I « WWW CHANGES OBSERVED IN SOME OF THE, CHARACTERIETECS 9F THE MELK FAY MEMBRANE MATERIAL DURING ITS PREPARATION 7519515 for ”19 Degree of M. S. MECHEMR 515ml UNIVERSWY Shegmarh Remember-g 1957 THESIS LIBRAR Y Michigan Ststc UHiVCf’aiiy CHANGES OBSERVED IN SOME OF THE CHARACTERISTICS OF THE MILK FAT MEMBRANE MATERIAL DURING ITS PREPARATION By Sherman Rosenberg AN ABSTRACT Submitted to the College of Agriculture of Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Dairy 19 57 Approved Q W 0' ABSTRACT SHERMAN ROSENBERG The primary reason for this study was to observe some of the changes which occur to the fat globule membrane dur- ing the isolation procedure. Comments were given concern- ing yield and general composition. Yields of 1.11 - 1.44 grams of total membrane material per 100 grams of fat and 0.59 gram of membrane protein per 100 grams of fat were obtained. The soluble protein frac- tion composed 46 percent and the insoluble fraction 54 per- cent of the membrane proteins. A distribution curve for the nitrogen in successive washed creams showed that less than the theoretical amount was lost as a result of repeated dilutions and separations of the original cream. This is due to the fact that mem- brane as well as plasma nitrogen was measured. Washing with a sugar solution rather than plain water resulted in an increase in the separation efficiency and consequently an increase in the yield of membrane material. Separation and washing at 38° C. resulted in a higher re- covery of lipoprotein complex than did working at 4° C. Ash determinations on the membrane proteins yielded lower results than those obtained by other researchers. Enzyme activities were reduced quite drastically dur- ing washing. Warm separated and washed creams retained more activity than the cold counterparts. Loss of activity after .‘“(‘1?‘.: m “Wm" ‘T ‘w \“I “~"'\‘."“./‘ JIBQII‘JXCJ. 3111411.. .24. J. .*J._J-s._)J_JrLU salting—out the membrane material with ammonium sulfate was quite pronounced. Use of organic solvents also inhibited activity or destroyed the enzymes. CHANGES OBSERVED IN SOME or THE CHARACTERISTICS OF THE MILK FAT MEMBRANE MATERIAL DURING ITS PREPARATION by ‘Sherman Rosenberg A THESIS Submitted to the College of Agriculture of Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Dairy 1957 /’7’5gs @,4$$3 ACFTOWLBDGmLKTS The author is sincerely grateful to Dr. J. R. Brunner, Associate Professor of Dairying, for his ready advice and aid during the experimental work and for his assistance in the preparation of this manuscript. Words cannot express the degree of indebtedness. Acknowledgment is also due to Dr. T. I. Hedrick, Asso— ciate Professor of Dairying, for being instrumental in the author's having come to Michigan State University and for his cooperation in allowing the writer free run of the dairy plant and aid of its staff.' The writer is grateful to Miss Lenore Ho, who did many of the total solids and fat deter- minations. The funds and facilities provided by the Uni- versity and Michigan Agricultural Experiment Station are sincerely appreciated. A general "Thank you" is certainly in order for the en~ tire staff of the Department of Dairy for their encourage- ment. Each and every person helped the author by contribu— ting to the general feeling of intimacy within these walls which surely is necessary for one to produce at his best. The writer's greatest thanks are due to his wife, Lillie, who never conplained about he long hours the author spent away from home, and who was ready always at the correct time with that necessary word of encouragement. TABLE OF COLT; 4.15 . o HUI} INTRODUCTION . . . . . . . . . . . . . . . . . . . REVIEW OF LITERATURE . . . . . . . . . . . . . . . . . EXPERIMENTAL PROCEDURE . . . . . . . . . . . . . . . . COCOKN Isolation of Membrane Proteins . . . . . . . . . Analytical Methods . . . . . . . . . . . . . . . 10 Fat and total solids Ash . . . . . . . . . . . . . . . . . . . . lO Alkaline phosphatase . . . . . . . . . . . . ll Xanthine oxidase . . . . . . . . . . . . . . ll IitrOgen . . . . . . . . . . . . . . . . . . 15 RESULTS . . . . . . . . . . . . . . . . . . . . . . . 15 Yields . . . . . . . .'. . . . . . . . . . . . . 15 Nitrogen . . . . . . . . . . . . . . . . . . . . 15 Fat and Total Solids . . . . . . . . . . . . . . 16 Ash . . . . . . . . . . . . . . . . . . . . . . . 18 Enzyme Activity . . . . . . . . . . . . . . . . . 18 DISCUSSION . . . . . . . . . . . . . . . . . . . . . . 31 Yields . . . . . . . . . . . . . . . . . . . . . 31 NitrOgen . . . . . . . . . . . . . . . . . . . . 33 Fat and Total Solids . . . . . . . . . . . . . . 35 Ash . . . . . . . . . . . . . . . . . . . . . . . 59 Enzyme Activity . . . . . . . . . . . . . . . . . 4O SUMMARI AND CONCLUSION . . . . . . . . . . . . . . . . 45 LITERATURE CITED .. g o o o o o o o o o o o o o o o o o 48 ii LIST TABLE 1. 5. IO. YIELD OF LAILRIAL ARA: I LII OI" 31331333 CLLTLET 333139 03333 13303311031 133 3“‘* 0033331 or .3333; CCKPCSlTICI, Ian, VARILUS PIISLJ CE .LIJIIno I; iyjbl’HA'. :AIICII (Trial 1A) COIfl2081TIC:I, ASII, VALE“ U5 II‘IALSISQ OI" TLI I5 I RE} AIII‘II‘IL (Trial 18) COMPOSITICII, ASE, VARIOUS PHAULS CF TLINS PREPARATION (Trial 2A) COMPOSITION, ASH, VARIOUS PHASES OE TEINS PREPARATION (Trial 2B) COIII’OOII Iva, ASH, VARIOUS PIL’IQLS OE TEINS FELLAEJI'I‘ION AND WASI'IED "JITII A 5A) COMPOSITILN, ASH, VARIOUS PHASES CF TEINS PREPARATION (Trial 5B) COMPOSITION, ASL, VARIOUS PHASLS OF FAI GLOBULL LILLLBLmHL IIIU'ELIIHS PP Lbl-AIULitlUI“ MILK SEPARAIBD A'T LIIK FAT OF TABLLS AID FIGURE - ‘QTI'~ ATP JLIIIILI...) I‘...m)I..> L2I' 1L} :BL’I—u IIulI LULLJLJ. *J J. LLC I“: .0 CF LIIK, 3313 LILK, 3331:, . A ', 31‘ .1 .32": i? "1?» 3 V‘IUIII. IUIAJJ IIIILIL'J-..“ ‘ T‘?‘ - f " LILLII... [III I) LIIZ. ' IL“... Cm TI JlTI CE IKE ‘L‘ Ill-J E‘ ak: ‘11—‘03? L} [LL loLi—JLIJFJLI‘LJ I: .Lfio- USIEG LILK 533333133 AT 40 c. AIID BIIZJ..1LLJ ACII J III. UT TILL ’1‘:IA.J .1. TAT 010431,} .LL'J LIL); LBLIJILI :1 ELIO- ..I31.:IIK 3-133.. . AT 530 c AIID .JIIZJLI-J ICICIJ. .lVIJ.|:1.I 0.1." TILL TAIL E “NAT GLOBU LL MIA—JAIIlgiLtLA E }*{C)— Uslre mlIK b.3333133 AT 40 c. AND ELZYEE ACTIVITY or 1:3 :33 331 GLOBULL 331.“.33133 PRO— Uslge MILK 333133133 AT 53° AIID LIIZI IIL ACTIV i‘I‘Y OE ‘I‘IIE ILL FA T GLUBULE LUMBRATH TRO- 3313s KILK 333333.33 AT 4° 0. 