Symposium review: Fat globules in milk and their structural modifications during gastrointestinal digestion

The fat globules in milk are unique oil droplets that are stabilized by a specific and structurally complex membrane, the milk fat globule membrane (MFGM). In the last decade, excellent progress has been made on studying the structure of the milk fat globules and the MFGM and how common processing treatments affect these structures to deliver dairy products with improved functional properties. Although the digestion of milk fat to deliver energy and lipid-soluble nutrients is essential for survival of the neonate, there is little understanding of the complex processes involved. The structural alterations to fat globules during gastrointestinal processing affect the way in which milk fat is digested, absorbed, and metabolized. The packaging of these globules within the MFGM or in other forms may affect the bioaccessibility of raw or processed milk fat globules; in turn, this may affect access of the gastrointestinal enzymes to the globules and, therefore, may influence the rate and extent of lipid digestion. This review focuses on recent advances in understanding milk fat globules during gastrointestinal digestion, including the effects of processing on their bioavailability and the kinetics of lipid digestion. Possible effects of the dairy matrix on lipid digestion and physiological responses are briefly described.     

Native vs. damaged milk fat globules: membrane properties affect the viscoelasticity of milk gels

The storage modulus G' of rennet and acid milk gels filled with milk fat globules was measured as a function of the fat globule surface composition (native milk fat globule membrane, caseins and whey proteins, or a mixture of the three due to mechanical treatments) and surface area (i.e., the fat globule size). By different technological procedures, it was possible to obtain fat globules of constant surface composition but various sizes, and vice-versa, which had never been done. For both rennet and acid gels, a critical fraction of the fat globule surface covered by caseins and whey proteins was identified (approximately 40%), beyond which G' increased. Below this threshold, the gel viscoelasticity was unaffected by mechanical treatments. When the diameter of native milk fat globules decreased, the G' of rennet gels increased slightly, whereas that of acid gels decreased sharply. For both types of gels, G' increased when the diameter of partially disrupted fat globules decreased. For recombined globules completely covered with caseins and few whey proteins, G' increased with fat globule surface area for rennet gels whereas it decreased for acid gels. With the help of confocal microscopy and in the light of general structural differences between rennet and acid gels, a mechanism is proposed for the effect of fat globule damage and diameter on G', depending on the interactions the globules can undergo with the casein network.     

Buffalo vs. cow milk fat globules: Size distribution,zeta-potential,compositions in total fatty acids and in polar lipids from the milk fat globule membrane

Although buffalo milk is the second most produced milk in the world, and of primary nutritional importance in various parts of the world, few studies have focused on the physicochemical properties of buffalo milk fat globules. This study is a comparative analysis of buffalo and cow milk fat globules. The larger size of buffalo fat globules, 5 vs. 3.5 μm, was related to the higher amount of fat in the buffalo milks: 73.4 ± 9.9 vs. 41.3 ± 3.7 g/kg for cow milk. Buffalo milks contained significantly lower amount of polar lipids expressed per gram of lipids (0.26% vs. 0.36%), but significantly higher amount of polar lipids per litre of milk (+26%). Buffalo and cow milk fat globule membranes contain the same classes of polar lipids; phosphatidylethanolamine, sphingomyelin (SM) and phosphatidylcholine (PC) being the main constituents. A significant higher percentage of PC and lower percentage of SM were found for buffalo milks. The fatty acid analysis revealed that saturated fatty acids, mainly palmitic acid, trans fatty acids, linolenic acid (ω3) and conjugated linolenic acid were higher in buffalo milk than in cow milk. Such results will contribute to the improvement of the quality of buffalo milk-based dairy products.     

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.     

Technical note: Characterizing individual milk fat globules with holographic video microscopy

We used in-line holography to create images of individual milk fat globules in diluted samples of milk. Analyzing these images with the exact Lorenz-Mie light scattering theory then yields the droplets’ radii with nanometer resolution and their refractive indexes to within one part in a thousand. This procedure rapidly and directly characterizes both the quantity and quality of fat in milk.     

