Emulsifying Properties of Bovine Milk Fat Globule Membrane in Milk Fat Emulsion: Conditions for the Reconstitution of Milk Fat Globules

A procedure for the reconstitution of milk fat globules (MFG) stabilized with milk fat globule membrane (MFGM) was developed. MFG was reconstituted by homogenizing a mixture of 1% MFGM and 25% milk fat at 45°C and at pH 7.0 for 1 min. The emulsifying properties of MFGM were evaluated by emulsifying activity (EA), emulsion stability (ES), whippability and foam stability. Of the variables affecting the reconstitution of MFG, prolonged homogenization decreased EA and ES. About 25% milk fat gave maximal EA and ES, increasing the MFGM concentration increased both EA and ES, which were also influenced by the pH level. Foam disappeared at >30°C.     

Physicochemical Properties of Milk Fat Emulsions Stabilized with Bovine Milk Fat Globule Membrane

Milk fat globules (MFG) were reconstituted with milk fat globule membrane (MFGM) and milk fat (MF). Viscosity of the reconstituted MFG was highest at pH 5.0 and 4 min emulsifying, and rose with an increase of MFGM between 40–80 mg/g fat. Adsorbed protein/unit fat increased at acid pH with increase of MFGM. The composition of proteins adsorbed on the surface of MFG was not influenced by factors of reconstitution. The size and specific surface area of globules were influenced by emulsifying time, MFGM and MF concentrations, and pH. The size range of MFG prepared by standard method was 0.9–17 μm in diameter. Median diameter was 5 μm and specific surface area was 15,600 cm²/cm³ of emulsion.     

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。     

Simple Method for Separate Determination of Three Flavins in Bovine Milk by High Performance Liquid Chromatography

Milk, dialyzed milk and milk fat globule membrane (MFGM) (1.0g each) were boiled 3 min to inactivate pyrophosphatase. Release of flavins bound to milk proteins and deproteinization were performed simultaneously by Pronase digestion for 1 hr at 45°C. After centrifugation, the extracted flavins were analyzed by HPLC, using a C18 column at 40°C connected to a spectrofluorometer. Recovery of FAD, FMN and riboflavin was 97–99% from whole milk, 95–100% from dialyzed milk, and 99-100% from MFGM. The method is simple, rapid, sensitive and reproducible. Bovine milk contained 202 μg of total flavins per 100g milk, of which FAD was 13.8, FMN 4.3, and RF 81.9mol%(n=6).     

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 Effect of Minor Components on Milk Fat Microstructure and Mechanical Properties

ABSTRACT: Although minor polar components affected the kinetics of milk fat crystallization, its structure and mechanical properties remained unchanged. At 5 °C, a granular microstructure was observed, while at 20 °C, a combination of granular and spherulitic microstructures were observed. By 27.5 °C, only spherulites were present after 24 h. There were no differences observed in the fractal dimension (D) determined by microscopy or rheology for anhydrous milk fat, milk fat triacylglycerols and MF‐TAGs with added diacylglycerols at 5 °C. There was, however, a strong temperature effect on D, increasing from 1.87 at 5 °C to 1.98 at 22.5 °C. The yield force and storage modulus were unaffected by removal of minor components.     

不同均质工艺处理对巴氏杀菌乳脂肪球膜蛋白质组成的影响

乳脂肪球大小和膜蛋白组分的变化决定了脂肪球稳定性,从而影响巴氏杀菌乳在贮藏期内的品质稳定性。本研究通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和液相色谱-质谱联用法分析不同预热温度（50、60、70?℃）和不同均质压力（20、30、40、45?MPa）对脂肪球膜蛋白的影响,并通过稳定性分析仪探究脂肪球膜蛋白变化与稳定性的关系。结果表明,均质工艺对脂肪球膜蛋白种类无显著影响,但对脂肪球膜蛋白的含量有一定影响：黄嘌呤氧化还原酶（xanthine dehydrogenase/oxidase,XO）、嗜乳脂蛋白（butyrophilin,BTN）、脂肪滴结合蛋白（adipophilin,ADPH）在预热60 ℃时的含量最低,黏液素（mucin 1,MUC1）在预热60 ℃时的含量最高,脂肪酸结合蛋白（fatty acid binding protein,FABP）、组织糖蛋白高碘酸烯夫6/7（periodic acid Schiff 6/7,PAS6/7）的含量随着预热温度的升高而减少,组织糖蛋白高碘酸薛夫IV（cluster of differentiation 36,CD36）的含量随着预热温度的升高而增多;XO、PAS6/7、BTN、ADPH、FABP在均质压力40 MPa时含量最低,CD36的含量随均质压力的增加而减少;脂肪球膜蛋白构成中,牛血清白蛋白（bovine serum albumin,BSA）、αs1-酪蛋白（casein,CN）和β-CN含量增加,其中BSA含量增加最明显。综上,推测均质工艺导致αs1-CN、β-CN和BSA组分参与了脂肪球膜的构建,并对脂肪球稳定性产生了积极的影响。     

