might indicate that the ratio of membrane protein to fat surface is relatively constant. ROBERT HOWARD JACKSON ABSTRACT Characterization and identification of protein fractions were based on electrophoretic, spectrophometric, and ultracentrifugal analyses. The protein fractions which were isolated from the homogen­ ized milk fat-globule membrane weres 1* Insoluble membrane protein. This protein presented a reddishbrown, mucoidal appearance. After solubilization with peracetic acid, it was electrophoretically homogeneous. The characteristics of this protein were identical to the insoluble membrane protein of the nohomogenized fat-globule membrane. Z• Solublemembrane protein. of this Investigations indicated that a portion protein forms a complex with the alpha-casein adsorbed on the homogenized fat-globule membrane. only in the presence of milk-fat. This complex seemed to form The remainder of the soluble membrane protein was isolated by denaturing the heat-labile, adsorbed proteins at 90° C. for 30 minutes, leaving the soluble membrane protein in a relatively pure form. 3- Casein. A complex seemed to be formed with alpha-casein and a portion of the soluble membrane protein, in the presence of milkfat. The protein-lipid interaction gave rise to an electrophoretic component with a mobility slightly less than that of alpha-casein. Also, the mobility of alpha-casein was decreased, and the ratio of alpha- to beta- casein was abnormally large. 4, Heat-labile prctein(s) . Although these proteins were not isolated ABSTRACT ROBERT HOWARD JACKSON and identified, they were considered to be whey proteins. In general, the fat-globule membrane proteins did not appear to be completely dissociated from the fat globule when the membrane was disrupted by homogenization. The increased surface area created by homogenization was covered, in part, by casein and by unidentified whey proteins. The results of this research did not indicate that the resurfacing of fat globules subsequent to homogenization was limited to any specific milk proteins, but that a possible lipid-protein complex was formed as a result of the treatment. PROTEINS OF THE HOMOGENIZED MILK FAT-GLOBULE MEMBRANE By Robert Howard Jackson A THESIS Submitted to the School for Advanced Graduate Studies of Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHI Department of Dairy 1959 ProQuest Number: 10008624 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest ProQuest 10008624 Published by ProQuest LLC (2016). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 4 8 1 0 6 - 1346 ACKNOWLEDGMENTS The author wishes to express his appreciation to Dr. J. R. Brunner, Professor of Daiiy, for directing this research and for his suggestions and counsel. Grateful acknowledgement is also due to the Michigan State Uni­ versity and the Michigan Agricultural Experiment Station far the funds and facilities which made this research possible. To Dr. W. N. Mack, the appreciation of the writer is extended for his efforts in providing for the altracentrifugal analyses. The author is most grateful to his wife, Betty Jean, for her inspiration and encouragement throughout the course of this study. TABLE OF CONTENTS Page INTRODUCTION 1 REVIEW OF LITERATURE 3 The Proteins of the Nonhomogenized Milk Fat-Globule Membrane 3 Emulsif ication of Butterfat in Various Solutions of Milk Origin 6 The Proteins of the Homogenized Milk Fat-Globule Membrane 7 The Effect of Heat on the Protein Adsorbed on the Milk Fat-Globule Membrane 9 Protein-Lipld Interaction in Homogenized Milk 9 EXPERIMENTAL PROCEDURE 10 Analytical Methods 10 Electrophoresis 10 Ultracentrifugal analyses 11 Paper-partition chromatography 12 Ultraviolet analyses 12 Procedure for the Isolation of Homogenized Milk Fat-Globule Membrane Proteins 13 Preparation of Homogenized Milk Fat-Globule Membrane Proteins 13 Determination of the Total Amount of Protein Remaining on the Homogenized Milk-Fat Globule 15 Experimentation with the Fraction Precipitated at pH k.6 15 Various Chemical and Physical Treatments Used in the Separation of the Casein-complex Heat 15 15 ii Page Urea 16 Ethyl ether 16 Rennet 16 Reconstruction of the Casein-complex with Selected-component Systems 17 Homogenization of a mixture of calcium caseinate and membrane-containing serum 17 Homogenization of a mixture of calcium caseinate, membrane-containing serum, and butteroil 18 Homogenization of a mixture of alpha-casein, soluble membrane protein, and butteroil 18 Homogenization of a mixture of alpha-casein and butteroil 18 Various Chemical and Physical Treatments of Casein and the Soluble Membrane Protein 19 Ethanol-ethyl ether treatment of casein 19 Peracetic acid treatment of casein 19 Homogenization of isolated casein and isolated whey proteins 19 Precipitation of the soluble membrane protein at pH 4,6 20 Precipitation of the soluble membrane protein and alpha-casein at pH 4 ,6 20 Electrophoretic analysis of casein isolated from homogenized milk 20 EXPERIMENTAL RESULTS 23 Effect of Heat on the Total Amount of Frotein Remaining on the Homogenized Milk-Fat Globule 23 Experimentation with the Fraction Precipitated at pH 4,6 23 The Effect of Various Chemical and Physical Treat­ ments in the Separation of the Casein-complex iii 23 Page Heat 23 Urea 23 Ethyl ether 24 Rennet 24 Reconstruction of the Casein-complex with Selected-component Systems 24 Homogenization of a mixture of calcium caseinate and membrane-containing serum 24 Homogenization of a mixture of calcium caseinate, membrane-containing serum, and butteroil 25 Homogenization of a mixture of alpha-casein, soluble membrane protein, and butteroil 25 Homogenization of a mixture of alpha-casein and butteroil 25 26 Analyses of Fractions Ultracentrifugal characteristics 26 Paper-partition chromatography 26 Ultraviolet analyses 27 The Effect of Various Chanical and Physical Treatments of Casein and the Soluble Membrane Protein 27 Effect of an ethanol-ethyl ether treatment on the electrophoretic characteristics of casein 27 Effect of a peracetic acid treatment on the electrophoretic characteristics of casein 27 Effect of homogenization on the electro­ phoretic characteristics of isolated casein and isolated whey proteins 27 Precipitation of the soluble membrane protein at pH 4.6 27 iv Page Precipitation of the soluble membrane protein and alpha casein at pH 4.6 28 Electrophoretic analysis of casein isolated from homogenized milk 28 43 DISCUSSION Effect of Heat on the Total Amount of Protein Remaining on the Homogenized Milk-Fat Globule 43 Proteins of the Homogenized Milk Fat-GlobuleMembrane 45 Insoluble Fracti on (I) 45 Soluble Fraction (II) 46 Casein-complex Fraction (III) 46 The effect of various chemical and physical treatments in the separation of the caseincomplex 47 Heat 47 Urea 47 Ethyl ether 47 Rennet 48 Reconstruction of the casein-complex with selected-component systems 48 Homogenization of a mixture of calcium caseinate and membrane-containing serum 48 Homogenization of a mixture of calcium caseinate, membrane-containing serum, and butteroil 49 Homogenization of a mixture of alphacasein, soluble membrane protein, and butteroil 49 Homogenization of a mixture of alphacasein and butteroil 50 Soluble Minus Casein Fraction 50 v (IV) Page Soluble Minus Casein and Heat-denatured Protein Fraction (V) 51 SUMMARY 53 CONCLUSIONS 56 LITERATURE C U E D 57 vi LIST OF* FIGURES FIGURES 1. 