Microfiltration of buttermilk and washed cream buttermilk for concentration of milk fat globule membrane components

Buttermilk, the by-product from butter manufacture, has gained much attention lately because of the application potential of its milk fat globule membrane (MFGM) components as health ingredients. Microfiltration (MF) has been studied for buttermilk fractionation because of its ability to separate particles from dissolved solutes. However, the presence in this by-product of skim milk solids, especially casein micelles, restricts concentration of MFGM. The use of cream washed with skim milk ultrafiltrate to produce buttermilk with lower casein content was studied as well as fractionation of this buttermilk by MF. Results have shown that washing the cream prior to churning yields buttermilk with 74% less protein than normal cream buttermilk. Analysis of the protein profile of washed cream buttermilk revealed that caseins and whey proteins were the main classes of proteins removed. The MF of washed cream buttermilk resulted in permeation fluxes 2-fold higher than with normal cream buttermilk. The second separation of the cream induced high losses of phospholipids in the skim phase. However, retention of remaining phospholipids in washed cream buttermilk by the MF membrane was higher resulting in a phospholipids concentration factor 66% higher than that of normal cream buttermilk. The results presented in this study highlight the impact of casein micelles on the separation of MFGM components as well as their effect on permeation flux during MF.     

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.     

Thermocalcic aggregation of milk fat globule membrane fragments from acid buttermilk cheese whey

Fragments originating from the milk fat globule membrane (MFGM), which is rich in polar lipids and membrane-specific proteins, are gaining interest for their functional and nutritional properties. Acid buttermilk cheese whey was used as a source for MFGM purification, because its MFGM content is more than 5 times higher than that of standard rennet whey. Because polar lipids are the main constituent of the MFGM and only occur in membranous structures, the polar lipid content was taken as a parameter for the total MFGM fragment content. The process of thermocalcic aggregation was evaluated on its recovery of MFGM fragments in the pellet. This method, originally intended for whey clarification and defatting, is a combination of calcium addition, a pH increase, and a thermal treatment. The influence of pH (6.5 to 8), temperature (40 to 70°C), and calcium concentration (0.1 to 0.24 g/100 g) on the pellet mass and dry matter (DM) content and on recovery of protein and polar lipids (and thus indirectly on MFGM fragments) was investigated by means of a response surface Box-Behnken orthogonal design. Reduced quadratic models were fit to the experimental data and were found to be highly significant. No outliers were observed. The recovery of MFGM fragments was found to be highly dependent on the pH, and less dependent on temperature and calcium addition. Next to MFGM proteins, whey proteins were also found to be involved in the formation of aggregates. Optimal conditions were found at 55°C, pH 7.7, and 0.205 g of calcium/L of whey. Under these conditions, 91.0% of the whey polar lipids were recovered in a firm and compact pellet of only 7.86% of the original whey mass, with a polar lipid concentration of 8.34% on pellet DM. Washing with water and centrifugation of the pellet was successful because after one washing step, virtually all sugars were removed, whereas 75.9% of the whey polar lipids could still be recovered. As such, the polar lipid content of the washed pellet increased to 10.70% on a DM basis. However, a second washing step resulted in serious losses of MFGM material.     

Effect of dietary fat and lactation status on insulin binding to bovine milk fat globule membranes

Lactating cows were used to examine the relationship between lactation status and insulin binding to milk fat globule membranes. Variables evaluated were daily milk yield, stage of lactation, breed, age, lactation number, daily milk fat and protein yields, milk fat and protein percentages, breeding status, body weight, body weight.75, and mammary health. Milk yield was correlated with insulin binding and accounted for 20% of the binding variability. No other variables were related to insulin binding. Insulin binding to milk fat globule membranes increased with supplemental dietary fat up to 4% added fat in the diet dry matter. Milk fat globule membranes may provide a useful model for assessing insulin receptor regulation in the mammary gland. Sources of variation in insulin binding to mammary membranes remain to be identified.     

