Milk Fat Globule Membrane Fractions Research Articles

Application of pressure homogenization on whole human milk pasteurized by high hydrostatic pressure: Effect on protein aggregates in milk fat globule membrane and skim milk phases

This study explored the impact of homogenization (at pressures of 16, 30, and 45 MPa) on both raw and high hydrostatic pressure (HHP)-treated human milk (HM). It focused on protein compositions and binding forces of soluble and insoluble fractions for both milk fat globule membrane (MFGM) and skim milk. Mild homogenization of HHP-treated milk increased lactoferrin (LF) levels in the insoluble fractions of both MFGM and skim milk, due to insoluble aggregation through hydrophobic interactions. Intense homogenization of HHP-treated milk decreased the LF level in the MFGM fractions due to the LF desorption from the MFGM, which increased LF level in the insoluble skim milk fraction. Homogenized-HHP treated milk showed noticeably higher casein (CN) level at the MFGM compared to homogenized-raw milk, attributed to HHP effect on CN micelles. Overall, the combined use of HHP and shear-homogenization should be avoided as it increased the biological proteins in insoluble fractions.

Variation in both proteome and N-glycoproteome of goat MFGM over lactation

Glycoproteins have been associated in numerous biological functions and has gained much interest for research. This study comprehensively compare proteome and N-glycoproteome of goat milk fat globule membrane (MFGM) over lactation by N-glycosylated peptides enrichment combined with nanospray LC-MS/MS on Q Exactive™ HF plus mass spectrometer. A total of 1639 proteins and 557 N-glycoproteins were identified in goat MFGM fraction. There were 232 proteins and 106 N-glycoproteins were significantly different in goat MFGM between colostrum and mature milk. Most of the differentially expressed proteins were associated with protein processing in endoplasmic reticulum and epstein-barr virus infection, while majority of significantly different N-glycoproteins were engaged in complement and coagulation cascades and lysosome, etc. Furthermore, MFGM proteins were found to be involved in PI3K-Akt signaling pathway, suggesting the potential hypoglycemic effect of goat MFGM protein. Both proteome and N-glycoproteins of caprine MFGM changes with the increase of lactation. The study provides the real changes of N-glycoproteins and insights into biological function of MFGM protein.

Partitioning of the milk fat globule membrane between buttermilk and butter serum is determined by the thermal behaviour of the fat globules

Isolation of the industrially interesting milk fat globule membrane (MFGM) components from dairy streams is challenging, and a full exploitation of their benefits can be gained by better understanding of the behaviour of the fat globule membrane fraction during milk fat processing. In this study, microfiltration of the cream before churning and the comprehensive compositional analysis of the process streams revealed new insights on MFGM partitioning during the phase inversion in butter making. After removal of the smallest fat globules by microfiltration, a reduced phospholipid content was reflected in the buttermilk, but not in the butter serum. Regardless of the cream washing, buttermilk and butter serum lipids were different in PL-to-fat ratio, phospholipid composition, degree of unsaturation and melting behaviour. We suggest that partitioning of the MFGM fraction between butter and buttermilk is a direct result of the physico-chemical properties of the fat globules, partly related to the globule size.

Comparative Proteomics of Milk Fat Globule Membrane (MFGM) Proteome across Species and Lactation Stages and the Potentials of MFGM Fractions in Infant Formula Preparation.

Milk is a lipid-in-water emulsion with a primary role in the nutrition of newborns. Milk fat globules (MFGs) are a mixture of proteins and lipids with nutraceutical properties related to the milk fat globule membrane (MFGM), which protects them, thus preventing their coalescence. Human and bovine MFGM proteomes have been extensively characterized in terms of their formation, maturation, and composition. Here, we review the most recent comparative proteomic analyses of MFGM proteome, above all from humans and bovines, but also from other species. The major MFGM proteins are found in all the MFGM proteomes of the different species, although there are variations in protein expression levels and molecular functions across species and lactation stages. Given the similarities between the human and bovine MFGM and the bioactive properties of MFGM components, several attempts have been made to supplement infant formulas (IFs), mainly with polar lipid fractions of bovine MFGM and to a lesser extent with protein fractions. The aim is thus to narrow the gap between human breast milk and cow-based IFs. Despite the few attempts made to date, supplementation with MFGM proteins seems promising as MFGM lipid supplementation. A deeper understanding of MFGM proteomes should lead to better results.

In Vitro and In Vivo Anti-inflammatory Activity of Bovine Milkfat Globule (MFGM)-derived Complex Lipid Fractions.

