Using lipase activity to probe milkfat globule membranes (MFGM) or interfaces

Measurement of lipoprotein lipase activity (as free fatty acid change or accumulation) is accepted as indicative of milkfat globule membrane (MFGM) disruption. However, measurement is confounded by variables unrelated to the MFGM, e.g., microbial quality. To resolve this, a modified approach to using lipase activity to probe MFGM was developed. Methanol (1%, w/w) and lipoprotein lipase (2%, w/w, raw milk) were added to pasteurised milk samples resulting in formation of methyl esters that therefore served as a new and unique signal for lipase gaining access to interfacial fat. Analysis conditions minimised product formation to minimise sample perturbation. The method could detect 1% (w/w) of homogenised milkfat globules blended with native globules, with linear response to 10% (w/w) and saturation above 20% (w/w). The approach was applied to assess MFGM in samples subjected to disruptive treatments (shear, aeration) under different conditions; findings were consistent with previous research, but with greater sensitivity.     

Bovine milk fat globule membrane proteome

Milk fat globule membranes (MFGM) were isolated from the milk of mid-lactation Holstein cows. The purified MFGM were fractionated using 1-dimensional SDS gels. Tryptic peptides from gel slices were further fractionated on a micro-capillary high performance liquid chromatograph connected to a nanospray-tandem mass spectrometer. Analysis of the data resulted in 120 proteins being identified by two or more unique peptide sequences. Of these 120 proteins, 71% are membrane associated proteins with the remainder being cytoplasmic or secreted proteins. Only 15 of the proteins identified in the cow MFGM were the same as proteins identified in previous mouse or human MFGM proteomic studies. Thus, the bulk of the proteins identified are new for bovine MFGM proteomics. The proteins identified were associated with membrane/protein trafficking (23%), cell signalling (23%), unknown functions (21%), fat transport/metabolism (11%), transport (9%), protein synthesis/folding (7%), immune proteins (4%) and milk proteins (2%). The proteins associated with cell signalling or membrane/protein trafficking may provide insights into MFGM secretion mechanisms. The finding of CD14, toll like receptor (TLR2), and TLR4 on MFGM suggests a direct role for the mammary gland in detecting an infection.     

The fat globule membrane in ice cream

A method for determining the average thickness of the membrane around the fat globules in homogenised ice cream mix from the mix viscosity is examined. The difficulties with this approach, such as the non-Newtonian behaviour of the mix, the polydisperse nature of the fat globule size distribution and the contribution of the micellar casein to the volume fraction of the aqueous phase, are discussed. Values of 60 to 90 nm for the effective “hydrodynamic” thickness have been obtained for ice cream mixes of 9% w/w vegetable fat and 10.5% w/w skim milk solids. It is suggested from these values, and from the interpretation of electron micrographs of similar mixes, that part of the micellar casein is attached to fat globules, thus increasing the effective volume fraction of the fat and accounting for the abnormally high viscosities of these mixes.     

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

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

Physical and chemical properties of milkfat and phytosterol esters blends

Over the past few decades, consumption of butter has declined due to its high content of saturated fatty acids and cholesterol. Opposingly, phytosterol esters (PE) have been used as a food ingredient for their effectiveness in lowering plasma cholesterol levels. The objective of this work was to evaluate the effects of the addition of PE on the physical and chemical properties of milkfat (MF). The fatty acid and sterol compositions were determined by gas chromatography (GC). A constant speed texture analyser was used to evaluate consistency. Solid fat content (SFC) was determined by nuclear magnetic resonance (NMR) and crystal structure was observed with polarized light microscopy. Results showed that β-sitosterol (33.5%), campesterol (23.6%), and stigmasterol (14.9%) were the most abundant sterols in the PE blend. Blends containing MF and PE presented a softer consistency than pure MF at low temperatures. Despite the higher SFC, PE caused a decrease in the SFC of MF. The images of MF, PE, and their blends obtained by polarized light microscopy showed the influence of temperature on crystal size and number. This method revealed the particular crystalline structure of PE, formed by a solid, dense crystal network, with amorphous crystals of varied sizes, different from crystal structures generally observed in fats. The high SFC and low consistency of PE may indicate a weak crystal network, probably due to the presence of different molecular structures.     

