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Recently, much interest has been raised for the characterization of signaling molecules carried by extracellular vesicles (EVs), which are particularly enriched in milk (mEVs). Such interest is linked to the capability of EVs to cross biological barriers, resist acidification in the gastric environment, and exert modulation of the immune system, mainly through their microRNA (miRNA) content. We characterized the small-RNA cargo of colostrum EVs (colosEVs) and mEVs from Italian Mediterranean buffalo through next generation sequencing. Colostrum (first milking after birth) and milk (day 50 of lactation) were sampled from seven subjects from five farms. ColosEVs and mEVs were subjected to morphological characterization, followed by high-depth sequencing of small RNA libraries produced from total RNA. The main difference was the amount of EV in the two samples, with colostrum showing 10 to 100-fold higher content than milk. For both matrices, miRNA was the most abundant RNA species (95% for colosEVs and 96% for mEVs) and three lists were identified: colosEV-specific, mEV-specific and shared most expressed. Gene ontology (GO) enrichment analysis on miRNA targets highlighted many terms related to the epigenetic, transcriptional and translational regulations across the three lists, with a higher number of enriched terms for colosEV-specific miRNAs. Terms specific to colosEVs were related to "cell differentiation" and "microvillus assembly", while for mEV "cardiac and blood vessel development" and "mitochondria" emergerd. Immune modulation terms were found for both sample-specific miRNAs. Overall, both matrices carry a similar molecular message in terms of biological processes potentially modulated into receiving cells, but there is significant difference in the abundance, with colostrum containing much more EVs than milk. Moreover, colosEVs carry molecules involved in signal transduction, cell cycle and immune response, as for mEVs and EVs of other previously characterized species, but with a special enrichment for miRNAs with epigenetic regulation capacities. These beneficial characteristics of colosEVs and mEVs are essential for the calf and could also be exploited for the therapeutic purposes in humans, although further studies are necessary to measure the sanitization treatment impact on EV conservation, especially in buffalo where milk is consumed almost exclusively after processing. [ABSTRACT FROM AUTHOR]

Milk is one of the most nutritionally complete foods and plays an important role in the human diet. Buffalo milk represents 15% of worldwide milk production and is an important source of bioactive compounds. Buffalo milk has a great market in the Mediterranean area, and dairy products, such as Mozzarella and Ricotta di Bufala Campana, obtained with the Italian Mediterranean buffalo milk, are acknowledged with the Protected Designation of Origin (PDO). This study aimed to characterize, using high-throughput sequencing of the 16S rRNA gene, the milk core microbiome of water buffalo rises in the Amaseno Valley included in the Mozzarella PDO region. The principal features of the core and the auxiliary buffalo milk microbiome are the predominance of Firmicutes and Lactococcus, one of the most important lactic acid bacteria (LAB) taxa in the dairy industry. The comparative analysis of the core microbiomes indicated that the milk of the Italian Mediterranean Buffalo and other mammals share the presence of Streptococcus-affiliated OTUs (operational taxonomic units). Our data also demonstrated that the core microbiome of milk samples collected from PDO and non-PDO regions differ in the number and type of taxa. Key points: • Buffalo milk and their derivate products are becoming more popular worldwide. • Dairy locations and practice management affect the structure of the milk microbiota. • Next-generation sequencing (NGS) analysis allows to identify the features of the Italian Buffalo milk microbiome. [ABSTRACT FROM AUTHOR]