Project description:The quality and authenticity of milk are of paramount importance. Cow milk is more allergenic and less nutritious than ewe, goat, or donkey milk, which are often adulterated with cow milk due to their seasonal availability and higher prices. In this work, a silicon photonic dipstick sensor accommodating two U-shaped Mach-Zehnder Interferometers (MZIs) was employed for the label-free detection of the adulteration of ewe, goat, and donkey milk with cow milk. One of the two MZIs of the chip was modified with bovine κ-casein, while the other was modified with bovine serum albumin to serve as a blank. All assay steps were performed by immersion of the chip side where the MZIs are positioned into the reagent solutions, leading to a photonic dipstick immunosensor. Thus, the chip was first immersed in a mixture of milk with anti-bovine κ-casein antibody and then in a secondary antibody solution for signal enhancement. A limit of detection of 0.05% v/v cow milk in ewe, goat, or donkey milk was achieved in 12 min using a 50-times diluted sample. This fast, sensitive, and simple assay, without the need for sample pre-processing, microfluidics, or pumps, makes the developed sensor ideal for the detection of milk adulteration at the point of need.
Project description:κ-casein (κ-CN) is one of the key components in bovine milk, playing a unique role in the structuration of casein micelles. It contains in its chemical structure up to sixteen amino acid residues (mainly serine and threonine) susceptible to modifications, including glycosylation and phosphorylation, which may further be formed during milk processing. In this study, changes in post-translational modification (PTM) of κ-CN during bovine milk fermentation were investigated. One-to-five-day fermented milk samples were produced. A traditional bottom-up proteomics approach was used to establish a multiple-reaction monitoring (MRM) method for relative quantification of κ-CN PTM. Endoproteinase Glu-C was found to efficiently digest the κ-CN molecule. The developed LC-MS method was validated by performing assessments of linearity, precision, repeatability, reproducibility, limit of detection (LOD), and limit of quantification (LOQ). Among the yielded peptides, four of them containing serine and threonine residues were identified and the unmodified as well as the modified variants of each of them were relatively quantified. These peptides were (1) IPTINTIASGEPTSTTE [140, 158], (2) STVATLE [162, 168], (3) DSPE [169, 172], and (4) INTVQVTSTAV [180, 190]. Distribution analysis between unmodified and modified peptides revealed that over 50% of κ-CN was found in one of its modified forms in milk. The fermentation process further significantly altered the composition between unmodified/modified κ-CN, with glycoslaytion being predominant compared to phosphorylation (p < 0.01). Further method development towards α and β-CN fractions and their PTM behavior would be an asset to better understand the changes undergone by milk proteins and the micellar structure during fermentation.
Project description:Halari donkey breed is one of the indigenous breeds of India and its population is rapidly decreasing. The Jenny milk is more similar to human milk, exhibits a wide range of probiotic diversity and hypo-allergenicity, especially among infants suffering from cow and buffalo milk protein allergy. Some studies indicated low levels of κ-casein fraction of casein protein in donkey milk. The k-casein gene was not amplified from the DNA derived from the milk somatic cells of Halari donkeys. The Halari donkey milk has low somatic cells count. We report the first isolation of DNA from milk somatic cells of Halari donkeys with subsequent characterization of k-casein gene. Through our work, we showed that the milk somatic cells can be used as a non-invasive source for DNA isolation towards molecular studies. It also proved the presence of k-casein gene in Halari donkey milk by its amplification from isolated DNA.
Project description:Caseinomacropeptide (CMP) is released from bovine kappa-casein after rennet treatment and is one of the major peptides in whey protein isolate. CMP has in vitro anti-inflammatory and antibacterial activities. CMP has two major amino acid sequences with different modifications, including glycosylation, phosphorylation and oxidation. However, no previous work has provided a comprehensive profile of intact CMP. Full characterization of CMP composition and structure is essential to understand the bioactivity of CMP. In this study, we developed a top-down glycopeptidomics-based analytical method to profile CMP and CMP-derived peptides using Orbitrap mass spectrometry combined with nano-liquid chromatography with electron-transfer/higher-energy collision dissociation. The liquid chromatography-tandem mass spectrometry (LC-MS/MS) spectra of CMPs were annotated to confirm peptide sequence, glycan composition and other post-translational modifications using automatic data processing. Fifty-one intact CMPs and 159 CMP-derived peptides were identified in four samples (one CMP standard, two commercial CMP products and one whey protein isolate). Overall, this novel approach provides comprehensive characterization of CMP and CMP-derived peptides and glycopeptides, and it can be applied in future studies of product quality, digestive survival and bioactivity.
