effect of homogenization pressure on the milk

1997 J Dairy Sci 80:2732–2739 2732

Received October 7, 1996.Accepted May 5, 1997.

Effect of Homogenization Pressure on the MilkFat Globule Membrane Proteins

M. E. CANO-RUIZ and R. L. RICHTERTexas A&M University,

Department of Animal Science,College Station 77843-2471

ABSTRACT

The effect of high pressure homogenization on themilk fat globule membrane proteins was investigated.Milk with 1.5 or 3.0% milk fat was heated in vats atlow or high temperature (65°C for 30 min or 85°C for20 min), homogenized, cooled, and centrifuged toseparate the cream and serum phases. The amount ofprotein load per surface area increased as homogeni-zation pressure increased but decreased with heattreatment. The composition of the proteins formingthe milk fat globule membrane in homogenized milkwas not affected by homogenization pressure or fatconcentration, but significant differences in the com-position of the milk fat globule membrane werecaused by the heat treatment that was applied beforehomogenization. The milk fat globule membrane pro-teins in homogenized milk were composed of nativemembrane proteins, caseins, a-lactalbumin, and b-lactoglobulin. Caseins represented about 70% of theproteins in the milk fat globule membrane. In milkheated at 85°C for 20 min, the ratios of adsorbed a-lactalbumin and b-lactoglobulin relative to adsorbedcaseins were higher than in milk heated to 65°C.( Key words: high pressure homogenization, fat glo-bules, caseins)

Abbreviation key: MFGM = milk fat globule mem-brane, dvs = mean diameter for volume surface, G =mass of protein adsorbed per surface area, OW = oil-water.

INTRODUCTION

The native membrane that surrounds milk fat glo-bules consists of a complex mixture of proteins,glycoproteins, enzymes, phospholipids, triglycerides,and other minor components (17). This milk fat glo-bule membrane ( MFGM) acts as a natural emulsify-ing agent that prevents flocculation and coalescence

of milk fat globules and also protects the fat againstenzymatic action (25).

Agitation, cooling, natural creaming, and lipolysisduring the processing of milk alter the MFGM, butheat treatment and homogenization produce thegreatest changes in the MFGM. Heat treatment ofmilk causes changes in the MFGM by promoting in-teractions between plasma proteins and nativeMFGM components (6, 8, 11, 22). It is not clearwhether the interactions result from direct bindingbetween denatured serum proteins and native MFGMproteins through intermolecular disulfide bonds (6,22) or by displacement of polypeptides in the nativeMFGM by milk serum proteins (8) . Homogenizationof milk causes a reduction of fat globule size and aconcurrent increase in the milk fat surface area,which alters the original MFGM because the concen-tration of native MFGM is insufficient to cover the fatsurfaces that are formed during homogenization. Ad-sorption of new material from the milk serum at theoil-water ( OW) interface occurs to cover this increasein surface area. The new MFGM consists of nativeMFGM plus adsorbed plasma proteins, with casein asthe dominant group, but not at the exclusion of wheyproteins (7, 10, 17, 22, 25).

Several studies detail the composition of theMFGM of homogenized and unhomogenized milk (1,3, 12, 13, 15, 16, 18, 21, 22), but reports of the effectof homogenization pressure on the MFGM material ofmilk have not been published. Our objective was toevaluate the effect of high homogenization pressureon the composition of MFGM proteins in milk.

MATERIALS AND METHODS

Materials

Fresh raw milk was obtained from the Texas A&MUniversity Dairy Farm (College Station) and wasseparated using a cream separator (De Laval 104;The Laval Separator Co., Poughkeepsie, NY). Milkfat concentrations of 1.5 and 3.0% were obtained byblending the skim milk and cream.

