Enhancing the extraction efficiency, functional activity, and freeze-stability of milk-derived extracellular vesicles via rennet coagulation combined with polyethylene glycol precipitation.

Milk-derived extracellular vesicles (MEV) hold considerable promise for immune regulation and nutrient delivery; however, their scalable production and long-term storage stability present major challenges. This study compared 2 whey pretreatments-rennet (EC 3.4.23.4) coagulation and acetic acid precipitation-combined with polyethylene glycol (PEG) precipitation or ultracentrifugation (UC) for MEV isolation. The yield, physicochemical properties, and storage stability of the extracted MEV were systematically evaluated. Results indicated that the PEG-rennet-coagulated (R) method demonstrated optimal performance, yielding a high particle concentration with enrichment efficiency comparable to the UC-R group, and it was enriched with higher levels of total RNA and key microRNAs (miR-30a-5p, miR-21-5p, miR-148a). Functionally, MEV from the PEG-R group significantly enhanced the proliferation of Caco-2 cells. They also effectively attenuated LPS-induced TLR4/NF-κB pathway activation, thereby suppressing NF-κB p65 nuclear translocation. This ultimately led to a significant reduction in proinflammatory factors TNF-α, IL-6, and IL-1β levels. After 30 d storage at -20°C, the PEG-R group exhibited superior retention of vesicle integrity, RNA content, and anti-inflammatory activity. In conclusion, the PEG-R method balances high yield, bioactivity, and storage stability, offering a robust strategy for producing MEV as functional food ingredients or delivery systems.