Fat globule membranes in ewes' milk: The main enzyme activities during lactation

A trial was carried out on seven Massese ewes, from which individual milk samples were taken during lactation. All the enzyme activities investigated (oxidase activity of xanthine oxidoreductase, gamma-glutamyltranspeptidase, alkaline phosphatase and 5′-nucleotidase) were affected by the lactation phase, with the exception of the dehydrogenase activity of xanthine oxidoreductase (XOR). A higher oxidase activity (XO) of XOR was found when the diameter of milk fat globules was smaller. In addition, the absence of XO in colostrum led to the hypothesis that its antibacterial role is independent of its activity. The increase in alkaline phosphatase activity during lactation requires further research to better define the criteria for pasteurised sheep milk.     

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。     

Distribution of xanthine oxidase and xanthine dehydrogenase activity in bovine milk: Physiological and technological implications

The aim of this study was to evaluate the distribution of xanthine oxidoreductase (XOR) and its two forms, xanthine oxidase (XO) and xanthine dehydrogenase (XD), in milk fractions. XOR associated with milk phospholipid membranes was found to be distributed among an intra-membranous pool in which it takes the form of a mixture of XO and XD, with a clear predominance of XD, and a free pool of XO, of which 33% is found in the outer surface of milk fat globule membrane, 20.5% in the outer surface of whey membrane particles, and the remaining 46.7% in apparent solution. The inner-membrane XOR may play a nonenzymatic role in fat secretion, whereas extra-membranous XO is freely available for a role in the innate gland immune system and may affect milk quality.     

Oxidation of aldehydes by xanthine oxidase located on the surface of emulsions and washed milk fat globules

The enzymatic activity of xanthine oxidase (XO) located within the native and recombined milk fat globule membrane (MFGM) on emulsion surfaces was investigated after successive washing with deionised water (DW) or simulated milk ultrafiltrate (SMUF). DW was more effective than SMUF at removing non-MFMG proteins from the globule surface. Surface XO activity on a protein basis increased after three washes due to loss of MFGM proteins to the serum phase. Recombined emulsions had higher surface enzymatic activity than washed native fat globule samples. The oxidation of acetaldehyde, 3-methylbutanal, 2-methylpropanol and 2-methylbutanal to the corresponding acids by XO on the globule surface was analysed. A higher proportion of aldehydes were depleted at higher XO activity when the initial aldehyde concentration was constant, and for higher initial concentrations of aldehydes. The recovery of aldehydes and acids was above 79%, indicating that the depleted aldehydes were mostly converted to the corresponding acids.     

Interactions of fat globule surface proteins during concentration of whole milk in a pilot-scale multiple-effect evaporator

The changes in milk fat globules and fat globule surface proteins during concentration of whole milk using a pilot-scale multiple-effect evaporator were examined. The effects of heat treatment of milk at 95 degrees C for 20 s, prior to evaporation, on fat globule size and the milk fat globule membrane (MFGM) proteins were also determined. In both non-preheated and preheated whole milk, the size of milk fat globules decreased while the amount of total surface proteins at the fat globules increased as the milk passed through each effect of the evaporator. In non-preheated samples, the amount of caseins at the surface of fat globules increased markedly during evaporation with a relatively small increase in whey proteins. In preheated samples, both caseins and whey proteins were observed at the surface of fat globules and the amounts of these proteins increased during subsequent steps of evaporation. The major original MFGM proteins, xanthine oxidase, butyrophilin, PAS 6 and PAS 7, did not change during evaporation, however, PAS 6 and PAS 7 decreased during preheating. These results indicate that the proteins from the skim milk were adsorbed onto the fat globule surface when the milk fat globules were disrupted during evaporation.     