CANO-RUIZ AND RICHTER 2733

Journal of Dairy Science Vol. 80, No. 11, 1997

Heat Treatment and Homogenization

Milk was heated in vats at low or high tempera-tures (65°C for 30 min or 85°C for 20 min) in a waterbath equipped with an agitator. Immediately afterheat treatment, milk was homogenized at the respec-tive heating temperature in a high pressurehomogenizer (Mini-Lab type 8.30H; APV Rannie Inc.,St. Paul, MN) at 30, 60, and 90 MPa. The sampleswere cooled to 4°C immediately after homogenization,and 0.02% sodium azide was added to preserve themilk before extraction of the MFGM.

Particle Size Analysis

The mean diameter for volume-surface ( dvs) andspecific surface area were determined in milk samplesafter homogenization using a light-scattering appara-tus (Coulter LS 130; Coulter Corporation, Miami,FL).

Determination of Protein Load

The concentration of protein in the homogenizedmilk samples and in the serum phase of the samplesafter centrifugation [10,500 × g for 30 min at 20°Cwith addition of 28.6 g of sucrose/100 g of milk (24)]was determined using the micro-Kjeldahl method(2) . The percentages of milk fat remaining in theserum phase after centrifugation and in the milksamples were determined using the Mojonniermethod for ether extraction (4) . The mass of ad-sorbed protein was calculated as the difference be-tween the amount of protein measured in the serumphase and the amount of protein present in the origi-nal sample. The protein load ( G) was calculated asmilligrams of protein per square meter of fat surfacearea, using the formula

G =( – )po ps rf

sa( – )fo fs

where

G = protein load (milligrams of protein persquare meter of milk fat),

po = protein content of milk (milligrams of pro-tein per gram of milk),

ps = protein content of the serum (milligramsof protein per gram of milk),

rf = density of milk fat (0.916 gram per mil-liliter at 20°C),

sa = specific surface area of milk fat (squaremeters of milk fat per milliliter of milkfat),

fo = fat fraction of the milk (grams of milk fatper gram of milk), and

fs = fat fraction of the serum (grams of milkfat per gram of milk).

The specific surface area of the milk fat was calcu-lated from the particle size analysis.

Separation of MFGM Proteins

The MFGM proteins of homogenized milk sampleswere obtained following the method of Kanno andKim ( 9 ) with modifications. Cream was separatedfrom the homogenized milk by centrifugation at10,500 × g for 30 min and 20°C after addition of 28.6g of sucrose/100 g of milk to increase the difference inthe density between the fat and serum phases. Creamwas washed three times with deionized water at 6700× g for 10 min at 20°C. The washed cream was dilutedto approximately 30% fat and churned. The butter-milk and butter granules were collected separately,and the butter granules were melted at 40°C andcentrifuged (6700 × g for 5 min at 20°C) to recoverthe serum from the butter oil. The buttermilk andbutter serum were combined and centrifuged for 15min at 3000 × g at 4°C. The MFGM proteins wereconcentrated from the aqueous phase by acidificationof the serum phase to pH 4.8 with 0.01N HCl toprecipitate the membrane proteins. The precipitatewas allowed to form for 30 min at room temperature(25°C) and then was collected by centrifugation(10,500 × g for 30 min at 4°C). The floating creamlayer was removed using a Pasteur pipette. Distilledwater was added to the precipitate and pH was ad-justed to 6.8 using 0.01N NaOH.

Quantification of Individual Proteins

The Coomassie Plus Protein Assay Reagent (PierceCo., Rockford, IL) was used to determine proteinconcentrations of the MFGM samples prior to elec-trophoresis. Ten micrograms of MFGM proteins fromeach sample were loaded on each gel. Analysis bySDS-PAGE under reducing and nonreducing condi-tions was conducted using the method of Laemmli(14) and a Novex Xcell Mini-Cell and Novex precastTris-glycine polyacrylamide gels of 18% acrylamideconcentration and 8 to 16% gradient acrylamide con-centration (Novex Co., San Diego, CA). Samplebuffer with 0.5 M Tris·HCl, 20% glycerol, 10% SDS,and 0.1% bromophenol blue was added 1:1 (vol/vol)to the MFGM solution to denature the samples fornonreducing conditions. b-Mercaptoethanol (5.0%)was added to the sample buffer to denature the sam-


HIGH HOMOGENIZATION PRESSURE2734

TABLE 1. Protein load ( G) and mean diameter for the volume-surface (dvs) in milk under different experimental conditions.

a,b,cMeans within a column for each variable without a commonsuperscript differ ( P < 0.05).