12.7m SUCROSE QULUI IL’II (Trial AED ENZYME ACTIVITY OF THE IHD LAT GLOb 3ULIJ LHIJLAIQ I80— UQIEG MILK QLIAHAILD AT 380 AID bIblhu AC'I .LVI“V OF Th5 iHE gCAET IAIIVLLI CCKTROLLEI 380 C. (Trial 4) iii .fiID AXD IRCTLIU USIII G 26 27 29 50 FIGURE I. Effect of successive washings on the total nitrogen content of cream. iv INTRODUCTION The fact that milk fat exists in the form of tiny glob- ules three to six microns in diameter has been known for ap- proximately 250 years. But not until Ascherson (1840) did his classical work on olive oil in contact with egg white was it postulated that these globules are surrounded by a "membrane" complex. Present knowledge classifies this ma— terial as a protein-phospholipid complex. The origin of the membrane is obscure. Mulder (1947) believed that when a fat globule is detached from the secre- ting cell, it drags along with it a thin film of material representing a complicated system of substances such as pro- teins, enzymes, phospholipids, carbohydrates, and other bio- logical substances. Sommer (1952) postulated that the triglyceride molecule exists independently in the secreting cell. However the lipid cannot remain by itself in solution, and thus it joins with others to form fat globules. As this progresses, phos- pholipids attach to the globule, but since the former are both hydrophilic and lipophilic, they cannot be absorbed completely by the fat. Therefore the phospholipids project out from the globule surface. The proteinaceous material may become associated with the globule at this same time. Eventually the fat globule will "grow" to a size such that _ 1 - it is just large enough for the phospholipid and protein ma- terial to completely envelOpe it in a single layer, thus ef- fectively sealing the globule from further growth. Obviously the origin of the fat globule membrane may be explained by the physiologist as well as the chemist. But since the membrane has been shown to be closely associated with flavor and palatability problems in milk (Palmer, 1944), knowledge of its chemical and physical characteristics is needed. This manuscript deals in particular with the protein materials of the membrane. Researchers believed for some time that only a single protein existed in the membrane- complex, or else that it was homogeneous. However Herald (1956) was able to classify this material into two portions, differentiated by their solubility in a dilute salt solu- tion. Electrophoretic studies showed each of these frac— tions to be heterogeneous, patterns of both consisting of two or more peaks. This present study deals with the detailed preparation of the membrane proteins. The procedure employed is given in some detail in the body of this thesis. The biological material is characterized by its chemical composition at various steps in its preparation. Also, compositional dif- ferences found when isolating membrane material from warm and cold separated cream were observed. REVIEW OF LITERATURE Two distinct approaches have been used for separating membrane material from the plasma phase and two for removing membrane from the lipid phase of milk. Because of the na- ture of the work involved, these will be discussed concur- rently. Eliminating the aqueous or plasma phase involves wash- ing the cream with water. VBltz (1904) and Abderhalden and V81tz (1909) used extensively a method whereby cream was al— lowed to rise through a column of water. These workers used a 60 centimeter column and removed the washed fat globules after 12, 24, 48, 72, and 96 hours. Although this is quite effective, it has the disadvantage of carrying particles from the plasma along with the cream. The product was fil— tered at 50 — 70° 0., after which the dried residue was ex- tracted by means of boiling ether. Titus, Sommer, and Hart (1928) employed a similar tech- nique except that their water column was twice as high; be- fore filtration they mixed the washed cream with one-third its volume of 95 percent ethanol; and their method of fil- ter residue purification was more involved. The second method of washing cream was devised and used by Storch (1897). Even though it came chronologically before that of V3ltz (1904), this technique has proven to be the -3- more pOpular means of removing milk plasma from around the fat globule. Cream, after being separated from the skim milk, was diluted back to its original volume of whole milk and separated again. This was repeated four or five times. Removing fat from the membrane was achieved by organic sol— vent extraction according to V61tz and Titus gt g1. (1928). The washed cream was shaken in strong alcohol followed by the addition of ether and benzene. A gelatinous precipi- tate was filtered from the aqueous phase, washed with strong alcohol and ether, and dried in air at room temperature. Again there was the problem of obtaining a product the com- position of which had been altered from that of the origi- nal membrane. In this case the procedure involved the loss of membrane phospholipids along with the milk fat. Palmer and Samuelsson (1924) used this method of re- peated dilutions and separations for washing the fat glob- ules. Then, in order to keep mechanical loss of membrane lipids to a minimum, these researchers churned the washed cream, the agitation from this procedure being sufficient to rupture the membrane. The 1ip0protein complex was re- covered from the buttermilk and the melted and separated butter. Jenness and Palmer (1945) published an elaborate pro- cedure for membrane protein isolation. Included was their mode of dialyzing the membrane-containing serum in order to concentrate said membrane. The final step consisted of ex- tracting phospholipids and a high-melting triglyceride frac- tion from the membrane material by means of ethanol and ethyl ether, leaving essentially nothing but protein. A modification of this technique was employed by Hare, Schwartz, and Weese (1952). They mixed the cream washings and buttermilk after churning and used acetone to precipi- tate the membrane material. The dry precipitate (moisture removed by centrifugation) was washed with absolute alcohol, a 1:4 alcohol-ether mixture, and finally anhydrous ether to remove the lipid portion of the membrane. Brunner, Duncan, and Trout (1955a) used a method simi- lar to that of Hardy and Gardiner (1910) for extracting the lipoidal materials. They first treated the membrane with a cold ethanol-ether mixture to separate the lipids from protein and then extracted the former with 400 C. ether. Varying amounts of membrane material have been isolated by different workers. Palmer and Wiese (1955) found 0.66 - 0.89 gamma per 100 grams of fat. Jenness and Palmer (1945) reported 0.71 - 1.20 grams of membrane substance per 100 grams of fat from different breeds and at various stages of lactation. Herald (1956) prepared 1.27 grams per 100 grams of milk fat. Storch (1897) estimated 58 grams per 100 grams of fat, a value which is obviously high. The large error was due possibly to his observations of membrane material under the microscope. In another section of his article, Storch claims that butter churned from fresh cream contains 7 percent membrane by weight. At the other extreme Schwarz and Fischer (1957) reported only 0.12 gram per 100 grams of fat. This low figure is attributed to the use of physi- ological saline for washing the cream, a procedure which would have removed some globulin protein. The milk fat was separated from the membrane by means of ether extraction; thus much of the lipid portion would have been removed. Again we find varying reports for the amount of protein in the membrane. Rimpila and Palmer (1955) found 0.46 - 0.71 grxnn per 100 grams of fat. Jenness and Palmer (1945) reported 0.58 - 0.86 gIRHE per 100 grams of fat. Herald (1956) obtained a value of 0.51 grwu: of membrane protein per 100 grams of milk fat. Schwarz and Fischer (1957) re- ported O.12 gpx3m per 100 grams of fat, a figure not only decidedly low when compared to other published quantities of membrane protein but also incompatible with their