Composition of milk fat from cows selected for milk fat globule size and offered either fresh pasture or a corn silage-based diet

The objective of this study was to examine the synthesis and composition of milk produced by dairy cows that secrete either small milk fat globules (SMFG) or large milk fat globules (LMFG), and to study their response to diets known to alter milk composition. Four groups of 3 multiparous dairy cows were assigned to 2 isoenergetic feeding treatments: a corn silage treatment supplemented with soybean meal, and fresh pasture supplemented with cereal concentrate. The 4 groups comprised 2 groups of 3 dairy cows that produced SMFG (3.44 μm) and 2 groups of 3 dairy cows that produced LMFG (4.53 μm). The SMFG dairy cows produced higher yields of milk, protein, and calcium. Nevertheless, their milk had lower fat and protein contents. Both SMFG and LMFG cows secreted similar amounts of milk fat; therefore, higher globule membrane contents in milk fat were observed in SMFG cows. Higher calcium mineralization of the casein micelles in SMFG cows suggests that it may be possible to improve cheese-making properties even if the lower protein content may lead to lower cheese yields. The SMFG cows secrete milk fat with a higher concentration of monounsaturated fatty acids and a lower concentration of short-chain fatty acids. They also have a higher C18:1/C18:0 ratio than LMFG cows. This suggests that SMFG cows have more significant fatty acid elongation and desaturation. The pasture treatment led to an increase in milk and protein yields because of increased energy intake. It also resulted in lower milk fat yield and fat and protein contents. The pasture treatment led to a decrease in milk fat globule size and, as expected, an increase in monounsaturated and polyunsaturated fatty acid contents. However, it induced a decrease in the protein content, and in calcium mineralization of casein micelles, which suggests that this type of milk would be less suitable for making cheese. This study also shows that there is no correlation between the cows, based on milk fat globule size and diet. These results open up possibilities for improving milk fat quality based on milk fat globule size, and composition. The mechanisms involved in milk fat globule secretion are still to be determined.     

Effect of Lenient Steam Injection (LSI) heat treatment of bovine milk on the activities of some enzymes,the milk fat globule and pH

This study investigated the effects of Lenient Steam Injection (LSI) treatment at temperatures 70–150 °C on the enzymatic activities of the indigenous milk enzymes alkaline phosphatase, lactoperoxidase (LPO), xanthine oxidase (XO), lipoprotein lipase (LPL) and plasmin in comparison with two reference heat treatments of 63 °C for 30 s and of 72 °C for 15 s by indirect heating. Milk fat globule (MFG) size distributions and pH were also monitored. Alkaline phosphatase, LPO, XO and LPL activities decreased with increasing LSI temperature. Plasmin activity was increased at temperatures <80 °C and decreased at temperatures above 90 °C. Milk fat globule homogenisation was observed at temperatures above 110–130 °C.     

牛乳和双峰驼乳中乳脂球膜蛋白的差异分析

为阐明双峰驼乳中乳脂球膜蛋白（milk fat globule membrane proteins,MFGMP）的组成以及与牛乳MFGMP之间的差异,以牛乳和双峰驼乳中的MFGMP为研究对象,采用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳、双向电泳和液相色谱-串联质谱（liquid chromatograph-tandem mass spectrometry,LC-MS/MS）技术对其中的高丰度蛋白质进行表征,并对双峰驼乳MFGMP提取方法进行选择。结果显示,双峰驼乳和牛乳MFGMP中含量最高的蛋白组分依次为嗜乳脂蛋白、黄嘌呤氧化还原酶/脱氢酶、乳凝集素（MFG-E8或PAS6/7）、脂肪分化相关蛋白（ADRP或PLIN2）,3 种方法分析双峰驼乳和牛乳中MFGMP中高丰度蛋白组分的结果具有一致性。基于LC-MS/MS技术对双峰驼乳的MFGMP进行鉴定,并将对鉴定出的蛋白质进行基因本体功能注释和京都基因与基因组百科全书路径分析,发现这些蛋白主要是细胞膜、线粒体、内质网膜等组分,参与的生物途径有翻译和翻译的起始、氧化还原过程、细胞内蛋白质转运、蛋白质折叠、细胞氧化还原稳态等,具有结合活性和分子催化等分子功能。     