Comparison of emulsifying properties of milk fat globule membrane materials isolated from different dairy by-products

Emulsifying properties of milk fat globule membrane (MFGM) materials isolated from reconstituted buttermilk (BM; i.e., BM-MFGM) and BM whey (i.e., whey-MFGM), individually or in mixtures with BM powder (BMP) were compared with those of a commercial dairy ingredient (Lacprodan PL-20; Arla Foods Ingredients Group P/S, Viby, Denmark), a material rich in milk polar lipids and proteins. The particle size distribution, viscosity, interfacial protein, and polar lipids load of oil-in-water emulsions prepared using soybean oil were examined. Pronounced droplet aggregation was observed with emulsions stabilized with whey-MFGM or with a mixture of whey-MFGM and BMP. No aggregation was observed for emulsions stabilized with BM-MFGM, Lacprodan PL-20, or a mixture of BM-MFGM and BMP. The surface protein load and polar lipids load were lowest in emulsions with BM-MFGM. The highest protein load and polar lipids load were observed for emulsions made with a mixture of whey-MFGM and BMP. The differences in composition of MFGM materials, such as in whey proteins, caseins, MFGM-specific proteins, polar lipids, minerals, and especially their possible interactions determine their emulsifying properties.     

Membrane-rich milk fat diet provides protection against gastrointestinal leakiness in mice treated with lipopolysaccharide

Milk fat globule membrane is a protein-lipid complex that may strengthen the gut barrier. The main objective of this study was to assess the ability of a membrane-rich milk fat diet to promote the integrity of the gut barrier and to decrease systemic inflammation in lipopolysaccharide (LPS)-challenged mice. Animals were randomly assigned to one of 2 American Institute of Nutrition (AIN)-76A formulations differing only in fat source: control diet (corn oil) and milk fat diet (anhydrous milk fat with 10% milk fat globule membrane). Each diet contained 12% calories from fat. Mice were fed diets for 5 wk, then injected with vehicle or LPS (10 mg/kg of BW) and gavaged with dextran-fluorescein to assess gut barrier integrity. Serum was assayed for fluorescence 24 h after gavage, and 16 serum cytokines were measured to assess the inflammatory response. Gut permeability was 1.8-fold higher in LPS-challenged mice fed the control diet compared with the milk fat diet. Furthermore, mice fed the milk fat diet and injected with LPS had lower serum levels of IL-6, IL-10, IL-17, monocyte chemotactic protein (MCP)-1, interferon (IFN)-γ, tumor necrosis factor (TNF)-α, and IL-3 compared with LPS-injected mice fed the control diet. The results indicate that the membrane-rich milk fat diet decreases the inflammatory response to a systemic LPS challenge compared with corn oil, and the effect coincides with decreased gut permeability.     

Size distribution of fat globules in human colostrum, breast milk, and infant formula

Only a few results are available on the size of human milk fat globules (MFG), despite its significance regarding fat digestion in the infant, and no data are available at <24 h postpartum (PP). We measured the MFG size distribution in colostrum and transitional human milk in comparison with fat globules of mature milk and infant formula. Colostrum and transitional milk samples from 18 mothers were collected regularly during 4 d PP and compared with mature milk samples of 17 different mothers and 4 infant formulas. The size distribution was measured by laser light scattering. For further characterization, the ζ-potential of some mature MFG was measured by laser Doppler electrophoresis. The MFG diameter decreased sigmoidally in the first days. At <12 h PP, the mode diameter was 8.9 ± 1.0 μm vs 2.8 ±0.3 μm at 96 h PP. Thus, the surface area of MFG increased from 1.1 ±0.3 to 5.4 ±0.7 m²/g between colostrum and transitional milk. In mature milk, the MFG diameter was 4 μm on average and increased with advancing lactation, whereas the droplets in infant formula measured 0.4 μm. The ζ potential of mature MFG was −7.8 ± 0.1 mV. The fat globules are larger in early colostrum than in transitional and mature human milk and in contrast with the small-sized fat droplets in infant formula. Human MFG also have a low negative surface charge compared with bovine globules. These structural differences can be of nutritional significance for the infant.     

