Bovine milk exosome proteome

Exosomes are 40-100 nm membrane vesicles of endocytic origin, secreted by cells and are found in biological fluids including milk. These exosomes are extracellular organelles important in intracellular communication, and immune function. Therefore, the proteome of bovine milk exosomes may provide insight into the complex processes of milk production. Exosomes were isolated from the milk of mid-lactation cows. Purified exosomes were trypsin digested, subjected offline high pH reverse phase chromatography and further fractionated on a nanoLC connected to tandem mass spectrometer. This resulted in identification of 2107 proteins that included all of the major exosome protein markers. The major milk fat globule membrane (MFGM) proteins (Butyrophilin, Xanthine oxidase, Adipophilin and Lactadherin) were the most abundant proteins found in milk exosomes. However, they represented only 0.4-1.2% of the total spectra collected from milk exosomes compared to 15-28% of the total spectra collected in the MFGM proteome. These data show that the milk exosome secretion pathway differs significantly from that of the MFGM in part due to the greatly reduced presence of MFGM proteins. The protein composition of milk exosomes provides new information on milk protein composition and the potential physiological significance of exosomes to mammary physiology.     

Farm animal milk proteomics

Milk is one of the most important nutrients for humans during lifetime. Farm animal milk in all its products like cheese and other fermentation and transformation products is a widespread nutrient for the entire life of humans. Proteins are key molecules of the milk functional component repertoire and their investigation represents a major challenge. Proteins in milk, such as caseins, contribute to the formation of micelles that are different from species to species in dimension and casein-type composition; they are an integral part of the MFGM (Milk Fat Globule Membrane) that has being exhaustively studied in recent years. Milk proteins can act as enzymes or have an antimicrobial activity; they could act as hormones and, last but not least, they have a latent physiological activity encoded in their primary structure that turns active when the protein is cleaved by fermentation or digestion processes. In this review we report the last progress in proteomics, peptidomics and bioinformatics. These new approaches allow us to better characterize the milk proteome of farm animal species, to highlight specific PTMs, the peptidomic profile and even to predict the potential nutraceutical properties of the analyzed proteins.     

Feast or famine: evidence for mixed capital–income breeding strategies in Weddell seals

Evolved patterns of resource expenditure for reproduction have resulted in a life history continuum across species. A strictly capital-breeding strategy relies extensively on stored energy for reproduction, whereas income breeding uses energy acquired throughout the reproductive period. However, facultative income breeding has been shown in some classically capital-breeding animals, and was originally thought to provide a nutritional refuge for smaller females incapable of securing sufficient reserves during pre-partum foraging. We examined milk composition and milk output for the Weddell seal to determine to what degree lactation was aided by food intake, and what factors contributed to its manifestation. Milk composition was independent of maternal post-partum mass and condition, but did change over lactation. Changes were most likely in response to energetic and nutritional demands of the pup at different stages of development. During early lactation, females fasted and devoted 54.9% of total energy loss to milk production. Later in lactation 30.5% more energy was devoted to milk production and evidence suggested that larger females fed more during lactation than smaller females. It appears that Weddell seals may exhibit a flexible strategy to adjust reproductive investment to local resource levels by taking advantage of periods when prey are occasionally abundant, although it is restricted to larger females possessing the physiological capacity to dive for longer and exploit different resources during lactation. This supports the assumption that although body mass and phylogenetic history explain most of the variation in lactation patterns (20–69%), the remaining variation has likely resulted from physiological adaptations to local environmental conditions. Our study confirms that Weddell seals use a mixed capital–income breeding strategy, and that considerable intraspecific variation exists. Questions remain as to the amount of energy gain derived from the income strategy, and the consequences for pup condition and survival. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

New method for the determination of protein N-linked homocysteine

Homocysteine (Hcy) is incorporated into protein via a reaction of the thioester Hcy–thiolactone with ε-amino group of a protein lysine residue. This reaction leads to impairment and alteration of protein’s function and has been implicated in atherothrombotic disease. However, the data regarding N-linked Hcy content in proteins are limited, mostly due to a lack of facile assays. Here I describe a new sensitive assay for the determination of protein N-linked Hcy and demonstrate its utility for individual proteins and biological fluids. N-linked Hcy is liberated from proteins by acid hydrolysis and converted to Hcy–thiolactone, which is then purified and quantified by high-performance liquid chromatography on a cation exchange column. The quantification is by fluorescence after postcolumn derivatization with o-phthaldialdehyde. Using this assay, the levels of N-linked Hcy in individual pure proteins were found to vary from as high as 0.470–0.515 mol/mol protein for human and equine ferritins to as low as 0.00006 mol/mol protein for chicken lysozyme. Hemoglobins from a variety of species contained more N-linked Hcy than did corresponding albumins (0.0127–0.0828 vs. 0.0027–0.0086 mol/mol). Normal human plasma and milk were found to contain submicromolar concentrations of protein N-linked Hcy, whereas cow milk and whey contained micromolar concentrations of protein N-linked Hcy.     