2. 3. 4. Page PROCEDURE FOR ISOLATING THE PROTEINS ASSOCIATED WITH THE HOMOGENIZED MILK-FATGLOBULE.................. 22 ELECTROPHORETIC PATTERNS WHICH ARE REPRESENTATIVE OF THE VARIOUS FRACTIONS ISOLATED BY THE FRACTION­ ATION SCHEME SHOWN IN FIGURE 1 ......................... 29 THE EFFECT OF HEAT AND UREA ON THE CASEIN -COMPLEX FRACTION (III).................................... 30 THE EFFECT OF ETHYL ETHER AND RENNET ON THE CASEINCOMPLEX FRACTION ( H I ) ................................ 31 5. ELECTROPHORETIC PATTERNS OF SEIECTED-COMPONENT SYSTEMS............................................. . ..32. .. 6. THE EFFECT CF VARIOUS CHEMICAL AGENTS AND HOMOGENIZATION IN MILK ON THE ELECTROPHORETIC CHARACTERISTICS OF CASEIN................................................ 7. ELECTROPHORETIC PATTERNS OF ISOLATED CASEIN AND ISOLATED WHEY PROTEINS BEFORE AND AFTERHOMOGENIZATION. 8 . SEDIMENTATION VELOCITY DIAGRAMS OF THE SOLUBLE MINUS CASEIN FRACTION (IV) AND THE SOLUBLE MINUS CASEIN AND HEAT-DENATURED PROTEIN FRACTION (V).......... ........ 9. 10. 11. 33 34 35 EXTRAPOLATION OF SEDIMENTATION VELOCITY CONSTANTS TO ZERO CONCENTRATION FOR THE SOLUBLE MINUS CASEIN FRACTION (IV)......................................... 36 EXTRAPOLATION OF SEDIMENTATION VELOCITY CONSTANTS TO ZERO CONCENTRATION FOR THE SOLUBLE MINUS CASEIN AND HEAT-DENATURED PROTEIN FRACTION (V)................... 37 ULTRAVIOLET SPECTROGRAM OF THE SOLUBLE MINUS CASEIN AND HEAT-DENATURED PROTEIN FRACTION (V) AND THE SOLUBLE MEMBRANE PROTEIN AFTER 30 MINUTES AT 90°C vtl 38 LIST OF TABLES TABLE Page 1. TOTAL PROTEIN REMAINING WITH THE HOMOGENIZED MILK-FAT GLOBULE WITH INCREASING HEAT TREATMENT.... 39 2. MOBILITIES AND AREAS CF THE ELECTROPHORETIC COMPONENTS SHOWN IN FIGURES II THROUGH VII....... 40 3. SEDIMENTATION VELOCITY CONSTANTS FOR THE SOLUBIE MINUS CASEIN FRACTION (IV) AND THE SOLUBLE MINUS CASEIN AND HEAT-DENATURED PROTEIN FRACTION (V) , CORRECTED TO 20° C ................................ 42 viii INTRODUCTION Homogenization had its inception at the beginning of the twentieth century# but did not receive consumer acceptance for nearly twenty-five years. The homogenization of fluid milk, to produce a smoother, more palatable commodity, has since become a fundamental process in the dairy industry. Essentially, homogenization is a mechanical method of reduc­ ing the milk fat-globule in size and increasing it in number. A large amount of research has been devoted to determining the optimum conditions for producing the best homogenized milk. Unfortun­ ately, there has been very little research directed toward a most impor­ tant area affected by the process - the fat/serum interface. Lipolysis and sunlight flavor are enhanced, while Copper-induced oxidation is retarded by an increase in fat surface following homogeni­ zation. These are but a few of the reactions indicating that the fat/ serum interface is an important site in the chemical and physical modi­ fication of homogenized milk. Such defects as "free fat" in whole milk powder and "chalky" flavor in fluid homogenized milk might be explained by the presence or absence of adsorbed materials on the homogenized fatglobule membrane. Only when these adsorbed materials are identified and their arrangement on the homogenized fat-globule is established, will we gain an insight into the problems inherent in homogenizing milk. In this research, the principal objective was to isolate, character­ ize, and identify the proteins of the homogenized milk-fat globule. Also, the effect of heat was studied on the total amount of protein 1 associated with the milk fat after homogenization. The results of these investigations are presented and discussed in the body of this manu­ script . 2 REVIEW OF LITERATURE Tte I r a t e l m at %>iAamaafUi3afta Milk Fat-Globule Membrane. Since Ascherson (1840) first proposed that a membrane surrounds the fat globule of bovine milk, there has been an ever-increasing compila­ tion of evidence to support his original theory. The bulk of this evidence has been submitted within the past fifty years by researchers using techniques ranging from the visual observations of stained fatglobule membranes by Storch (189?) to the electrophoretic studies of Brunner, Lillevik, Trout and Duncan (1953c). Storch was the first to use a washing technique in isolating fatglobule membrane materials. His fat-globule membrane had a nitrogen content of 1^.76$ and contained an associated substance capable of reducing Fehling's solution. He believed that this membrane, which he called "albuminoid” , differed from any previously recognized albuminoid of milk. Wiegner (191^+) , calculating from viscosity measurements, reported that the nonhoraogenized fat globule adsorbed Z% of the casein of milk. Using a technique in which milk was treated with water which was saturated with chloroform, Hattori (1925) isolated the material surrounding milk fat globules and fomnd it to be principally protein. This protein, which he called "haptein", differed from any known milk protein on the basis of its solubility and chemical composition. "Haptein” was reported to have a low nitrogen content and a cystine content exceeded only by keratin. Palmer and Weise (1933) revealed that 3 the material most closely adsorbed on the fat globule surface is a mixture of phospholipids and proteins. Furthermore, they contended that this protein had both a hydrophilic and a hydrophobic nature and a phosphorous, nitrogen, and sulfur content unlike any other milk protein. Titus, Sommer, and Hart (1928) believed the membrane protein to be closely related to, if not identical with, casein, based on its nitrogen distribution characteristics, specific rotation, and sulfur, phosphorous, and tryptophane content. Moyer (l9*K)) concluded from electrokinetic studies that the fat-globule membrane protein differed from other known milk proteins, and that much of the confusion of previous research in this area could be attributed to the incomplete removal of other milk plasma proteins from those occurring on the surface of fat globules. Mulder (19^9) postulated that the surface layer of fat globules should not be regarded as having been adsorbed from milk plasma. In­ stead, he offered the conjecture that it consisted of components of the protoplasm of the cells of the lactiferous glands as well as substances adsorbed from the plasma of milk. Mulder (1957) further postulated that the composition of the surface