Milk Fat Globule Membrane Substances Inhibit Mouse Intestinal β-Glucuronidase

The β-glucuronidase inhibitory action of milk fat globule membrane (MFGM) was studied. By increasing the MFGM concentration to 0.2%, nearly 90% of the β-glucuronidase activity was inhibited. The trypsin-derived digests of MFGM inhibited β-glucuronidase activity by ～ 42%. The inhibitory effect of MFGM on intestinal β-glucuronidase of mouse was studied. We administered MFGM (groups I: 5%; group II: 10%; group III: 20%) to mice and investigated the time course of changes in fecal β-glucuronidase activity. In group I and II, enzyme activity had decreased 15–20% on the 5th day of treatment, but in group III, a decrease in enzyme activity was seen with an inhibition rate of 50%, showing a marked intracolonic efficacy of MFGM. Thus MFGM may have potential in explaining, treating or preventing intestinal cancers.     

The Effect of Milk Processing on the Microstructure of the Milk Fat Globule and Rennet Induced Gel Observed Using Confocal Laser Scanning Microscopy

ABSTRACT: Confocal laser scanning microscopy (CLSM) was successfully used to observe the effect of milk processing on the size and the morphology of the milk fat globule in raw milk, raw ultrafiltered milk, and standardized and pasteurized milk prepared for cheese manufacture (cheese-milk) and commercial pasteurized and homogenized milk. Fat globule size distributions for the milk preparations were analyzed using both image analysis and light scattering and both measurements produced similar data trends. Changes to the native milk fat globule membrane (MFGM) were tracked using a MFGM specific fluorescent stain that allowed MFGM proteins and adsorbed proteins to be differentiated on the fat globule surface. Sodium dodecyl sulfate polyacrylamide gel electrophoresis confirmed the identity of native MFGM proteins isolated from the surface of fat globules within raw, UF retentate, and cheese-milk preparations, whereas only casein was detected on the surface of fat globules in homogenized milk. The microstructure, porosity, and gel strength of the rennet induced gel made from raw milk and cheese-milk was also found to be comparable and significantly different to that made from homogenized milk. Our results highlight the potential use of CLSM as a tool to observe the structural details of the fat globule and associated membrane close to its native environment.     

母乳和牛乳中乳脂肪球膜蛋白质的差异分析

为比较乳脂肪球膜（milk fat globule membrane,MFGM）蛋白在母乳和牛乳中的差异,采用有机试剂提取法,从母乳和牛乳中提取分离出MFGM蛋白,经皮尔斯TM去垢小柱去除十二烷基硫酸钠后,通过液相色谱-质谱联用技术检测分析,检测结果根据特异性肽段匹配数不小于1筛选后,在母乳和牛乳中分别检测到863 种和454 种蛋白,其中二者共有175 种,分别独有688 种和279 种。将母乳中差异表达的688 种蛋白质按PANTHER数据库进行蛋白质的Gene Ontology（GO）功能注释,这些蛋白主要是细胞部分、细胞器、细胞膜等组分,参与的生物途径有新陈代谢过程、生物调节、刺激性反应、免疫系统过程、再生作用等,具有催化、黏合、转运、酶调节等多种分子功能。另外,母乳中的这些差异蛋白参与氨酰tRNA生物合成、致病性大肠杆菌感染、脂肪酸代谢、丙酸代谢、甾体合成、酸降解等都是牛乳中的乳脂肪球膜蛋白不具有的功能。通过分析得出母乳和牛乳中的MFGM蛋白具有明显差异,虽然有部分组成和功能的重叠,但在丰度和代谢路径上,牛乳MFGM蛋白不能替代母乳MFGM蛋白。     