乳蛋白生物活性肽的序列及其功能

乳蛋白经消化产生的肽类除了具有营养作用外,还具有多种生物活性,包括阿片肽和阿片拮抗肽活性及免疫调节,抗高血压、抗血栓、抗菌抗病毒,促进矿质元素吸收,防止腹泻等作用。本文对近几年发现的乳蛋白生物活性肽的序列结构及功能做了综述。     

Effects of type of fat in the diet on iron bioavailability assessed in suckling and weanling rats

Differences in iron bioavailability from human milk and milk formulas may in part be due to differences in lipid composition. We investigated the short and long term effects of diets based on different fats [corn, coconut, olive, or soy oil, human milk fat (HMF) and a formula fat blend (FF)] on iron absorption in rats. Suckling rat pups dosed with 59Fe-labeled diets containing different fat sources were killed after 6 h, and blood and individual tissues were counted. Iron availability was estimated by % 59Fe in blood. Pups dosed with a more saturated fat (coconut oil) had a higher % 59Fe in blood than those fed other fat sources. Weanling rats were used to determine iron bioavailability from fat sources using both the hemoglobin repletion method and whole body counting. Hemoglobin regeneration was significantly higher for rats fed the HMF diet (8.4 +/- 0.5 g/dl) than from the FF diet (6.5+/-0.6 g/dl) or the corn oil diet (less saturated) (6.4 +/- 0.3 g/dl). Rats fed diets based on coconut oil (more saturated) had significantly higher % 59Fe retention (61.6 +/- 1.4) than rats fed diets based on FF (49.8 +/- 3.4). There was a significant positive association between oleic acid in the diet and oleic acid in the intestinal mucosa (r = 0.95, p < 0.05) and between linoleic acid in the diet and linoleic acid in the intestinal mucosa (r = 0.97, p < 0.05) suggesting that the dietary treatment altered the fatty acid composition of the brush border membrane. Our results suggest that saturated fats may increase iron absorption and that part of this may be achieved by changes in the fatty acid composition of the intestinal mucosa. Hemoglobin regeneration and % 59Fe retention data suggest that differences in iron absorption from infant diets may in part be due to differences in fat composition. Therefore, lipid composition of infant formulas should also be taken into consideration as a factor influencing iron bioavailability.     

Purification and characterization of β-galactosidase from probiotic Pediococcus acidilactici and its use in milk lactose hydrolysis and galactooligosaccharide synthesis

β-galactosidase is a commercially important enzyme that was purified from probiotic Pediococcus acidilactici. The enzyme was extracted from cells using sonication and subsequently purified using ammonium sulphate fractionation and successive chromatographies on Sephadex G-100 and Q-Sepharose. The enzyme was purified 3.06-fold up to electrophoretic homogeneity with specific activity of 0.883 U/mg and yield of 28.26%. Molecular mass of β-galactosidase as estimated by SDS-PAGE and MALDI-TOF was 39.07 kDa. The enzyme is a heterodimer with subunit mass of 15.55 and 19.58 kDa. The purified enzyme was optimally active at pH 6.0 and stable in a pH range of 5.8–7.0 with more than 97% activity. Purified β-galactosidase was optimally active at 50 °C. Kinetic parameters K_m and V_max for purified enzyme were 400 µM and 1.22 × 10⁻¹ U respectively. Its inactivation by PMSF confirmed the presence of serine at the active site. The metal ions had different effects on enzyme. Ca²⁺, Mg²⁺ and Mn²⁺ slightly activated the enzyme whereas NH₄⁺, Co²⁺ and Fe³⁺ slightly decreased the enzyme activity. Thermodynamic parameters were calculated that suggested that β-galactosidase is less stable at higher temperature (60 °C). Purified enzyme effectively hydrolysed milk lactose with lactose hydrolysing rate of 0.047 min⁻¹ and t_1/2 of 14.74 min. This is better than other studied β-galactosidases. Both sonicated Pediococcus acidilactici cells and purified β-galactosidase synthesized galactooligosaccharides (GOSs) as studied by TLC at 30% and 50% of lactose concentration at 47.5 °C. These findings indicate the use of β-galactosidase from probiotic bacteria for producing delactosed milk for lactose intolerant population and prebiotic synthesis. pH and temperature optima and its activation by Ca²⁺ shows that it is suitable for milk processing.     

Nature of micellar calcium phosphate in cows' milk as studied by high-resolution electron microscopy

The nature of the inorganic calcium phosphate in the casein micelle of cows' milk has been studied by high-resolution electron microscopy. No periodic lattice spacings could be imaged, and diffraction patterns were of the diffuse amorphous type. Short-range order of less than 15 Å may be present, but the results indicate that there is no long-range order in micellar calcium phosphate.     

Penicillin-binding protein 3 of Streptococcus pneumoniae and its application in screening of β-lactams in milk

The soluble form of penicillin-binding protein 3 (sPBP3^∗) from Streptococcus pneumoniae was expressed in Escherichia coli as a six-histidine fusion protein. The protein was purified and used to develop a microplate assay in direct competitive format for the detection of penicillins and cephalosporins in milk. The assay was based on competitive inhibition of the binding of horseradish peroxidase-labeled ampicillin (HRP–Amp) to the sPBP3^∗ by free β-lactam antibiotics in milk. Under optimized conditions, most of the β-lactam antibiotics (11 penicillins and 16 cephalosporins) could be detected at concentrations corresponding to the maximum residue limits (MRLs) set by the European Union. Analysis of spiked milk samples showed that acceptable recoveries ranged from 74.06 to 106.31% in skimmed milk and from 63.97 to 107.26% in whole milk, with coefficients of variation (CVs) less than 16%. With the high sensitivity and wide-range affinities to penicillins and cephalosporins, the developed assay based on sPBP3^∗ exhibited the potential to be a screening assay for fast detection of β-lactam antibiotics in milk.