MicroRNAs (miRNAs) are small non-coding RNAs with unique functions. Their presence in human milk raises the possibility of accumulation along the food chain. Buffalo milk extracellular vesicles, as other milks, are a known source of dietary miRNAs. However, information on the digestive stability of miRNAs remains limited, which is a prerequisite for understanding their in vivo functionalities.

To this end, the presence of widely conserved miRNAs:

miR-10a-5p, miR-24-3p, miR-25-3p, miR-26a-5p, miR-27b-5p, miR-33a-5p, miR-103a-3p, miR-125b-5p, miR-130a-3p, miR-133a-3p, miR-138-5p, miR-139-5p, miR-141-3p, miR-148a-3p, miR-153-3p, miR-199a-3p, miR-223-3p

were assessed in isolated extracellular vesicles extracted from buffalo milk. The miR-10a-5p, miR-24-3p, and miR-130a-3p were not detected in raw buffalo milk.

Therefore, gastrointestinal digestion was simulated using INFOGEST 2.0, and extracellular vesicles were extracted from the digest. Apart from particle numerosity, which differed significantly—from 1.2 × 10¹¹ ± 5.3 × 10⁹ particles/mL in raw milk to 9.53 × 10¹⁰ ± 1.2 × 10⁹ particles/mL in digested milk—the extracted extracellular vesicles showed no structural differences before and after digestion.

The miRNA cargo exhibited a similar pattern, with the following exceptions:

miR-141-3p and miR-153-3p: slightly increased
miR-223-3p: substantially increased
miR-148a-3p: decreased
miR-33a-3p: no longer detectable after digestion

Bioinformatics analysis of the 13 miRNAs detected post-digestion indicated concerted targeting of neural and immunological pathways, including MHC-mediated antigen processing and presentation.

These findings highlight the potential of milk, through its EV-miRNA fraction, to impact inflammatory responses in neurodevelopmental processes of the offspring and, by extension, dairy consumers. However, relevant in vitro and in vivo studies are needed to demonstrate post-digestion transfer of these nucleic acids from dietary sources and their effects on target tissues.

Work Packages Involved

WP1 – Management and reporting
WP2 – Creation of new feed, experimental groups and sample collection
WP3 – Milk nutrient analysis, extracellular vesicle characterization and digestomic assessment
WP4 – Dissemination and open science activities

Tasks Involved

Project coordination and reporting
Development of innovative buffalo feed formulations
Experimental design and sample collection
Milk nutrient analysis
Isolation and characterization of extracellular vesicles and microRNA cargo
Digestomic analysis using INFOGEST protocol
Organ-on-chip assays to assess intestinal impact
Data integration, dissemination and open-science activities

People involved