Numerous health related properties have been reported for bovine milk fat globule membrane (MFGM) and its components. Here we present novel data on the in vitro and in vivo anti-inflammatory activity of various MFGM preparations which confirm and extend the concept of MFGM as a dietary anti-inflammatory agent. Cell-based assays were used to test the ability of MFGM preparations to modulate levels of the inflammatory mediators IL-1β, nitric oxide, superoxide anion, cyclo-oxygenase-2, and neutrophil elastase. In rat models of arthritis, using MFGM fractions as dietary interventions, the phospholipid-enriched MFGM isolates were effective in reducing adjuvant-induced paw swelling while there was a tendency for the ganglioside-enriched isolate to reduce carrageenan-induced rat paw oedema. These results indicate that the anti-inflammatory activity of MFGM, rather than residing in a single component, is contributed to by an array of components acting in concert against various inflammatory targets. This confirms the potential of MFGM as a nutritional intervention for the mitigation of chronic and acute inflammatory conditions.

In vitro Fermentation of Digested Milk Fat Globule Membrane From Ruminant Milk Modulates Piglet Ileal and Caecal Microbiota.

Lipids in milk are secreted as a triacylglycerol core surrounded by a trilayer membrane, the milk fat globule membrane (MFGM). This membrane, known to have important roles in infant brain and intestinal development, is composed of proteins, glycoproteins, and complex lipids. We hypothesized that some of the beneficial properties of MFGM are due to its effects on the gastrointestinal microbiota. This study aimed to determine the effect of a commercial phospholipid concentrate (PC) and enriched bovine, caprine, and ovine MFGM fractions on ileal and hindgut microbiota in vitro. Digestion of PC and MFGMs was conducted using an in vitro model based on infant gastric and small intestine conditions. The recovered material was then in vitro fermented with ileal and caecal inocula prepared from five piglets fed a commercial formula for 20 days before ileal and caecal digesta were collected. After each fermentation, samples were collected to determine organic acid production and microbiota composition using 16S rRNA sequencing. All substrates, except PC (5%), were primarily fermented by the ileal microbiota (8–14%) (P < 0.05). PC and caprine MFGM reduced ileal microbiota alpha diversity compared to ileal inoculum. Caprine MFGM increased and PC reduced the ileal ratio of firmicutes:proteobacteria (P < 0.05), respectively, compared to the ileal inoculum. Bovine and ovine MFGMs increased ileal production of acetic, butyric, and caproic acids compared to other substrates and reduced the proportions of ileal proteobacteria (P < 0.0001). There was a limited fermentation of bovine (3%), caprine (2%), and ovine (2%) MFGMs by the caecal microbiota compared to PC (14%). In general, PC and all MFGMs had a reduced effect on caecal microbiota at a phylum level although MFG source-specific effects were observed at the genus level. These indicate that the main effects of the MFGM in the intestinal microbial population appears to occur in the ileum.

Polar lipid, ganglioside and cholesterol contents of infant formulae and growing up milks produced with an alpha lactalbumin-enriched whey protein concentrate

The minor lipids of human milk fat, mainly originating from the milk fat globule membrane (MFGM), provide several nutritionally important components, including gangliosides, cholesteryl esters and polar lipids (PLs; e.g., phospholipids and sphingolipids). These components are typically lower in infant formula (IF) than in human milk, since the former primarily contain vegetable oils as lipid sources. In this study, IF and growing up milk (GUM) prototypes containing a specific α-lactalbumin-enriched whey protein concentrate (80% protein) were produced to have higher levels of cholesterol, gangliosides and PLs than prototypes containing whey protein concentrate (35% protein). In the case of the enriched IF, PLs (46 mg 100 mL−1), gangliosides (2.20 mg 100 mL−1) and cholesterol (10.0 mg 100 mL−1) levels were all similar to those reported for human milk, demonstrating that appropriate ingredient selection can be used to achieve a lipid profile closer to human milk without the need for specialised MFGM fractions.