Native vs. damaged milk fat globules: membrane properties affect the viscoelasticity of milk gels

The storage modulus G' of rennet and acid milk gels filled with milk fat globules was measured as a function of the fat globule surface composition (native milk fat globule membrane, caseins and whey proteins, or a mixture of the three due to mechanical treatments) and surface area (i.e., the fat globule size). By different technological procedures, it was possible to obtain fat globules of constant surface composition but various sizes, and vice-versa, which had never been done. For both rennet and acid gels, a critical fraction of the fat globule surface covered by caseins and whey proteins was identified (approximately 40%), beyond which G' increased. Below this threshold, the gel viscoelasticity was unaffected by mechanical treatments. When the diameter of native milk fat globules decreased, the G' of rennet gels increased slightly, whereas that of acid gels decreased sharply. For both types of gels, G' increased when the diameter of partially disrupted fat globules decreased. For recombined globules completely covered with caseins and few whey proteins, G' increased with fat globule surface area for rennet gels whereas it decreased for acid gels. With the help of confocal microscopy and in the light of general structural differences between rennet and acid gels, a mechanism is proposed for the effect of fat globule damage and diameter on G', depending on the interactions the globules can undergo with the casein network.     

Effect of the fat globule membrane on in vitro digestion of milk fat globules with pancreatic lipase

The rate and extent of in vitro lipid digestion in raw and recombined milk were investigated by determining the release of fatty acids in simulated intestinal fluid containing pancreatic lipase. Changes in the globule size, surface charge and microstructure of fat globules during digestion were examined. In the absence of bile extract, the rate of lipid digestion was slower in raw milk than in recombined milk, suggesting that the composition of the milk fat globule membrane influences the rate of lipid hydrolysis in milk. Flocculation of fat globules occurred in the early stages of digestion; the globules then coalesced to form large particles, from within which triacylglycerols were removed. However, in the presence of bile extract, the changes in the size and microstructure of the fat globules during digestion were different. Bile extract may therefore affect physicochemical interactions of fat globules and hence alter the lipolysis during the digestion.     

Buffalo vs. cow milk fat globules: Size distribution,zeta-potential,compositions in total fatty acids and in polar lipids from the milk fat globule membrane

Although buffalo milk is the second most produced milk in the world, and of primary nutritional importance in various parts of the world, few studies have focused on the physicochemical properties of buffalo milk fat globules. This study is a comparative analysis of buffalo and cow milk fat globules. The larger size of buffalo fat globules, 5 vs. 3.5 μm, was related to the higher amount of fat in the buffalo milks: 73.4 ± 9.9 vs. 41.3 ± 3.7 g/kg for cow milk. Buffalo milks contained significantly lower amount of polar lipids expressed per gram of lipids (0.26% vs. 0.36%), but significantly higher amount of polar lipids per litre of milk (+26%). Buffalo and cow milk fat globule membranes contain the same classes of polar lipids; phosphatidylethanolamine, sphingomyelin (SM) and phosphatidylcholine (PC) being the main constituents. A significant higher percentage of PC and lower percentage of SM were found for buffalo milks. The fatty acid analysis revealed that saturated fatty acids, mainly palmitic acid, trans fatty acids, linolenic acid (ω3) and conjugated linolenic acid were higher in buffalo milk than in cow milk. Such results will contribute to the improvement of the quality of buffalo milk-based dairy products.     

Proteolysis of milk fat globule membrane proteins during in vitro gastric digestion of milk

The influence of gastric proteolysis on the physicochemical characteristics of milk fat globules and the proteins of the milk fat globule membrane (MFGM) in raw milk and cream was examined in vitro in simulated gastric fluid (SGF) containing various pepsin concentrations at pH 1.6 for up to 2 h. Apparent flocculation of the milk fat globules occurred in raw milk samples incubated in SGF containing pepsin, but no coalescence was observed in either raw milk samples or cream samples. The changes in the particle size of the fat globules as a result of the flocculation were dependent on the pepsin concentration. Correspondingly, the physical characteristics of the fat globules and the composition of the MFGM proteins in raw milk changed during incubation in SGF containing pepsin. The major MFGM proteins were hydrolyzed at different rates by the pepsin in the SGF; butyrophilin was more resistant than xanthine oxidase, PAS 6, or PAS 7. Peptides with various molecular weights, which altered with the time of incubation and the pepsin concentration, were present at the surfaces of the fat globules.     