人乳的磷脂组成与脂肪球结构

采用核磁共振磷谱(~(31)P-NMR)测定不同泌乳期、不同胎龄的人乳磷脂组成,结果显示足月儿和早产儿人乳中,磷脂的主要组成均为鞘磷脂(SM)、磷脂酰胆碱(PC)、磷脂酰乙醇胺(PE)、磷脂酰乙醇胺缩醛磷脂(EPLAS)、磷脂酰肌醇(PI)和磷脂酰丝氨酸(PS),其中SM含量最高,其次为PC和PE,PS和PI含量最低。总磷脂含量在相同胎龄不同泌乳期以及相同泌乳期不同胎龄间均无显著性差异。在磷脂组成上,足月儿人乳中PI在初乳和过渡乳中的含量分别为(4.14±0.42)%和(3.66±0.66)%,显著高于成熟乳中的(2.79±0.09)%;早产儿人乳中PC在初乳和过渡乳中的含量分别为(30.74±2.03)%和(29.40±2.37)%,显著高于成熟乳的(27.55±2.42)%,此外EPLAS含量随泌乳期的延长逐渐降低,PE含量逐渐升高,PE+EPLAS在不同的泌乳期无显著性差异。另外,足月儿和早产儿的脂肪球结构无明显差异,磷脂构成的膜包裹体积平均粒径约为5μm的脂肪球。     

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).     

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。     

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.     

Isolation of milk fat globule membrane (MFGM) material by coagulation and diafiltration of buttermilk

Due to the functional potential of milk fat globule membrane (MFGM), its isolation from buttermilk is receiving increasing attention by the food industry. However, extraction of MFGM proteins from buttermilk is challenging because of the high levels of serum proteins. In this study, a two-step approach was applied to obtain a MFGM isolate. First, native casein micelles from buttermilk were removed by rennet-induced coagulation. Next, the buttermilk whey obtained was filtered to remove the residual whey proteins. Purified MFGM isolate was collected after six diafiltration steps. The yield of MFGM proteins in the isolates was determined using sodium dodecylsulphate-polyacrylamide gel electrophoresis. In the MFGM isolates, 70% of peripheral membrane proteins were obtained, which is much more effective in comparison with recent isolation methods like cream washing or filtration of buttermilk. Further investigations on casein coagulation conditions of buttermilk may reduce losses, especially of integral MFGM proteins during renneting.     

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。     

Oil/fat blending strategy for improving milk fat globule membrane stability and its effect on fatty acid composition

This study examined the effect of blending with cumin essential oil (CEO) on the stabilisation of milk fat globule membrane (MFGM) emulsions, as well as the particle migration rate, fatty acid composition and phospholipid content after pasteurisation. Blending the MFGM with 20% CEO at pH 6.6 (CM4) led to a decrease in the creaming rate. With increasing CEO ratio, the creaming measurements increased to a 30 mm height with 20% CEO at pH 5.5 (CM3) from 25 mm with 10% CEO at pH 5.5 (CM2). The CM4 migration rate was 0.55 mm/h, and this sample showed no significant forward movement during implementation. The droplet size increased with increasing CEO ratio from 1.69 to 2.07 µm. A total of 20% CEO at pH 6.6 (CM4) was the best blending condition for MFGM stabilisation. This study showed enhanced creaming rate and droplet size, indicating greater destabilisation of the MFGM emulsion, when the CEO ratio was increased.     

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.     

Native milk fat globule size and its influence on whipping properties

The objective of this study was to investigate the influence of native milk fat globule size on the aeration of high fat dairy products with regard to maximum firmness time, gas inclusion and foam stability. The results showed that whipping time to maximum firmness was inversely proportional to mean fat globule size for both unhomogenised and slightly homogenised (2 MPa) creams. Additionally, increasing native mean fat globule size of the creams resulted in increased overrun. No significant differences in serum drainage were found between creams with different native milk fat globule size. Furthermore, when creams with native large mean fat globules were homogenised, the results showed that the maximum firmness time was in accordance with the mean fat globule size of non-aggregated creams. In the present study, cream fractionation was achieved by creaming or in a cost effective and fast manner using a modified centrifugal separator.     

Effect of pressure treatment of recombined whole milk on fat globule membrane composition and acid gelation functionality

Recombined whole milk was prepared by pressure treating skim milk (200 to 600 MPa/30 min) then homogenizing with milkfat (HPHO) or by homogenizing milkfat with skim milk then pressure treating the recombined whole milk (HOHP). β-Lactoglobulin denaturation increased at higher pressures. Low levels of α-lactalbumin were denatured at 600 MPa only. Denaturation was similar in the HPHO and HOHP milk. The HPHO milk had statistically similar levels of total protein, higher levels of whey protein and κ-casein and lower levels of α_s-casein adsorbed to the fat globules compared with the HOHP milk. The HPHO milk had a higher proportion of β-casein directly at the interface at all pressures and a higher proportion of κ-casein and a lower proportion of denatured whey proteins at pressure up to 400 MPa than the HOHP milk. Acid gels prepared from the HOHP milk had higher final G′ and yield stresses than those from the HPHO milk. These differences are discussed in relation to the compositions of the proteins adsorbed to the fat globules and how these interact during acidification.     