Project description:Caseins and many other secretory proteins are phosphorylated during their transport through the secretory pathway by a protein kinase present within Golgi compartments. Molecular analysis of the Golgi casein kinase (GCK) has not been possible since it has not been purified to homogeneity or been cloned. Previous attempts have been made to purify GCK activity from mammary gland Golgi fractions, but these have not resulted in extensive purification of the enzyme. In the present study, we have demonstrated that substantial amounts of GCK activity, assayed using a specific peptide substrate, can be detected as a soluble form in bovine milk, and we have used milk as a source for purification. A purification protocol was established that allowed>80000-fold purification to a specific activity of GCK (approx. 700 nmoles/min per mg of protein) far higher than previously achieved. These findings cast doubts on previous claims for purification of GCK activity. In addition, ion-exchange chromatography resolved two closely eluting peaks of activity, suggesting the existence of two related, but distinct, GCK activities.
Project description:β-casein is a primary protein in milk, and its variants have been associated with changes in the protein content of bovine milk. However, there has been little research focused on the effects of β-casein variants on milk metabolites. In the present study, dairy cows producing milk with β-casein variant A1/A1 (A1), A2/A2 (A2), and their heterozygote A1/A2 (A12) were screened by a high-resolution melting method. Individual milk samples were then collected from each of the cows, and the milk metabolites were separated and analyzed using nuclear magnetic resonance spectroscopy- and liquid-chromatography mass spectrometry-based metabolomics techniques. Differences in metabolites among the variant groups were evaluated by multivariate statistical analysis. The relative abundances of methionine, proline, and α-lactose were the highest in β-casein variant A2 milk, whereas choline, glycine, citric acid, and cyclic adenosine monophosphate (cAMP) showed the highest abundances in variant A1 milk. Metabolic pathways analysis indicated that the differential metabolites between variants A1 and A2 were involved in pantothenate and coenzyme A biosynthesis, butanoate metabolism, and valine, leucine, and isoleucine biosynthesis. Our results reveal the differences in milk metabolites among the β-casein variants A1, A2, and the heterozygote. These findings, thus, provide novel insights into the effects of β-casein variants on milk metabolites, facilitating further research into the mechanism of the biosynthesis of milk components in the mammary gland and the potential physiological function of milk associated with β-casein variants.
Project description:Although many studies have been conducted on the components present in human breast milk (HM), research on the differences of chemical metabolites between HM, bovine milk (BM) and formula milk (FM) is limited. This study was to explore the chemical diversity of HM, BM and FM by metabolomic approaches. GC-TOFMS and UPLC-QTOFMS were applied to investigate the metabolic compositions in 30 HM samples, 20 FM samples and 20 BM samples. Metabolite profiling identified that most of the non-esterified fatty acids, which reflected the hydrolysis of triglycerides, were much more abundant in HM than those in FM and BM, except for palmitic acid and stearic acid. The levels of tricarboxylic acid (TCA) intermediates were much higher in FM and BM than those in HM. Each type of milk also showed its unique composition of free amino acids and free carbohydrates. In conclusion, higher levels of non-esterified saturated fatty acids with aliphatic tails <16 carbons, monounsaturated fatty acids and polyunsaturated fatty acids and lower levels of TCA intermediates are characteristic of HM, as compared with FM and BM. The content of non-esterified fatty acids may reflect the hydrolysis of triglycerides in different milk types.
