Distribution and carbohydrate structures of high molecular weight glycoproteins, MUC1 and MUCX, in bovine milk

High molecular weight glycoproteins, MUC1 and MUCX, originating from bovine milk, were compared with regard to their distribution in milk fat and skim milk fractions and for presence of carbohydrate structures. Polymorphic MUC1, which migrated into 6% resolving SDS-PAGE gels, was found in both milk fat globule membrane and skim milk phases of bovine milk. In contrast, MUCX, appearing as a non-polymorphic single band in 3% polyacrylamide stacking gels, was present only in the skim milk fraction. Peptide-N-glycosidase F digestion studies indicated that MUC1 and MUCX possessed N-glycans with MUC1 containing more N-glycans than MUCX. Exoglycosidase digestion studies revealed the existence of abundant terminal sialic acid residues in both MUC1 and MUCX. Lectin-binding studies showed that MUCX likely possessed more complex carbohydrate structures than MUC1. The complex carbohydrate structures carried by both MUC1 and MUCX suggest that they may have potential to bind a wide spectrum of pathogenic microorganisms. If that proves to be the case in vivo, such structures could have a role in preventing or reducing some infectious diseases.     

Purification of MUC1 from bovine milk-fat globules and characterization of a corresponding full-length cDNA clone

The highly glycosylated protein MUC1 was purified from bovine milk-fat globule membranes by a procedure involving detergent extraction, ion-exchange chromatography and reverse-phase chromatography. The identity of the purified mucin protein was confirmed by N-terminal sequencing and partial amino acid sequences obtained by peptide mapping. The complete amino acid sequence of MUC1 was determined by cloning and sequencing the corresponding bovine mammary gland cDNA, which was shown to encode a protein of 580 amino acid residues comprising a cleavable signal peptide of 22 residues. The deduced amino acid sequence demonstrated structural features characteristic for mucins, including an extracellular tandem repeat region with 11 partially conserved repeats (20 amino acids each), a membrane-proximal SEA module, a transmembrane domain, and a cytoplasmic C-terminal region. Monosaccharide composition determinations suggested significant structural differences between O-linked glycans of MUC1 originating from either bovine or human milk. Interspecies differences of the consensus repeat sequence in MUC1 and the physiological functions are discussed.     

Major proteins of the goat milk fat globule membrane

Fat is present in milk as droplets of triglycerides surrounded by a complex membrane derived from the mammary epithelial cell called milk fat globule membrane (MFGM). Although numerous studies have been published on human or bovine MFGM proteins, to date few studies exist on MFGM proteins from goat milk. The objective of this study was thus to investigate the protein composition of the goat MFGM. Milk fat globule membrane proteins from goat milk were separated by 6% and 10% sodium dodecyl sulfate-PAGE and were Coomassie or periodic acid-Schiff stained. Most of MFGM proteins [mucin-1, fatty acid synthase, xanthine oxidase, butyrophilin, lactadherin (MFG EGF-8, MFG-E8), and adipophilin] already described in cow milk were identified in goat milk using peptide mass fingerprinting. In addition, lectin staining provided a preliminary characterization of carbohydrate structures occurring on MFGM proteins from goat milk depending on α_S1-casein genotype and lactation stage. We provide here first evidence of the presence of O-glycans on fatty acid synthase and xanthine oxidase from goat milk. A prominent difference between the cow and the goat species was demonstrated for lactadherin. Indeed, whereas 2 polypeptide chains were easily identified by peptide mass fingerprinting matrix-assisted laser desorption/ionization-time of flight analysis within bovine MFGM proteins, lactadherin from goat milk consisted of a single polypeptide chain. Another striking observation was the presence of caseins associated with MFGM preparations from goat milk, whereas virtually no caseins were found in MFGM extracts from bovine milk. Taken together, these observations strongly support the existence of a singular secretion mode previously hypothesized in the goat.     

Invited review: Bovine milk fat globule membrane as a potential nutraceutical

For the last 15 yr, a great deal of knowledge has been accumulated on health beneficial factors, protein and nonprotein, of bovine milk fat globule membrane (MFGM). Among the health-beneficial components of the MFGM are cholesterolemia-lowering factor, inhibitors of cancer cell growth, vitamin binders, inhibitor of Helicobacter pylori, inhibitor of beta-glucuronidase of the intestinal Escherichia coli, xanthine oxidase as a bactericidal agent, butyrophilin as a possible suppressor of multiple sclerosis, and phospholipids as agents against colon cancer, gastrointestinal pathogens, Alzheimer's disease, depression, and stress. All of the above compel us to consider bovine MFGM as a potential nutraceutical.     