report of 0.12 gram of membrane material per 100 grams of fat. Their values are valid only if one considers the fat glob— ule membrane to be composed exclusively of protein, a fact which is strongly disputed by the literature on this subject. Roland (1956) found 0.44 - 2.22 grams of protein per 100 grams of fat, basing his figures on a formula devised by himS elf o Herald (1956) also reported a procedure for fraction- ating the membrane protein into soluble and insoluble frac- tions. As this technique was followed rather closely in the work reported in this manuscript, the details will be given under the experimental procedure. Suffice it to say that a different means of concentrating the membrane mate- rial was used (by Herald). Instead of dialyzing the mem- brane-containing serum, saturated ammonium sulfate was added to a final concentration of 55 percent. This "salted out" the entire membrane substance, not the protein portion alone, because of the strong physical attraction and chemical bonds between components of the membrane. Herald estimated that the soluble fraction contained 44 percent and the insoluble fraction 56 percent of the original membrane protein. King (1955) wrote a brilliant, comprehensive review of this field of research which cites all but the most recently published work in the area. EXPLRIEENTAL PROCEDURE Isolation of Membrane Proteins Fifty gallon lots of relatively fresh, raw, mixed herd cow's milk were separated at 40 C. and 580 C. using a De- Laval Model No. 590 Standardizing-Clarifier. The cream from each separation was diluted back to its original volume with tap water of the same temperature. he wash water was re— moved from the cream by means of the separator used above. This washing-separation process was repeated for a total of three to five times. The final washed cream was allowed to stand overnight at 50 C. Both the high and low temperature washed creams were treated identically. The cream was brought to 15 - 140 C. and batch-churned in large glass jars placed horizontally in a mechanical shaker and agitated to rupture the fat glob— ule membrane. The combined butter and buttermilk was warmed to 580 C. and run through a DeLaval Model No. 9 Laboratory Separator. The membrane-containing serum was collected and the butteroil discarded. Some of the warm separated cream achieved a plastic condition after overnight storage, and it was necessary to dilute it with distilled water at 15° c. in order to obtain churning consistency. The serum was adjusted to room temperature and a sat- umwrted ammonium sulfate solution was slowly added with _ 8 _ stirring until a final salt concent*ation of 2.2 Q was reached. Salted-out membrane material was concentrated by centrifuging at 2,000 R.P.fi. in a Universal Ko. 2 Centri- fuge. The membrane substance was finally concentrated in a Model 83-1 Servall Centrifuge by running at 25,300 x G for 50 minutes. A solution of 55 percent ethanol in ethyl ether at -50 C. was added to the membrane material in the prOportion of 100 milliliters to 50 grams of concentrated membrane. All ether used in the preparation was tested for formation of peroxides according to Weissberger, Iroskauer, Riddick, and Toops (1955). The mixture was held at 00 to -50 C. and agitated for 15 minutes. Subsequently it was observed that for large quantities of membrane substance, the ratio of ethanol-ether to membrane must be increased; otherwise, the final ethanol concentration is insufficient to break the lipid-protein bonds. Following the cold ethanol treatment, the mixture was adjusted to —200 C. and filtered. The al- cohol-treated membrane material was washed five times with 200 - 600 milliliter portions of -200 C. ethyl ether. Fi- nally, the residual membrane lipids were extracted with three 250 - 800 milliliter portions of ether at 500 C. SOIVent separation was achieved by centrifugation. The pro- teins were held overnight under a vacuum of 28 inches to re- move residual ether. 10 Separation of the soluble from insoluble protein frac- tion was accomplished by the addition of successive 150 — 450 milliliter portions of 0.02 g sodium chloride solution. Following agitation at 2° C. for approximately 2 hours the suspension was centrifuged at 25,000 x G for 45 minutes. The supernatant from each extraction was drawn off and pooled. Extractions were continued until the supernatant gave a neg- ative biuret test. No more than four extractions were re— quired. The supernatant contained the "soluble" fraction while the residue in the bottom of the tube was the "insolu- ble" fraction. All procedures were carried out in glass or stainless steel containers. Analytical Methods Fat and total solids. Fat and total solids were de- termined by the method of Mojonnier and Troy (1925). In cases where only a small amount of a given sample could be used for testing purposes, a modified extraction technique was employed for determining fat. The extraction was car- ried out in a test tube using 0.25 - 0.50 gram of sample, 1 - 2 milliliters water, 1 milliliter concentrated ammonium hydroxide, 2 milliliters ethanol (two extractions), 5 milli- liters ethyl ether, and 5 milliliters petroleum ether. Agh. Samples to be ashed were exhaustively dialyzed against tap water, distilled water, and redistilled water. ll Concentrated materials first were suspended in distilled water. Total solids determinations were run on the dialyzed samples. Fifty gram samples were dried on a steam bath and then placed in a muffle furnace at 550 - 6000 C. overnight. Alkaline phosphatase. The activity of this enzyme was determined according to the procedure of Sanders and Sager (1947). In every case, the protein precipitant used was 5.0 grams of zinc sulfate and 0.6 gram of cupric sulfate made up to 100 milliliters with distilled water. Optical density was read at 610 millimicrons in a Bausch and Lomb "Spectronic 20" Colorimeter.g Calculatiens were based on the method of Little, DellaMonica, Custer, and Rudd (1956b) so that units of activity for both enzymes studied would be of the same order. The activity found was converted from optical density to units according to a standard curve with the equation x - 2y = 0. Ten and five-tenths units of activity is equivalent to the release of 1.0 x 10-7 moles of phenol. Xanthine oxidase. A slightly modified method of Zittle 25 31. (1956a) was used for determining xanthine oxidase activity. This method of assay is based on the fact that tetrazolium salts are good oxidation-reduction indicators. They are colorless, soluble compounds in the oxidized form and colored, water insoluble, oxygen stable formazans in 12 the reduced state. In this test triphenyltetrazolium chlo- ride is reduced by the action of xanthine oxidase. The principal reagents are a 0.5 M phosphate buffer to keep the reaction close to pH 7.5, 0.005 E xanthine to act as sub- strate for the enzyme, and 0.05 E triphenyltetrazolium chlo- ride. Nitrogen gas was bubbled through the tube containing the reactants in order to prevent oxygen from inhibiting the reduction of the tetrazolium salt. To insure that the nitrOgen was pure, three columns of Benedict's (1912) oxy- gen absorbent were placed in the nitrogen line. The assay was conducted at 50° C. and the triphenyltetrazolium allowed to be in contact with the enzyme for exactly 10 minutes. Mustakallio, Ahos, and Autio (1955) reported that triphenyl— tetrazolium chloride in solution is photosensitive and be- comes red. They suggested using this salt only under con- dition of total darkness. But because of the nature of this particular test, Zittle (1956b) suggested using a room with a constant light source. The reduced triphenyltetrazolium was taken up in toluene and Optical density was read at 485 millimicrons in the "Spectronic 20." The equation for the standard curve run for the xanthine oxidase had the equation 6x - 5y - % = 0. One unit of activity is equivalent to the formation of 0.5 x 10-7 moles of the reduced triphenyltetrazolium. 