Surface Of Milk Fat Globules Research Articles

Structure, Biological Functions, Separation, Properties, and Potential Applications of Milk Fat Globule Membrane (MFGM): A Review.

The milk fat globule membrane (MFGM) is a thin film that exists within the milk emulsion, suspended on the surface of milk fat globules, and comprises a diverse array of bioactive components. Recent advancements in MFGM research have sparked a growing interest in its biological characteristics and health-related functions. Thorough exploration and utilization of MFGM as a significant bioactive constituent in milk emulsion can profoundly impact human health in a positive manner. Scope and approach: This review comprehensively examines the current progress in understanding the structure, composition, physicochemical properties, methods of separation and purification, and biological activity of MFGM. Additionally, it underscores the vast potential of MFGM in the development of additives and drug delivery systems, with a particular focus on harnessing the surface activity and stability of proteins and phospholipids present on the MFGM for the production of natural emulsifiers and drug encapsulation materials. MFGM harbors numerous active substances that possess diverse physiological functions, including the promotion of digestion, maintenance of the intestinal mucosal barrier, and facilitation of nerve development. Typically employed as a dietary supplement in infant formula, MFGM's exceptional surface activity has propelled its advancement toward becoming a natural emulsifier or encapsulation material. This surface activity is primarily derived from the amphiphilicity of polar lipids and the stability exhibited by highly glycosylated proteins.

The extracellular region of bovine milk butyrophilin exhibits closer structural similarity to human myelin oligodendrocyte glycoprotein than to immunological BTN family receptors.

Bovine butyrophilin (BTN1A1) is an abundant type I transmembrane glycoprotein exposed on the surface of milk fat globules. We have solved the crystal structure of its extracellular region via multiple wavelength anomalous dispersion after incorporation of selenomethionine into the bacterially produced protein. The butyrophilin ectodomain exhibits two subdomains with immunoglobulin fold, each comprising a β-sandwich with a central disulfide bridge as well as one N-linked glycosylation. The fifth Cys residue at position 193 is unpaired and prone to forming disulfide crosslinks. The apparent lack of a ligand-binding site or receptor activity suggests a function predominantly as hydrophilic coat protein to prevent coagulation of the milk fat droplets. While there is less structural resemblance to members of the human butyrophilin family such as BTN3A, which play a role as immune receptors, the N-terminal bovine butyrophilin subdomain shows surprising similarity to the human myelin oligodendrocyte glycoprotein, a protein exposed on the surface of myelin sheaths. Thus, our study lends structural support to earlier hypotheses of a correlation between the consumption of cow milk and prevalence of neurological autoimmune diseases and may offer guidance for the breeding of cattle strains that express modified butyrophilin showing less immunological cross-reactivity.

The effect of high-pressure homogenisation on the size of milk fat globules and MFGM composition in sweet buttermilk and milk

Effects of high-pressure homogenisation (HPH, 100 MPa) at different temperatures (4–60 °C) or conventional homogenisation (20 MPa, 60 °C) on size of milk fat globules (D32) and content of protein and phospholipids in milk fat globule membrane (MFGM) in standardised milk, buttermilk and a mixture of milk and buttermilk were compared. HPH decreased D32 and promoted the adsorption of casein at ≥ 4 °C, β-lactoglobulin in milk and buttermilk at ≥ 40 °C and ≥20 °C, respectively, and α-lactalbumin at 60 °C, on the surface of milk fat globules. The content of milk proteins adsorbed on MFGM increased with increasing temperature and pressure in milk, and, to a lesser extent, in buttermilk. The content of phospholipids in buttermilk was 2-fold higher than in milk. HPH influenced the content of sphingomyelin (increased) and phosphatidylcholine (decreased) in MFGM in milk. Conventional homogenisation increased the phosphatidylethanolamine content in MFGM in buttermilk.

The adhesion of homogenized fat globules to proteins is increased by milk heat treatment and acidic pH: Quantitative insights provided by AFM force spectroscopy

The rheological properties and microstructure of dairy gels involve the connectivity between milk fat globules (MFG) and casein micelles that is affected by technological processes such as milk homogenization and heat treatment. The underlying mechanisms require further quantification of the interactions at the nanoscale level to be fully understood and controlled. In this study, we examined the adhesion of homogenized MFG to milk proteins and evaluated the role of ultra-high temperature (UHT) heat treatment and pH. The combination of physico-chemical analysis, rheology and microscopy observations at different scale levels associated to atomic force microscopy (AFM) force spectroscopy were used. AFM experiments performed at the particle scale level showed that adhesion of individual homogenized MFG to milk proteins (1) is increased upon acidification at pH 4.5: 1.4 fold for unheated samples and 3.5 fold for UHT samples, and (2) is enhanced by about 1.7 fold at pH 4.5 after UHT heat treatment of milk, from 176 pN to 296 pN, thanks to highly-reactive heat-denatured whey proteins located at the surface of MFG and caseins. The increased inter-particle adhesion forces accounted for more connected structures and stiffer UHT milk acid gels, compared to unheated-milk gels. Using a multiscale approach, this study showed that heat treatment of milk markedly affected the interactions occurring at the particle's surface level with consequences on the bulk structural and rheological properties of acid gels. Such findings will be useful for manufacturers to modulate the texture of fermented dairy products through the tailoring of heat-induced complexation of proteins and the connectivity of homogenized MFG with the protein network. This work will also contribute in a better understanding of the impact of process-induced changes on the digestibility and metabolic fate of proteins and lipids.

