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.     

制备乳脂肪球膜磷脂-维生素A脂质体的工艺优化

目的:制备可添加于婴儿液态配方乳的乳脂肪球膜磷脂-维生素A脂质体,并优化其制备参数。方法:本实验采用薄膜水合-高压均质法,应用乳脂肪球膜磷脂为膜材,制备维生素A脂质体,在单因素实验基础之上,探讨制备脂质体过程中磷脂浓度、主药与磷脂比例、胆固醇与磷脂比例、维生素E占磷脂百分比浓度、反应温度等制备因素对形成脂质体后包封率的影响规律。采用二次旋转正交组合实验方法设计实验,经SAS软件与Matlab软件处理数据得到配方原料指标和反应温度对脂质体包封率的影响。结果:优化得到制备脂质体工艺参数:磷脂浓度7.43%,磷脂与主药比例28.95∶1,磷脂与胆固醇比例为6.53∶1,维生素E占磷脂百分比浓度为0.83%,反应温度59.58℃,得到优化后最高包封率为88.77%,脂质体在扫描电子显微镜下观察,其超微结构为球状囊泡。结论:本实验首次以乳脂肪球膜磷脂作为膜材包埋维生素A制备脂质体,将传统的薄膜水合法与高压均质法相结合,成功制备出可添加于婴儿食品的可食性脂质体。     

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.     

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.     

Impact of cream washing on fat globules and milk fat globule membrane proteins

Milk proteins, contained within the aqueous phase surrounding fat globules, should be removed before analysis of the composition of the native milk fat globule membrane (MFGM). The effect of the conditions applied during washing of cream on MFGM integrity has not been fully studied, and factors potentially effecting a modification of MFGM structure have not been systematically assessed so far. In this study, a cream separator was used to investigate the impact of cream washing on milk fat globule stability and the corresponding loss of MFGM proteins. Flow velocity, fat content, and type of washing solution were varied. Particle size measurements and protein analyses were carried out after each washing step to determine fat globule coalescence, removal of skim milk proteins, and efficiency of MFGM isolation. Significant differences in fat globule stability and protein amount in the MFGM isolates were measured using different washing conditions.     

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.     

Chemical and structural characterisation of almond oil bodies and bovine milk fat globules

Lipids in almonds are present as oil bodies in the nut. These oil bodies are surrounded by a membrane of proteins and phospholipids and are a delivery vehicle of energy in the form of triglycerides, similarly to the more studied bovine milk fat globule membrane. Chemical, physical and microscopic analyses revealed major differences in the composition and structure of almond oil bodies and bovine milk fat globules. The lipids of both natural emulsions differed in degree of unsaturation, chain length, and class. The almond oil body membrane does not contain any cholesterol or sphingomyelin unlike the bovine milk fat globule membrane. Therefore, the phospholipid distribution at the surface of the oil bodies did not present any liquid-ordered domains. The membranes, a monolayer around almond oil bodies and a trilayer around bovine fat globules, may affect the stability of the lipid droplets in a food matrix and the way the lipids are digested.     

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。     

β-Casein-phospholipid monolayers as model systems to understand lipid-protein interactions in the milk fat globule membrane

Phospholipid-protein monolayer films were studied as model systems to mimic the structure of the native bovine milk fat globule membrane (MFGM) and to understand lipid-protein interactions at the surface of the globule. Phospholipids extracted from bovine raw milk, raw cream, processed milk and buttermilk powder were spread onto the air-water interface of a Langmuir trough, β-casein was then added to the sub-phase, and Langmuir-Blodgett films were studied by epifluorescence microscopy and atomic force microscopy. In all films, β-casein was responsible for clustering of the sphingomyelin- and cholesterol-rich microdomains into larger platforms. This suggests that the same phenomenon may happen at the surface of the milk fat globule, where specific MFGM proteins may cause aggregation of microdomains.     

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.     

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 heating whey proteins in the presence of milk fat globule membrane extract or phospholipids from buttermilk

The objective of this study was to determine the contribution of phospholipids from buttermilk as a nucleus in the heat-induced aggregation of whey proteins. Solutions of whey proteins (5%, w/v) were adjusted to pH 4.6 or 6.8 and then heated at 65 or 80 °C for 25 min with or without 1% (w/v) of milk fat globule membrane (MFGM) extract or phospholipid powder. The aggregation mechanisms were characterised using analysis with Ellman's reagent, one-dimensional gel electrophoresis, thin-layer chromatography, and three-dimensional confocal laser-scanning microscopy. Three-dimensional images showed protein/phospholipid interactions in the presence of MFGM extract or phospholipids, and thin-layer chromatography plates showed no trace of free phospholipids after 20 min at pH 4.6. Overall, the results demonstrate that phospholipids from buttermilk were involved in the formation of protein aggregates through the MFGM fragments at a low temperature, whereas phospholipids could interact directly with the proteins at a higher temperature (80 °C).     