Omics analysis reveals variations among commercial sources of bovine milk fat globule membrane

Milk fat globule membrane (MFGM) is a glycosylated, protein-embedded, phospholipid fraction that surrounds triglycerides in milk. Commercial bovine sources have recently come to the market as a novel food ingredient and have been added to various products, including infant formula. Considering that MFGM is a heterogeneous mixture of fat, protein, and carbohydrate, it can be expected that variations among MFGM products exist. For this reason, our aim was to characterize the composition of commercial MFGM samples through a combination of proteomic and lipidomic analyses. Six bovine milk fractions, represented as MFGM fractions or phospholipid fractions, were obtained from various commercial sources. Additionally, the MFGM samples were compared with 2 infant formulas, a standard formula as well as a premium formula containing MFGM. For proteomic analysis, bottom-up data-dependent liquid chromatography-tandem mass spectrometry (LC-MS/MS) was performed on each MFGM fraction, and nearly a thousand proteins were identified across all samples, with 364 of them having different abundance across the samples tested. One hundred twelve proteins differed by a fold-change of 10 or greater, 14 by a fold-change of 50, and 2 by a fold-change of 100 in at least 1 pair, suggesting large differences in the proteins present in these fractions. Even though the classical MFGM proteins were enriched in the MFGM fractions, the relative protein composition varied considerably, and all contain an abundance of milk (casein and whey) proteins. Lipidomic analysis identified a total of 393 lipid species across both positive and negative ionization modes, with the major classes detected being triglycerides, sphingomyelins, and several phospholipids. Across all samples, triglycerides comprised at least 50% of total lipids, with phosphatidylcholine and sphingomyelin being the second and third most abundant lipid classes, respectively. These findings demonstrate the heterogeneous nature of various bovine commercial MFGM fractions. This variation must be considered when evaluating and describing potential functional benefits of these products shown in clinical trials.

Impact of industrial cream heat treatments on the protein composition of the milk fat globule membrane.

The impact of cream processing on milk fat globule membrane (MFGM) was assessed in an industrial setting for the first time. Three creams and their derived MFGM fractions from different stages of the pasteurization procedure at a butter dairy were investigated and compared to a native control as well as a commercial MFGM fraction. The extent of cross-linking of serum proteins to MFGM proteins increased progressively with each consecutive pasteurization step. Unresolved high molecular weight aggregates were found to consist of both indigenous MFGM proteins and β-lactoglobulin as well as αs1- and β-casein. With regards to fat globule stability and in terms of resistance towards coalescence and flocculation after cream washing, single-pasteurized cream exhibited reduced sensitivity to cream washing compared to non- and double-pasteurized creams. Inactivation of the agglutination mechanism and the increased presence of non-MFGM proteins may determine this balance between stable and non-stable fat globules.

Milk Fat Globule Membranes: Effects on Microbiome, Metabolome, and Infections in Infants and Children.

Dietary supplementation with bovine milk fat globule membrane (MFGM) concentrates has recently emerged as a possible means to improve the health of infants and young children, or defense against infections. We identified 5 double-blind, randomized, controlled trials (DBRCT) exploring the effects of supplementing the diet of infants and children with bovine MFGM concentrates on infections. We reviewed 3 studies which found a protective effect against infections at different ages during infancy and early childhood. Two of them have reported effects on the metabolome, and 1 study also on the microbiome and lipidome. MFGM supplementation had moderate, albeit interesting, effects on the oral and fecal microbiome, fecal and serum/plasma metabolome, and serum and erythrocyte membrane lipidome, which also are reviewed. We conclude that studies on MFGM supplementation during infancy and childhood indicate positive effects on the defense against infections and other outcomes, but more high-quality DBRCTs with well-defined MFGM fractions and outcome measures are needed before firm conclusions can be drawn.

In vivo anti-hypercholesterolemic effect of buttermilk, milk fat globule membrane and Enterococcus faecium FFNL-12

The present study was undertaken to evaluate the hypocholesterolemic activity of buttermilk, milk fat globule membrane (MFGM) and Enterococcus faecium FFNL-12 in rat model. Thirty-sixth male Abino rats were divided into six groups. The first one (coded as G1) was fed a standard diet containing 10% corn oil as fat source while remaining five (coded G2 to G6) were fed hypercholesterolemic diets in which oil was replaced with animal grease. Animals subjected to treatment G1 served as healthy control while those in G2 were assigned as hypocholesterolemic animals which did not receive any treatment. The remaining experimental groups were designed to assess the hypocholesterolemic effect of intragastric adminstartion of dose of 109 CFU/Kg body weight of Enterococcus faecium FFNL-12 (G3), Enterococcus faecium FFNL-12/butter milk (G4), buttermilk (G5) and milk fat globule membrane (MFGM). After four weeks, animals were evaluated in relation to growth, fecal pH, organs weight, serum lipid profile, antioxidant activity of liver tissue, liver and heart function and liver histopathological architecture. Results revealed that animals fed hypercholesterolemic diet (G2-G6) had significantly lower faecal pH and liver weight compared with those fed standard diet (G1). Treatments applied to animals fed hypercholesterolemic diet with the above mentioned additions (G3 to G6) appeared to improve both cardiac and hepatic functions, serum lipid profile and glucose concentration and liver histopathological architecture compared with animals subjected to G2 treatment. In most cases, treatment with MFGM appeared to be the most effective to avoid adverse effects associated to feeding hypercholesterolemic diet. MFGM fraction as well as E. faecium FFNL-12/buttermilk combination were effective in reducing serum lipids and glucose levels to the normal range. This combination also had potential antioxidant activity and ability to improve liver and heart functions.

