Goat and cow-milk based infant formulas contain extracellular vesicles with different miRNA and protein cargo

While the presence of extracellular vesicles (EVs) and their cargo miRNAs is well established in human, cow and goat milk, their presence in infant formulae (IFs) was less documented. Particles whose EV nature was subsequently confirmed (size, microscopy) were isolated from whole goat milk powder (WGMP), from two IFs based on goat milk and from two based on cow milk using size exclusion chromatography. The qPCR analyses of RNA extracted from these EVs show that they contain mRNAs specifying major milk proteins and mammary epithelial cell markers, demonstrating the mammary origin of EVs. The miRNA profile and proteome of EVs were determined by small-RNA sequencing and TimsTOF Pro LC-MS, respectively. We identified a total of 1142 and 1162 proteins in goat and cow milk derived IFs, respectively, including EV-specific markers (as CD9, CD63) and proteins involved in vesicular trafficking. More than 350 miRNAs were identified and revealed that the expected milk miRNAs were among the most abundant in IFs. Among the 20 most abundant miRNAs, 16 were found in all IFs and WGMP, and 12 are described as the most abundant in human milk. They were predicted to influence cell life, protein and nucleic acid processes, or may be linked to inflammation. Differential analysis of miRNomes showed the type of milk (goat vs cow with 80 differential miRNAs) had the greatest impact, followed by the whey protein:casein ratio. This study demonstrates that EVs are present in milk-based IFs, which are a source of miRNAs. Further studies on their potential biological relevance for infants are warranted.