Comparison of the antipathogenic effect toward Staphylococcus aureus of N-linked and free oligosaccharides derived from human,bovine, and goat milk

N-linked oligosaccharides (N-glycans) derived from milk were recently found to be antipathogenic. This study compares the antimicrobial activity of N-linked glycans and free oligosaccharides from human, bovine, and goat milk against Staphylococcus aureus. Milk N-glycans showed a bactericidal/bacteriostatic effect on the pathogen when compared to free milk oligosaccharides, evidenced by the clear zone from the halo assay, with the order of human milk >goat milk >bovine milk. None of the free milk oligosaccharide samples were bactericidal/bacteriostatic, despite its positive results in growth curve and minimum inhibitory concentration (MIC) assays which are believed to be related to hyperosmosis. Both N-glycans and free milk oligosaccharides can reduce the adhesion of Staphylococcus aureus to Caco-2 cells, however, N-glycans worked significantly more effective than free milk oligosaccharides. Structural analysis of all free oligosaccharide and N-glycan samples showed the obvious interspecies differences, and the structure/function relationship of the respected N-glycans is of interest for future study. The significant bactericidal/bacteriostatic activity possessed by human, bovine, and goat milk N-linked glycans holds great potential as a novel substitute for antibiotics.     

Characterization of bovine neutrophil gelatinase-associated lipocalin

A protein of relative molecular mass of approximately 25,000 was purified from bovine colostrum by cation-exchange and size-exclusion chromatography. The N-terminus of the protein matched the sequence predicted by the National Center for Biotechnology Information for the bovine homolog of human neutrophil gelatinase-associated lipocalin, a glycoprotein of relative molecular mass 25,000 belonging to the family of lipocalins. The protein was further designated as bovine neutrophil gelatinase-associated lipocalin (bNGAL). Sodium dodecyl sulfate-PAGE of enzymically deglycosylated bNGAL indicated that the intact protein bears one N-linked glycan. Monosaccharide and mass spectrometric analyses of released N-linked carbohydrates revealed the presences of complex- and hybrid-type glycans, with galactose substituted with N-acetylgalactosamine. This substitution is typical for glycoproteins expressed in the bovine mammary gland. A specific ELISA revealed bNGAL concentrations in plasma and mature milk of about 0.05 and 1 μg/mL, respectively, whereas values as high as 51 μg/mL were measured in colostrum. Thus, we have isolated and characterized a novel bovine (milk) protein that is a new member of the lipocalin family.     

Bovine milk glycome

Bovine milk oligosaccharides have several potentially important biological activities including the prevention of pathogen binding to the intestinal epithelial and as nutrients for beneficial bacteria. It has been suggested that milk oligosaccharides are an important source of complex carbohydrates as supplements for the food and the pharmaceutical industries. However, only a small number of structures of bovine milk oligosaccharides (bMO) are known. There have been no systematic studies on bMO. High-performance mass spectrometry and separation methods are used to evaluate bMO, and nearly 40 oligosaccharides are present in bovine milk. Bovine milk oligosaccharides are composed of shorter oligomeric chains than are those in human milk. They are significantly more anionic with nearly 70%, measured abundances, being sialylated. Additionally, bMO are built not only on the lactose core (as are nearly all human milk oligosaccharides), but also on lactose amines. Sialic acid residues include both N-acetyl and N-glycolylneuraminic acid, although the former is significantly more abundant.     

Distribution and fatty acid content of phospholipids from bovine milk and bovine milk fat globule membranes

The phospholipid (PL) content was determined comparatively in the milk fat globule membrane (MFGM) and whole milk including their fatty acid profiles. The possible role of milk PLs in defence against pathogens was also addressed. The MFGM and whole milk showed a similar distribution of PL species; however, the fatty acid contents of the PL species were different. Total PL from MFGM showed a decrease in C18:0 content in parallel with an increase in C18:1 and C18:2 and very long-chain fatty acid (more than C20) content. No significant differences in the fatty acid content of phosphatidylcholine and sphingomyelin from either source were found. However, the phosphatidylethanolamine from MFGM had more C18:1 and C18:2 and less C14:0 and C16:0 than that from whole milk. A similar but less pronounced result was found for phosphatidylserine/phosphatidylinositol. Enterotoxigenic Escherichia coli strains failed to bind to PL, which had been previously separated by high-performance thin-layer chromatography.     

