Abstract
Aim:Small extracellular vesicles (sEVs) and their RNA cargo are not exclusively derived from endogenous synthesis but can also be absorbed from milk and gut bacteria. Given the high rate of bacterial fermentation in the gastrointestinal tract of ruminants, we hypothesized that preparations of bovine milk sEVs (BMEs) contain bacterial mRNAs whose bioavailability in humans remains unknown. Methods: BMEs were purified from chilled antibiotics-treated raw milk (RM) and store-bought skim milk (SBM) using sequential ultracentrifugation. BMEs from RM were treated with RNase to remove RNA adsorbed to the BME surface. BMEs from SBM were treated (SBM+) or not treated (SBM-) with RNase. mRNAs were identified by RNA sequencing analysis and mapping to the bovine genome and bacterial reference. The bioavailability of bacterial mRNA was assessed by RNA sequencing analysis of plasma collected before and 4 h after consuming one liter of cow's milk in humans. Results: Approximately 50% of the mRNA sequencing reads were non-bovine in BMEs from RM, SBM+, and BM-. Up to two-thirds of the non-bovine contigs mapped to microbial transcriptomes, including bacteria, viruses, and fungi. The levels of 17 bacterial mRNAs from Escherichia coli and Cutibacterium acnes were significantly higher after milk consumption compared to before milk consumption, but the number of reads was too low to confidently draw the conclusion that microbial mRNAs in milk are bioavailable in humans. Conclusions: BMEs prepared by ultracentrifugation contain bacterial mRNAs that are not bioavailable in humans.