The surface properties of milk fat globules govern their interactions with the caseins: Role of homogenization and pH probed by AFM force spectroscopy

The surface of milk fat globules consists of a biological membrane rich in polar lipids and glycoproteins. However, high shear stress applied upon homogenization disrupts the membrane and leads to the adsorption of casein micelles, as the major protein fraction of milk. These changes in the interface properties could affect the interactions between native or homogenized milk fat globules and the surrounding protein matrix, at neutral pH and upon acidification. In this study, macroscale rheometry, microscopic observations, nanoscale AFM-based force spectroscopy and physico-chemical analysis were combined to examine the interfacial composition and structure of milk fat globules and to evaluate their interactions with casein micelles. We showed that the surface properties of milk fat globules (biological membrane vs. caseins) and pH govern their interactions with casein micelles. The adhesion between individual fat globules and casein micelles was higher upon homogenization, especially at acid pH where the work of adhesion increased from 3.3 x 10-18 to 14 x 10-18 J for native and homogenized fat globules, respectively. Consequently, casein-coated homogenized fat globules yield stiffer milk acid gels. These findings cast light on the importance of colloidal particle’s surface properties and pH on their connectivity with the surrounding matrix, which modulates the bulk microstructure and rheological properties with potential functional consequences, such as milk lipid digestion.

Change in interfacial properties of milk fat globules by homogenization and thermal processing plays a key role in their in vitro gastrointestinal digestion

The effects of homogenization, homogenization plus thermal processing including high temperature short time (HHTST) or ultra-high temperature (HUHT) treatment on digestion and interfacial properties of milk fat globules during simulated gastrointestinal digestion were investigated. Compared to raw milk fat globules, homogenization and thermal processing increased the initial digestion rate of milk fat globules. Homogenization increased the release of middle- and long-chain fatty acids, HHTST increased the release of unsaturated fatty acids, and HUHT increased the release of all types of fatty acids under gastrointestinal digestion. After homogenization, milk proteins (caseins and whey proteins) increased but glycosylated molecules decreased, both of which were at the surface of milk fat globules. The amount of glycosylated molecules at the surface of HHTST milk fat globules and the amount of denatured milk proteins at the surface of HUHT milk fat globules were higher than those of homogenized milk fat globules. During gastrointestinal digestion, fat globules in HHTST milk possessed higher amount of glycosylated molecules and more integrated phospholipid layers, and HUHT milk fat globules had more filamentous phospholipid layers, and less integrated phospholipid layers and glycosylated molecules than those of homogenized milk fat globules. Therefore, the changes in composition and microstructure of glycosylated molecules and phospholipid layers at the surface of milk fat globules induced by homogenization and thermal processing might contribute to the digestion difference of milk fat globules.

Lipid domains in the milk fat globule membrane: Dynamics investigated in situ in milk in relation to temperature and time.

The microstructure of the milk fat globule membrane (MFGM) is still poorly understood. The aim of this study was to investigate the dynamics of the MFGM at the surface of milk fat globules in relation to temperature and time, and in relation to the respective lipid compositions of the MFGM from bovine, goat and sheep milks. In-situ structural investigations were performed using confocal microscopy. Lipid domains were observed over a wide range of temperatures (4-60°C). We demonstrated that rapid cooling of milk enhances the mechanisms of nucleation and that extended storage induces lipid reorganization within the MFGM with growth, leading to circular lipid domains. Diffusion of the lipid domains, coalescence and reduction in domain size were observed upon heating. Different MFGM features could be related to the respective cholesterol/sphingomyelin molar ratio in the three milk species. These structural changes may affect the interfacial properties of the MFGM, with consequences for the functional properties of fat globules and the mechanisms of their digestion.