layer varied from globule to globule, depending upon the disturbance of the layers surrounding the fat globule. In this same publication, Mulder professed that the degree of disturb­ ance of the membrane layers was proportional to the adsorption of material from the serum. Hare, Schwartz, and Weese (1952) , using a microbiological assay technique, showed that the membrane protein contained more phenylalanine, threonine, and glycine and less aspartic acid than any of the major milk proteins. Brunner, Duncan, and Trout (l953b), using a similar technique, 4 shewed the amino acid composition of the nonhomogenized fat-globule mem­ brane to be of the same order. Brunner et al. (1953c) presented the first data on the electrophoretic behavior of isolated* nohomogenized membrane proteins which revealed from one to three electrophoretic components. Brunner, Duncan, Trout, and MacKenzie (1953a) showed the nonhomogenized fat-globule membrane protein to have a sedimentation diagram featuring one principal, homogeneous component with an of 7 .3 - They classified this protein as a globulin in nature. Herald and Brunner (1957) Isolated and characterized the nonhomo­ genized fat-globule membranes as having soluble and insoluble entities in 0.02 M NaCl and/or water. Furthermore, these researchers found car­ bohydrate associated with the soluble fraction. The insoluble fraction was classified tentatively as a pseudokeratin, based on it solubility and chemical properties. The soluble fraction was analyzed electrophor- etically by Herald and Brunner (1958) in various buffers, ranging in pH values from 2.05 to 8 .79 . and showed one small and two large migrating boundaries. After treating the insoluble fraction with various protein- solubilizing agents, these workers shewed a single, electrophoretic peak with sodium-sulfide-treated samples, but one small and two large migrat­ ing boundaries in detergent-treated samples. Sasaki, Koyama, and Nishikawa (1956b) postulated that the fat-globule membrane is electro­ phoretic ally similar to whey proteins or whey proteins less beta- lactoglobulin. Morton (195^) produced electron micrographs showing milk microsomes which were adsorbed to the membrane protein surrounding the fat globule. These microsomes were shown to be brown in color and to contain 22$ 5 lipid, 10$ nucleic acid, a haemochromogen, alkaline phosphatase, xanthine oxidase, diaphorase, and DFN cytochrome £, reductase. Zittle, DellaHonica, Custer, and Rudd (195&) found only 0.5$ nucleic acid in the microsomes of milk and defined them as parcels of enzymes cemented to­ gether with nucleic acid and phospholipid. The enzymes given most consideration in this study were alkaline phosphatase and xanthine oxidase. The monograph of King (1955) should be given special recogni­ tion as a complete and comprehensive review of research on the milk fatglobule membrane. gS«Asi£lsM i , o,ii of Butterfat in lasl&aL Solutions sL Milk °rigln Weise and Palmer (1932) emulsified butterfat in buttermilk and solutions of calcium caseinate, lactalbumin, globulin, phospholipid, and globulin plus phospholipid. Using chumability as an index, they maintained that buttermilk most closely replaces the substances adsorbed on the fat globules in cream. By comparing the material which did not wash off the fat globule, after emulsification in sweet rennet-whey, skiramilk, and buttermilk, with the natural fat globule membrane, Rimpila and Palmer (1935) showed the materials to differ in percent and propor­ tion of phospholipids and protein and in their nitrogen distribution characteristics. They also concluded that in the synthesis of milk, the natural fat-globule membrane Is not derived from milk plasma. Sasaki and Koyama (1956a) emulsified butterfat in solutions of radio­ active casein and radioactive whey proteins. Two washings removed casein and the beta-lactoglobulin of the whey protein, but the remainder of the whey proteins was still associated with the fat after four 6 washings. These workers then concluded that the fat-globule membrane does not contain casein or beta-lactoglobulin, but some whey proteins plus a possible unknown or specific protein. sL ih& HprcaK/snl&ad MU k F a J k -P lo b u ie M gafrgaiw , By applying the mathematical formula, 4/3TTr^, Trout, Halloran, and Gould (1935) calculated that homogenization increased the surface of fat globules by a factor of 5 or 6 . They also observed that the stability of milk proteins toward alcohol decreased after homogenization. Doan (1938) arxi Sommer (19^6) felt that the degree of destabilization increased with increasing fat contents. Doan (1938) found milk to be more easily coagulated after homogenization. He explained this phenomenon by stating that "a greater amount of casein is adsorbed on the expanded amount of fat globule surface", and since this casein is fixed, it is non-motile, which is the first stage of coagulation. Wiegner (191*0 assumed that the adsorbed protein film of homogenized milk had a density of 1.4, Using this value, he calculated from viscos­ ity measurements that is homogenized milk, 25$ of the casein was ad­ sorbed by the fat. theories. Sullam (l9^l) disagreed with the generally-accepted H© maintained that homogenization increased the stability of milk proteins. Chambers (193&) suspected that casein or other polar constituents were adsorbed by the fat in proportion to the increased fat surface made available for such adsorption by the homogenization of the milk. Sammis (191*0 found that homogenized cream appeared thicker than ordinary cream of the same fat content and was more difficult to churn. He also reported "that homogenization affects the casein in ways 7 which cannot be fully explained at present”. Sommer (19^ 6) stated that, quantitatively, there may be differences between the adsorbed material of homogenized fat globules as compared with globules as they exist naturally in milk. Weinstein, LiUevik, Duncan, and Trout (l95l) reported that the development of activated flavor in homogenized milk may be attributed to the selective rearrar^jement of the fat-globule membrane following homogenization. The con­ stituents which give rise to activated flavor may be adsorbed on the increased fat surface, which is the reactive site for the development of this off-flavor. Trout (1950) believed that the protein material of plasma was adsorbed on homogenized fat globules and that this adsorbed membrane is different from the membrane of the original fat globules. In other words, the newly created fat globules of homogenized milk are resurfaced. Brunner et (1953b) showed marked differences In the amino acid composition of homogenized and nonhomogenized fat-globule membranes. From this, they concluded that the adsorbed layer on homogenized fat globules appears to be different from the adsorbed layer on normal fat globules. Brunner et al. (1953c) based their interpretations on an