13 Nitrogen. The samples tested were fractionated accord- ing to the method of Rowland (1958). Nitrogen was deter- mined by means of a modification of the technique employed by Menefree and Overman (1940). Size of the Kjeldahl sample and amount of concentrated sulfuric acid used for digestion were determined by the nature of the sample and the speci- fic protein for which one was analyzing. In general, 10 milliliters of nitrOgen-free concentrated sulfuric acid and 5 grams of sodium sulfate-mercuric oxide (14:1) mixture were added to the sample. Digestion was allowed to proceed for 15 minutes after the solution became clear and color- less. The contents of the Kjeldahl flask were allomed to cool, the sides rinsed with distilled water, and the solu- tion redigested for an additional one-half hour. One hun- dred fifty milliliters of distilled water and 40 milliliters of 50 percent sodium hydroxide plus sodium thiosulfate were added to the cooled contents. Approximately 100 milliliters were distilled into a flask containing 50 milliliters of use solution boric acid and a few drOps of methyl red-methylene blue indicator. The receiving solution was made up as fol- lows: 1 pound of reagent grade boric acid was dissolved in 10 liters of distilled water; the use solution contains 2 Parts of the above diluted in 5 parts of water. The dis- ‘tillate was titrated with 0.05 N hydrochloric acid. Nitro- gen was calculated as follows: 14 ml. HCl x N HCl x .014 gm. eq. x 1000 mg. N/gm. sample mg. N/gm. sample x 100 mg./% N 0 moo'rétN x 100 milliliters of hydrochloric acid used in mg. N/lOO gm. fat Wher 6 ml . HCl titration N H01 = normality of hydrochloric acid .014 = milliequivalent weight of Nitrogen gnu eq. = grams of sample to which the aliquot taken is equivalent. REéULTd Yields The final preparation was carried out with the DeLaval flkydléal No. 9 Laboratory Separator, since its smaller size would permit a closer accounting of the materials and also allow for smaller losses due to milk, cream, etc. being held in the bowl. {Twenty-five pounds of 25 percent cream were obtained from 169 pounds of milk. After the first washing, 1? pounds °f7 35GB. percent cream remained. Succeeding washings changed the yield and percent fat only slightly, though it is ob- ViIDIIEs when looking at the final washed cream-~15.5 pounds of 31 percent fat-~that pure-1y mechanical loss plays a part in the total picture which is obtained of the membrane. {Dilea ‘yields from this preparation are tabulated in Table 1. From this isolation 1.11 - 1.44 grams of 1ip0protein mat erial per 100 grams of fat were obtained. The final yield of membrane protein was 0.59 gram per 100 grams of rat - The soluble- fraction appeared to make up 46 percent of the total proteins and the insoluble portion 54 percent. Nitrogen The results of a nitr0gen distribution study on one Se - 1‘3— es of washed creams are shown in Tables 2 and 5. -15.. 16 Determinat ions made for total nitrogen, casein nitrogen, non-casein nitrogen, proteose-peptone nitrOgen, and non- protein nitrogen all displayed a sharp decrease after the first Washing. From this point the various fractions lost nitrogen quite gradually. In fact, casein nitrOgen leveled Off 50 that the values for the final three washed creams Were C"Onstant . The amount of protein in each washed cream was calcu— l ated. These values followed the nitrogen curve (see Fig— ur e I) and are similar to those reported by Rimpila and F 511516: (1955). Fat and Total Solids Tables 4 to l0 inclusive show, as one would expect, that the total solids and fat percentages in cream range higher from the warm than from the cold separations and waghings. Conversely, the total solids content of the skim milk of the cold separations were slightly higher than from warm skimming. The first cold washing showed a marked de- crease in solids content, while successive washing of the c:old. separation showed an increase. 0n the other hand, the 1:015-‘u211 solids and fat content of the warm preparations in- creased markedly as a result of the first dilution and sep- aration, reaching a maximum at this point in the washing 1) I‘o'izedure. Looking at the compositional data from another 1? viewpoint, the solids-not—fat of cold washed creams dropped after the first washing, whereas it increased after the first war-m washing. Thus we have two distinct situations. After the first 4° C. washing the cream generally decreased in 13013511 solids and fat content, while the solids-not-fat increased. proportionally. But the first warm washed cream usually showed an increase in the concentration of all three ComPon‘i‘llts. In one trial a 12.7 percent sucrose solution was used for chd-washing the cream in order to increase the specific gravity of the mixture for separating. Data in Table 8 Show that in this case a cream of considerably higher 1501331 solids content than the other cold separations was obtained, but With succeeding washing the cream's solids percentage he Vex, came within more than 15 percent of that from the 5&0 ti - Q adverse effect on the plasma during washing, skim milk C. separation. To be certain that the sugar solution had <1:LZLuted to four times its volume with 12.7 percent sucrose was agitated and centrifuged. The casein was unaffected. 1\‘rerbhrally, the increase in total solids was due to sugar rt.0111 the wash solution entering the plasma. But the fat C=°Jiltent also increased, indicating; that sugar has some ef- fect in preventing the loss of fat during the washing op- e bat ion. 18 Membra‘ine—containing serum had a total solids range of 0.8 - 2.4 percent. The sugar-washed preparation did not Show a solids content out of proportion to the other trials. Approximately one-half of the total solids was fat. Con- centrated membrane ranged from 56.4 - 42.5 percent total SOIidS- Solids-not-fat at this phase was in the range from 27'2 ‘ 35.5 percent. The membrane proteins were approxi- mately 5 percent fat. In two out of three cases the soluble protein fraction from the cold separation was lower in total so ‘ lids and solids-not-fat than the corresponding warm prep- aratioh SOlids The cold insoluble fraction was higher in total and solids-not-fat content than the warm preparation. Ash 3?or most trials the ash content was highest in the mem- brahe~containing serum, the controlled trial yielding 5-43 Percent total ash on the dry weight basis. The soluble