Identification of ABCA1 and ABCG1 in milk fat globules and mammary cells--implications for milk cholesterol secretion

The ATP-binding cassette (ABC) transporters ABCA1 and ABCG1 play an important role in cellular cholesterol homeostasis, but their function in mammary gland (MG) tissue remains elusive. A bovine MG model that allows repeated MG sampling in identical animals at different functional stages was used to test whether 1) ABCA1 and ABCG1 protein expression and subcellular localization in mammary epithelial cells (MEC) change during the pregnancy-lactation cycle, and 2) these 2 proteins were present in milk fat globules (MFG). Expression and localization in MEC were investigated in bovine MG tissues at the end of lactation, during the dry period (DP), and early lactation using immunohistochemical and immunofluorescence approaches. The presence of ABCA1 and ABCG1 in MFG isolated from fresh milk was determined by immunofluorescence. The ABCA1 protein expression in MEC, expressed as arbitrary units, was higher during the end of lactation (12.2 ± 0.24) and the DP (12.5 ± 0.22) as compared with during early lactation (10.2 ± 0.65). In contrast, no significant change in ABCG1 expression existed between the stages. Throughout the cycle, ABCA1 and ABCG1 were detected in the apical (41.9 ± 24.8 and 49.0 ± 4.96% of cows, respectively), basal (56.2 ± 28.1 and 54.6 ± 7.78% of cows, respectively), or entire cytoplasm (56.8 ± 13.4 and 61.6 ± 14.4% of cows, respectively) of MEC, or showed combined localization. Unlike ABCG1, ABCA1 was absent at the apical aspect of MEC during early lactation. Immunolabeling experiments revealed the presence of ABCA1 and ABCG1 in MFG membranes. Findings suggest a differential, functional stage-dependent role of ABCA1 and ABCG1 in cholesterol homeostasis of the MG epithelium. The presence of ABCA1 and ABCG1 in MFG membranes suggests that these proteins are involved in cholesterol exchange between MEC and alveolar milk.     

Effect of nonthermal technologies on the native size distribution of fat globules in bovine cheese-making milk

Milk-fat globule membranes are susceptible to damage by mechanical and thermal processes. This damage is translated into alterations of milk fat structure and functionality of cheese-making milk. The objective of this work was to evaluate the effect of pulsed electrical fields (PEF), high hydrostatic pressure (HHP), and conventional thermal treatments on fat globule size distribution and ζ-potential. Milk was processed by HHP at 400 and 500 MPa for 0–20 min, and with PEF at 36 kV/cm and 42 kV/cm up to 64 pulses. The ζ-potential of HHP and PEF treated milk were − 15.47 mV and − 14.63 mV respectively. HHP treatments induced fat globules flocculation, increasing their mass moment mean diameter. Although PEF processing did not modify the true mean diameter of MFG, it induced small globules to clump together, causing an apparent increment in the population of larger milk-fat globules.

Industrial relevance

The market for traditional raw dairy products has increased in recent times in several regions of the world due to their unique flavor and texture attributes. However, the potential negative implications of consuming raw products limit the growth of this market segment. Manufacture of raw-like cheese from thermally pasteurized milk is not feasible, among other things, because of milk fat globule membrane damage caused by elevated temperatures. Nonthermal food preservation technologies offer the potential to produce milk technically suitable for the industrial manufacture of microbiologically safe raw-like dairy products.     

Changes in the surface protein of the fat globules during ultra-high pressure homogenisation and conventional treatments of milk

Disruption of fat globules upon homogenisation provokes a reduction of their size and a concomitant increase in their specific surface area. In order to overcome this phenomenon, the milk fat globule membrane (MFGM) adsorbs non-native MFGM proteins. The aim of the present study was to examine the effects of UHPH conditions (temperature and pressure) on the milk fat globule and the surface proteins by comparison with conventional treatments applied in the dairy industry. Transmission electron microscopy and SDS-PAGE revealed major differences. In UHPH-treated milk, casein micelles were found to be adsorbed on the MFGM in a lesser extent than in conventional homogenisation–pasteurisation. However, greater adsorption of directly bonded casein molecules, released by UHPH through the partial disruption of casein micelles, was observed especially at high UHPH pressures. Adsorption of whey proteins on the MFGM of conventionally homogenised–pasteurised milk was mainly through intermolecular disulfide bonds with MFGM material, whereas in UHPH-treated milk, disulfide bonding with both indirectly and directly adsorbed caseins was also involved.     