Project description:This study aims to see if probiotic bacteria from human milk could ameliorate oral cow's milk sensitization. The probiotic potential of the SL42 strain isolated from the milk of a healthy young mother was first determined. Rats were then randomly gavaged with cow's milk casein without an adjuvant or assigned to the control group. Each group was further subdivided into three groups, with each receiving only Limosilactobacillus reuteri DSM 17938, SL42, or a phosphate-buffered saline solution. Body weight, temperature, eosinophils, serum milk casein-specific IgE (CAS-IgE), histamine, and serum S100A8/A9 and inflammatory cytokine concentrations were measured. The animals were sacrificed after 59 days; histological sections were prepared, and the spleen or thymus weights, as well as the diversity of the gut microbiota, were measured. On days 1 and 59, SL42 abridged systemic allergic responses to casein by dropping histamine levels (25.7%), CAS-specific IgE levels (53.6%), eosinophil numbers (17%), S100A8/9 (18.7%), and cytokine concentrations (25.4-48.5%). Analyses of histological sections of the jejunum confirmed the protective effect of probiotic bacteria in the CAS-challenged groups. Lactic acid bacteria and Clostridia species were also increased in all probiotic-treated groups. These findings suggest that probiotics derived from human milk could be used to alleviate cow's milk casein allergy.
Project description:We have previously generated transgenic cattle with additional copies of bovine β- and κ casein genes. An initial characterisation of milk produced with a hormonally induced lactation from these transgenic cows showed an altered milk composition with elevated β-casein levels and twofold increased κ-casein content. Here we report the first in-depth characterisation of the composition of the enriched casein milk that was produced through a natural lactation. We have analyzed milk from the high expressing transgenic line TG3 for milk composition at early, peak, mid and late lactation. The introduction of additional β- and κ-casein genes resulted in the expected expression of the transgene derived proteins and an associated reduction in the size of the casein micelles. Expression of the transgenes was associated with complex changes in the expression levels of other milk proteins. Two other major milk components were affected, namely fat and micronutrients. In addition, the sialic acid content of the milk was increased. In contrast, the level of lactose remained unchanged. This novel milk with its substantially altered composition will provide insights into the regulatory processes synchronizing the synthesis and assembly of milk components, as well as production of potentially healthier milk with improved dairy processing characteristics.
Project description:Arginine, a semi-essential functional amino acid, has been found to promote the synthesis of casein in mammary epithelial cells to some extent. Data from mouse indicated that microRNA (miRNA) are important in regulating the development of mammary gland and milk protein synthesis. Whether there are potential links among arginine, miRNA and casein synthesis in bovine mammary gland is uncertain. The objective of the present work was to detect the effects of arginine supplementation on the expression of miRNA associated with casein synthesis in mammary tissue and mammary epithelial cells (BMEC). The first study with bovine mammary epithelial cells (BMEC) focused on screening for miRNA candidates associated with the regulation of casein production by arginine. The BMEC were cultured with three different media, containing 0, 1.6 and 3.2 mM arginine, for 24 h. The expression of candidate miRNA was evaluated. Subsequently, in an in vivo study, 6 Chinese Holstein dairy cows with similar BW (mean ± SE) (512.0 ± 19.6 kg), parity (3), BCS (4.0) and DIM (190 ± 10.3 d) were randomly assigned to three experimental groups. The experimental cows received an infusion of casein, arginine (casein plus double the concentration of arginine in casein), and alanine (casein plus alanine, i.e., iso-nitrogenous to the arginine group) in a replicated 3 × 3 Latin square design with 22 d for each period (7 d for infusion and 15 d for washout). Mammary gland biopsies were obtained from each cow at the end of each infusion period. Results of the in vitro study showed differences between experimental groups and the control group for the expression of nine miRNA: miR-743a, miR-543, miR-101a, miR-760-3p, miR-1954, miR-712, miR-574-5p, miR-468 and miR-875-3p. The in vivo study showed that arginine infusion promoted milk protein content, casein yield and the expression of CSN1S1 and CSN1S2. Furthermore, the expression of miR-743a, miR-543, miR-101a, miR-760-3p, miR-1954, and miR-712 was also greater in response to arginine injection compared with the control or alanine group. Overall, results both in vivo and in vitro revealed that arginine might partly influence casein yield by altering the expression of 6 miRNAs (miR-743a, miR-543, miR-101a, miR-760-3p, miR-1954, and miR-712).