Variable dvs G

( mm) (mg/m2)Pressure30 MPa 0.45a 6.12c

60 MPa 0.25b 9.79b

90 MPa 0.19c 11.88a

Temperature65°C 0.35a 9.69a

85°C 0.25b 8.84b

Fat concentration1.5% 0.28a 9.24a

3% 0.32a 9.28a

ples for reducing conditions. Samples were heated inboiling water for 2 min prior to being loaded on gels.The premixed molecular mass marker (BoehringerManheim Co., Indianapolis, IN) and milk proteinstandards a-CN, k-CN, a-LA, and b-LG (SigmaChemical Co., St. Louis, MO) were applied to eachgel. To eliminate variations between gels, 0.5 mg ofBSA (Pierce Co., Rockford, IL) was loaded on eachgel as an internal standard. A regression equation todetermine protein concentration in gels was devel-oped by applying 0.05, 0.10, 0.25, 0.50, and 1.00 mg ofBSA in duplicate on the acrylamide gels. Gels werestained with Coomassie blue solution and destainedovernight in a mixture of 30% methanol and 10%acetic acid. Images of the gels were obtained using ascanner (HP ScanJet IIcx; Hewlett-Packard Co., PaloAlto, CO). The relative concentration of the polypep-tides that were positive by Coomassie blue was calcu-lated from the relative peak areas in the gel imagesusing gel image analysis software (23) and theregression equation obtained from the BSA stan-dards.

Statistical Analysis

All experiments were conducted in duplicate ortriplicate using a complete factorial experimental de-sign. The data were analyzed for main effects and forall two-way interactions using the general linearmodels procedure and the least significant differencemeans test of SAS (20).

RESULTS AND DISCUSSION

Protein Load

In order to separate the cream from the milkhomogenized at high pressures by centrifugation, su-crose (28.6 g of sucrose/100 g of milk) was added toincrease the difference in densities between the fatand the serum phases. Even with the addition ofsucrose, not all of the milk fat could be separatedfrom the homogenized samples by centrifugation,probably because of the increase in density of thehomogenized milk fat globules after homogenization.The fat globules that are formed during homogeniza-tion are covered with plasma proteins to coat theincreased surface area caused by homogenization (7,16, 17, 21, 22), and their density could increase to adensity near that of the serum phase, especially forthe smaller fat globules, making it difficult toseparate all of the fat from milk by centrifugation.The fraction of milk fat remaining in the serum phaseafter centrifugation was taken into account for the

calculation of G, and, therefore, the G obtainedrepresents only the values for the fraction of fat glo-bules that were separated using centrifugation.

Table 1 shows the statistical analysis of G and dvsof homogenized milk under different experimentalconditions. The G increased as homogenization pres-sure increased but decreased as heat treatment in-creased. The dvs of homogenized milk decreased ashomogenization pressure and heat treatment in-creased. An inverse relationship existed between Gand the milk fat globule size. Walstra and Oortwijn(26) used collision theory to predict that largercasein micelles were preferentially adsorbed oversmall micelles, and this preference was morepronounced for small fat globules, which caused the Gfor the smaller fat globules to be increased. Sharma etal. (21) also reported that G increased as fat globulesize decreased. Those researchers obtained G of 6.56and 8.14 mg/m2 for dvs of 0.48 and 0.30 mm, respec-tively, for milk with 4.0% milk fat homogenized at 17MPa and 52°C. These values were similar to the G of6.12 mg/m2 that were obtained in this experiment formilk homogenized at 30 MPa, which had a dvs of 0.45mm. The G on the fat globule surfaces increased sig-nificantly at the higher homogenization pressures.The G for samples with dvs of 0.25 and 0.19 mm was9.79 and 11.88 mg/m2, respectively, for milkhomogenized at 60 and 90 MPa (Table 1). Tomas etal. (24) homogenized milk at 35 MPa and 52°C andobtained G of 11 mg/m2 in milk emulsions when thefat to protein mass ratio was <4 and G of 6.6 mg/m2