PTO” tein fraction had a slightly higher ash content than the in soluble portion, 1.80 percent as opposed to 1.51 percent. Enzyme Activity —Enzyme activities were not followed solely for the sap: e of studying the effects of various physical and chemi— Cal actions upon alkaline phosphatase and xanthine oxidase. The Cic‘bivities were used also as a means of observing l9 destructiC>IZL or loss of membrane. Tables 4 through 10 show the graduiiil. loss of these enzymes in terms of sample size and grams of fat. On the average, cream beginning: with 146 \uuts 0f 1phosphatase activity per gram of sample dropped to 76 units.; xanthine oxidase activity fell from El to 7 units Per granl. -A1Aso one can see the relative effects of cold and warm Separation and washing. Although the data were somewhat enrartj_c, the general picture shows that with both enzymes, the <1(Dld washed membrane holds the phosphatase and xanthine ”lilacs e more tightly than the warm washing during the ear- jpart of the procedure. But by the fourth dilution and sexPellr‘ation there seems to be greater activity in the warm CI§EEEiIns. Most of the activity assays performed on the latter :plléiises of the preparation proved to be unsatisfactory. How- EE‘TGEJ? the author found that both phosphatase and xanthine ()lth<1ase activity were lost to a considerable extent as a l§€353111t of the salting-out step and ethanol-ether treatment.. 4(£1530 observed was the fact reported by Zittle (1956a) that IIlajority of the alkaline phosphatase was concentrated in 1: klea soluble protein fraction. 20 TABLE 1 YIELD (:LF MATERIAL AT VARIOUS Pflndns OF THE PREPARATION OF MILK FAT GLOBULL MEMBRAEE PROTEINS W Phase Yield Composition Total Solids— __‘___‘~__ Solids Fat not-Fat Whol e (poundS) (%) (%) (% Ore Milk 169 12.7 5.9 8.8 was? 25 32.4 24.9 7.5 Washed Cream 1 17 57.9 56.4 1.5 wa5311f3apo< osmaam nmd soapamomaoo mmmnm Asa Hoaeev .o o4 as easemeaam qus eaHo: onesmsmana anmaomm QZ are so MBHeaaoe anemaa ems .rma .oneHnomsoo é mqmda 25 .0HDSHO0CH firm was mapsaom Q00 mo mHmwn Go w0woasofl000 cm m.a onmm 00a 5a.a a.5 a.a 0.0 soaoooem qfl0pohm mansaomnH nama 6.5 comma mm 50.0 5.m 0.0 0.5 soaoosam eaoooam oapoaom mm 4.0 III III .mo.a 0.50 H.m m.n0 msaopoam ostenaos ea a.m oer 05m eo.m H.04 H.Hm 0.05 osoenaos eoooospIaosonpm 5a 3.5 05m mm III 0.5m 0.4a m.me eaten I802 ©0p09pQ0oqou 560 5.4 coma 65 00.0 5.0 5.a 4.0 esaom 00a Inflmps00I0n0Hpa0E mean .0\moaoev Amen .0\opaesv ma om mom ama III 0.5 0.54 o.mm m eooao sonata rm 5a 5mm 54a III m.aa o.m¢ 0.4m m eooao norms; ama m0 mmm maa III 0.0 m.0¢ H.5m a goose oonmoa and a5 mm: aom III 5.5 0.0: m.am aooao omoa 0.5 III III III 0.5 5.0 4.0H xaaa seam mam 0H III III III H.m m.m e.ma Mafia oaona some .mxooasschnasxooaesc moon .mxooaoova.aexooaeac has ac Aav 5a 000©on 0mflgpn0x 000u0rmmo£m 0GHH0MH< anohd pmmlpoq 90M meadow Isnssv Imeaaom annoy .Hpfl>wpo< 0Ehmsm n04 mafipflmomaoo 000nm Ama Hoaeev .o orm as naeamsmam maHn eras: eoaeeraammm omHaeomm Mafifimfid: mADmOQU 84% flua m0 mmmsmm Dbaanfi> Ema m sense no aaaeaeos enemas one .moa .onaHoomaoo 2 .80000808080 088830 pmoa hp0>flpo0 08huGM0 a e s e ma.a 0.00 0.m 0.40 ooaoooem 800ponm 0HQ9H008H 0 0 0 0 III H.H H.0 N.H doHpo0Hm geopoem oansaom 0 0 0 0 III III 0.4 III 0800ponm 08089802 0 0 0 0 III m.0m 0.m n.00 08080808 eoosoepIaoeanpm 000 00 a 0 III 0.00 0.ma 0.H4 00000 I802 00p08p800800 00m0 0a 00008 00 III 0.0 5.H m.0 80000 00a I800p800I08089802 A080 .0\meaqov A080 .m\ooaqnv NH m.4 III III III H.m 0.0m ©.H¢ 4 80080 000003 III III III III III m.¢ 5.0m 0.H4 m 80080 000003 04 ma III III III 5.0 m.mm 0.0m 0 80000 000008 H0 00 000 45 III 0.4 n.0m 0.0m a 88000 000000 000 5m mom mma III 0.5 0.0m 0.H4 e0000 owNH m.m comma Hm III “.0 m.0 m.m Mafia 8080 400 4.5 00mm Haa III 0.0 m.m n.0a 8H0: oaone hp0w .mkmwflunvn.a8\0pflssp hung .n\mpflnsvn.H8\mpm880 “fl ARV AW .wfi mooeawo 08000008 08808000000 meaanaas amsoae psaIpoe 008 meaaom I88040 Ioeaaom awnoe Huabflpod 08808m 804 deflpfimom8o0 000nm A40 800050 .0 o4 04.043404000 8440 eszn 83404000800 08440000 04080088 0400040 040 sea 00 080400 anoarse 104 80 naaeaa 4 arenas 044 .004 .00054008200 PF'IK w 84m<8 00 .000000000 N0m 000 0090000 900 00 0000p 00 00000500000 .0000000m009 000050 pmoa hp0>0po0 08%08fi0 0 0 0 0 00.0 0.00 0.0 0.00 00000000 80oponm mansaoqu 0 0 0 0 mm.a 0.m m.0 0.m 00000000 000p00m 0Hnsaom 0 0 0 0 00.0 4.00 0.0 0.00 00000000 00000000 0 0 0 0 0III 0.00 0.00 0.0 00000000 0000000I0o00000 0 0 0 0 III 0.0 0.0 0.00 00000 I800 000000000800 400 0.0 0004 00 00.0 0.0 0.0 0.0 00000 000 IQHG pflo Olwflwh @052 A000 .0\000000 0000 .0\000000 Ha H.m 0nd 000 III N.o 0.00 0.00 0 80000 000003 00 ON 0mm 000 III 0.0 H.m0 0.00 m 80000 000003 mm 00 mmm mwa III 0.m 0.00 m.00 N 80000 000003 0m mm III III III 0.0 0.00 H.m0 0 80000 000003 00 00 mom 000 III 0.0 0.00 0.00 00000 0000 0.0 00000 000 III 0.0 0.0 4.0 0000 0000 0mm 0.0 00mm Had III 0.0 m.m m.NH 0005 00003 0000 .0\0000000.H0\000000 0000 .0\0000000.00\000000 A0 00 000 000 0000000 00000800 000p000000m 00000000 000000 p0hlpoq 00m 000Hom I00000 I000000 00000 000>0000 08008m £04 800000om800 000nm I‘HIH IIIIHll J 000 000000 .0 000 00 000400000 0000 00000 00000000000 00000000 00000000 0000000 000 400 00 000400 0000000 000 00 00030000 000000 00: .000 .00000000000 0 0400s .manmaommfi Rém and mansfiom fiw¢ Ho mammp no dmpmasoamon .QOHpmnammHm wuflhsw pmoa mpfl>flpow mflhxqmm ) w 000a ONH ;D.H 0.0H m.H ¢.ma GQflpomHm Qflspoum manzaomnH a m 0000a mma om.0 w.m m.0 0.¢ GoapomHm nflmponm mansaom w m 00: mma 00.H $.0m .m 0.00 mmflopoum mumnpama a 0 mm: moa 9:0.H m.¢¢ o.m 0.0: mqmgnamz dmpwmaplaoqwnpm n d d 0 III m.mm n.n B.H# mnmhn IEou dwpmnpqoquo smma 0.5 oomxa 9a Ho.a 0.3 9.0 m.H asumm mafl udflwpmoo mmmnnama mmw .m\mpflqs0 Ammm .w\mpfiasv mm 0H mo 0m nu: m.m $.w¢ o.mm t aano dogmas 0m ta 00H 00 III m.m m.>¢ m.mm m awono donmma m m an: III In: m.w m.@# n.¢m m ammpo omnmma 00 mm II: III III m.d #.m¢ ©.mm H Emmno vmnmma 00H md mm mma III 0.0 m.a¢ N.0d ammno wmmm 0.0 0000: 00H II: 0.0 #.0 3.0 MHHE afixm w0¢ da 0m0¢ m¢H III m.m ©.m w.ma xawa oaoga hpmw .wxmpmqgvh.fiaxmpflqs~ “paw .m\mpflusvn.flg\npflqsp my; flfi A& ha mmmwflmo mnanpcdx mmwpmngmonm QQHmead Ammonc pmmupon pmm mcaaom umgq40 umwflaom Hmpmm th>Hpo< mfimnmm £m< moapflmomaoo mmmnm “mm Hmflnev audaga,n anemone n§.ma 4 maHe namm azq as maapaaon qawqmq ma .nom .onaHnomaoo . \ . m flAmdB 0 ®HQDHOWQH «xi km 000 mapsaom £00 90 00009 no 0000030000 :L 9 .00000000000 000050 pmoa hpfl>flpom mahuqmm 0 0 000m 000 00.0 0.00 0.0 0.00 00000000 0000000 mandaoqu 00mm 00 00000 000 00.0 0.0 0.0 0.0 00000000 0000000 0000000 N0 H0 000 00 000.0 m.00 0.0 0.00 00000000 00000000 0 0 0000 000 00.0 0.00 0.0 0.00 00000000 000000puaoq0000 000 00 u.: u-u u-- 0.00 0.0 0.00 00000 1802 umpwhmeomoo 000m 00 0000 00 00.0 0.0 0.0 0.0 00000 000 IqflmeOQImanDamE 0000 .0\000000 A000 .0\000000 0.0 0.0 000 000 nus 0.0 0.00 0.00 0 00000 000000 an- In- 000 000 In: 0.0 0.00 0.00 0 00000 000000 nun nu- :u- :u: 1:: m.m 0.00 0.00 N 00000 000000 00 00 000 000 nu- 0.0 0.00 0.00 0 00000 000000 00 00 00m 000 :n- 0.0 0.00 0.00 00000 0000 0.0 00000 000 unn 0.0 m.0 0.0 0000 0000 000 00 0000 000 :n: 0.0 0.0 0.m0 0000 00000 0000 .0x0000000.H0\0p0000 0000 .0\0000000.H00000000 000 0 00 00 0000000 mqflnpcmm mmapwnmmona 000000H0 mwsond pwmlpos pmm mdaaom :00000 :00000m H0000 Npfl>flpod mahuqm n00 Q0Hpflmomaoo mmwnm A00 000000 .0 000 00 000000000 0000 00000 00000000000 00000000 Maxquflfi AHDnoaw nzh rma mo flflnfiflm flDQHmd fine 0 04000 00 000>He00 000000 000 .000 .00H0H000000 000000000 000 500 0095000 000 00 00009 no 000 00.90000 // (D. .20000000000 w00000 0000 mp0>0pom ofihmcmm 0 .0 0000 000 00.0 0.00 0.0 0.00 00000000 aflopopm mansaomQH : 0 0000 0m 00. 