驴初乳与常乳乳脂球膜蛋白质组的对比分析

为阐明驴初乳与常乳中乳脂球膜蛋白的差异,利用蛋白质组学技术对二者进行蛋白质组差异分析。在驴初乳和驴常乳中分别鉴定到216 种和215 种乳脂球膜蛋白,并在其中筛选出40 种差异蛋白,包括15 种差异表达蛋白和25 种特异表达蛋白。将驴初乳与常乳中的差异蛋白进行生物信息学分析发现,差异蛋白主要参与的细胞组成为细胞外泌体、胞外囊泡、胞外细胞器等;主要参与的生物过程为对外部刺激的反应过程、细胞增殖过程、血管形态生成过程等;主要参与的分子功能为金属离子结合、阳离子结合、钙离子结合等。此外,这些差异蛋白主要参与补体和凝血级联,为生成IgA而形成的肠道免疫网络等代谢通路。利用蛋白质网络互作分析发现,差异蛋白中存在具有高连接度的关键乳脂球膜蛋白因子。本研究有助了解驴乳脂球膜蛋白的生物学特性,并为驴乳中乳脂球膜蛋白的营养学研究及相关配方乳粉的研发提供理论基础。     

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。     

STORAGE STABILITY OF MILKFAT GLOBULE MEMBRANE

To investigate changes in structure and function of milk fat globule membrane (MFGM) components due to oxidation of membrane lipids, freeze-dried MFGM were stored under controlled temperature and relative humidity (RH) conditions. It was found that while membranes underwent extensive lipid oxidation when stored under air, fluorescent compounds were formed. These compounds exhibited fluorescence with excitation maxima at 350 nm and emission maxima at 440 nm; which were similar to those of the fluorescent substances derived from the reaction of oxidized fatty acids and primary amines. Formation of high molecular weight proteins was detected by SDS-PAGE in samples stored under air, but not in samples stored under nitrogen and was also affected by time and relative humidity. Activity of xanthine-oxidase, an important prooxidative agent, was greatly influenced by temperature and complete inactivation was observed when MFGM's were stored at 37°C, 50% RH for 1 day.     

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。     

Influence of the Colloidal Structure of Dairy Gels on Milk Fat Fusion Behavior: Quantification of the Liquid Fat Content by In Situ Quantitative Proton Nuclear Magnetic Resonance Spectroscopy (isq 1H NMR)

Dairy gels (DG), such as yoghurts, contain both solid and liquid fats at the time of consumption, as their temperature rises to anything between 10 and 24 °C after being introduced into the mouth at 4 °C. The mass ratio between solid and liquid fats, which depends on the temperature, impacts the organoleptic properties of DG. As the ordinary methods for determining this ratio can only be applied to samples consisting mainly in fat materials, a fat extraction step needs to be added into the analytical process when applied to DG, which prevents the study of the potential impact of their colloidal structure on milk fat fusion behavior. In situ quantitative proton nuclear magnetic resonance spectroscopy (isq ¹H NMR) was investigated as a method for direct measurements in DG: at temperatures between 20.0 and 70.0 °C, the liquid fat content and the composition of triacylglycerols of the liquid phase (in terms of alkyl chains length) were determined. Spectra of isolated milk fat also enable the quantification of the double bonds of triacylglycerols. Statistical tests showed no significant difference between isolated milk fat and milk fat inside a DG in terms of melting behavior: the fat globule membrane does not seem to have a significant influence on the fat melting behavior.     

Fat Content of Milk and Cream and Effects on Fat Globule Stability

Milk fat globules with crystalline fat content were sheared in defined flow conditions in order to ascertain the critical strain rate, which led to destabilization of the fat globules. Based on a fluid-mechanical model which considered the influence of the dispersed phase, a critical shear rate was defined. The model predicted a linear decrease in destabilization shear rate coupled with an increase in fat volume concentration. This interrelationship was confirmed in experiments. Fat globule stability, as related to shear strain rate, increased at decreasing temperatures. Critical shear rates for milk and cream should be calculated at a fat content not exceeding 45% for temperatures between 5 and 20°C. Any motion at higher fat content had a destabilizing effect on fat globules.     