Detection of bovine milk adulteration in caprine milk with N-acetyl carbohydrate biomarkers by using 1H nuclear magnetic resonance spectroscopy

In a return to tradition, the popularity of caprine milk is on the rise. However, particularly in countries with developed dairy industries based on bovine milk, there is the risk of adulteration with bovine milk, which is a cheaper alternative. Thus, a rapid, robust, and simple method for the detection of bovine milk added to caprine milk is necessary, and ¹H nuclear magnetic resonance spectroscopy appears to provide a solution. A matrix of 115 pure and artificially adulterated pasteurized milk samples was prepared and used to discover biomarkers of bovine milk that are independent of chemical and biological variation caused by factors such as genetics, diet, or seasonality. Principal component analysis and orthogonal projections to latent structures discriminant analysis of pure bovine milk and pure caprine milk revealed spectral features that were assigned to the resonances of 4 molecules. Of these, the peaks corresponding to protons in the N-acetylglucosamine and N-acetylgalactosamine acetyl moieties showed significant applicability for our method. Receiver operating characteristic curve analysis was used to evaluate the performance of the peak integrals as biomarkers of adulteration. This approach was able to distinguish caprine milk adulterated with 5% of bovine milk with 84.78% accuracy and with 10% of bovine milk an excellent 95.65% accuracy. This study demonstrates that N-acetyl carbohydrates could be used as biomarkers for the detection of bovine milk in caprine milk and could help in protecting caprine milk authenticity.     

母乳和牛乳中乳脂肪球膜蛋白质的差异分析

为比较乳脂肪球膜（milk fat globule membrane,MFGM）蛋白在母乳和牛乳中的差异,采用有机试剂提取法,从母乳和牛乳中提取分离出MFGM蛋白,经皮尔斯TM去垢小柱去除十二烷基硫酸钠后,通过液相色谱-质谱联用技术检测分析,检测结果根据特异性肽段匹配数不小于1筛选后,在母乳和牛乳中分别检测到863 种和454 种蛋白,其中二者共有175 种,分别独有688 种和279 种。将母乳中差异表达的688 种蛋白质按PANTHER数据库进行蛋白质的Gene Ontology（GO）功能注释,这些蛋白主要是细胞部分、细胞器、细胞膜等组分,参与的生物途径有新陈代谢过程、生物调节、刺激性反应、免疫系统过程、再生作用等,具有催化、黏合、转运、酶调节等多种分子功能。另外,母乳中的这些差异蛋白参与氨酰tRNA生物合成、致病性大肠杆菌感染、脂肪酸代谢、丙酸代谢、甾体合成、酸降解等都是牛乳中的乳脂肪球膜蛋白不具有的功能。通过分析得出母乳和牛乳中的MFGM蛋白具有明显差异,虽然有部分组成和功能的重叠,但在丰度和代谢路径上,牛乳MFGM蛋白不能替代母乳MFGM蛋白。     

Glycosidase activities in bovine milk over lactation

The presence and activity of glycosidases in Irish bovine milk over three months of lactation was investigated. A low level of variation between animals was observed and the highest level of glycosidase activity was present in colostrum, decreasing through transitional milk production to minimal but constant levels in mature milk. N-acetyl-β-d-glucosaminidase, α-l-fucosidase, α-galactosidase and N-acetyl-neuraminidase appear to be the most biologically relevant glycosidases in bovine milk. The elevated levels of enzymatic activity in colostrum suggests the milk glycosidases may play a role in the digestion of bovine milk glycans in the infant mammal, possibly acting as substitutes for bacterial glycosidases prior to colonisation by the gut microflora, which are involved in in vivo oligosaccharide metabolism in the lower gastrointestinal tract.     