0.0 0.0 0.0 00000000 :0mpohm mansflom 0 0 0000 000 000.0 0.00 0.0 0.00 00000000 00000000 0 0 000 000 u-n 0.00 0.0 0.00 00000000 0000000u0000000 0 0 na: nun in: m.0m 0.00 m.00 00000 IE 2 UmpMHpnmonoo 0 0 00000 000 00.0 0.0 0.0 0.0 00000 000 IQHm PQO Olmflwhpfimé 0000 .0\000000 0000 .0\000000 0 0 0mm 00 nun 0.m 0.00 0.00 0 00000 000000 0 0 000 000 an: m.m 0.m0 0.00 0 00000 000000 0 0 00m 00 In: 0.0 0.00 0.00 0 00000 000000 0 0 0mm 00 nun 0.m 0.00 0.00 N 00000 000000 0 0 00m 00 us: 0.0 0.00 0.00 0 00000 000000 000 00 000 0N0 In: 0.0 0.0m 0.m0 00000 0 000mm 0N0 In: 0.0 m.0 0.0 0000 000m 00m 0. 0 0000 000 1:: 0.0 0.0 0.m0 0000 00000 0000 .0\0000000.H0\000000 0000 .0N0000000.00\000000 00 Q 00 AQV mmmdflxo mcflgpqmw mmMpanMOnm wd000000 005000 90m: pod 90m mUHHom 100000 -000000 00000 hw0>flpo¢ mawnna 000 nowpwmomaoo mmmnm A0 000000 .0 000 00 040000040 0000 00000 00000000000 00000000 000m0000 0000000 000 00009 0:00 00300004040000 00;0 00 0400 ..H 0.0)...304b 3 .05 OH flqmda 00 00000000 000000 000 .000 .00000000000 DldCUdleK Yields The results from this study indicated a concentration of 1.11 - 1.44 grams of membrane material per 100 grams of fat. These values compare favorably with data reported by others. Palmer and Wiese (1933) isolated 0.66 - 0.89 gram of membrane substance per 100 grams of fat; Jenness and Pal- mer (1945) reported 0.71 - 1.20 grams per 100 grams of fat; and Herald (1956) found 1.27 grams per 100 grams of fat. Storch (1897) and Schwarz and Fischer (1937) isolated 38 grams and 0.12 gram of lipoprotein material per 100 grams of fat, respectively--values which are not coincident with other data. Also it should be noted that the values reported by Jenness and Palmer do not include the high-melting trigly— ceride fraction of the membrane founl by Palmer and Wiese. Nevertheless the data of the latter indicates values some- what lower than those of Jenness and Palmer. A yield of 0.59 gram of membrane protein per 100 grams of fat was isolated in the present work. This is close to the 0.51 gram per 100 grams of milk fat reported by Herald (1956). Rimpila and Palmer (1935) and Jenness and Palmer (1945) found 0.46 - 0.71 gram and 0.38 - 0.86 gram of mem- brane protein per 100 grams of fat, respectively. Roland (1956) calculated the mean of a number of determinations to _ 31 _ ‘-’.r“d- 32 be 1.15 grams of protein per 100 grams of fat. He based this figure on his formula X : lg-g) loo—r) where x = percent nitrogen in protein n = percent nitrOgen in fat-free liquid e = percent nitrogen in skim milk f = percent fat in cream and 7.4x = percent protein in membrane. Even with the exag- gerated figure of 0.12 gram of membrane protein per 100 grams of fat reported by Schwarz and Fischer (1937), the amounts of lipoprotein material and membrane protein would indicate that workers in this field are fairly well agreed on the amount of these substances which is present in nor- mal cow's milk. Since Herald (1956) was the first to fractionate the membrane proteins according to their solubility in dilute salt solution or water, an extensive comparison with results found in this present study cannot be made. Forty—six per- cent of the membrane protein was found in the supernatant solution while 54 percent remained as the insoluble frac— tion. Herald's results were almost identical. His soluble fraction accounted for 44 percent and the insoluble portion* 56 percent of the total membrane protein. Brunner, Duncan, Trout, and Mackenzie (1953b) provi- sionally classified the entire fat globule membrane protein \r! \N as one of a globulin-like nature. Interestirgly, Herald (1956), on the basis of sedimentation velocity and other properties, tentatively cl ssified his soluble proteins as globulin in nature. 0n the basis of their solubility in 0.02 g sodium chloride, ODL might go so far as to call them euglobulins. Herald also concluded that the insoluble f‘ac— tion should be provisionally classified as a "pseudoh ratin." Nitrogen Table 3 and Figure I show the theoretical and actual effects of the washing procedure on the total nitro;en con- tent of the cream. Theoretically the plasma (and its nitro- gen) of the cream should be reduced by three-fourths after each washing. The curve in Figure I showing the expected distribution of nitrogen over five cream washings was cal- culated on the basis of the results obtained in this study. As can be seen in Table 10, the plasma content of the vari- ous washed creams was not constant, so the theoretical curve was based on the changing values rather than on the original 24.9 percent cream. Since fat is hydrOphobic, it cannot be diluted by the water, and one assumes that the plasma in the first washed cream has been reduced to 25 per- cent of its value in the original cream. Therefore the to- tal nitrogen content of the plasma portion of the first washed cream should be only one-fourth that of the original. 34 The aqueous phase of the second washed cream then would have 25 percent of the nitroben which was in the Irevious washing, and so forth. Brunner gt al. (1953a) showed a nitrogen curve for non- homogenized milk similar to the "Actual h" in Figure I.. Af— ter the second washing the nitrOQen content is slightly higher than the expected level. Then the actual value lev-' els off so that the curve is practically flat. ‘he theoret- ical curve also levels off but can never become horizontal to the abscissa since the nitrogen would continue to be lost until only an infinite amount remained. Therefore the ac— tual total nitrogen content of the washed creams has in- creased in comparison to the expected values. This is due to the fact ‘hat not only plasma nitrogen but also mem ran nitrogen has been measured. Palmer (1944) pointed out that washing cream probably removes from the membrane all but the most tenaciously held materials. The curves in Figure I bear out this hypothesis. It is interesting to note that Brunner £3 al. (1953a) found that the protein of homOgenized milk membrane differed mark— edly from that of nonhomogenized milk. Trout (1950) recog— nized the probability of such a change when he mentioned that due to the effects of homogenization, one ay ,resume ". . . that a film of protein material is adsorbed to the surfaces of the . . . fat globules.” Trout (1957) also mentioned the possibility thxt some of the original nembrane might be enveloped by the fat globule during homogenization. This could account for the change in amino acid composition reported by Brunncr et a1. All of the above mentioned is yet another indication that the membrane proteins obtained by means of the rather harsh treatsents used in this prepa— ration procedure arectmwped materially from th natural membrane proteins. A final note on the amount of protein found in the fat globule membrane might be made. A comparison between the results of Rimpila and Palmer (1935) and those from the present study showed the latter to be slightly lower in ab- solute value, bet the trend was similar. For the sake of uniformity, protein values were calculated on the basis that an average nitrOLen content is 15.68 percent per protein. The author realizes that such a figure must be used criti— cally, but as already stated, this provides a common ground for discussion. Also the present calculation assumes (since it was not specifically stated) that Rimpila and Palmer com— puted their protein results to 15.68 grams of nitrOLen. Fat and Total Solids When Storch (1897) first devised and used the method of ,,4- a- repeated dilutions and separations, he found that the cream from each washing had a considerably smaller amount of fat c.