electrophoretic comparison of the two membranes and proposed that the membrane of homogenized milk was composed of adsorbed milk-plasma proteins in addition to the fat-membrane protein complex of the original fat globules. Brunner e£ &1,. (1953a) compared the sedimentation velocity diagrams of solutions of the homogenized and nonhoraogenized fat-raembrane proteins. The homogenized membrane showed three, or possibly four peaks, which were identified as casein, Kekwick's whey protein, and an altered lactoglobulin (beta-lactoglobilin or a complex of two or 8 more constituent proteins). The Effect of Heat op the Protein Adsorbed sm Milk Fat-Globule Membrane Lowenstein and Gould (195*0 showed a decreased adsorption of protein on the fat globule with increased heat. Itfhen whole milk was heated momentarily to 40° C. and 62° C. for 30 minutes, and 82° C. for 15 minutes, the amount of protein in the membrane material was 21*86 , 15.5*+» and 7.70 percent, respectively. intoragtaga in Hgntpgqaiaftti Fox, Caha, Holsinger, and Pallansch (1959) found that conditions existing at the homogenizer valve produced a fat-protein complex, and that the principal protein, if not the only one, involved in the inter­ action is the casein micelle. Litman (1955) reported a fat-protein complex or "scum" formation in whole milk powder. Of the 3*$ protein in the complex, 82^ to 96^ consisted of casein and denatured whey protein. The fat isolated from the complex had a high melting point, which may be due to oxidation, or the fat may be similar to the highmelting triglyceride isolated from the fat-globule membrane. He further stated that the amount of complex was directly related to the pre-heat treatment of processing. 9 EXPERIMENTAL 'PROCEDtKE Electrophoresis. All electrophoretic data were obtained with a Perkin-Elraer Model 38-A Electrophoresis Unit. Protein solutions were dialyzed against standard buffers for at least 36 hours with no less than two 400 milliliter buffer changes* Electrophoretic runs were begun after a temperature equilibrium of the protein solution and the icewater bath at 1° C. was reached. Electrophoretic mobilities were calculated using the following equation: ai (cm2f, volt”1 * sec"1) = d .a tc t i r m where: d s distance boundary traveled a = cross-sectional area of the cell k = conductivity cell constant = 0*8491 t = time In seconds i = current In amperes r = resistance of buffer in ohms, and m = magnification factor of the optical system The distance (d) was measured from the initial boundary* and all mobil­ ities were calculated by this method. Potential gradient or field strength was calculated from the following equation: F = i/aK 10 where: F s potential gradient 1 = current in amperes a = cross-sectional area of the cell, and K » specific conductivity of the buffer protein solution Since all the electrophoretic analyses were conducted under similar conditions# potential gradient was not considered a variable factor. Relative areas under individual electrophoretic peaks were obtained by: (l) averaging three planimeter readings of the area beneath the peak or (2) drawing a perpendicular line from the lowest point between peaks, cutting-out the area, and weighing the individual segments. All interpretations of electrophoretic patterns were made on the descending channel. Ultracentrifugal analyses. Sedimentation velocity data were obtained with a Spinco Model E analytical centrifuge operated under the conditions indicated with the presentation of results. Proteins were carried in Veronal buffer solutions of pH 8 #7 and ionic strength of 0 .1 . These solutions were prepared and analyzed at three concentration levels, varying between 0# and 2P, and their values plotted and extra­ polated to zero concentration. The method of least squares was used to obtain a linear plot of the data, according to Federer (1955)• Calculations were made from the following equation: S (uncorrected) - . d » m _____ *K)*t *rps^-r where: d = distance migrated in centimeters 11 m = magnification factor of the instrument - 1.7 *K) = ^7T2 t = time in seconds r * distance from center of rotor to a position at the center of a component, which it would be if an exposure were made half-way between the pair of peaks used for a calculation. 5*7 = distance in centimeters from the center of the rotor to the second margin of the counterbalance hole. The S (uncorrected) was corrected to S20 from a table of conversion factors (n£/n2(P given by Svedberg and Pedersen (19^ 0)• The sedimenta­ tion velocity constants were uncorrected for viscosity. Pa per-part it ion chromatography. Unidimensional, descending, paper- partitlon chromatography was used to identi^r carbohydrate moities associated with the heat-stable fraction isolated by the fractionation scheme used in this research. system in a ^ An anyl alcohol: pyridine^ water solvent 3< 2 ratio carried the sugars over unbuffered Whatman #1 cellulosic filter paper for contact periods ranging from 32 to *K) hours. Protein hydrolyzates were obtained by dissolving 50 milligrams of protein in 10 milliliters of 2 N HC1, placing the solution in a boiling water bath for 3 hours, and filtering-out the extraneous residue. Chromatograms were developed after spraying with 2$ triphenyltetrazolium chloride in an equal volume of 1 N NaOH, according to Block, Durrum, and Zweig (1955)• Ultraviolet analyses. Distilled water solutions of proteins with concentrations of 0 .05^ were examined in the ultraviolet spectrum ranging 12 from 220 to 3*4-0 millimicrons, using a Beckman Model DK -2 Spectro­ photometer . Pressure for tj^e Isolation £f Homogenized Milk P^t-GlQi)ule Membrane Proteins Essentially, the isolation scheme used in this research was similar to the procedure developed by Herald and Brunner (195?) in their work with the nonhomogenized fat-globule membrane proteins. Since additional proteins were present on the homogenized fat-globule membrane, the isola­ tion scheme was extended to provide for their fractionation. The method of fractionation and isolation employed in this study is shown in Figure 1. Preparation of Homogenized Milk Fat-Globule Membrane Proteins Fresh, raw milk, with approximately 3*5$ fat and 12$ total solids, was the starting material in all of the preparations. The milk was subjected to one of several selected heat treatments, and then homogen­ ized at 2500 p.s.i. and 58° C. in a Manton-Gaulin Model K homogenizer. The homogenized milk was separated at 60° C. in a Model 518 DeLaval Laboratory Separator after the addition of 3$ (w/w) sucrose, which increased the specific gravity differential of the fat and the serum. The homogenized fat, with its newly-formed surfaces, was washed three times with three volumes each of 3$ sucrose solution and water, both at 40° C. The washed homogenized cream was churned between 50° C. and 58° C., after chilling below these temperatures overnight. Separa­ tion of the fat