Impact of milking frequencies on the level of free fatty acids in milk, fat globule size, and fatty acid composition

The aim of the present study was to study the effect of milking cows 4 times daily on free fatty acids (FFA) in the milk compared with milking twice daily. An experiment was performed during 2 wk in which half udders in 11 cows were milked 2 or 4 times daily. Milk yield was measured, and milk was analyzed for fat content, FFA, fatty acid composition, fat globule size, and activity of γ-glutamyl transpeptidase. Concentration of FFA was greater (1.49 mEq/100 g of fat) in milk from half udders milked 4 times daily than in milk from the half udders milked twice daily (1.14 mEq/100 g of fat). Further, it was noted that milk from the half udder milked 4 times daily contained milk fat globules with larger average diameters. Increased milking frequency increased milk yield by 9% compared with the udder half milked twice daily, but fat content and fat yield were not affected. The results are of importance for further understanding the mechanisms behind the increased content of FFA that is frequently observed in automatic milking systems.     

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.     

Transfer of dietary zinc and fat to milk--evaluation of milk fat quality, milk fat precursors, and mastitis indicators

The present study demonstrated that the zinc concentration in bovine milk and blood plasma is significantly affected by the intake of saturated fat supplements. Sixteen Holstein cows were used in a 4 × 4 Latin square design with 4 periods of 12 d, and 4 dietary treatments were conducted. A total mixed ration based on corn silage, grass-clover silages, and pelleted sugar beet pulp was used on all treatments. A high de novo milk fat diet was formulated by adding rapeseed meal and molasses in the total mixed ration [39 mg of Zn/kg of dry matter (DM)], and a low de novo diet by adding saturated fat, fat-rich rapeseed cake, and corn (34 mg of Zn/kg of DM). Dietary Zn levels were increased by addition of ZnO to 83 and 80 mg of Zn/kg of DM. Treatments did not affect daily DM intake, or yield of energy-corrected milk, milk fat, or milk protein. The high de novo diet significantly increased milk fat percentage and milk content of fatty acids with chain length from C6 to C16, and decreased content of C18 and C18:1. Treatments did not influence milk free fatty acids at 4°C at 0 or 28 h after milking. The average diameter of milk fat globules was significantly greater in milk from cows offered low de novo diets. Furthermore, the low de novo diet significantly increased the concentration of nonesterified fatty acids and d-β-hydroxybutyrate in blood plasma, the latter was also increased in milk. Treatments did not affect the enzyme activity of lactate dehydrogenase and N-acetyl-β-d-glucosaminidase in milk or the activity of isocitrate dehydrogenase and malate dehydrogenase in blood plasma. The low de novo diet significantly increased plasma Zn and milk Zn content, whereas dietary Zn level did not in itself influence these parameters. This indicates that the transfer of fat from diet to milk might facilitate transfer of Zn from diet to milk.     

Preparation of liposomes from milk fat globule membrane phospholipids using a microfluidizer

The isolation of milk fat globule membrane (MFGM) material from buttermilk on a commercial scale has provided a new ingredient rich in phospholipids and sphingolipids. An MFGM-derived phospholipid fraction was used to produce liposomes via a high-pressure homogenizer (Microfluidizer). This technique does not require the use of solvents or detergents, and is suitable for use in the food industry. The liposome dispersion had an average hydrodynamic diameter of 95 nm, with a broad particle-size distribution. Increasing the number of passes through the Microfluidizer, increasing the pressure, or reducing the phospholipid concentration all resulted in a smaller average liposome diameter. Changing these variables did not have a significant effect on the polydispersity of the dispersion. Electron microscopy showed that the dispersions formed had a range of structures, including unilamellar, multilamellar, and multivesicular liposomes. The composition of the MFGM phospholipid material is different from that of the phospholipids usually used for liposome production in the pharmaceutical and cosmetic industries. The MFGM-derived fraction comprises approximately 25% sphingomyelin, and the fatty acids are primarily saturated and monounsaturated. These differences are likely to affect the properties of the liposomes produced from the phospholipid material, and it may be possible to exploit the unique composition of the MFGM phospholipid fraction in the delivery of bioactive ingredients in functional foods.     