for the fat to protein mass ratio was 13. Oortwijn andWalstra (18) obtained a G of about 9 mg/m2 withmilk emulsions that had been homogenized at 10MPa and 40°C with fat to protein mass ratios be-tween 4 and 10. These values were higher than thevalues obtained in this experiment and those reportedby Sharma et al. (21).



TABLE 2. Percentage distribution of proteins in the milk fat globule membrane (MFGM) ofhomogenized milk under different experimental conditions.

a,bMeans within a column for each variable without a common superscript differ (P < 0.05).

NativeVariable membrane Caseins b-LG a-LA

Pressure30 MPa 13.14ab 67.57a 6.80a 4.76a

60 MPa 12.61b 69.34a 6.32a 4.76a

90 MPa 13.49a 66.74a 6.87a 4.90a

Temperature65°C 12.33b 68.02a 5.10b 4.13b

85°C 13.83a 67.74a 8.22a 5.48a

Fat concentration1.5% 14.21a 68.30a 6.11a 4.71a

3.0% 11.95b 67.47a 7.21a 4.90a

Reducing conditionsNo 16.06a 65.20b 4.77b 4.63a

Yes 10.10b 70.57a 8.56a 4.98a

Figure 1. The SDS-PAGE of milk fat globule membrane (MFGM) proteins of milk with 3.0% fat separated using 18% acrylamide gelsand reducing conditions. a. Lane 1, molecular mass markers of 200, 116, 97.4, 66.2, and 39.2 kDa (top to bottom); lane 2, BSA standard;lane 3, as-CN standard; lane 4, k-CN standard; lane 5, b-LG standard; lane 6, a-LA standard; and lanes 7 to 10, MFGM proteins of milkwith 3.0% fat, heated at 65°C for 30 min and homogenized at 0, 30, 60, and 90 MPa. b. Lane 1, molecular mass markers: 200, 116, 97.4,66.2, and 39.2 kDa (top to bottom); lane 2, BSA standard; lane 3, as-CN standard; lane 4, k-CN standard; lane 5, b-LG standard; lane 6, a-LA standard; and lanes 7 to 10, MFGM proteins of milk with 3.0% fat, heated at 85°C for 20 min, and homogenized at 0, 30, 60, and 90MPa.

The fat to protein ratios used in this experiment(0.47 and 0.94) did not affect the amount of G or dvs(Table 1). Oortwijn and Walstra (18) reported that Gwas little affected by the fat to protein ratio until theamount of protein was insufficient to coat the surfaceof the lipid properly. The milk fat to protein ratios

used in this case were <1, and the amount of proteinwas apparently sufficient to coat the fat globulesproperly at all homogenization pressure conditions.

Heat treatment affected G (Table 1) and is relatedto denaturation of proteins and the higher kineticenergy associated with increased temperature.



Caseins apparently are adsorbed at the OW interfaceas casein micelles in homogenized milk (5, 10, 21,25). High heat treatments can cause whey proteins toassociate with casein micelles via the formation ofdisulfide groups between denatured whey proteinsand the caseins, especially k-CN (22, 25), whichwould increase the total amount of protein absorbed ifcasein molecules were absorbed as intact caseinmicelles. However, G decreased as the heat treatmentof the milk increased (Table 1). High heat treatmentcould cause direct adsorption of denatured b-LG anda-LA (19) that do not interact with the k-CN duringhomogenization at the OW interface, limiting the sur-face available for casein micelles to be adsorbed andtherefore decreasing the total amount of protein load.Also, the disintegration of casein micelles into sub-micelles and a-CN and b-CN monomers by highhomogenization pressures might possibly havedecreased the total amount of protein that was ad-sorbed at the fat globule surfaces. Sharma et al. (21),using electron microscopy, found that the milk fatglobule surfaces were covered with intact caseinmicelles and that partial spreading of casein micellesoccurred. Those researchers also observed smallerparticles, which probably resulted from disintegrationof casein micelles, and a thin layer of protein, whichwas probably whey proteins. Also, the higherhomogenization temperature and the resultant higherkinetic energy may have caused more spreading ofthe casein micelles over the OW interface (25).