and sera at 38° C. was completed in a Model 9 DeLaval Laboratory Separator. The milk fat was discarded and the membrane- 13 containing serum was salted-out when the solution was adjusted to 2.2 M The membrane material was concentrated by centrifuging in a Model SS-1 Servall Centrifuge for 30 minutes at 25,000 x G. Two hundred milliliters of 35$ (v/v) ethanol in ethyl ether at 0° C. to -5° 0. were added to each 50 grams of concentrated, homogenized membrane material. the cold. The mixture was agitated 15 minutes and filtered in The residue was washed 5 times with ethyl ether at 0° C. to -5° 0 . and extracted 3 times with ethyl ether at 25° C. for 10 , 5 * and 5 minutes. Residual ether was removed overnight under 16 inches of vacuum. The homogenized membrane-proteins were extracted h times with 0.02 M NaCl and centrifugally separated for 30 minutes at 25,000 x G. The residue was the insoluble membrane protein of the nonhomogenized fatglobule membrane, which was designated in this research as the Insol­ uble Fraction (I). Peracetic acid was used to solubilize this protein. The supernatant contained a number of proteins which were separated by repeatedly adjusting the pH of the solution to ^.6 and centrifuging at 25.000 x G. for 30 minutes. The residue was redissolved in a dilute NaOH solution with an adjusted pH of 9*0. Electrophoretically, the residue appeared to be casein; however, the pattern showed a component with a mobility slightly less than that of alpha-casein. The super­ natant was comprised of at least one heat-stable and one heat-labile protein. The heat-labile fraction was removed by centrifugation at 25.000 x G for 30 minutes following a heat treatment at 90° C. for 30 minutes. 1^ Dptgj^ijyrtjp.n sL the. Total Amo tint o£ Protein ReiaaiAWg. £U the. Homogenized Ki^k-Fat Globule Washed homogenized cream was prepared in exactly the same manner as illustrated in Figure 1 , with the exception that 8$ (w/w) sucrose was used in the initial separation and the 5 subsequent washings. This amount of sucrose was taken into account in all calculations. The washed cream was analyzed for fat and total solids by the method of Mojonnier and Troy (1925) and nitrogen by a modification of the method of Menefree and Overman (19^ 0). Since the fat content of the washed cream ranged between 40$ and 55$. it was necessary to use 30 milliliters of nitrogen-free HgSO^ to digest the 5 gram sample. Sixty milliliters of 50$ NaOH was used to neutralize the sample prior to distillation. The milk used in this portion of the research was analyzed for serum protein denaturation by heat according to a slightly modified method of Kuramoto, Coulter, Jenness, and Choi (1959)• The modification entailed the use of a 16.67 gram sample (12.00$ T. S.) which was equivalent to a 2 gram sample of dry milk. Experimentation wjth the. Fractto.h Precipitated at oH ^.6 The isolation of this fraction has been discussed and the scheme is shown in Figure 1 . Various Chemical and Phvs ic al Treatment s Used in the Separation of t M Ca??in-.Copiplex Heat. A test tube containing 20 milliliters of a 1.0$ (w/w) water solution of the Casein-complex Fraction (III) was heated for 30 15 minutes at 90° C. in a water bath. The solution was centrifuged at 25*000 x G. for 30 minutes, after which the supernatant was dialyzed against buffer in lieu of electrophoretic analysis. Urea♦ The Casein-complex Fraction (HI) was solubilized in 6.6 urea and agitated for 18 to 2^ hours. The molarity of the urea and the pH of the solution was adjusted to 1.0 and ^* 6 , respectively. After centrifuging at 25*000 x G. for 30 minutes, the residue was washed with distilled water to remove the urea present. After dialyzing against water and then buffer, the protein was analyzed electrophoretically. The Casein-complex Fraction (III) was further fractionated with urea according to the method of Hipp, Groves, Custer, and McMeekin (1952). The primary purpose of this experiment was the separation of the unidentified electrophoretic component at some level of urea molarity, for subsequent identification. Ethyl ether. Three grams of the Casein-complex Fraction (III) were agitated with 50 milliliters of ethyl ether at room temperature. After centrifuging to reclaim the protein, the residual ether was removed under vacuum, and the protein was prepared for electrophoretic analysis. Rennet. Thirty milliliters of a 3$ (w/w) water solution of the Casein-complex Fraction (III) was prepared and agitated for 20 minutes with 0.5 milliliters of a solution of commercial rennet and water, mixed in a ratio of 1 to 20. The temperature and pH of the solution was 30° C. and 8.0, respectively. After agitation, a dilute solution of Ca CI2 was added drop-wise until flocculation no longer occurred. The precipitate was concentrated by centrifhgation at 25*000 x G. for 16 30 minutes and redispersed with dilute NaGH. The solution was then dialyzed against buffer before being analyzed electrophoretically. Reconstruction of the Casein-complex with Selected-Component Systems This segment of the research necessitated the preparation of the following materials: 1 *) Membrane -containing serum: Fresh, raw nonhomogenized milk was fractionated according to the technique of Herald and Brunner (1957) to obtain membrane-containing serum. This serum can also be called washed- cream buttermilk, which is rich in the soluble and insoluble nonhomogen­ ized fat-globule membrane proteins. 2 .) Calcium caseinate: Three liters of a (w/w) solution of sodium caseinate, at pH 7*5» was dialyzed against 10 gallons of pasteur­ ized skimmilk for three days. This yielded a casein solution whose protein approached that of casein in its native state. Homogenization of a mixture of calcium caseinate and membranecontaining serum. Six hundred milliliters of the calcium caseinate solution and 300 milliliters of the membrane-containing serum were mixed and heated for 30 minutes at 60° C. and homogenized at this temperature at 3000 p.s.i. Two-stage homogenization was used throughout this research; pressure at the first and second stages being 2500 and 500 p.s.i., respectively. After homogenization, the mixture was salted-out with (NH^^SO^, treated with organic solvents, extracted with 0.02 M NaCl and adjusted to pH k.6 according to the isolation scheme employed in this research to obtain the Casein-complex Fraction (HI). 