Production of a novel ingredient from buttermilk

The presence of material derived from the milk fat globule membrane (MFGM) makes buttermilk (the byproduct of butter making) distinct from any other dairy product. Membrane filtration of commercial buttermilk was carried out to obtain isolates rich in MFGM material. The separation of MFGM from the skim milk proteins present in commercial buttermilk was carried out by the addition of sodium citrate followed by microfiltration through a membrane of 0.1-microm nominal pore size. The sodium citrate caused the dissociation of casein micelles and allowed permeation of a large proportion of the skim-milk derived proteins through the membrane. This process successfully concentrated MFGM material in the retentate, and demonstrated that membrane filtration can be employed to produce MFGM fractions from commercial buttermilk. The utilization of MFGM isolates from buttermilk is of increasing importance in light of recent studies suggesting the role of phospholipids in many health-related functions: buttermilk is an untapped resource of these functional components.     

牛乳脂肪球膜组分分离鉴定及其功能性研究进展

乳脂肪球膜（milk fat globule membrane,MFGM）是牛乳中脂肪球外表面包裹着的一层膜,在牛乳中的含量非常微小。但由于其具有良好的生理活性以及潜在的食品加工应用潜力,关于其展开的基础研究和应用基础研究受到广泛关注。本综述介绍MFGM的分离制备技术、结构、组分鉴定分析、生理活性及其应用特性的研究进展,以期为MFGM在食品工业尤其是在功能性食品中的应用提供一定的理论支持。     

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.     

Invited review: Milk fat globule membrane—A possible panacea for neurodevelopment,infections, cardiometabolic diseases,and frailty

Milk is an evolutionary benefit for humans. For infants, it offers optimal nutrients for normal growth, neural development, and protection from harmful microbes. Humans are the only mammals who drink milk throughout their life. Lipids in colostrum originate mostly from milk fat globule membrane (MFGM) droplets extruded from the mammary gland. The MFGM gained much interest as a potential nutraceutical, due to their high phospholipid (PL), ganglioside (GD), and protein contents. In this review, we focused on health effects of MFGM ingredients and dairy food across the life span, especially on neurodevelopment, cardiometabolic health, and frailty in older adults. The MFGM supplements to infants and children reduced gastrointestinal and respiratory tract infections and improved neurodevelopment due to the higher content of protein, PL, and GD in MFGM. The MFGM formulas containing PL and GD improved brain myelination and fastened nerve conduction speed, resulting in improved behavioral developments. Administration of MFGM-rich ingredients improved insulin sensitivity and decreased inflammatory markers, LDL-cholesterol, and triglycerides by lowering intestinal absorption of cholesterol and increasing its fecal excretion. The MFGM supplements, together with exercise, improved ambulatory activities, leg muscle mass, and muscle fiber velocity in older adults. There are great variations in the composition of lipids and proteins in MFGM products, which make comparisons of the different studies impossible. In addition, investigations of the individual MFGM components are required to evaluate their specific effects and molecular mechanisms. Although we are currently only beginning to understand the possible health effects of MFGM products, the current MFGM supplementation trials as presented in this review have shown significant clinical health benefits across the human life span, which are worth further investigation.     

Milk fat globule membrane glycoproteins: Valuable ingredients for lactic acid bacteria encapsulation?

The membrane (Milk Fat Globule Membrane – MFGM) surrounding the milk fat globule is becoming increasingly studied for its use in food applications due to proven nutritional and technological properties. This review focuses first on current researches which have been led on the MFGM structure and composition and also on laboratory and industrial purification and isolation methods developed in the last few years. The nutritional, health benefits and techno-functional properties of the MFGM are then discussed. Finally, new techno-functional opportunities of MFGM glycoproteins as a possible ingredient for Lactic Acid Bacteria (LAB) encapsulation are detailed. The ability of MFGM to form liposomes entrapping bioactive compounds has been already demonstrated. One drawback is that liposomes are too small to be used for bacteria encapsulation. For the first time, this review points out the numerous advantages to use MFGM glycoproteins as a protecting, encapsulating matrix for bacteria and especially for LAB.     

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.     

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.     

乳脂肪球膜功能特性的研究进展

乳脂肪以脂肪球的形式存在, 乳脂肪球膜是包裹在乳脂肪球周围的三层生物薄膜, 具有很高的营养价值。随着食品科学研究的深入和分离技术的发展, 乳脂肪球膜中的活性成分及其功能作用正在逐渐被揭示。乳脂肪球膜是含有蛋白质、磷脂、鞘脂、神经节苷脂、胆碱、唾液酸和胆固醇的混合物, 这些成分是具有重要功能的食品成分, 应用于配方食品生产。本文综述了乳脂肪球膜中常见的蛋白质、脂质及其生物活性, 综述了近年来乳脂肪球膜及其成分在改善肠道健康、改善大脑发育、改善肥胖及相关并发症、改善老年人虚弱、抗癌、抗氧化和缓解疲劳等方面的体内研究和临床研究进展, 并讨论了其可能的作用机制, 以期为乳脂肪球膜配料的研发及其在配方食品中的应用提供借鉴和参考。