Invited review: Anti-adhesive properties of bovine oligosaccharides and bovine milk fat globule membrane-associated glycoconjugates against bacterial food enteropathogens

The prevalence of the main raw milk and raw milk–derived dairy product enteropathogens (Campylobacter, Shiga toxin-producing Escherichia coli, Listeria, and Salmonella) is higher than the number of epidemiological cases related to ingesting these foodstuffs. Bovine milk oligosaccharides and milk fat globule membrane (MFGM)-linked glycoconjugates interact with foodborne enteropathogens to inhibit their adhesion to intestinal cells and tissues. This review examines the main mechanisms and strategies used by enteropathogens to adhere to their target, details the anti-adhesive properties of MFGM against enteropathogens and enterotoxins, assesses the integrity of bacteria–MFGM complexes during dairy product manufacture and digestion, and discusses the potential for using these macromolecules and glycoconjugates in foods for public health.     

Sialyloligosaccharides in human and bovine milk and in infant formulas: variations with the progression of lactation

Several lines of research support a role for human milk oligosaccharides in the defense of breast-fed infants against pathogens. Some ofthese oligosaccharides contain at least one moiety of sialic acid and are, thus, termed sialyloligosaccharides. These constitute a significant component (>1 g/L) of human milk. It is well established that milk composition varies among species, and previous reports have indicated that one ofthe differences between human and bovine milk is precisely their contents of sialyloligosaccharides. Because most infant formulas are manufactured with bovine milk components, it follows that formula-fed and breast-fed infants ingest dissimilar quantities of these carbohydrate structures. To ascertain these differences and their impact along lactation, the contents of oligosaccharide-bound sialic acids and major sialyloligosaccharides in samples of human and bovine milk (obtained at different lactation stages) were determined. In addition, infant formulas were assayed for their sialyloligosaccharide contents. Seven sialyloligosaccharides were identified in human milk; namely, 3'-sialyl-3-fucosyllactose and sialyllacto-N-tetraoses (a and b+c), the predominant structures at all lactation stages. Five sialyloligosaccharides were identified in bovine milk, of which 6'-sialyllactosamine and 3'-sialyllactose were the most abundant. In addition, sialyloligosaccharides in human and bovine milk decreased along lactation, and infant formulas did not contain significant amounts of sialyloligosaccharides. The results point to the general conclusion that regarding both qualitative and quantitative aspects, milk from humans and cows and infant formulas have different oligosaccharide contents. In this sense, bottle-fed infants are subject to reduced sialyloligosaccharide intake as compared to breast-fed infants.     

Impact of different milk fat globule membrane preparations on protein composition,xanthine oxidase activity,and redox potential

The protein composition, redox potential (E_h), and xanthine oxidase (XO) content and activity were determined for anhydrous milk fat emulsions containing milk fat globule membrane (MFGM) fractions derived from either buttermilk or commercial sources of bovine α-serum, β-serum, and buttermilk powder (BMP). Caseins were the dominant proteins in the MFGM fractions isolated from BMP and buttermilk, whereas fractions from α- and β-serum contained higher amounts of membrane proteins. The XO content and activity was >70-fold and >700-fold higher in α- and β-serum samples, respectively, compared with the BMP fraction. The E_h values of the recombined emulsions were highest for α-serum (196 mV), and β-serum (169 mV), followed by BMP (131 mV). These positive values contrasted with the highly negative E_h of the buttermilk emulsion (−580 mV). This study demonstrates how milk-processing methods can alter the composition and functionality of the MFGM.     

Lipid rafts in the bovine milk fat globule membrane revealed by the lateral segregation of phospholipids and heterogeneous distribution of glycoproteins

This study reveals the lateral organisation of the milk fat globule membrane (MFGM). Using confocal laser scanning microscopy (CLSM) and a lipid soluble molecule, an exogenous phospholipid and two lectins as fluorescent probes we located triacylglycerols in the core of fat globules and investigated the organisation of the polar lipids and glycoproteins of the MFGM, in situ in milk. Lipid rafts corresponding to the lateral segregation of sphingolipids in liquid-ordered phases surrounded by liquid-disordered domains composed by the glycerophospholipids were observed in the MFGM. These lipid rafts which correspond to rigid sphingolipid-rich domains have a circular shape at room temperature. CLSM experiments revealed that glycoproteins and glycolipids are heterogeneously distributed around fat globules and that they are not located in the lipid rafts. The characterisations performed by in depth thin sectioning of fat globules and in dynamic as a function of time revealed chemical and structural heterogeneities in the MFGM. Schematic 3D and 2D representations of the MFGM are proposed and discussed. The physiological and nutritional consequences of the lateral organisation of polar lipids and glycoproteins in the MFGM are discussed but remain to be elucidated.     