17 The mixture was analyzed electrophoretically after sufficient dialysis against Veronal buffer of pH 8.6 and ionic strength of 0.1. Homogenization of & mixture of calcium caseinate, membranecontaining serum, and butteroil. One thousand milliliters of calcium caseinate solution was mixed with 1000 milliliters of membranecontaining serum and 100 milliliters of fresh butteroil. The mixture was heated to 60° C.t for 30 minutes and homogenized at 3000 p.s.i. After homogenization, the isolation scheme shown in Figure 1 was followed to obtain the material which precipitated at pH 4.6. action s£ 1 m a t u re. of alpha-casein, soluble membrane orotetn. and butteroil. Two hundred milliliters of a solution of soluble membrane protein was mixed with a solution of 600 milliliters of alpha-casein and 40 milliliters of fresh butteroil. The total solids of the soluble membrane solution was 0 .93$ and that of the alpha-casein solution was 0.68$. The mixture was heated to 60° C. and held for 30 minutes, after which it was homogenized 3 times at 3000 p.s.i. at 60° C. The solution was cooled, adjusted to pH 4.6, and centrifuged at 25,000 x G. for 30 minutes. Lipids were extracted in the cold according to the isolation scheme. The residual ether was removed under vacuum, and a sample of the protein was prepared for electrophoretic analysis. Homogenization of a mixture of alpha-cas_ein and butteroil. The same procedure was followed as in the preceding experiment, with the exception that no soluble membrane protein was included. 18 la?faqff ShaaiP.aX a M Physical Treatments of Casein i M the Soluble Membrane Protein lthaftQl^e.ihyl flthsc -treatment £f casein. Isoelectrically precip­ itated casein was prepared and divided into two parts. The first was analyzed electrophoretically as a control, and the second was treated with organic solvents as indicated by Herald and Brunner (1957)• Eive grams of casein were added to 10 milliliters of 35$ ethanol in ethyl ether at 0° C. to -5° C. and agitated for fifteen minutes. The mixture was filtered and washed 5 times with ethyl ether at approximately 0° C. and extracted 3 times with ethyl ether at room temperature for 10 , 5 * and 5 minutes. Residual ether was removed under vacuum, and the casein prepared for and analyzed electrophoretically. Peracetic acid treatment of casein. Enough isoelectrically prec­ ipitated casein was added to a 3$ (v/v) solution of peracetic acid to make a protein solution slightly greater than 1$ (w/v) in concentration. The mixture was agitated for 18 to 2b hours, until all the protein particles were dissolved. Centrifugation at 1^,000 x G. for 15 minutes yielded a residue which was transferred to filter paper and washed 3 times with cool distilled water. The residue was put into solution with the aid of a few drops of dilute NHj^OH. The solution was prepared for electrophoresis by dialysis against distilled water and buffer. Homogenization qL isolated £3§.5jn afid isolated whgy proteins. Casein was isoelectrically precipitated from samples of the same lot of raw skimmilk before and after homogenization. The milk was homogenized at 2000 p.s.i. first stage and 500 p.s.i. second stage at 55° C. These two casein samples, together with their supernatants which contained 19 whey proteins, were analyzed electrophoretically. This investigation provided a comparison of the electrophoretic patterns of casein and whey proteins before and after homogenization. qL i M soluble, membrane nrotein 4*6. The pH of a 0.95$ solution of soluble membrane protein was adjusted to 4.6 x-jith dilute HC 1 and centrifuged at 9200 x G. for 10 minutes. The percentage total solids of the supernatant was determined. of the soluble membrane protein &t pH 4.6. Equal volumes of solutions of 0.95$ soluble membrane protein and 0.98$ alpha-casein were mixed. The pH of the solution mixture was adjusted to 4.6 with dilute HC1 and centrifuged at 9200 x G. for 10 minutes. The total solids of the supernatant was determined. Electrophoretic analysis of b (residue) SOLUBLE MINUS CASEIN-COMPLEX (iy)a>1? (supernatant) 1. Heat to 90° C. for 30 min. 2 . Precipitate removed by centrifu­ gation at 25*000 x G. for 30 min. SOLUBLE MINUS CASEIN AND HEAT-DENATURED PROTEIN (V)a,b (supernatant) a See Glossary for description of isolated fractions b See Figure II for electrophoretic patterns of isolated frac­ tions. FIGURE 1 . Procedure for isolating the proteins associated with the homogenized milk-fat globule. 22 EXPERIMENTAL RESULTS s & to rt ol H gai on the. T o ta l Arcavffit. o f P ro te in RgBaAni.gR on %h& Homogenized M ilk -F a t G lobule The data in Table 1 show that the total amount of protein remain­ ing with the homogenized milk-fat globule decreased as the temperature was increased. At 60°, 70°, and 80° C. for 30 minutes, the amount of protein associated with 100 grams of homogenized fat was 2 ,27 , 1 .60 , and 1.31 grams, respectively. As might be expected, the whey protein nitrogen values for the milk decreased with increasing heat treatment. (Table l). Exoerimentatlon wjth the F ra c tio n Pr.<5fllpiAftt^4 & t ^16 The Effect of Various Chemical and Physical Treatments in the Separation of the Casein-complex Heat. Heating the Casein-complex Fraction (ill) for 30 minutes at 90° C. did not markedly affect any of the electrophoretic components, as shown in Figure 3* The only apparent affect was the decreased mobilities of the components of the complex after heating. The electro­ phoretic mobilities and relative areas of the peaks are listed in Table 2 . Urea. After solubilizing the Casein-complex Fraction (HI) in 6.6 M urea, the molarity of the solution was adjusted to 1.0, and the aggregated protein was concentrated by centrifugation. 23 The electro- phoretic characteristics of the protein were not affected by the treatment. Electrophoretic patterns of the complex before and after urea treatment are shown in Figure 3 . The electrophoretic mobilities and relative peak areas are given in Table 2 . Using the urea fractionation technique of Hipp ^t al. (1952) , the protein isolated at ^.7 M urea is shown in Figure 6 . Based on electro­ phoretic mobilities, the two observed components were the first two of the Casein-complex Fraction (HI), in order of decreasing mobility. Mobilities and relative peak areas are listed in Table 2 . Ethyl ether. The electrophoretic patterns of the Casein-complex Fraction (IH) before and after being treated with ethyl ether at room temperature are shown in Figure h. area by 22 .6$ (Table 2). Component 2 was reduced in relative The electrophoretic mobilities of the compon­ ents, listed in Table 2 , of the ether-treated sample were somewhat less than the same components in the untreated sample. Rennet. Addition of rennet to the Casein-complex Fraction (ill) did not affect the typical electrophoretic pattern. A comparison of the patterns before and after rennet treatment are shown in Figure Calculated electrophoretic mobilities and relative peak areas are given in Table 2 . Reconstruction of the Casein-complex with Selected-Component Systems Homogenization containing se£yffl. £ mW'XSL.e <2? qalai.Uft ggffi&n&te. membrane-, This experiment was conducted in anticipation of this selected-component system producing an electrophoretic pattern similar to that of a typical Casein-complex Fraction (III). The pattern shown in Figure 5 is a typical casein pattern, whose only deviation from a normal pattern was manifested by