Project description:Milk-derived extracellular vesicles (mEVs) have been proved to play a critical role in intercellular communication, mainly through the microRNAs (miRNAs) that they carry, to regulate biological functions of the target cells. Given miRNAs are evolutionarily conserved, EVs present in commercial milk may play a role in the physiology and health consumers. It is therefore essential to know the effects of technological treatments such as skimming and spray drying on the EV content of milk powders and on the cargo of bioactive molecules, in particular miRNAs, that they convey. Since goat’s milk or goat milk based formulas are considered as a healthy alternative for infants with cow’s milk sensitivities, including allergy, we undertook to analyze the EV content of skimmed and unskimmed goat's milk powders and to characterize their RNA content, in particular their miRNomes. mEVs were isolated using an optimized protocol based on Size Exclusion Chromatography (SEC) and compared regarding morphology, number and size by Transmission Electron Microscopy (TEM) and Nanoparticle Tracking Analysis (NTA). Their RNA and protein content were determined and their miRNomes established, using RNA sequencing. In this study we demonstrated that goat milk powders, skimmed or not upstream the spray drying treatment, contained many mEVs, ranging from 5.4 1011 to 2.5 1012 particles per mL of reconstituted milk, with an average size between 136.8 and 160.6 nm. We also demonstrated that mEVs carried significant amounts of RNA, including miRNAs. Using RT-qPCR, mRNAs encoding five of the major milk proteins were detected, suggesting that mEVs originated from mammary epithelial cells. We established the goat milk powder miRNome by identifying 351 miRNAs of which 233 are common to the 262 miRNAs previously profiled in raw goat milk. The 20 most abundant miRNAs (TOP 20) account for 80% of the total reads and the hierarchy of this TOP 20 miRNAs is somewhat overturned when comparing goat milk powder and raw goat milk. Surprisingly, whereas the comparison of raw from cow and goat milk confirmed the prevalence of miR-148a, miR-21-5p and miR-26a/miR-30a-5p, let-7a-5p and let-7f, which occupied ranks 1 and 2, respectively, in powders, were relegated to ranks 6 and 10 and 5 and 11 in raw goat and cow milk, respectively. Conversely to what was previously reported, we provide evidence that: i) EVs of typical morphology are present in goat milk powders; ii) mEVs survived the technological processes used to produce the powders; iii) their miRNA cargo is protected from degradation even though their miRNomes are not an exact mirror of miRNomes of EVs derived from fluid raw milk.
Project description:We have reported that microRNAs are present in human, bovine, and rat milk whey. Milk whey miRNAs were resistant to acidic condition and to RNase. Thus, milk miRNAs were thought to be present packaged into membrane vesicles like exosome. However, body fluid miRNAs have been reported that there are in different forms. To clarify which miRNAs species are exist in exosome and which species are exist in another form, we used bovine raw milk and purified total RNA from exosome fraction and ultracentrifugated supernatant fraction, and analyzed by miRNA microarray.
Project description:Mastitis, the inflammation of the mammary gland, is one of the most prevalent diseases in dairy farming worldwide. Unfortunately, the disease is most often present in a subclinical type with no clear symptoms. The sooner the infection is detected, the less opportunities for the disease to progress and the more treatment options remain available. Milk microRNA (miRNA) encapsulated in extracellular vesicles (EV) have been proposed as potential biomarkers of different mammary gland conditions, including subclinical mastitis. However, little is known about the robustness of EV analysis regarding sampling time-point or natural infections. In order to estimate the reliability of EV measurements in raw bovine milk, we first evaluated the changes in EV size, concentration and miRNA cargo during three consecutive days. Then, we compared milk EV differences from natural infected quarters with high somatic cell count (SCC) with their healthy adjacent quarters with low SCC and quarters from uninfected udders. We found that milk EV miRNA cargo is very stable along three days and that infected quarters do not induce relevant changes in milk EV of adjacent healthy quarters, making them suitable controls. We observed cow-individual changes in immunoregulatory miRNA in quarters with chronic subclinical mastitis, pointing towards infection-specific alterations. Finally, we proposed bta-miR-223 as a potential indicator of subclinical mastitis prognosis in raw milk.
Project description:Here we studied the glycation of bovine milk proteins by lactose as dominant sugar in milk and hexoses using tandem mass spectrometry (CID and ETD mode). In a bottom-up proteomics approach after enriching glycated peptides by boronate affinity chromatography, first we could identify 260 lactosylated peptides corresponding to 124 lactosylation sites in 28 bovine milk proteins in raw milk, raw colostrum, three brands of pasteurized milk, three brands of UHT milk, and five brands of infant formula. The same regular and additionally two lactose-free milk products (pasteurized and UHT milk) where lactose is enzymatically cleaved into the more reactive hexoses were analyzed in terms of hexosylation sites that resulted in identification of 124 hexosylated tryptic peptides corresponding to 86 glycation sites in 17 bovine milk proteins. In quantitative terms glycation increased from raw milk to pasteurized milk to UHT milk and infant formula, i.e., with the harsher processing conditions. Lactose-free milk contained significantly higher hexosylation degrees than the corresponding regular milk product.
Project description:We have reported that microRNAs are present in human, bovine, and rat milk whey. Milk whey miRNAs were resistant to acidic condition and to RNase. Thus, milk miRNAs were thought to be present packaged into membrane vesicles like exosome. However, body fluid miRNAs have been reported that there are in different forms. To clarify which miRNAs species are exist in exosome and which species are exist in another form, we used bovine raw milk and purified total RNA from exosome fraction and ultracentrifugated supernatant fraction, and analyzed by miRNA microarray.
Project description:We report the application of miRNA next generation sequencing (NGS) for the analysis of impact of processing on miRNA in human breast milk, donated by 3 volunteers. MiRNA content of total and exosomal fraction was compared between unprocessed milk and sample subjected to either Holder (thermal) pasteurization (HoP) or elevated pressure processing (HPP). NGS reads were mapped to miRBase in order to obtain miRNA counts. Then, we analyzed differences in the miRNA abundance and function between raw and processed material. It was observed that both processing methods reduce number of miRNA reads and HoP is significantly more detrimental to miRNA than HPP.
2019-12-19 | GSE142282 | GEO
Project description:16S donkey milk
| PRJNA838247 | ENA
Project description:16S amplicon sequencing of raw milk and biofilm from raw milk depending on temperature
Project description:We investigated the effect of feeding mice a Total Western Diet formulated using the 50th percentile daily intake levels for macro and micronutrients from the National Health and Nutrition Examination Survey (NHANES) with 0, 2, 5, or 10% added raw potato starch on the cecal microbiome (16S) and cecum, proximal and distal colon gene expression by RNASeq analysis.