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.     

Milk fat globule membrane – a source of polar lipids for colon health? A review

The milk fat globule membrane (MFGM) surrounds fat globules, protects them against lipolysis and disperses the milk fat in the milk plasma. Besides their structural and emulsifying roles, in vivo and in vitro studies have demonstrated that phospholipids and sphingolipids of MFGM possess cancer risk‐reducing properties. Several reports attribute its chemopreventive activity to products of sphingomyelin hydrolysis, which affect multiple cellular targets that control cell growth, differentiation and apoptosis. With knowledge on the potential health benefits of MFGM lipids and proteins, dairy industries could in the future address their research in developing new functional dairy products enriched in beneficial MFGM components.     

Effect of the size and interface composition of milk fat globules on their in vitro digestion by the human pancreatic lipase: Native versus homogenized milk fat globules

Although the bioavailability of dietary lipids is of primary importance in human nutrition and health, the mechanisms involved in lipid digestion are not fully understood and are of growing interest. The objective of this study was to determine the effect of the size of milk fat globules and of the composition of their interface on the activity of the human pancreatic lipase (PL). Native milk fat globules of various sizes covered by their biological membrane (MFGM) and homogenized fat globules of various sizes covered by milk proteins were prepared from whole milk and underwent lipolysis by the human PL with colipase and bile salts. A lag phase preceding the hydrolysis of milk TAG occurred with all native milk fat globules samples but not with homogenized milk samples. The kinetic parameters of human PL were determined by measuring the enzyme activity either after the lag phase for native milk fat globules samples or immediately after the addition of the enzyme for homogenized milk samples. The catalytic efficiency of human PL is 4.6-fold higher on small (1.8 μm) than large (6.7 μm) native milk fat globules, related to a 3.6-fold larger available surface. Despite the 25-fold larger available surface, milk TAG from homogenized milk are only 2-fold better hydrolyzed compared to native milk fat globules, as a possible result of a less favourable interface covered by milk proteins. The potential mechanisms involved in native vs. homogenized milk fat globules digestion by the human PL are discussed. Our study highlights the crucial role of the MFGM in the efficient digestion of milk fat globules and brings new insight for the design of dairy products and infant formulas.     

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.     

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.     

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.     

Symposium review: The relevance of bovine milk phospholipids in human nutrition—Evidence of the effect on infant gut and brain development

This paper reflects the concepts reviewed during the presentation in the Joint MILK/Lactation Biology Symposium at the ADSA 2018 Annual Meeting. Our intention is to update the concepts and advances in the area of research regarding milk phospholipids or polar lipid fraction as part of a dairy ingredient used today in nutritional studies that focus on gut health as well as brain development of infants. Although processing advances have allowed the production of novel ingredients rich in milk fat globule membrane (MFGM) components, mostly monitored by phospholipid concentration and presence of membrane proteins, there is wide variability in their composition and structure. Furthermore, we aimed to include in the phospholipid fraction of milk nanovesicles designated as milk exosomes, which are secreted into milk by different secretion mechanisms than those of the fat globules but are also made up of a unique mixture of polar lipids. We consider imperative the study of polar lipid-derived structures from milk regarding composition and structure to gain insights into their biological effect in human health. Nevertheless, and tolerating the differences in composition and concentration of their components, studies supplementing the diet of infants with polar lipids (i.e., MFGM components) have shown significant advances in several areas of human health and well-being. Here we present a summary of the important components of MFGM and milk exosomes as well as an overview of the effects on gut health and brain and cognitive development when added to the diet of infants.     

Ultrasonication retains more milk fat globule membrane proteins compared to equivalent shear-homogenization

Ultrasonication, like common shear homogenization, can reduce the milk fat globule size and may change the milk fat globule membrane (MFGM). This work compared the effect of ultrasonication to equivalent shear homogenization on MFGM proteins and lipid-derived volatile components. Results showed that treating milk with ultrasound at 35 kJ/L would realize a similar size distribution of the milk fat globules as shear-homogenization at 20 MPa. Proteomics analysis revealed that in total 192 MFGM proteins were identified and quantified and a number of these proteins were lost after both treatments; however, more MFGM proteins remained after ultrasonication than after shear-homogenization. SDS-PAGE results showed that milk plasma proteins, and especially caseins, were absorbed on the milk fat globules after both treatments. In addition, the amount of the volatile free fatty acids increased after both treatments.Industrial relevance: Ultrasonication, as an innovative food processing technology, in comparison to traditional homogenization, was shown to equally efficiently decrease the MFG size, but lead to less damage to native MFGM proteins, which may be due to its longer homogenization time window. These results increased knowledge on the biochemical changes of milk fat globules after their size reduction and showed that ultrasonication could be used as a novel approach to improve dairy product quality.     