Adsorption of Bile Salts to Milk Phospholipid and Phospholipid–Protein Monolayers

The adsorption of bile salts to milk phospholipid and phospholipid-protein monolayers at the air-water interface was studied under simulated intestinal conditions using a Langmuir trough, epifluorescence microscopy, and atomic force microscopy. Surface pressure changes were affected by temperature, initial surface pressure, and bile composition. The rate of addition of bile salts and the initial surface pressure of the monolayers had an impact on the microstructure of the mixed monolayers. The presence of proteins in monolayers at different ratios did not affect the surface pressure change upon addition of bile. However, at 20 °C, the addition of bile to phospholipid and phospholipid-protein monolayers led to different features with branching and clustering of liquid-ordered domains and possible formation of bile salt-rich areas within liquid-ordered domains. This study provides a basic understanding of the interfacial changes occurring at the surface of milk fat globules and milk phospholipid liposomes during their passage in the duodenum.

Post-weaning increases in the milk-fat globule EGF-factor VIII on fat globules in mouse milk and in the uptake of the fat globules by HC11 mammary epithelial cells

Milk fat globules (MFGs) secreted by lactating mammary gland are unique lipid surrounded by a phospholipid bi-layer. We report here post-weaning changes in MFG EGF factor VIII (MFG-E8) and annexin V-accessible phosphatidyl-l-serine on the surface of MFGs. The MFG content in milk markedly decreased to about one-half within 2 days after forced weaning, despite a slight increase in milk protein content. Immunofluorescence-staining of MFGs using anti-MFG-E8 and annexin V indicated that MFG-E8 was present on some, but not all, MFGs before weaning, whereas most of MFGs were MFG-E8-positive and annexin V-negative after weaning. Free MFG-E8 with binding activity to phosphatidyl-l-serine was present abundantly in the post-weaning milk, and indeed exhibited binding to MFGs in pre-weaning milk. MFGs were taken up by HC11 mouse mammary epithelial cells in vitro, and those from post-weaning milk were remarkable for such cellular uptake. Moreover, the uptake of MFGs by the cells was inhibited by an anti-MFG-E8 antibody. Taken together, these findings suggest that MFG-E8 plays a critical role in regulation of MFG dynamics after weaning or during the suckling interval through the control of MFG-epithelial cell interaction in lactating mammary glands.

Studies on the structure of milk fat globule membrane.

Milk fat globule membrane was solubilized with sodium dodecyl sulfate and mercaptoethanol and the membrane proteins were separated by SDS-polyacrylamide gel electrophoresis. The membrane preparations contained three major size classes of polypeptide of 155,000, 62,500 and 43,500 daltons. At least five glycopeptides were separated of which two stained intensely with periodic acid-Schiff reagent, but poorly with coomassie blue. Trypsin hydrolysis of whole cream and isolated milk fat globule membrane revealed major differences in the rates of protein hydrolysis. Many of the membrane proteins of whole cream resisted proteolysis compared with the same proteins in the isolated membrane. Two glycopeptides were resistant to trypsin digestion in either preparation. Treatment of whole cream with neuraminidase led to the release of at least 70% of the protein-bound sialic acid. Whole cream and isolated membrane samples were iodinated with 125I in the presence of lactoperoxidase and hydrogen peroxide. The membrane proteins were significantly more accessible to lactoperoxidase-125I i in isolated membrane compared with the proteins of whole cream. Polypeptides of molecular weight 43,500 and approximately 48,000 daltons were predominantly labelled in whole cream and could be eluted from the fat globules with magnesium chloride (1.5m). The results strongly suggest that the proteins of milk fat globule membrane are asymmetrically arranged in the membrane and that most of the protein-bound sialic acid is present on the external surface of milk fat globules.

Structure and Synthesis of Milk Fat. VI. Unity of the Phospholipids in Milk1,2

Summary While recent findings have emphasized the complexity of milk phospholipids (P-lipids), as revealed by the classes present and their fatty acid compositions, results of this study indicate that these P-lipids occur in units of relatively homogeneous lipid composition. Disposition of the P-lipids was studied using cream separation, churning, and ultra-centrifugation to fractionate the lipids in milk. The classes of P-lipids present in the various milk fractions were revealed by thin-layer chromatography (TLC), and the fatty acid composition of total P-lipids in the fractions was determined by gas chromatography (GC). The results demonstrated the same classes of P-lipids (mainly cephalin, lecithin, and sphingomyelin) in similar proportions with much the same total fatty acid composition in milk, skimmilk, cream, and buttermilk. This unity also was detected in fractions derived by freezing and sawing tubes of milk or buttermilk that had been centrifuged in a manner (25,000× g for 6 hr) to yield differentiation of the P-lipids by sedimentation. In both milk and buttermilk the bulk of the P-lipids tended to deposit with a particle that settled mainly in the upper level of the casein layer. The evidence is consistent with the concept of a membrane of fairly homogeneous lipid composition in milk serum and on the surface of milk fat globules. Current evidence suggests that this membrane is derived at least in part from the cytoplasmic membrane in the apical region of the secreting cell. Some affinity of unsaturated lipids for protein phase was noted and may be relevant to lipid oxidation and enzymatic phenomena.