Composition of the MFGM Proteins

Differences in the composition of the MFGM pro-teins were not visible among homogenization pres-sures of 30, 60, or 90 MPa within heat treatments orin milks with different concentrations of milk fat.However, differences in the concentration of wheyproteins were observed between heat treatments of 65and 85°C (Figure 1) and between reducing and non-reducing conditions of electrophoresis. Bands for pro-teins with low molecular mass in the region of 20 to11 kDa were observed in the gels of the MFGMproteins of milk samples heated at 65°C under non-reducing and reducing conditions, but these bandswere not present in the gels of milk samples heated at85°C. The mobility of these bands approximately cor-responds to the g fraction of caseins (molecularmasses of 20.5, 11.8, and 11.6 kDa), which might be aresult of plasmin activity on b-CN. This proteolyticenzyme is associated with casein micelles and couldhave survived the heat treatment at 65°C, but not at85°C (25), and be associated with the casein micelles

on the milk fat globule surface. This would explainthe presence of the g-CN in the gels of MFGM pro-teins of milk with lower heat treatment, but not inthe gels of MFGM proteins of milk with the higherheat treatment. In gels of MFGM proteins of milkheated at 85°C and under reducing electrophoresisconditions (Figure 1b), the density of the bands of a-LA and b-LG increased compared with the density ofthe bands of these proteins in milk heated at 65°C(Figure 1a).

The percentage distribution of the proteins formingthe MFGM in milk after homogenization is shown inTable 2. These values represent the composition ofthe membrane proteins of the fraction of the fat glo-bules that were separated under the experimentalconditions. There were no significant differences inthe relative amounts of different plasma proteins ad-sorbed in the OW interface because of homogenizationpressure or milk fat concentration. However, signifi-cant differences were found that were due to heattreatment and reducing conditions during electropho-resis.

Caseins were adsorbed preferentially over wheyproteins at the OW interface and represented about70% of the proteins in the MFGM after homogeniza-tion (Table 2). Several authors (18, 21, 22, 25) havereported preferential adsorption of caseins over wheyproteins at the OW interface in homogenized milk.Sharma et al. (21) reported that caseins constitutedapproximately 90% of the MFGM proteins in recom-bined milk. In recombined milk, milk fat is emulsifiedinto skim milk, and the MFGM material is formedonly by serum proteins. In contrast, in this experi-ment, cream was combined with skim milk, and milkfat globules retained some of the native membraneproteins. Approximately 10% of the proteins in theMFGM formed during homogenization were proteinsfrom the native membrane (Table 2). Keenan et al.(10) found that about 10% of the surface ofhomogenized fat globules in milk was covered by theirnatural membranes. Differences in the concentrationof caseins were only observed after their concentra-tions were determined using reducing and nonreduc-ing electrophoresis (Table 2). This result indicatedthat some caseins interacted with original membraneproteins, whey proteins, or both. These interactionswere probably disulfide linkages because the additionof b-mercaptoethanol caused the increase in the per-centage of caseins in the membrane. Keenan et al.(10) found that casein micelles are associated withmembrane-like material at the interface between fatglobules and the serum phase.

Homogenization pressure did not affect the per-centage of the proteins from the native membrane



TABLE 3. Percentage distribution of individual casein proteins in the milk fat globule membrane(MFGM) of homogenized milk under different experimental conditions.

a,bMeans within a column for each variable without a common superscript differ (P < 0.05).