an increase of the peak area in the position normally occupied by gamma casein. The relative concen­ tration of alpha- to beta-casein was in order (Table 2). SoWQEWnisaUpn oL a atofcqrg. Of calcium caseinate, membranecontaining serum, and butteroil. The selected-component system employed in this experiment differed from the preceding by the incorporation of butteroil. The relative concentration of alpha- to beta-casein is higher than in normal casein. The electrophoretic pattern of this homogenized mixture is shown in Figure 5» and the mobilities and relative peak areas are listed in Table 2 . HQaQiPLenis.a^iop & nfatara soluble, membrane ££&- tein and butteroil. The electrophoretic pattern of the precipitate of the solution mixture at pH 4.6 did not show the ratio of peak areas that was expected. Before homogenization, the ratio of alpha-casein to soluble membrane protein was much lower than the same ratio after homo­ genization, as determined from areas under electrophoretic peaks. The supernatant, at pH 4.6, had a total solids content of 0.106$, of which 0 .025$ was fat. In the precipitate, the leading electrophoretic peak, which was alpha-casein, had a descending mobility of 5 .8 . This value is considerably lower than the corresponding value of 6.5 for the same alpha-casein used as a control under identical conditions. The electro­ phoretic pattern of this complex is shown in Figure 5. Homogenisation sL & BLAslagg. &oha-QAs.eift butteroil. The alpha-casein precipitated at pH 4.6 and treated with organic solvents showed a characteristic lipid spike in its electrophoretic pattern 25 (Figure 5)* However, its electrophoretic mobility was not decreased, as in the preceding experiment (Table 2). The same alpha^-casein was used in this and the preceding experiment. Analyses of Fractions Ultracentrifugal characteristlcs. Sedimentation velocity studies were made on two fractions: the Soluble Minus Casein Fraction (IV) and the Soluble Minus Casein and Heat-denatured Protein Fraction (V). A diagram of the Soluble Minus Casein Fraction (XV) shown in Figure 8 t demonstrates the presence of three molecular species. Figure 9 shows the extrapolation of S20 sedimentation values at three concen­ tration levels to obtain an S£q for each component at zero concentration. Also shown in Figure 8 are sedimentation velocity diagrams of the Soluble Minus Casein and Heat-denatured Protein Fraction (V). were two components in this fraction. There Figure 1C shows the S20 values extrapolated to zero concentration for both components. Table 4 lists the sedimentation velocity constants for all the components in both fractions. Paper- part it ion chromatography. The Soluble Minus Casein and Heat- denatured Protein Fraction (V) shewed three definite carbohydrate moities. A fourth carbohydrate spot appeared frequently but could not be accounted for in all the chrormtograms. Identification of chondro- samine, glucosamine, and mannose was made by comparing the Hf values of known carbohydrates on the same chromatogram. spotted alone and in mixtures. The known sugars were When discernible, the fourth sugar had an Rf value identical to that of glucose. 26 Ultraviolet analyses. Figure 11 shews the spectrophotometric pattern of the Soluble Minus Casein and Heat-denatured Protein Fraction (V) in the ultraviolet region of the spectrum. In the same figure, a pattern of the soluble membrane protein is shown for comparison. The soluble membrane protein was heated for 30 minutes at 90° C. prior to being analyzed. The coincidence in the positions of the small absorption peaks should be noted. Ite Effftgt jq£ Various Chejrd.Qal Physical Treatments Caaeifc SJQd tfcfi Spjubj^ Membrane Pr<2fceiT) Effect Qf ar* ethanol-ethyl ether treatment on the, electrophoretic characteristics of casein. The electrophoretic pattern of whole casein after treatment with ethanol and ethyl ether is shown in Figure 6 . No major alteration of the components was electrophoretically evident. The mobilities of the peaks appearing in the treated sample were slower than those in the control sample. Table 2 lists the mobilities and relative areas of the electrophoretic peaks# Effect of a peracetic acid t r e a t y o n the. electrophoretic char­ acteristics of casein. Treating whole casein with peracetic acid did not appreciably affect its electrophoretic pattern, although it appears slightly atypical in the relative concentration of alpha- to b etacasein. (Fig ure 6 , Table 2) Effect homogenization 2H the e^ectrapfrpRe.t,characterist ics isolated casein and. 3&ey RCQ.tei.ns. There v as no change in the electrophoretic patterns of either casein or whey proteins before and after homogenization. (Figure 7, Table 2) Precipitation ja£ the fiolvftjLfi fflePfcrailQ protein a$L pH 4*£. 27 The super- natant of the solution adjusted to pH *+.6 had a total solids value of 0.71#* With the original solution having a total solids content of 0.95#. calculations show that 25.2# of the soluble membrane protein will precipitate at pH 4.6. Precipitation gf thg. soluble membrane protein a M pH *+.6 . The total solids of the supernatant of the solution adjusted to pH 4.6 was 0.35#* Calculations made from this value show that 63.8# of the total protein was precipitated. Assuming complete precipitation of the alpha-casein and 25.2# precipitation of the soluble membrane protein, the 36.2# protein remaining in solution was completely assigned to the soluble membrane protein. That is, the 74.8# soluble membrane protein, not precipitated at pH 4.6 and diluted by a factor of 2 , gave 37.*$ total solids in the supernatant. ElQgt-XP.Rhpretjg, analysis. of casein isolate^ f r m homogenized milk. The electrophoretic pattern of casein isoelectrically precipitated from homogenized milk does not indicate clearly the presence or absence of a complex formation in this preparation (Figure 6). The electrophoretic mobilities of the components, listed in Table 2 , are in order with those of whole casein from nonhomogenized milk and do not seem to be deterred by a complex formation. On the other hand, the concentration ratio of the alpha- to beta- casein components, in Table 2 , is too large and the small component behind the alpha-casein peak may not be merely a contam­ inant of the preparation. 28 ASCENDING INSOLUBLE FRACTION (I) a. DESCENDING 4------- SOLUBLE FRACTION (II) CASEIN-COMPLEX (III) SOLUBLE MINUS CASEIN (IV) SOLUBLE MINUS CASEIN AND HEAT-DENATURED PROTEINS (V) FIGURE 2. Electrophoretic patterns which are representative of the various fractions isolated by the fractionation scheme shown in Figure 1 . 29 TREATMENT ASCENDING » DESCENDING <------- CONTROL FOR HEAT TREATMENT VERONAL; pH 8 .6; CONC., 1.00% HEATED AT 90° C. FOR 30 MINUTES w /L -A w VERONAL; pH 8.7; CONC., 1.35* I CONTROL FOR UREA TREATMENT VERONAL; pH 8.7; CONC.