乳脂肪球膜的特性、开发及在模拟母乳脂肪球结构中的应用

乳脂肪球膜（milk fat globule membrane,MFGM）是包裹在天然乳脂肪球外部的3 层膜状结构,然而牛乳基和大豆基婴儿配方奶粉缺少MFGM,脂肪球结构与母乳存在较大差异,因此添加外源MFGM以及制备与母乳脂肪球结构接近的婴儿配方奶粉成为了近期的研究焦点。本文综述了MFGM的相关特性和生产开发途径,以及牛乳MFGM在仿母乳脂肪球结构乳液和婴儿配方奶粉中的应用。体外模拟婴儿胃肠道消化实验以及啮齿动物体内实验结果表明,仿母乳脂肪球结构乳液和婴儿配方奶粉能够促进婴儿脂肪消化并且改善脂质代谢过程。     

婴儿配方乳粉脂质母乳化的研究进展

人乳脂肪是最复杂的天然脂质混合物之一,具有独特的脂肪酸组成、分布和许多复杂脂质。乳脂肪几乎提供着新生婴儿所需的一半能量,同时还会影响婴儿日后的代谢方式和整体发育。因此,随着对母乳脂肪及婴儿生长需求研究的深入,以母乳脂质组成为模板,对婴儿配方乳粉脂质进行母乳化模拟,可以缩小母乳与婴儿配方乳粉喂养婴儿之间的差异。目前,婴儿配方乳粉脂质母乳化主要集中在脂肪酸组成模拟、sn-2棕榈酸结构脂的应用以及乳脂肪球膜成分的添加方面。另外,利用乳脂肪球膜包裹脂滴实现脂肪球结构模拟的相关研究也在逐步开展。本文着重对这几个方面的研究进展进行总结,在关注婴儿营养需求的同时,为婴儿配方乳粉的研发提供理论依据。     

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.     

The Macrostructure of Milk Lipids: The Fat Globules

The aim of this review article is to summarize the information available related to milk fat globules (MFGs) in order to highlight their contribution to the nutritional and technological characteristics of milk and dairy products. The macrostructure of milk lipids is composed of globules made up of triglycerides with different melting points, enveloped by a biological membrane from the mammary epithelial cell. In milks of different animal species, there are different-sized MFGs, ranging from diameters of less than 0.2 μm to a maximum of 15 μm. The average diameter and the number of globules are affected by endogenous, physiological, and exogenous factors. The size of the globules in turn affects the qualitative characteristics of milk and cheese. In addition, the average diameter of the globules and their surface that is exposed to the digestive system affect fat digestibility in different ways. Finally, the components of the MFG membranes have been shown to take part in the secretion process of globules and to have a beneficial effect on human health. In conclusion, by acting on factors influencing the dimensions of the fat globules and by increasing the content of the milk membrane could help adapt milk production to specific consumer targets and improve milk nutritional properties.     

Stability of milk fat globule membrane proteins toward human enzymatic gastrointestinal digestion

The milk fat globule membrane (MFGM) fraction refers to the thin film of polar lipids and membrane proteins that surrounds fat globules in milk. It is its unique biochemical composition that renders MFGM with some beneficial biological activities, such as anti-adhesive effects toward pathogens. However, a prerequisite for the putative bioactivity of MFGM is its stability during gastrointestinal digestion. We, therefore, subjected MFGM material, isolated from raw milk, to an in vitro enzymatic gastrointestinal digestion. Sodium dodecyl sulfate PAGE, in combination with 2 staining methods, Coomassie Blue and periodic acid Schiff staining, was used to evaluate polypeptide patterns of the digest, whereas mass spectrometry was used to confirm the presence of specific MFGM proteins. Generally, it was observed that glycoproteins showed higher resistance to endogenous proteases compared with non-glycosylated proteins. Mucin 1 displayed the highest resistance to digestion and a considerable part of this protein was still detected at its original molecular weight after gastric and small intestine digestion. Cluster of differentiation 36 was also quite resistant to pepsin. A significant part of periodic acid Schiff 6/7 survived the gastric digestion, provided that the lipid moiety was not removed from the MFGM material. Overall, MFGM glycoproteins are generally more resistant to gastrointestinal digestion than serum milk proteins and the presence of lipids, besides glycosylation, may protect MFGM glycoproteins from gastrointestinal digestion. This gastrointestinal stability makes MFGM glycoproteins amenable to further studies in which their putative health-promoting effects can be explored.     