Inhibitory effects of human and bovine milk constituents on rotavirus infections

Among etiologic agents, rotavirus is the major cause of severe dehydration diarrhea in infant mammals. In vitro and in vivo studies have indicated that the human milk-fat globule protein lactadherin inhibits rotavirus binding and protects breast-fed children against symptomatic rotavirus infection. The present work was conducted to evaluate the effect of lactadherin, along with some other milk proteins and fractions, on rotavirus infections in MA104 and Caco-2 cell lines. It is shown that human, and not bovine, lactadherin inhibits Wa rotavirus infection in vitro. Human lactadherin seems to act through a mechanism involving protein-virus interactions. The reason for the activity of human lactadherin is not clear, but it might lie within differences in the protein structure or the attached oligosaccharides. Likewise, in our hands, bovine lactoferrin did not show any suppressive activity against rotavirus. In contrast, MUC1 from bovine milk inhibits the neuraminidase-sensitive rotavirus RRV strain efficiently, whereas it has no effect on the neuraminidase-resistant Wa strain. Finally, a bovine macromolecular whey protein fraction turned out to have an efficient and versatile inhibitory activity against rotavirus.     

Fat globules selected from whole milk according to their size: Different compositions and structure of the biomembrane,revealing sphingomyelin-rich domains

Milk fat globules are unique delivery systems for biologically active molecules in the gastrointestinal tract. However, their properties have not yet been fully investigated. In this study, we performed a comparative analysis of the polar lipid and fatty acid compositions of milk fat globules as a function of their size and investigated the structure of the milk fat globule membrane (MFGM). An optimised process of microfiltration was used to select the small milk fat globule (SMFG; 1.6 μm) fractions and the large milk fat globule (LMFG; 6.6 μm) fractions from the same initial whole milks (4.2 μm). The SMFG-fractions contained significantly (i) higher amounts of polar lipids, 8.9 ± 0.9 vs 2.7 ± 0.3 mg/g fat for LMFG-fractions and 6.3 ± 0.5 mg/g fat for whole milks, (ii) lower relative proportions of phosphatidylcholine and sphingomyelin in the MFGM, (iii) higher amounts of C12:0, C14:0, C16:0, C18:1 trans, C18:2 c9 tr11, and lower amounts of C18:0 and C18:1 c9 than did LMFG-fractions and whole milks. Whatever the size of native milk fat globules, the biophysical characterisation performed in-situ, using confocal laser scanning microscopy, showed heterogeneities in the MFGM. The lateral segregation of sphingomyelin in rigid liquid-ordered domains, surrounded by the fluid matrix of glycerophospholipids in the liquid-disordered phase, was revealed. The heterogeneous distribution of glycolipids and glycoproteins was also observed in the MFGM. A new model for the structure of the MFGM is proposed and discussed. The physical, chemical and biological consequences, (i) of the differences in milk fat globule compositions according to their size and (ii) of the specific structure of the MFGM due to sphingomyelin remain to be elucidated.     

Rapid,quantitative analysis of 3′- and 6′-sialyllactose in milk by flow-injection analysis–mass spectrometry: Screening of milks for naturally elevated sialyllactose concentration

Non-protein-bound oligosaccharides are important bioactive components of cow milk, with potential human-health benefits such as stimulation of the growth of beneficial gut bacteria and defense against pathogens. In bovine milk, the majority of oligosaccharides are sialylated; 3′-sialyllactose (3′-N-acetylneuraminyl-d-lactose; 3′-SL) is the predominant sialylated oligosaccharide, followed by 6′-sialyllactose (6′-N-acetylneuraminyl-d-lactose; 6′-SL). Both 3′-SL and 6′-SL have antimicrobial activity. As bovine milk products such as infant formula can be an important component of the human diet, and the concentrations of 3′-SL and 6′-SL are lower in bovine milk compared with human milk, we aimed to identify cows that naturally produce higher concentrations of sialyllactose in their milk. Milk from such cows could be used to produce foods with an increased sialyllactose content, potentially providing increased health benefits. We speculated that cows overexpressing 3′-SL and 6′-SL would exist at low frequency in the population and, to allow their efficient identification, we developed a novel assay for 3′-SL and 6′-SL utilizing flow-injection analysis-mass spectrometry, which could be used for high-throughput analysis of milk samples. We then determined 3′-SL and 6′-SL concentrations in milk samples from 15,507 cows from Friesian, Jersey, and Friesian-Jersey crossbred animals. We found 329 cows with concentrations of 3′-SL or 6′-SL >2-fold higher than the mean, 26 cows with concentrations of 3′-SL or 6′-SL >3-fold higher than the mean, and 1 cow with concentrations of 3′-SL >4-fold higher than the mean. Although these outliers were observed across the 3 groups of cows, breed had a strong effect on mean 3′-SL and 6′-SL concentrations.     