Characterization of protein components of natural and heat-treated milk fat globule membranes

The proteins associated with the milk fat globule membrane (MFGM), isolated from early, mid and late season whole milks, were characterized using one- and two-dimensional SDS-PAGE under reducing and non-reducing conditions. In some experiments, MFGM separated from fresh whole milk was suspended in simulated milk ultrafiltrate (SMUF) and heated at various temperatures and times. SDS-PAGE under reducing conditions followed by staining with Coomassie blue showed the presence of about 37 protein bands, ranging in molecular weight from 15 to 200 kDa. SDS-PAGE under non-reducing conditions showed only about 25 distinct bands and the intensity of xanthine oxidase and butyrophilin bands was much less, while the intensity of PAS 6/7 band was similar compared with the reduced SDS-PAGE. Two-dimensional SDS-PAGE showed that the protein complexes that remained at the top of non-reducing gel were resolved into mostly xanthine oxidase and butyrophilin with a small proportion of PAS 6. These results indicate that xanthine oxidase and butyrophilin may be complexed via intermolecular disulfide bonds in the natural MFGM, although it is not possible to differentiate the individual protein distributions within these aggregates. It was found that the total protein content (mg/g fat) of MFGM and the percentage of xanthine oxidase and butyrophilin in early and late season MFGM were higher than that of mid season MFGM. In heated samples (above 60°C), xanthine oxidase and butyrophilin interacted further to form higher molecular weight protein complexes, while PAS 6/7 was relatively heat stable.     

Fractionation of Anhydrous Milk Fat by Superficial Carbon Dioxide

Milk fat was fractionated with supercritical CO₂ (SC-CO₂) into 8 fractions at temperatures of 50° and 7O°C, over a pressure range of 100–350 bar. Two fractions (L1 and 2) were liquid, 3 fractions (11–3) were semi-solid and 3 fractions (Sl-3) were solid at 20°C. The peak melting temperature progressively increased (9.7° to 38.3°C) from fraction L1 to S3. The concentration of short chain (C24-C34) triglycerides decreased from fraction L1 to S3 while that of long chain (C42-C54) triglycerides increased gradually. The medium chain triglycerides were more concentrated in fractions L2 and 11–3. The proportion of short (C4-C8) and medium (ClO-C12) chain fatty acids decreased and that of long (C14-C18) chain fatty acids increased gradually from fraction Ll-S3. The weight average molecular weights and geometric mean-carbon number of milk fat fractions were in the range from 625.6 to 805.0 and 34.2 to 47.6, respectively, in comparison to 729.3 and 41.O, respectively, for native milk fat, suggesting SC-CO₂ effected a fair degree of molecular weight separation.     

The impact of cream churning conditions on xanthine oxidase activity and oxidation–reduction potential in model emulsion systems

Six types of milk fat globule membrane (MFGM) fractions were isolated from fresh raw cream using various churning conditions at different temperature (10, 15 or 20 °C) and pH values (5.5 or 6.6) and characterised for protein composition, xanthine oxidase (XO) content and activity, and oxidation–reduction potential (E_h). A greater proportion of non-membrane proteins was found at higher temperatures and higher cream pH, and the XO content decreased significantly under these conditions. XO activity was greater at lower temperatures and pH, yielding a more positive E_h value for the emulsions. Storage at 4 °C for 14 d significantly reduced the Eh of the emulsions to between −320 mV and −580 mV for low pH/low temperature, and high pH/high temperature emulsions, respectively. This study suggests that it is possible to obtain MFGM fractions with specific Eh reducing ability by altering churning conditions.     

Use of ultrafiltration and supercritical fluid extraction to obtain a whey buttermilk powder enriched in milk fat globule membrane phospholipids

Whey buttermilk, a by-product from whey cream processing to butter, is rich in milk fat globule membrane (MFGM) constituents, which have technological and potential health properties. The objective of this work was to produce a dairy ingredient enriched in MFGM material, especially phospholipids, from whey buttermilk. Whey buttermilk was concentrated by ultrafiltration (10×) and subsequently diafiltered (5×) (10 kDa molecular mass cutoff membrane) at 25 °C and the final retentate was spray-dried. The whey buttermilk powder was submitted to supercritical extraction (350 bar, 50 °C) using carbon dioxide. The membrane filtration removed most of the lactose and ash from the whey buttermilk, and the supercritical extraction extracted exclusively non-polar lipids. The final powder contained 73% protein and 21% lipids, of which 61% were phospholipids. This ingredient, a phospholipids-rich dairy powder, could be used as an emulsifier in different food systems.