Composition and Structure of the Bovine Milk Fat Globule Membrane—Some Nutritional and Technological Implications

Milk fat is dispersed in milk as small, spherical globules, stabilized in the form of emulsion by its surrounding membrane. This membrane, called the milk fat globule membrane (MFGM), is created in the secretory cells of the mammary gland, and represents an ordered and unique biophysical system. This review characterizes the main milk fat globule components, their structure, and intracellular origin. The milk fat globule membrane has many potentially bioactive components. These are discussed in terms of their health effects for the native and processed globules. Because of their functional and nutritional properties, MFGM components can be used as valuable ingredients in the manufacture of new functional foods.     

The Microstructure and Physicochemical Properties of Probiotic Buffalo Yoghurt During Fermentation and Storage: a Comparison with Bovine Yoghurt

The physicochemical and rheological properties of yoghurt made from unstandardised unhomogenised buffalo milk were investigated during fermentation and 28 days of storage and compared to the properties of yoghurt made from homogenised fortified bovine milk. A number of differences observed in the gel network can be linked to differences in milk composition. The microstructure of buffalo yoghurt, as assessed by confocal laser scanning microscopy (CLSM) and cryo scanning electron microscopy (cryo-SEM), was interrupted by large fat globules and featured more serum pores. These fat globules have a lower surface area and bind less protein than the homogenised fat globules in bovine milk. These microstructural differences likely lead to the higher syneresis observed for buffalo yoghurt with an increase from 17.4 % (w/w) to 19.7 % (w/w) in the weight of whey generated at days 1 and 28 of the storage. The higher concentration of total calcium in buffalo milk resulted in the release of more ionic calcium during fermentation. Gelation was also slower but the strength of the two gels was similar due to similar protein and total solids concentrations. Buffalo yoghurt was more viscous, less able to recover from deformation and less Newtonian than bovine yoghurt with a thixotropy of 3,035 Pa.s^?1 measured for buffalo yoghurt at the end of the storage, at least four times higher than the thixotropy of bovine yoghurt. While the titratable acidity, lactose consumption and changes in organic acid concentrations were similar, differences were recorded in the viability of probiotic bacteria with a lower viability of Lactobacillus acidophilus of 5.17 log (CFU/g) recorded for buffalo yoghurt at day 28 of the storage. Our results show that factors other than the total solids content and protein concentration of milk affect the structural properties of yoghurt. They also illustrate the physicochemical reasons why buffalo and bovine yoghurt are reported to have different sensory properties and provide insight into how compositional changes can be used to alter the microstructure and properties of dairy products.     

Technical difficulties and future challenges in isolating membrane material from milk fat globules in industrial settings – A critical review

Research on the milk fat globule and surrounding membrane began a century ago. Synthesis and secretion mechanisms of milk fat globules in mammary epithelial cells are well documented, but there is still controversy about the composition of the milk fat globule membrane (MFGM). In recent years, interest in isolating MFGM material has increased because of the presumed functional potential of the proteins and lipids. However, no standardised isolation procedure exists to our knowledge. Consequently, published reports on the MFGM composition differ significantly. Various isolation methods under different conditions were applied, and contradictory effects on the MFGM structure were obtained. In addition to compositional changes, losses of MFGM material also occur under particular conditions. This makes it difficult to compare reports on the composition of the isolated MFGM material. We therefore saw a necessity to critically review past and current literature with emphasis on the reported isolation methods and respective results.     

Effect of washing conditions on the recovery of milk fat globule membrane proteins during the isolation of milk fat globule membrane from milk

During the isolation of milk fat globule membrane (MFGM) from milk, washing is considered the most critical stage in which loss of MFGM components occurs. In this study, using a cream separator, the influence of washing on the recovery of MFGM proteins was investigated. The residue of non-MFGM proteins in the MFGM material obtained after washing was quantitatively determined using densitometric analysis of one-dimensional sodium dodecyl sulfate-PAGE after silver staining of the gel. Using deionized water as the washing solution did not increase the loss of MFGM proteins compared with other common salt solutions in terms of recovery of MFGM proteins and contamination with non-MFGM proteins. The increase in wash temperature from 38 to 46°C did not show a significant decrease in yield of MFGM proteins because of variation between the experimental replicates. Coalescence of fat globules occurs during isolation. To increase MFGM purity while maintaining a high MFGM protein recovery, using larger volumes of wash solution is more advisable rather than increasing the number of washings from 2 to 3.     