‘ “v . 56 than the cream from the previous separation. If he meant that the percentage of fat in the cream lessened, such re— sults are not corroborated by this present study, for the fat dropped after the first washing only; succeeding wash- ings showed a fairly constant level of fat. Although his work was performed at room temperature while the studies presented here were carried out at 40 and 580 0., ‘he milk fat in Storch's experiments was largely in a solid state and should have behaved somewhat similarly to the cold sep- aration of this report. Therefore one may assume Storoh (1897) meant that the total amount of fat in the cream decreased after each dilu- tion and separation. Using the figures from Trial 4 (Table 10), we find the following: Washed cream 1 2.81 kilOQrams of fat Washed cream 2 2.68 kilograms of fat Washed cream 5 2.55 kilograms of fat Washed cream 4 2.51 kilograms of fat Washed cream 5 2.20 kilograms of fat. These values do indicate that fat was lost with each suc- ceeding washing. Data from the second through fifth washed creams show gram of membrane protein per gram of fat ratio values of 0.0057, 0.015, 0.018, and 0.020. The ratio of protein to fat over the same period was 0.025, 0.020, 57 0.019, and 0.020. Jack and Dahle (1957) and Brunner gt a_. (1953a) also found that the nitroLen/fat ratio was fairly constant after the third washing, demonstrating that almost all of the plasma protein had been removed. Therefore one may presume that membrane material was lost to only a slight extent. Furthermore we may conclude that the lipoprotein complex still adsorbed on the fat globulo after the third washing will not be charged to any treat extent until the emulsion is broken by ". . . twelve or more . . ." (Rimpila and Palmer, 1955) successive washing U} Again referring to Storch (1897), one finds that he, too, used a strong sugar solution in some of his washin;s. He observed that using this technique left most of the mem- brane around the fat globule, whereas washing the cream with plain water resulted in the loss of a considerable part of the 1ip0protein complex. The sucros solution was used in the present study in order to increase the difference of specific gravity between the cream and wash solution to permit the physical separation of fat from wash water during cold washings. When water alone was used, we found it dif- ficult to obtain good yields of cream from operations at 40 c, A comparison of Tables 8 and 9 points out the advantage of a sugar wash. One must agree with Storch that much more fat and accompanying membrane material is lost with plain water washings. Skim milk washed with the same concentration sucrose solution was not affected, indicating that this pro- cedure did not change the results by preventing adsorbed casein from being released by the fat globule surface even though the specific gravities of the two are close. A su- crose solution was not used for any of the warm washings, since it was felt that the mechanical separation was effi- cient; therefore, no comparison can be made between the amount of membrane left under the different conditions dis— cussed. In all probability an even greater yield of lipo- protein would have been obtained from warm sugar—washed cream. The membrane proteins ‘fter the ethanol-ether treat- ment contained approximately 5 percent fat. This was prob- ably phospholipid from the lipoprotein complex which would have been removed during the early stages of the prepara- tion had a more drastic method than churning been used for freeing the membrane from the milk fat. However it was thought desirable to employ this technique so as to obtain proteins as close to those of the original membrane as pos- sible. The results in Tables 4 through 9 demonstrate that per- haps cold separation and washing of cream remOVeS more ad- sorbed material from the fat globule surface. This would agree with the findings of Jenness and Iclmer (1945) wherein they stated that both chilling and aging of cream tend to a d 59 increase the dissociation of the membrane material from the cream. The reason for this is not clearly understood, t}1eu h it is probably merely a physical phenomenon whereby the cold er te.‘aperature e uses the fat Clobule surface to become more "brittle” and lo so me into the plasma. Using this same line of reasoning, warm fat surfs e are quite pli— OJ able, si nee the fat is completely melted, and lipOprotein material is less likely to be washed off b1 the .Jcn ical action of the wash water or Separator. Ash Titus e: a_. (19 :2E) reported 5.11 percent ash in the membrane proteins, an Hare gt a1. (1952) found 3.22 percent. {erald (1956 ) noted a considerably higher percentage of ash, 4.27 percent. he presen study found an averaLe of 1.63 percent ash, quite a bit lower than other reported values. In the light of the lower enzyme activities reported for the latter phases of the preparation, the ash values are not as far out of line as they might appear at first glance. Her— ald, Brunner, and Bass (1957) made the comment that both protein fractions contained mineral elements in concentra- tions higher than reported for other milk proteins suggest- ing that the fat globule membrane may be the Spot at which the trace elements in milk are located. ’\ The ash values shown in Tables 4 through 14 indicate no definite pattern with regard to differences occurring in cold and warm washed creams. Enzyme Activity Enzyme activity results as conplete as might be desired were not obtained. This wa due partially to mechanical problems enCountered with the assay met‘ partly to loss of activity during the preparation. Zittle 32 al. (1956b) have shown the enzyme activity distribution through successive cream washings. The pr)— sent study agrees with their findings. On the avers“ percent of the original cream's phosphatase activity remains after four washings, while only 18 percent of the xanthine oxidase activity is present. Sharp (1940) commented that over 50 percent of the xanthine ox'dase originally present in the membrane is removed by washing. This indicates that alkaline phosphatase is more tenaciously held by the lipo— protein complex. Zittle gt a1. note that the loss is a mechanical one into the wash water rather than destruction of the enzymes. The large drop in xanthine