Distribution and fatty acid content of phospholipids from bovine milk and bovine milk fat globule membranes

The phospholipid (PL) content was determined comparatively in the milk fat globule membrane (MFGM) and whole milk including their fatty acid profiles. The possible role of milk PLs in defence against pathogens was also addressed. The MFGM and whole milk showed a similar distribution of PL species; however, the fatty acid contents of the PL species were different. Total PL from MFGM showed a decrease in C18:0 content in parallel with an increase in C18:1 and C18:2 and very long-chain fatty acid (more than C20) content. No significant differences in the fatty acid content of phosphatidylcholine and sphingomyelin from either source were found. However, the phosphatidylethanolamine from MFGM had more C18:1 and C18:2 and less C14:0 and C16:0 than that from whole milk. A similar but less pronounced result was found for phosphatidylserine/phosphatidylinositol. Enterotoxigenic Escherichia coli strains failed to bind to PL, which had been previously separated by high-performance thin-layer chromatography.     

Effects of pulsed electric field on fat globule structure,lipase activity,and fatty acid composition in raw milk and milk with different fat globule sizes

To modify the structure of milk fat globules (MFGs), raw milk (RM) with native MFGs, and milk with small (MS) and large (ML) MFGs, were subjected to moderate pulsed electric field (PEF) treatment at 9 and 16 kV·cm⁻¹ for 30 μs. The changes caused by PEF on MFG structure leading to MFG-MFG and MFG-protein aggregation were found to depend on the size, composition and/or previous damage caused by the separation and mixing processes during production of MS and ML, in addition to the intensity of the PEF treatment. Although, PEF treatment had a more pronounced effect on MFGM proteins of MS, these MFG were less affected by PEF due to significant adsorption of milk proteins. The structural changes caused by PEF were accompanied by changes in lipase activity and fatty acid (FA) profiles, i.e. increase in the proportion of C6:0, C8:0, C10 and some long-chain FAs and a decrease in medium and other long chain FAs.Industrial relevancePEF is gaining attention in the dairy industry. However, information regarding the effect of PEF processing on milk components and functionality is still limited. The present investigation contributes to understand the potential of PEF at moderate field intensities to modify the MFGs and consequences for milk stability, lipolysis and composition of FAs. The new insight is relevant in development of new products and ingredients containing milk fat.     

Effect of reverse osmosis and ultra-high-pressure homogenization on the composition and microstructure of sweet buttermilk

Buttermilk (BM), the by-product of butter making, is similar to skim milk (SM) composition. However, it is currently undervalued in dairy processing because it is responsible for texture defects (e.g., crumbliness, decreased firmness) in cheese and yogurt. One possible way of improving the incorporation of BM into dairy products is by the use of technological pretreatments such as membrane filtration and homogenization. The study aimed at characterizing the effect of preconcentration by reverse osmosis (RO) and single-pass ultra-high-pressure homogenization (UHPH) on the composition and microstructure of sweet BM to modify its techno-functional properties (e.g., protein gel formation, syneresis, firmness). The BM and RO BM were treated at 0, 15, 150, and 300 MPa. Pressure-treated and control BM and RO BM were ultracentrifuged to fractionate them into the following 3 fractions: a supernatant soluble fraction (top layer), a colloidal fraction consisting of a cloudy layer (middle layer), and a high-density pellet (bottom layer). Compositional changes in the soluble fraction [lipid, phospholipid (PL), protein, and salt], as well as its protein profile by PAGE analysis, were determined. Modifications in particle size distribution upon UHPH were monitored by laser diffraction in the presence and absence of sodium citrate to dissociate the casein (CN) micelles. Microstructural changes in pressure-treated and non-pressure-treated BM and RO BM particles were monitored by confocal laser scanning microscopy. Particle size analysis showed that UHPH treatment significantly decreased the size of the milk fat globule membrane fragments in BM and RO BM. Also, pressure treatment at 300 MPa led to a significant increase in the recovery of total lipids, CN, calcium, and phosphate in the BM soluble fraction (top layer) following ultracentrifugation. However, PL were primarily concentrated in the pellet cloud (middle layer), located above the pellet in BM concentrated by RO. In contrast, PL were evenly distributed between soluble and colloidal phases of BM. This study provides insight into the modifications of sweet BM constituents induced by RO and UHPH from a compositional and structural perspective.