Changes in bovine milk fat globule membrane proteins caused by heat procedures using a label-free proteomic approach

The milk fat globule membrane (MFGM) plays an important role in stabilizing fat in the aqueous phase, and the components of this membrane are involved in multiple biological functions. Here, we investigated changes in the protein composition of the MFGM fraction between raw and heated whole milk using a label free proteomic approach. In total, 612 MFGM-related proteins were identified in all groups. Compared with raw milk, the number of proteins that were not identified in the MFGM fraction was increased from pasteurized milk to ultra-high-temperature milk, whereas the number of milk proteins incorporated into the MFGM fraction was similar among heated groups. The abundances of several milk proteins (β-lactoglobulin and β-casein) were increased in the heated milk groups in a temperature-dependent manner. From our functional analysis, proteins that were not identified in the MFGM fraction of heated milk were mainly associated with multiple biological functions. These findings provided novel insights into the effects of heat procedures on MFGM protein components and their potential physiological functions, thereby yielding data on the appropriate heating procedures to use for raw milk.

Evaluation of six sample preparation procedures for qualitative and quantitative proteomics analysis of milk fat globule membrane.

Proteomic analysis of membrane proteins is challenged by the proteins solubility and detergent incompatibility with MS analysis. No single perfect protocol can be used to comprehensively characterize the proteome of membrane fraction. Here, we used cow milk fat globule membrane (MFGM) proteome analysis to assess six sample preparation procedures including one in-gel and five in-solution digestion approaches prior to LC-MS/MS analysis. The largest number of MFGM proteins were identified by suspension trapping (S-Trap) and filter-aided sample preparation (FASP) methods, followed by acetone precipitation without clean-up of tryptic peptides method. Protein identifications with highest average coverage was achieved by Chloroform/MeOH, in-gel and S-Trap methods. Most distinct proteins were identified by FASP method, followed by S-Trap. Analyses by Venn diagram, principal-component analysis, hierarchical clustering and the abundance ranking of quantitative proteins highlight differences in the MFGM fraction by the all sample preparation procedures. These results reveal the biased proteins/peptides loss occurred in each protocol. In this study, we found several novel proteins that were not observed previously by in-depth proteomics characterization of MFGM fraction in milk. Thus, a combination of multiple procedures with orthologous properties of sample preparation was demonstrated to improve the protein sequence coverage and expression level accuracy of membrane samples.

Polar lipid composition of the milk fat globule membrane in buttermilk made using various cream churning conditions or isolated from commercial samples

Milk fat globule membrane (MFGM) fractions were isolated from fresh raw cream at different temperatures and pH values. The polar lipid (PL) composition of these fractions, and of commercially produced α-serum, β-serum, and buttermilk powder samples, was determined. Lower temperature and pH during churning increased the total level of PLs in the MFGM fractions. Phosphatidylethanolamine (31–36% of total PLs), phosphatidylcholine (31–39%), and sphingomyelin (13–24%) were the major PLs in all MFGM fractions; phosphatidylserine (2–7%) and phosphatidylinositol (2–4%) were the minor ones. The β-serum fraction had the highest total PL content of the commercial samples (2.9 mg g−1 fat); buttermilk powder contained the least amount of total PL amongst all fractions (0.99 mg g−1). Processing temperature and pH can therefore influence the PL composition, likely due to how the treatments impact on the different PLs that are located within different regions within the MFGM trilayer structure.

The interfacial properties of various milk fat globule membrane components using Langmuir isotherms

Abstract In an attempt to understand the interfacial properties of various milk fat globule membrane (MFGM) components, their monolayers were characterized using a Langmuir film balance. MFGM fractions were separated into lipids (total and polar lipids) and defatted mass. The interfacial properties of these components were characterized at the air/water interface. The interfacial properties of MFGM were characterized at both the air/water and oil/water interfaces. Using the compression isotherm, the surface elasticity of MFGM components was also calculated. It was found that MFGM polar lipids showed relatively high surface elasticity which confirms their potential to help stabilize food systems.