Evaluation of cooling strategies for pumping of milk - impact of fatty acid composition on free fatty acid levels

Cooling strategies for pumping of raw milk were evaluated. Milk was pumped for 450 s at 31 degrees C, or pumped after cooling to 4 degrees C and subsequently subjected to various incubation times. Two types of milk were used; i.e. milk from cows fed a diet high in saturated fat supplements resulting in significantly larger milk fat globules than the other type of milk which comes from cows fed a low-fat diet that stimulates high de novo fat synthesis. The content of liquid fat was determined by low-field 1H NMR, which showed that milk from cows given the saturated fat diet also contained less liquid fat at both 4 degrees and 31 degrees C than the other type of milk. This can be ascribed to the differences in the fatty acid composition of the milk as a result of the fatty acid composition of the diets. After pumping of the milk at 31 degrees C, measurement of fat globule size distribution revealed a significant coalescence of milk fat globules in the milk obtained from the saturated fat diet due to pumping. Pumping at 4 degrees C or pumping the other type of milk did not result in coalescence of milk fat globules. Formation of free fatty acids increased significantly in both types of milk by pumping at 31 degrees C. Cooling the milk to 4 degrees C immediately before pumping inhibited an increased content of free fatty acids. However, when the milk was incubated at 4 degrees C for 60 min after cooling and then subjected to pumping, a significant increase in the formation of free fatty acids was observed in both types of milk. It is suggested that this increase in free fatty acids is caused by transition of polymorphic crystal forms or higher level of attached lipoprotein lipases to the milk fat globule before pumping.     

The manufacture of miniature Cheddar-type cheeses from milks with different fat globule size distributions

A novel 2-stage gravity separation scheme was developed for fractionation of raw, whole bovine milk into fractions enriched in small (SFG) or large (LFG) fat globules. The volume mean diameter of fat globules in SFG, LFG or control (CTRL) milk was 3.45, 4.68 and 3.58 microm, respectively. The maximum in storage modulus (index of firmness) decreased with increasing fat globule size for rennet-induced gels formed from SFG, LFG or CTRL milks. Miniature (20 g) Cheddar cheeses were manufactured using each of the 3 milks. There were no significant (P > 0.05) differences in the pH, moisture and fat in dry matter levels between cheeses made using any of the 3 milks, however, the fat content of the cheese made using SFG milk was approximately 1% lower than that of cheese made using LFG or CTRL milk in each of the 2 trials. Image analysis of confocal scanning laser micrographs of the cheeses illustrated that the star volume of fat globules in the cheeses decreased significantly (P < or = 0.05) as the size of fat globules in the milks used for cheesemaking was reduced. This indicates that it is possible to manipulate the size distribution of fat globules in Cheddar cheese by adjusting the fat globule size distribution of the milk used for cheese-making. The concentration of free fatty acids (FFA) increased in all cheeses during ripening. At 120 d of ripening, the concentration of FFA varied significantly (P < or = 0.05 and P < or = 0.001 for trials 1 and 2, respectively) with fat globule size, with cheeses made in trial 2 from LFG, SFG or CTRL milks having total FFA levels of 3391, 2820 and 2612 mg/kg cheese, respectively.     