Inhibitory activities of bovine macromolecular whey proteins on rotavirus infections in vitro and in vivo

Rotavirus is a major cause of infantile viral gastroenteritis and can lead to severe and sometimes lethal dehydration. Previous studies have shown that breast-fed children are better protected against symptomatic infections, and that the milk fat globule protein lactadherin might be at least partly responsible for this effect. In vitro studies have shown that human lactadherin, in contrast to the bovine ortholog, could inhibit rotavirus infectivity, and that bovine MUC1 and a commercially available bovine macromolecular whey protein (MMWP) fraction proved to be effective. The present work describes the versatility of MMWP against the infection of 2 human intestinal cell lines (Caco-2 and FHs 74 Int) by 4 different rotavirus strains (Wa, RRV, YM, RF). Isolation of a protein fraction (CM3Q3) from MMWP that effectively inhibits rotavirus infectivity in vitro is documented. Purification was achieved by monitoring the rotaviral inhibitory activity in fractions obtained from 2 consecutive steps of ion-exchange chromatography. The major component of CM3Q3 was shown to be bovine IgG, and the attenuating capacity of this fraction is most properly linked to this component. The capacity of MMWP, MUC1, lactadherin, and the CM3Q3 fraction to inhibit the infectivity of the murine EMcN rotavirus strain was analyzed in adult BALB/c mice by using 2 different amounts of virus (10 and 100 times more than 50% the viral shedding doses). Only CM3Q3 was able to significantly affect the shedding of rotavirus in the stools of experimentally infected mice when the high viral dose was given. Detection of rotavirus-specific serum antibodies showed that the high dose infected all groups of mice. Experiments with the low dose of virus implied that all the tested milk proteins could affect the viral shedding in stools; in addition, use of MUC1, MMWP, and CM3Q3 prevented the appearance of serum viral antibodies. The advantages of using bovine immunoglobulins to induce passive immunity against rotavirus have been substantially investigated, although studies have mainly focused on the use of derivatives from immunized cows, especially colostrum. This report associates considerable activity against rotavirus infectivity with an ordinary whey product, suggesting that there might be alternatives to colostral-derived products.     

不同均质工艺处理对巴氏杀菌乳脂肪球膜蛋白质组成的影响

乳脂肪球大小和膜蛋白组分的变化决定了脂肪球稳定性,从而影响巴氏杀菌乳在贮藏期内的品质稳定性。本研究通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和液相色谱-质谱联用法分析不同预热温度（50、60、70?℃）和不同均质压力（20、30、40、45?MPa）对脂肪球膜蛋白的影响,并通过稳定性分析仪探究脂肪球膜蛋白变化与稳定性的关系。结果表明,均质工艺对脂肪球膜蛋白种类无显著影响,但对脂肪球膜蛋白的含量有一定影响：黄嘌呤氧化还原酶（xanthine dehydrogenase/oxidase,XO）、嗜乳脂蛋白（butyrophilin,BTN）、脂肪滴结合蛋白（adipophilin,ADPH）在预热60 ℃时的含量最低,黏液素（mucin 1,MUC1）在预热60 ℃时的含量最高,脂肪酸结合蛋白（fatty acid binding protein,FABP）、组织糖蛋白高碘酸烯夫6/7（periodic acid Schiff 6/7,PAS6/7）的含量随着预热温度的升高而减少,组织糖蛋白高碘酸薛夫IV（cluster of differentiation 36,CD36）的含量随着预热温度的升高而增多;XO、PAS6/7、BTN、ADPH、FABP在均质压力40 MPa时含量最低,CD36的含量随均质压力的增加而减少;脂肪球膜蛋白构成中,牛血清白蛋白（bovine serum albumin,BSA）、αs1-酪蛋白（casein,CN）和β-CN含量增加,其中BSA含量增加最明显。综上,推测均质工艺导致αs1-CN、β-CN和BSA组分参与了脂肪球膜的构建,并对脂肪球稳定性产生了积极的影响。