Heritabilities of measured and mid-infrared predicted milk fat globule size,milk fat and protein percentages,and their genetic correlations

The objective of this study was to estimate the heritability of milk fat globule (MFG) size and mid-infrared (MIR) predicted MFG size in Holstein cattle. The genetic correlations between measured and predicted MFG size with milk fat and protein percentage were also investigated. Average MFG size was measured in 1,583 milk samples taken from 254 Holstein cows from 29 herds across Canada. Size was expressed as volume moment mean (D[4,3]) and surface moment mean (D[3,2]). Analyzed milk samples also had average MFG size predicted from their MIR spectral records. Fat and protein percentages were obtained for all test-day milk samples in the cow's lactation. Univariate and bivariate repeatability animal models were used to estimate heritability and genetic correlations. Moderate heritabilities of 0.364 and 0.466 were found for D[4,3] and D[3,2], respectively, and a strong genetic correlation was found between the 2 traits (0.98). The heritabilities for the MIR-predicted MFG size were lower than those estimated for the measured MFG size at 0.300 for predicted D[4,3] and 0.239 for predicted D[3,2]. The genetic correlation between measured and predicted D[4,3] was 0.685; the correlation was slightly higher between measured and predicted D[3,2] at 0.764, likely due to the better prediction accuracy of D[3,2]. Milk fat percentage had moderate genetic correlations with both D[4,3] and D[3,2] (0.538 and 0.681, respectively). The genetic correlation between predicted MFG size and fat percentage was much stronger (greater than 0.97 for both predicted D[4,3] and D[3,2]). The stronger correlation suggests a limitation for the use of the predicted values of MFG size as indicator traits for true average MFG size in milk in selection programs. Larger samples sizes are required to provide better evidence of the estimated genetic parameters. A genetic component appears to exist for the average MFG size in bovine milk, and the variation could be exploited in selection programs.     

Lateral lipid organization of the bovine milk fat globule membrane is revealed by washing processes

Evidence for the asymmetric distribution of phospholipids in the milk fat globule membrane (MFGM) was obtained by applying 3 washing processes using aqueous solutions with different degrees of stringency (mild, intermediate, and intensive) to milk fat globule (MFG) surfaces in simulated milk ultrafiltrate buffer. We detected no change in the amount of cholesterol after the mild washing process; however, intensive washing yielded a relative enrichment of surface cholesterol with concomitant damage to the outer bilayer of the MFGM. This finding supports the hypothesis of repartitioning of cholesterol on MFG surfaces during mechanical treatments. An updated model system of lipid organization was developed according to the results of relative depletion of individual phospholipids, as analyzed by HPLC.     

Developmental changes in the milk fat globule membrane proteome during the transition from colostrum to milk

Shotgun proteomics, using amine-reactive isobaric tags (iTRAQ), was used to quantify protein changes in milk fat globule membranes (MFGM) that were isolated from d 1 colostrum and compared with MFGM from d 7 milk. Eight Holstein cows were randomly assigned to 2 groups of 4 cow sample pools for a simple replication of this proteomic analysis using iTRAQ. The iTRAQ labeled peptides from the experiment sample pools were fractionated by strong cation exchange chromatography followed by further fractionation on a microcapillary high performance liquid chromatograph connected to a nanospray-tandem mass spectrometer. Data analysis identified 138 bovine proteins in the MFGM with 26 proteins upregulated and 19 proteins downregulated in d 7 MFGM compared with colostrum MFGM. Mucin 1 and 15 were upregulated greater than 7-fold in MFGM from d 7 milk compared with colostrum MFGM. The tripartite complex of proteins of adipophilin, butyrophilin, and xanthine dehydrogenase were individually upregulated in d 7 MFGM 3.4-, 3.2-, and 2.6-fold, respectively, compared with colostrum MFGM. Additional proteins associated with various aspects of lipid transport synthesis and secretion such as acyl-CoA synthetase, lanosterol synthase, lysophosphatidic acid acyltransferase, and fatty acid binding protein were upregulated 2.6- to 5.1-fold in d 7 MFGM compared with colostrum MFGM. In contrast, apolipoproteins A1, C-III, E, and A-IV were downregulated 2.6- to 4.3-fold in d 7 MFGM compared with colostrum MFGM. These data demonstrate that quantitative shotgun proteomics has great potential to provide new insights into mammary development.     