Factors related to the formation of cytoplasmic crescents on milk fat globules

Cytoplasm from lactating cells is included with some milk fat globules at secretion. The objective was to search for factors causing this phenomenon. Globules bearing crescents of cytoplasm were selectively stained with the fluorescent dye, acridine orange, and their proportion in the globule population was obtained by counting from photomicrographs. Incidence of crescents on fat globules in milk samples of 50 human donors ranged from 1 to 29%, and the mean was 7.2%. Two bovine milk samples, both representing over 100 animals, contained 1% or less of globules with crescents. Globules in individual milkings of five beef cows showed the same low proportion of crescents. In addition to species, genetic and diurnal factors influenced numbers of crescents. Two sisters showed evidence within and between lactations of a persistent high proportion (greater than 25%) of globules with crescents. Samples collected in the a.m. contained a lower percentage of globules with crescents (6.5%) than those obtained in the p.m. (9.7%). Crescent incidence was not correlated with lipid or protein content of human milk, interval within a milking, days in lactation, or the donor's age. Evidence is presented to suggest that the concentration, distribution, and acylation of butyrophilin and xanthine oxidase, coat proteins of the apical plasma membrane, are important factors in globule crescent formation.     

Disruption of fat globules during concentration of whole milk in a pilot scale multiple-effect evaporator

The effects of heat treatment by direct steam injection (DSI) and concentration of whole milk using a pilot scale multiple-effect evaporator on fat globule size and the total milk fat globule membrane (MFGM) proteins were examined. In both nonpreheated and preheated whole milk, the size of milk fat globules decreased while the surface protein concentration of the fat globules increased as the milk passed through each effect of the evaporator. These results indicate that the fat globules were disrupted during DSI heating and evaporation and the proteins from the skim milk were adsorbed onto the fat globule surface.     

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.     

Free oil and rheology of Cheddar cheese containing fat globules stabilized with different proteins

Cheddar cheese was manufactured from recombined milk containing fat globules coated with alpha(s1)-CN (casein), alpha(s2)-CN, beta-CN, kappa-CN, alpha-lactalbumin, or beta-lactoglobulin. The effect of the coating on fat globule structure, free oil formation, and cheese rheology was investigated to determine if globule coating affected the physical structure of cheese. Fat globule size and shape were determined in cheese using confocal laser scanning microscopy, and the rheological properties measured by uniaxial compression after maturation for 35 and 70 d. Fat globules were elongated and clustered in the control cheese coated with native membrane material and in cheese where the globules were coated with alpha(s2)-CN, but were more circular and distinct than all others. Cheese containing globules coated with alpha(s2)-CN fractured at a lower strain and with a lower stress than other experimental cheeses. Free oil decreased in cheese as the stress at fracture of the cheese protein matrix increased. Strain at fracture increased as pH increased from 4.7 to 5.3. There was no correlation between free oil and fat globule circularity. Cheddar cheese aroma was not evident in experimental cheeses.     

Free and membrane-bound xanthine oxidase in bovine milk during cooling and heating

The effect of cold storage (5 C, 24 h) and heat treatment (60 C, 5 min) of milk on activities of free and membrane-bound xanthine oxidase has been studied. Both treatments enhanced total xanthine oxidase activity in milk. Activity of membrane-bound xanthine oxidase increased and free xanthine oxidase decreased in buttermilk while it increased in skim milk on cold storage. Heat of milk increased free and membrane-bound xanthine oxidase activities in both buttermilk and skim milk. The state of xanthine oxidase activity in skim milk from reconstituted milk, which was prepared by mixing xanthine oxidase inactivated skim milk and fresh cream, showed that only the free enzyme migrated from the cream phase to skim milk on cold storage. Very little xanthine oxidase activity was detectable in skim milk on heat treatment of the reconstituted milk sample. The overall increased activity of xanthine oxidase in milk during cold storage or heat treatment may not be due to the release of fat globule membrane enzyme to skim milk.     

冻藏条件对人乳脂质及挥发性风味物质的影响

以新鲜人乳为对照(0d),设置-18、-60℃和-60℃快速冷冻(-60℃(Q))3种冻藏条件分别冻藏60 d与180 d,通过测定脂质含量、非酯化脂肪酸(non-esterified fatty acids,NEFA)含量、脂质过氧化物(lipid peroxide,LPO)含量、脂肪酸组成、乳脂肪球微观结构及挥发性...