Studies have suggested that nanoscale extracellular vesicles (EV) in human and bovine milk carry immune modulatory properties which could provide beneficial health effects to infants. In order to assess the possible health effects of milk EV, it is essential to use isolates of high purity from other more abundant milk structures with well-documented bioactive properties. Furthermore, gentle isolation procedures are important for reducing the risk of generating vesicle artefacts, particularly when EV subpopulations are investigated. In this study, we present two isolation approaches accomplished in three steps based on size-exclusion chromatography (SEC) resulting in effective and reproducible EV isolation from raw milk. The approaches do not require any EV pelleting and can be applied to both human and bovine milk. We show that SEC effectively separates phospholipid membrane vesicles from the primary casein and whey protein components in two differently obtained casein reduced milk fractions, with one of the fractions obtained without the use of ultracentrifugation. Milk EV isolates were enriched in lactadherin, CD9, CD63 and CD81 compared to minimal levels of the EV-marker proteins in other relevant milk fractions such as milk fat globules. Nanoparticle tracking analysis and electron microscopy reveals the presence of heterogeneous sized vesicle structures in milk EV isolates. Lipid analysis by thin layer chromatography shows that EV isolates are devoid of triacylglycerides and presents a phospholipid profile differing from milk fat globules surrounded by epithelial cell plasma membrane. Moreover, the milk EV fractions are enriched in RNA with distinct and diverging profiles from milk fat globules. Collectively, our data supports that successful milk EV isolation can be accomplished in few steps without the use of ultracentrifugation, as the presented isolation approaches based on SEC effectively isolates EV in both human and bovine milk.
The aim of the present study was to study the effect of milking cows 4 times daily on free fatty acids (FFA) in the milk compared with milking twice daily. An experiment was performed during 2 wk in which half udders in 11 cows were milked 2 or 4 times daily. Milk yield was measured, and milk was analyzed for fat content, FFA, fatty acid composition, fat globule size, and activity of gamma-glutamyl transpeptidase. Concentration of FFA was greater (1.49 mEq/100 g of fat) in milk from half udders milked 4 times daily than in milk from the half udders milked twice daily (1.14 mEq/100 g of fat). Further, it was noted that milk from the half udder milked 4 times daily contained milk fat globules with larger average diameters. Increased milking frequency increased milk yield by 9% compared with the udder half milked twice daily, but fat content and fat yield were not affected. The results are of importance for further understanding the mechanisms behind the increased content of FFA that is frequently observed in automatic milking systems.
We examined the physical and chemical changes in milk during early lactation, and how these changes were affected by leaving one quarter unmilked in either the first or second milking, with the purpose of discriminating between colostrum and normal milk. Milk samples were collected from each quarter of 17 cows during the first 5 d after calving and then after about 7 d and 14 d. Samples were analysed for somatic cell count (SCC), fat, protein, casein, lactose, IgG1, colour, plasmin, pH and coagulation properties. Large variations occurred in both chemical and physical properties throughout the study period. Within six milkings, the concentration of casein decreased by 60%, IgG1 by 94%, and lactose increased by 34%. At milking number 6, rennet coagulation time was lowest and curd firmness was highest. The pH increased from 6·4 to 6·7 over the period of the experiment, and the colour changed from yellow (reddish) to white. Coagulation properties and the pH fell within the range of normal milk after five milkings. Measurement of colour and density appeared to be a potential method for detection of milk unsuitable for the dairy factory. Effects of omitting one quarter in one milking differed between milk components, but seemed to be of little importance to the physical properties.
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