Variable as2-CN as1-CN b-CN k-CN

Pressure30 MPa 7.98a 26.30a 23.36a 9.93a

60 MPa 8.49a 27.50a 24.40a 8.96a

90 MPa 7.18a 26.80a 24.44a 8.31a

Temperature65°C 7.50a 27.46a 23.29a 9.78a

85°C 8.27a 26.28a 24.84a 8.35a

Fat concentration1.5% 8.08a 26.48a 24.30a 9.43a

3.0% 7.68a 27.25a 23.84a 8.70a

Reducing conditionsNo 6.83b 26.84a 24.58a 6.94b

Yes 8.94a 26.89a 23.55a 11.19a

Figure 2. Interaction between temperature and reducing andnonreducing electrophoretic conditions on the percentage of b-LGand a-LA in the milk fat globule membrane (MFGM) ofhomogenized milk; b-LG, reducing ( — ⁄—); b-LG, nonreducing(---⁄---); a-LA, reducing ( — ÿ—); and a-LA, nonreducing (---ÿ---).

(molecular masses between 50 and 150 kDa) thatwas associated with the milk fat globules afterhomogenization, but differences in the distribution ofthese proteins were found that were due to heat treat-ment of the milk, milk fat concentration, and elec-trophoresis procedure (Table 2). The effects of tem-perature and the changes observed under differentelectrophoresis conditions on the distribution of na-tive membrane proteins can be explained by interac-tions of the native membrane proteins with plasmaproteins. More of the high molecular mass bands (50to 150 kDa) were found at 85 than at 65°C and whenthe protein distribution was determined using non-reducing conditions. When b-mercaptoethanol was ad-ded prior to electrophoresis, the percentage of theseproteins decreased, which indicated that these pro-teins interacted with plasma proteins and caused anapparent increase in the percentage of these proteins.Plasma proteins can form complexes with nativemembrane proteins by formation of disulfide linkagesand appear to increase the relative amount of nativemembrane proteins with high molecular masses inthe MFGM formed after homogenization. Houlihan etal. ( 8 ) found that it was possible for heat-denaturedserum proteins to interact with native membraneproteins.

The percentages of a-LA and b-LG in the MFGMwere affected by temperature, as was observed usingthe gels obtained under nonreducing and reducingconditions of electrophoresis (Table 2). However,homogenization pressure or milk fat concentration didnot affect the relative concentration of these proteinsin the MFGM of homogenized milk. Heat treatment ofthe milk to 85°C caused an increase in a-LA and b-LGas part of the MFGM after homogenization comparedwith the percentage of these proteins in milk heated

at 65°C (Table 2). When the concentrations of a-LAand b-LG were determined using reducing conditions(Table 2), the amount of b-LG forming part of theMFGM was higher than under nonreducing elec-trophoresis conditions (Figure 2), indicating that aninteraction occurred between this protein and thecaseins and native proteins from the original MFGM.The amount of a-LA that was associated with themembrane did not change whether analyzed by reduc-ing or by nonreducing electrophoresis conditions.However, an interaction between temperature andelectrophoresis conditions was found for a-LA. Thepercentage of a-LA decreased as the temperature in-creased from 65 to 85°C when the proteins were de-



TABLE 4. Comparison of ratios of plasma proteins in un-homogenized milk serum to the ratios of plasma proteins in themilk fat globule membrane in milk homogenized at 65 and 85°C.

1Walstra and Jenness (26).2From this work under reducing conditions during electrophore-

sis.3Whey protein ( a-LA and b-LG).

Unhomogenizedmilk serum1

Homogenizedmilk2

Ratios 65°C 85°C

as1-CN/b-CN 1.08 1.23 1.05as2-CN/b-CN 0.28 0.35 0.41as1-CN/as2-CN 3.85 3.55 2.54k-CN/b-CN 0.35 0.53 0.42a-LA/b-LG 0.38 0.39 0.68Caseins/WP3 5.81 9.09 3.54

termined using nonreducing electrophoresis condi-tions (Figure 2). When a-LA was determined usingreducing electrophoresis conditions, its percentage onthe MFGM proteins increased as the heat treatmentincreased to 85°C (Figure 2).