Proteolysis of milk fat globule membrane proteins during in vitro gastric digestion of milk

The influence of gastric proteolysis on the physicochemical characteristics of milk fat globules and the proteins of the milk fat globule membrane (MFGM) in raw milk and cream was examined in vitro in simulated gastric fluid (SGF) containing various pepsin concentrations at pH 1.6 for up to 2 h. Apparent flocculation of the milk fat globules occurred in raw milk samples incubated in SGF containing pepsin, but no coalescence was observed in either raw milk samples or cream samples. The changes in the particle size of the fat globules as a result of the flocculation were dependent on the pepsin concentration. Correspondingly, the physical characteristics of the fat globules and the composition of the MFGM proteins in raw milk changed during incubation in SGF containing pepsin. The major MFGM proteins were hydrolyzed at different rates by the pepsin in the SGF; butyrophilin was more resistant than xanthine oxidase, PAS 6, or PAS 7. Peptides with various molecular weights, which altered with the time of incubation and the pepsin concentration, were present at the surfaces of the fat globules.     

Filtration of milk fat globule membrane fragments from acid buttermilk cheese whey

The proteins and polar lipids present in milk fat globule membrane (MFGM) fragments are gaining attention for their technological and nutritional properties. These MFGM fragments are preferentially enriched in side streams of the dairy industry, like butter serum, buttermilk, and whey. The objective of this study was to recover MFGM fragments from whey by tangential filtration techniques. Acid buttermilk cheese whey was chosen as a source for purification by tangential membrane filtration because it is relatively rich in MFGM-fragments and because casein micelles are absent. Polyethersulfone and cellulose acetate membranes of different pore sizes were evaluated on polar lipid and MFGM-protein retention upon filtration at 40°C. All fractions were analyzed for dry matter, ash, lipids, proteins, reducing sugars, polar lipid content by HPLC, and for the presence of MFGM proteins by sodium dodecyl sulfate-PAGE. A fouling coefficient was calculated. It was found that a thermocalcic aggregation whey pretreatment was very effective in the clarification of the whey, but resulted in low permeate fluxes and high retention of ash and whey proteins. By means of an experimental design, the influence of pH and temperature on the fouling and the retention of polar lipids (and thus MFGM fragments), proteins, and total lipids upon microfiltration with 0.15 μM cellulose acetate membrane was investigated. All models were highly significant, and no outliers were observed. By increasing the pH from 4.6 to 7.5, polar lipid retention at 50°C increased from 64 to 98%, whereas fouling of the filtration membrane was minimized. A 3-step diafiltration of acid whey under these conditions resulted in a polar lipid concentration of 6.79 g/100 g of dry matter. As such, this study shows that tangential filtration techniques are suited for the purification of MFGM fragments.     

Development of the milk fat microstructure during the manufacture and ripening of Emmental cheese observed by confocal laser scanning microscopy

Changes in the physico-chemical properties and microstructure of milk fat globules were investigated during the manufacture and ripening of Emmental cheese. The measurement of fat globule size and apparent zeta-potential showed that they were slightly affected during cheese milk preparation, i.e. storage of cheese milk overnight at 4 °C and pasteurisation. After rennet-induced coagulation and heating of curd grains, coalescence caused the formation of large fat globules (i.e.>10 μm). The structure of fat in Emmental cheese was characterised in situ using confocal laser scanning microscopy (CLSM). The rennet-induced coagulation lead to the formation of a continuous network of casein strands in which fat globules of various sizes were entrapped. Heating of curd grains induced the formation of fat globule aggregates. Pressing of the curd grains resulted in the greatest disruption of milk fat globules, their coalescence, the formation of non-globular fat (free fat) and the release of the milk fat globule membrane (MFGM) material. This study showed that milk fat exists in three main forms in ripened Emmental cheese: (i) small fat globules enveloped by the MFGM; (ii) aggregates of partially disrupted fat globules and (iii) free fat, resulting from the disruption of the MFGM and allowing free triacylglycerols to fill voids in the protein matrix. The curd grain junctions formed in Emmental cheese were also characterised using CLSM: they are compact structures, rich in protein and devoid of fat globules.