Buffalo (Bubalus bubilis) colostrum and milk fat globule membrane fractions are potent antioxidants

Colostrum is the first liquid food for the new born baby, secreted after the parturition. After few days of maturation the colostrum turns to milk. The cream or fat fraction of milk consists of fat droplets composed primarily of triacylglycerols that are secreted from the apical surface of the mammary cells surrounded by cellular thin membranes membrane, the fat globule membrane (FGM), formed by proteins which have been suggested to be cholesterolemia-lowering factors, inhibitors of cancer cell growth, vitamin binders, antioxidant, bactericidal, suppressors of multiple sclerosis. FGM was isolated from buffalo colostrum and milk, the fat content was > 2-fold higher in the former than in the latter. The colostrum fat globule membrane (CFGM) fraction also showed significantly higher protein content than milk fat globule membrane (MFGM) extract. Further, the SDS-PAGE separation of the total proteins from the CFGM and MFGM showed differential banding pattern. Antioxidative property of the two FGM extracts was deliberated based on their free radical scavenging ability and reducing power. CFGM was found to exhibit a relatively higher antioxidant and reductive capacity than the MFGM fractions. The results obtained in the present study showcases the superior beneficial health effects of the CFGM over the MFGM.

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

The protein composition, redox potential (Eh), 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 Eh 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 Eh of the buttermilk emulsion (−580 mV). This study demonstrates how milk-processing methods can alter the composition and functionality of the MFGM.

N-glycosylation proteomic characterization and cross-species comparison of milk fat globule membrane proteins from mammals.

Glycosylation of proteins has been implicated in various biological functions and has received much attention; however, glycoprotein components and inter-species complexity have not yet been elucidated fully in milk proteins. N-linked glycosylation sites and glycoproteins in milk fat globule membrane (MFGM) fractions were investigated by combining N-glycosylated peptides enrichment and high-accuracy Q Exactive identification, to map the N-glycoproteome profiles in Holstein and Jersey cows, buffaloes, yaks, goats, camels, horses, and humans. A total of 399 N-glycoproteins with 677 glycosylation sites were identified in the MFGM fractions of the studied mammals. Most glycosylation sites in humans were classified as known and those in the other studied mammals as unknown, according to Swiss-Prot annotations. Functionally, most of the identified glycoproteins were associated with the 'response to stimulus' GO category. N-glycosylated protein components of MFGM fractions from Holstein and Jersey cows, buffaloes, yaks, and goats were more similar to each other compared with those of camels, horses and human. The findings increased the number of known N-glycosylation sites in the milk from dairy animal species, revealed the complexity of the MFGM glycoproteome, and provided useful information to further explore the mechanism of MFGM glycoproteins biosynthesis among the studied mammals.

Clinical Benefits of Milk Fat Globule Membranes for Infants and Children

The milk fat globule membrane (MFGM) in breast milk contains many bioactive components. Infant formulas traditionally have been devoid of the MFGM fraction, but dairy technology now has made the addition of bovine MFGM technically feasible. We identified 6 double-blinded randomized controlled trials exploring the effects of MFGM supplementation on the diets of infants or children. Results suggest that supplementation is safe and indicate positive effects on both neurodevelopment and defense against infections. MFGM supplementation of infant formula may narrow the gap in cognitive performance and infection rates between breastfed and formula-fed infants. Because of the small number of studies and the heterogeneity of interventions, more high-quality double-blinded randomized controlled trials are needed, with well characterized and clearly defined MFGM fractions, before firmconclusions on the effects of MFGM supplementation on the health and development of infants can be drawn.

Defatted bovine milk fat globule membrane inhibits association of enterohaemorrhagic Escherichia coli O157:H7 with human HT-29 cells

The bovine milk fat globule membrane (MFGM) is a source of food-derived glycans that can offer an approach to prevent Escherichia coli O157:H7 infection by inhibiting attachment of the pathogen to host cells. Such glycans may decrease the need for antibiotic treatment by acting as prophylactics. In this study, we generated a defatted bovine MFGM fraction, rich in proteins and glycoproteins, and demonstrated its ability to prevent the association of several enterohaemorrhagic E. coli O157:H7 strains with human colonic adenocarcinoma, HT-29 cells. This defatted MFGM fraction reduced bacterial association with HT-29 cells in a concentration dependent and strain specific manner. This study may present a new approach to mitigate the adverse health effects caused by E. coli infections in humans.