The statistical analysis of the percentage distribu-tion of the individual caseins forming the MFGM inhomogenized milk is shown in Table 3. The percent-ages of as1-CN and b-CN did not change whether thecomposition of the membrane was determined usingreducing or nonreducing electrophoresis conditions,but the percentages of as2-CN and k-CN increasedwhen they were determined using reducing condi-tions. Sharma et al. (21) used dissociating agentsand found that the dissociation of k-CN from the fatglobule surface increased from 25 to 50% with theaddition of b-mercaptoethanol, and the dissociation ofb-LG slightly increased under these conditions.

The distribution of caseins and whey proteins inthe MFGM of homogenized milk was compared withthe distribution of these proteins in the serum phaseof unhomogenized milk; the percentage of individualproteins in the membrane was determined using thesum of the plasma proteins in the MFGM duringreducing conditions as 100%. Ratios of plasma pro-teins in the MFGM of homogenized milk at 65 or 85°Cthat were calculated from the electrophoresis dataunder reducing conditions and the ratios of theseproteins in unhomogenized milk are presented in Ta-ble 4. The ratio of total casein to a-LA and b-LGincreased from 5.81 in unhomogenized milk to 9.09 inmilk that had been homogenized at 65°C, indicatingpreferential adsorption of caseins over whey proteinsafter homogenization at 65°C. Sharma et al. (21)found a casein to whey protein ratio of 8.83 in milk

homogenized at 17 MPa and 52°C. The ratio of caseinto whey protein decreased to 3.54 when the heattreatment was at 85°C. The ratio of a-LA to b-LG inthe MFGM of milk homogenized after heated treat-ment at 65°C was the same as in unhomogenizedmilk, but the ratio of these two proteins increased to0.68 in the MFGM of milk heated at 85°C and thenhomogenized, indicating preferential adsorption of a-LA over b-LG in the OW interface. Sharma and Dal-gleish (22) reported a ratio of a-LA to b-LG of 0.60 inthe MFGM of milk that was heated to 70 to 75°C afterhomogenization.

CONCLUSIONS

The SDS-PAGE analysis of the proteins in theMFGM of homogenized milk suggested two mechan-isms for the adsorption of plasma proteins at the fatglobule surface that were dependent on the heattreatment of the milk prior to homogenization. Heattreatment at 65°C did not significantly denaturewhey proteins, and caseins were adsorbed preferen-tially over whey proteins at the OW interface. Heattreatment of milk at 85°C denatured a-LA and b-LGto the extent that these proteins could form disulfidelinkages with other proteins. The b-LG mainly wasattached to k-CN in the casein micelles or to otherunidentified protein, and these complexes were ad-sorbed at the OW interface during homogenization.Because the G decreased when the heat treatment ofthe milk increased from 65 to 85°C, even when b-LGwas attached to the casein micelles, the binding sitebetween the fat globule surface and the plasma pro-teins possibly was through the b-LG and k-CN com-plex; spreading of this complex was an additionalpossibility. Also, the decrease in G as heating temper-ature increased could have been the result of spread-ing of the plasma proteins at the OW interface be-cause of the higher kinetic energy of the system. At85°C, the adsorption of a-LA and b-LG directly at thefat surface would limit the available surface for ad-sorption of caseins, which would reduce the ratio ofcaseins to whey proteins in the MFGM ofhomogenized milk.

ACKNOWLEDGMENTS

The authors acknowledge Dairy Management Inc.for its financial support and CONACYT (NationalCouncil of Science and Technology of Mexico) for itssupport through a scholarship for M. E. Cano-Ruiz.

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effect of homogenization pressure on the milk

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