Factors affecting the concentration of sphingomyelin in bovine milk

Sphingomyelin is a phospholipid located in the outer leaflet of the plasma membrane of most cells and is a component of the milk fat globule membrane. Sphingomyelin and its digestion products participate in several antiproliferative pathways that may suppress oncogenesis. Although milk and dairy products are important sources of sphingomyelin in the human diet, little is known about factors that influence sphingomyelin concentrations in milk fat or whether concentrations can be modified via the nutrition of cows. Sphingomyelin concentrations were determined in milk from Holstein and Jersey cows matched for parity and stage of lactation. Sphingomyelin was more concentrated in milk fat from Holstein cows than in milk fat from Jersey cows (1,044 vs. 839 μg/g of fat). Concentrations in whole milk did not differ because of greater milk fat content for milk from Jerseys. Differences between breeds may be related to the greater fat globule size in milk from Jerseys. Sphingomyelin content in whole milk increased with increasing days in milk because of associated increases in milk fat content. Regardless of breed, primiparous cows had greater amounts of stearic acid and less palmitic acid in sphingomyelin than did older cows. The sphingomyelin concentration in milk fat of cows in a commercial Jersey herd was lower for cows in their fourth or greater parity. Sphingomyelin content in whole milk was greater for cows in late lactation because of greater milk fat content. Feed restriction of multiparous Holstein cows to 37% of ad libitum dry matter intake increased milk fat content but did not affect milk sphingomyelin content or milk fat globule size. Supplementation of the diet with 4% soybean oil did not affect milk composition, sphingomyelin content, or milk fat globule size. Milk was sampled seasonally from 7 herds throughout Illinois during a 2-yr period. Sphingomyelin concentration in milk fat was greatest during summer and least during winter, but whole milk concentrations did not vary across seasons. We conclude that 1) sphingomyelin content of milk fat is greater in milk from Holsteins than that from Jerseys, 2) sphingomyelin content in whole milk increases with stage of lactation, and 3) sphingomyelin content of milk fat is greater during summer. However, efforts to produce milk with a greater sphingomyelin content through altering management and nutrition are unlikely to be successful.     

Cooling causes changes in the distribution of lipoprotein lipase and milk fat globule membrane proteins between the skim milk and cream phase

Lipoprotein lipase (LPL) activity and free fatty acid levels were studied in freshly milked, uncooled milk from individual Danish Holstein or Jersey cows, or after storage for up to 24 h at either a cooling temperature (4°C) or at the milking temperature (31°C). Upon cooling for up to 24 h, LPL activity increased in the cream phase, whereas the activity in the skim milk was steady, as observed for Jersey cows, or increased, as seen for the Holsteins. Storage at 31°C decreased the LPL activity in both the cream phase and the skim milk phase. The increase in free fatty acid levels was found to depend on LPL activity, incubation temperature, substrate availability, and incubation time. Furthermore, the migration of milk proteins between the skim milk phase and the cream phase upon cooling of milk from Jersey cows or from Danish Holstein cows was studied using proteomic methods involving 2-dimensional gel electrophoresis and mass spectrometry. Proteins associated with the milk fat globules were isolated from all milk fractions and analyzed. Major changes in the distributions of proteins between the skim milk phase and the cream phase were observed after cooling at 4°C for 4 h, where a total of 29 proteins between the 2 breeds was found to change their association with the milk fat globule membrane (MFGM) significantly. Among these, the MFGM proteins adipophilin, fatty acid-binding protein, and lactadherin, as well as the non-MFGM proteins β-casein, lactoferrin, and heat shock protein-71, were identified. Adipophilin, lactadherin, and lactoferrin were quantitatively more associated with the MFGM upon cold storage at 4°C, whereas β-casein, fatty acid-binding protein, and heat shock protein-71 were found to be less associated with the MFGM upon cold storage.