Characterization and lipidomics of extracellular vesicles derived from infant formula during storage: Insights from...

Milk-derived extracellular vesicles (EVs) are enriched with RNA, proteins, and lipids. Limited research has explored the changes in EVs from infant formula (IF) during storage, particularly the link between EV lipid alterations and flavor deterioration. Here, IF-derived EVs (IFEVs) were isolated via gradient ultracentrifugation and characterized using nanoparticle tracking analysis, transmission electron microscopy, western blot and untargeted lipidomics. Distinct differences emerged between IFEVs and raw milk-derived EVs (RMEVs), with RMEVs showing higher protein concentrations and particle yields. A total of 1814 lipid species across 37 classes were identified, dominated by sphingosines (20.98-80.74 %), triglycerides (5.41-31.48 %), and phosphatidylcholines (2.65-14.96 %). Quantitative descriptive analysis indicated declining acceptability, overall flavor, and milky odors across all IF stages with prolonged storage. Correlation analysis showed no statistically significant associations with the six flavor biomarkers. Storage duration (0-24 months) significantly altered IFEV lipid profiles, with quantitative shifts correlating to off-flavor via six volatile biomarkers (e.g., hexanal, nonanal) and seven hub lipids (VIP > 1, p < 0.05) identified through weighted lipid co-expression network analysis. Glycerophospholipid metabolism emerged as the key pathway mediating these changes, without inducing oxidative stress or immune dysfunction in cell models. These findings elucidate IFEVs' role in off-flavor formation, informing storage optimization and dairy product quality enhancement.