Effects of Different Processing on miRNA and Protein in Small Extracellular Vesicles of Goat Dairy Products.

Objectives: Small extracellular vesicles (sEVs) are nanosized vesicles with biological activities that exist in milk, playing functional roles in immunity, gut balance, and the nervous system. Currently, little is known about the impact of processing on milk sEVs.

Methods: In this study, sEVs were collected from raw goat milk (g-sEV), pasteurized goat milk (pg-sEV), and goat milk powder (-sEV) using a sucrose cushion centrifugation combined with qEV chromatography. Then, the sEVs were identified and compared using NTA, Western blot, and TEM. After extracting RNA and the total proteome from sEVs derived from different samples, the RNA was subjected to high-throughput sequencing, and peptide fragments were analyzed using mass spectrometry. Finally, GO and KEGG pathway analyses were performed on the results.

Results: The characterization results revealed a decrease in diameter as the level of processing increased. High-throughput sequencing results showed that all three types of small extracellular vesicles were found to be rich in miRNA, and no significant differences were observed in the most abundant sEV species. Comparing with g-sEV, there were 3938 and 4645 differentially expressed miRNAs in pg-sEV and -sEV, respectively, with the majority of them (3837 and 3635) being downregulated. These differentially expressed miRNAs were found to affect biological processes or signaling pathways such as neurodevelopment, embryonic development, and transcription. Proteomic analysis showed that there were 339 differentially expressed proteins between g-sEV and pg-sEV, with 209 proteins being downregulated. Additionally, there were 425 differentially expressed proteins between g-sEV and -sEV, with 293 proteins being downregulated. However, no significant differences were observed in the most abundant protein species among the three types of sEVs. Enrichment analysis indicated that the differentially expressed proteins were associated with inflammation, immunity, and other related processes.

Conclusions: These results indicate that extracellular vesicles have a protective effect on their cargo, while processing steps can have an impact on the size and quantity of the sEVs. Furthermore, processing can also lead to the loss of immune-related miRNA and proteins in sEVs.