oxilase activity may be because this enzyme is associated with the plasma as Fl wel as the fat globule surface. Cn the ther hand Zittle O et al. (1956a) found only a low level of activity in skim milk. 41 Rimpila and Talmer (1955) reported that 72 percent of th 9 Original alkaline phosphatase activity remain d after four washings, a figure somewhat higher than reported here (a)? by Zittle _e_t_ al. (1956b). The latter postulate the cause to be due to chilling of the original cream before The present study tends to support such a theory, was hi rig . for xvlien cream was separated and washed in the cold, 49 and 15 percent of the phosphatase and xanthine oxidase activity, respectively, remained, whereas 56 and 25 percent, respec- tively, were retained by the warm cream. Jenness and Pal- mer (1945) said that ". . . mere aging. of milk at low tem— perature followed by rewarming: before separation greatly de- creases the retention of protein and phospholipide during: waShirigj." This would explain to so:::e extent the reason for thls author's washed creams loosing; so much more enzyme than that or Rimpila and Palmer, for all milk used had been in for 16 — 48 hours at (J) the University Dairy's holding tank 0 5'5 C - before separation. But the warm washed cream re- ta' - . med more enzyme than the cold. Perhaps the rewarming allows a portion of the enzymes to be readsorbed onto the fat 810131116. surface, while separating. and washing; directly at the colder temperature permits a maximum amount to re- main ill the milk plasma. Lst of enzyme activity after tno churning process Iay be due to chemical as well as physical changes. there-as 42 L11‘ing separation, washing;- and churning, the cream came in contact with nothing that could destroy the enzyme or in- lbit activity, subsequent treatments conceivably were de- iQQntal to the enzyme. In one preparation the butter and Jad‘termilk, instead of being; held at room temperature until all was ready to be warmed and separated, was brought to approximately 380 C. and held there for from 20 to 90 min- utes before the butteroil was removed from the serum. Hei- ther xanthine oxidase nor alkaline phosphatase activity could be detected in subsequent stages of the preparation with the exception of a considerably reduced amount of ac- tivitayr for phosphatase in themembrane-containing serum. An explanation of this phenomenon is difficult. We know that phOSphatase is only 9b percent inactivated after being; held at 61.50 C. for 50 minutes (Kay and Graham, 1954), and Zittle 31; al. (1956a) have shown that xanthine oxidase is even nor: resistant to heat. In addition, the wori: of Z' .. - . . lttle et §_l_. was performed on skim milk, a low fat product, whi . . . , le t31:1e butter and buttermilk in the preparation under dis - . , t ,- , CUSSlon had a co.;;‘oinei‘tbe xanthin: oxidase having b.en removed. This in- dlCEft<3.3 that some of the phosphatase in milk is sore closely .1-' 1 a -. ‘ .-. ' ‘-°r~ ~\~. um .. ‘Alm mile as well as in creaa. “vain t1e sir“ sep- arhl+’ ..__ ‘n A ‘ ~ u o _. ‘7' - “Ave e1 and nesbed cream retained nor; enzyme actiVity than the - , C<3§1.d.products, 5e percent conpw led to 49 peroert of the F P210 (NI: ' 1' fi ' - - r? f) f- 'r‘ '1' x 4‘ v' "I " V" " ‘ 4" 1.; ‘ "" ' -“ J— " 1‘ J"‘-‘ ‘ 4~-<3gtase and a) percent as o;_oled b0 1, PELCemb 91 u-e 45;.1tj‘ 4 _‘ o 7 ,_ ' ' ._._ _ n J ‘r-lwae oridase. Loss of one 3a'1e actiVity alter nal "Jit11 .— V... , o I 0 _ ~ 0 - _ o C14~Lmonium sulfate was quite pronounced. has of Qfgtnlc ("f‘, ‘\ Owl—IEl- /‘ A _ _ _3 ,\ 0 +~r V 3.~.~ , _. .;_‘l -. 4- s alSo appeared to inhioit activity oi destroy these in J possibly concluie thet the membrane naterial of normal fall ’ 47 Should further enzyme studies of the preparation JiOld ex . . , . . . L _ °Ults Similar to those ootained in the oresent 101k, ne mi ” 8 milk is altered as a result of the techniques employed tslne isolation procedure. LIJQigiLLlLJJ V13. LID Abie¢h all 64., E. C1121 VBJtZ, 1]. I? l909. Beitrng zur henn.3ls der Zusa mcn etz n; un' der fotur der hfillen der Kile rLLelcien. \si gJ‘e-oe'” er's Ztschr. l’h’ fizl3-18(Origifial not seen Ki :13, 1955.) l 01. ClleuflL. ; C .1. t Oil 1);? 2. .fiLsscherson, F. H. 1840. (On the physiOIOgical utility of the fat and a new thcory of cell fornstien Easel o tleir COOpe r'tien anl suggested b” s “eral nkf' fzflrts.) niriz. Axnflt. I j'si ' 7: 44. (CriLiral not seen; cit 1.94“ ) 5. BCEe-dict, F. C. V“ . ’W m‘," ‘_ . v"\- x‘. ‘ ‘\ ‘ L1 .- r - 1 --‘ ‘ 'q. ‘r " , - . 1‘). L; o .L..‘ 0'. . .410 J {3.1.2}..- A- ~-.1 t - A .L. Q- 0 J..:..Lj- - , 91 In +0 114". lbbibQ-Bl. -Ij ,» ~~ ‘\ r\ A q "I I" " ”‘- ll“ >- 4. grulll GI." Jo R. , DUZVkaj‘, V. H. , JLAaL .L.J.O1-ut, U. “A. _r mm .. ‘1 :. .. AI. A ’ MA. \. .r .. .. l 571. He iat-Llouule “encich el ”OHMeLC;HLlMJd p A l..M-_l, an m r '- p '-n a:.& I ‘J-.1(JQLLJTL--L1 Ali-‘- .0 I. J...LL/ lbOlQ.—‘Ll‘v l... r.* , xv . v" ~. : 1 «1‘ 1" . r -, r 41- - . - .~ -'— and amino ,c-a CD.pO;ltle; 0. tie fa:— ‘v " 1 ‘3r". ‘1”. o J" P " 1. n h "N " l " “Unblsfle :"OLClWQ. FCC; RC“. 12.454-(2o 5 ‘ {\ _ i~ r .- In A'. J- n f- -, ° E3I‘L z-r, J. R., Duncan, G. u., lICU , u. m., end nae— ‘I: r. 4" lie ’1 ie, 1... 195:1). T:le folk-£101.14 1116 lilti..OI'3_I"lG Of 1‘01" ”.0 :C-B( 1:1 3:51 and hosoienized milk. III. Differences in the seliscntaticn li- ixgws of the f~t~ nesbrene proteins. rvel Res. lC.4u° "4. 'H‘iltkfly, W. 3. and Gardirer, (Lrs.) dtanlev .1910. Proteins of blo d ilasns. J. Physiol. 40:lmflii-l:ci. Jo II. , SCh‘.”LlrtZ, D. P. , anCL #6886, S. J. 2. Tl1e amino acid comp sition of the fat— globule membrane protein of milk. J. Dairv Sci. 22:615-19 ‘1’ Aerald, C. T. lo 6. Fractionation an” c11iracte rirzction of solu— ole and insclnule froteins of the milc fa‘ Llobule nenbrane. lh.D. Thesis, Iichiban State University, Last Laxs n3. _ 48 _ d “ Herald, 3. T., Brinincr, J. 3., and Bass, 5. T. 1957. A spectre iaphic analysis of two frotein fraction. of the fat-globule nonbr r;uie 01 normal coi's milk. J. Dairy Sci. 49:446. Jack 3. L. and Dahle, C. D. 7 The electrokinetic potential of milk fat. III. Rela tf; on to the fat globule "membrene." J. Dairy Sci. 29:639—45. \0v \3 ll. Jenness, R. and Painm, L. 5. 104 . Substances adsorbed on the fet globules in / < I I L I ream and their rel: tion to churning. V. Comyosition of the "membiane" and distri— bution of the adsorbed substances in churn- ing. J. Dairy Sci. 28:611—25. 120 Kay’, :0 Do and Gral’lm, I". 3., Jr. 1954. Phosghorus compounds of milk. VI. The ef— fect of heat on m'lk phos h.tase. A simple method for distin“tisnin+ raw from pasteur— ized milk, ra .. iro:1 p;steur zed cream, and butter nad- from ran cream from that made from pasteurized cream. J. Daiiy Res. 150 ICi_J:Lg3, II. 1955. The Milk Fat Globule Henbrenc and Some Asso- ciated Phenomena. Farnham Royal, England, Commonwealth Agricultural Bureeux. 99 p3. 14- Menefree, S. G. and Overman, O. R. 1940 A seninicro—Kjeldahl method for th deter- mination of total nitrOgen in milk. J. Dairy Sci. 22:1177—€.5. 15' Moéonriier, T. and Troy, H. C. 3.925. Technical Control of Dair; Irolucts. 2nd Ld. pp. 95-109, 122—126. Chicago, Hojon— nier Bros., Inc. 956 fp. 16°I‘h13L