— 1.00* SOLUBILIZED IN 6.6 M UREA FOR 2k HOURS VERONAL; pH 8.7; CONC., 1.36* FIGURE 3. The effect (h i ). heat and urea on the Casein-complex Fraction 30 TREATMENT ASCENDING DESCENDING CONTROL FOR ETHYL ETHER TREATMENT VERONAL; pH 8.5; CONC,, 0*1$% AGITATED WITH ETHYL ETHER FOR 30 MINUTES AT 2$° C. VHiONAL; pH 8«5l CONC*, 0*1$% CONTROL FOR RENNET TREATMENT wA -A** JL-A* VERONAL} pH 8.5; CONC., 0*793^ ACTED UPON BY RENNET VERONAL; pH 8 .6 ; C0NC.~1.00£ FIGURE U* The effect of ethyl ether and rennet on the Casein-coraplex Fraction (III)- 31 SELECTED-COMPONENT SYSTEMS AFTER HOMOGENIZATION ASCENDING I DESCENDING -> MIXTURE OF CALCIUM CASEINATE AND MEMBRANE-CONTAINING SERUM VERONAL; pH 8 .6 ; CONC., 0,97% MIXTURE OF CALCIUM CASEINATE, MEMBRANE-CONTAINING SERUM, AND BUTTEROIL VERONAL; pH 8 .6 ; CONC., 0.8$% MIXTURE OF ALPHA CASEIN, SOLUBLE MEMBRANE PROTEIN, AND BUTTEROIL VERONAL; pH 8 .6 ; CONC^-O.99^ MIXTURE OF ALPHA CASEIN AND BUTTEROIL VERONAL; pH 8 .6 ; CONC.— 0*95^ FIGURE 5. Electrophoretic patterns of selected-component systems 32 DESCENDING, TREATMENT CASEIN-COMPLEX PRECIPITATE IN U«7 M UREA VERQNALj pH 8*7| CONC., 0.90% ETHANOL-ETHYL ETHJ2i TREATMENT OF WHCLB CASEIN VERQNALj pH 8.7* CONC., 0.92% PERACETIC ACID TREATMENT OF WHOLE CASEIN VERONAL) pH 8.6; CONC., 0.9U* ISOELECTRICALLY PRECIPITATED CASEIN FROM HOMOGENIZED MILK ' j J i . J k i / VERONAL^ pH 8.7; CONC., 0.80* FIGURE 6 . The effect of various chemical agents and homogenization in milk on the electrophoretic characteristics of casein. 33 ASCENDING DESCENDING -> HGNHDMOGENIZED CASEIN VERONAL; pH 8.5; CONC,, 0.8U* HOMOGENIZED CASEIN 'waAb mht* VERONAL; pH 8.5; CONC., 0.8356 NONHOMOGENIZED WHET PROTEINS VERONAL; pH 8.5; CONC., O.8I4* HOMOGENIZED WHEY PROTEINS VERONAL; pH 8.5; CONC., 0.78* FIGURE 7. Electrophoretic patterns of isolated casein and isolated whey proteins before and after homogenization. 3^ 35 FIGURE 8 . Sedimentation velocity diagrams of the Soluble Minus Casein Fraction (IF) and the Soluble Minus Casein and Heat-denatured Protein Fraction (V). 8.0 7.0 7.28 6.0 i Sedimentation Constants (S20 = S20 x 10"^) I 5.0 3.0 - 2.0 1.0 0.8 1.2 1.6 2.0 Protein Concentration ($) FIGURE 9. Eixtrapolation of sedimentation ^elocdty constants to zero concentration for the Soluble Minus Casein Fraction . 36 8.0 - 7-0 ‘5.96 o rH m 5.0 IQ n o CM CO w to p k.O fl (0 p a o o § P P w 3.0 2.0 i.t> 0 0.8 1.2 Protein Concentration ($) FIGURE 10. Extrapolation of sedimentation velocity con­ stants to zero concentration for the Soluble Minus Casein and Heat-denatured Protein Fraction (V). 37 2.0 11. CO — o or I 10 o — cn CM — 00 CD CM CD LU LU CM Noiiddosav 38 Ultraviolet spectrogram of the Soluble Minus Casein and Heat-denatured Frotein Fraction (7) and the soluble membrane protein after 30 minutes at 90° C. ro FIGURE rO TABLE 1 TOTAL PROTEIN REMAINING WITH THE HOMOGENIZED MILK FAT-GLCBULE WITH INCREASING HEAT TREATMENT Observations 60° C. (30 min.) Heat treatment 70° C. 80° C. (30 min.) (30 mih.) Index of heat treatment Whey protein nitrogen (mg/g total solids) 8.1 6.7 Composition and yields, Volume of washed homogenized cream (ml)a ^20 Fat in washed homogenized cream (&) 610 730 i+9.9 55-1 19^.9 30^.4 ^02.2 52.2 55.1 59.8 Yield of total solids (g) 219.2 336.1 ^36.5 Kjeldahl nitrogen in cream (mg f) 165 126 116 Yield of fat (g) Total solids in washe^ homogenized cream ($) Total solids due to protein (#) <$ N x 6.38) 1> 0.6 0.2 Grams of protein/100 grams fat 2.27 1.60 1.31 a Prepared from 10 gallons of raw milk b Assume ^.53# total solids in sucrose 39 ro O o |H Vi £>-3- rH H on M3 o O rH • VO • -3 - av * CM o to o| on cC a) Cl. CM 5 • iH • • ■ O • CM CM • H r -t • • • • ("4 • VO VO VO CM O • O o rH oo VO • • • • m • «* • m • « • • CO O• -4" rH iH O• -3- ON ro vo VO • ON vo • CM rH • vo « • • ro • VO • • * o CM Cv, • O CM • vO • vo ro • -3- rH • 00 ON VO r-* c - On • ON rH VC m 0 cm co CM i—1 • ON VO -=}• VO VO CM On • r" O- o vo CM O • O rH ro rH • CM rH 4-3 ca rH © Pi • f—1 • CM H CO ro • O' CM VO • rH vO CO © OO • 0 H X • * (T> rH O I £3 +3 H Xi C © . °e o ii ^ CM • * CM ^r\ • CM • ■ • • -3" « ro r"N CM • r"\ • • • CO • • » • * • • • * 9 -J- • CO CO CO ro • ro toCM • C^ VO • cv CO • ro CO rH VO CO ro ro -3 • VO 00 o * VO vO O•n VO CM ro o CM • rH CO O -3 - CD © *r4 rH vO • ■3- -P •*■4 f H -r4 O O • i —1 -V • vO • CM ro VO XO • ro CM O • • • ro OO rv • VO ON • VO *o o sa CO c Vi © -P -P nJ a o ■H •P © V. o XCL I o V. -p o Pi VO © v 3 CkO *r4 CM © rH W ifO rH » rH Vi O o 03S - CM CM « t>CM w On o CM ON H • 0 tN- 0 cn i—i on 0 on VT\ CM CO 0 CM m d H CM d On IS- 0 0 rH • 00 0 rH rH 0 rH 0 CM 0 0 CM CO CM vn CM 0 CNvn •P A(D Ctf • o CO • H VO • rv ao o 0 o o VT\ 0 ON CO CJN O 0 rH 0 N d d CN- ON VO n- CO CM ON 0 0 0 0 0 0 0 0 0 CM d rH Cn • CM I>~ CM • CM CO CO 0 CO CO 0 on CM d VO CM 0 0 CO v r\ NO CO 0 v r\ o On 0 vn CM ON 0 vn vo cn 0 vo O CO 0 VO CM on • 0 VT\ VTN, NO • rH r - On 0 0 ON Vi O • • • ♦ vn I 0 rH a rH cn 0 0 0 • •—1 • 0 0 0 0 0 0 0 0 0 0 • • • • rH ON • rH VO d 0 d o ON 0 CM VO d d in in rH 0 vn o oo 0 vn vo 0 - CM CO 0 d rH 0 CM H CM on • ON rH • 0^ CH O rH d d 0 CM Q d0 CM X rH 1 o TJ S c c o o CM I 9 CH rH • ON • O n • • • CM • on CM • CO CM vo CN- NO 1—1 0 0 0 0 0 0 CO 03 CO O Cn cn d On • m rH a I js; in A4 O cti > P03 U CM e o CM 0 cn d d O o CO ON 0 vn vo CO 0 vn CM C^N 0 0 II 3 E-t +3 •rl 0 NO rH .H 00 cn 0 VO 0 vn 0 vn rQ o £ n G V. © -P P & 3 O os o •H P 03 *H O XO, O G -P o © © u n tlfl •rH (fci NO vn rH M *a TABLE 3 SEDIMENTATION VELOCITY CONSTANTS FOR THE SOLUBLE MINUS CASEIN FRACTION (IV) AND THE SOLUELE MINUS CASEIN AND HEAT-DENATURED PROTEIN FRACTION (V), CORRECTED TO 20° C.a Protein fraction Soluble Minus Casein and Heat-denatured Protein Fraction V. Soluble Minus Casein Fraction (IV). Concentration (^) Sedimentation velocity constants (S20”s2Q x ' °^ 1 Peak Number 2 1.90 5.62 3.13 1.81 5.69 3.62 .... 1.44 5.40 3.20 .... 1.11 5.43 2.88 .... 1.01 5.58 2 .60 .... 0.90 6.08 3.48 .... 0.83 5.77 2.90 .... 1.88 8.17 6.20 2.35 1.33 7.90 5.28 2.00 1.00 7.76 5.58 2.54 3 a All analyses were made in Veronal buffer; pH 8 .8 ; ionic strength; 0 1 . . 42 DISCUSSION With the isolation scheme used in this research, comparable pro­ tein fractions were nearly identical from preparation to preparation* In a biological system as complex as milk, this reproducibility was gratifying. However, some doubt remains as to the degree of denatura- tion imposed by the techniques of isolation. The use of organic sol­ vents to extract lipid materials from the proteins would probably draw the most crit icism. The effect of the cold ethanol-ethyl ether treat­ ment on the electrophoretic characteristics of casein was determined. Seemingly, the only effect of the solvents on the casein was the decreased electrophoretic mobilities of the components. No investiga­ tions of the effect of solvents on the other milk proteins were made, but it seems likely that the chemical and physical characteristics of some of the more labile species would be altered. Effect of H