Major proteins of the goat milk fat globule membrane

Fat is present in milk as droplets of triglycerides surrounded by a complex membrane derived from the mammary epithelial cell called milk fat globule membrane (MFGM). Although numerous studies have been published on human or bovine MFGM proteins, to date few studies exist on MFGM proteins from goat milk. The objective of this study was thus to investigate the protein composition of the goat MFGM. Milk fat globule membrane proteins from goat milk were separated by 6% and 10% sodium dodecyl sulfate-PAGE and were Coomassie or periodic acid-Schiff stained. Most of MFGM proteins [mucin-1, fatty acid synthase, xanthine oxidase, butyrophilin, lactadherin (MFG EGF-8, MFG-E8), and adipophilin] already described in cow milk were identified in goat milk using peptide mass fingerprinting. In addition, lectin staining provided a preliminary characterization of carbohydrate structures occurring on MFGM proteins from goat milk depending on α_S1-casein genotype and lactation stage. We provide here first evidence of the presence of O-glycans on fatty acid synthase and xanthine oxidase from goat milk. A prominent difference between the cow and the goat species was demonstrated for lactadherin. Indeed, whereas 2 polypeptide chains were easily identified by peptide mass fingerprinting matrix-assisted laser desorption/ionization-time of flight analysis within bovine MFGM proteins, lactadherin from goat milk consisted of a single polypeptide chain. Another striking observation was the presence of caseins associated with MFGM preparations from goat milk, whereas virtually no caseins were found in MFGM extracts from bovine milk. Taken together, these observations strongly support the existence of a singular secretion mode previously hypothesized in the goat.     

乳脂肪球膜(MFGM)的组成及生理特性

综述了MFGM的组成、结构、MFGM的分离纯化,并对MFGM对人类具有有益生理效应的组分进行了总结,为今后MFGM的研究开发和更广泛的应用提供了一定的应用参考.     

Physical,textural, and rheological properties of whipped cream affected by milk fat globule membrane protein

This work aims at improving the textural and whipping properties of whipped cream by the addition of milk fat globule membrane protein. The determination of particle size distribution and average diameter of whipped cream showed that the small particle size was shifted to a larger range after milk fat globule membrane protein was added. The average particle size (d_3,2) of whipped cream reached a maximum value of 5.05 µm at 1% milk fat globule membrane protein, while slowly decreased with increasing milk fat globule membrane protein levels from 2% to 5%. In addition, the partial coalescence of fat increased with the increase of milk fat globule membrane protein levels, and the correlation between the whipping time and the overrun of whipped cream was positive. The addition of milk fat globule membrane protein also altered the rheological behaviour of whipped cream, resulting in the increase of modulus G′ and the loss modulus G″. The results also indicated that higher milk fat globule membrane protein level decreased the serum loss of whipped cream while improved its stability. While milk fat globule membrane protein levels had no significant effect on viscosity, its increasing levels effectively improved the hardness, consistency, and viscosity of whipped cream.     

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.     

Modulation of immune function by milk fat globule membrane isolates

The nutritional value and characterization of minor milk components on mammalian immune function are not fully understood. The aim of this research was to test the ability of a milk fat globule membrane (MFGM) isolate to modulate murine immune function in vitro, by studying its effects on splenocyte proliferation, apoptosis, and cytokine production. Proliferation of spleen cells was not affected by the MFGM isolate; however, in the presence of polyclonal activators, the MFGM isolate suppressed cell proliferation. Results obtained by flow cytometry did not support programmed cell death as the cause of the MFGM immune-modulating capacity. A mode of suppression on the splenocyte activation process was suggested from a marked decrease in the production of IFN-γ and tumor necrosis factor-α cytokines, typical indicators of immune cell activation. The effect of MFGM on IL-4 secretion was significantly less than that for the other 2 cytokines. The activity exerted by the MFGM over concanavalin A-stimulated cells differed from that observed in cells treated with lipopolysaccharide, suggesting a different mode of action depending on the activator used. These results indicate the potential of MFGM extracts as functional ingredients with bioactive modulating capacity.     

乳脂肪球膜的组成与应用研究进展

综述了乳脂肪球膜的组成、分离提取方法与应用的研究进展,对有效利用乳脂肪球膜资源提供参考。     

菜籽蛋白水解物及其膜分离组分的体外抗肿瘤活性研究

应用胃肠道蛋白酶水解菜籽分离蛋白,并通过膜分离技术将水解物分离成4种不同分子量组分(1 kDa、1~3 kDa、3~5 kDa和5~10 kDa);研究了水解物和不同组分对HepG2、HeLa和MCF-7三种肿瘤细胞的体外增殖抑制作用及其氨基酸组成。结果表明:水解物及各组分对HepG2体外增殖具有较明显的抑制作用,而对HeLa和MCF-7体外增殖的抑制效果不明显;1kDa的组分对HepG2的抑制率最强(36.4%),其氨基酸组成中以谷氨酸、亮氨酸、天冬氨酸含量最高。研究认为,菜籽蛋白胃肠道蛋白酶水解物及其1 kDa的膜分离组分可能作为功能性成分用于抗肿瘤相关的功能食品和保健品的开发。     

乳脂酶法水解产物的微胶囊化

用喷雾干燥法对乳脂酶法水解产物进行微胶囊化,考察了乳化温度、加水量等因素对乳化液稳定性、粘度的影响,通过正交实验确定了最适条件为:加水量50%、乳化剂添加量2%、心壁比30%。     

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.     

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。     

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.     

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.