Optimisation and comparison of orthogonal methods for separation and characterisation of extracellular vesicles to investigate how representative infant milk formula is of milk

This study aimed to separate and characterise extracellular vesicles (EV) from infant milk formula (IMF) and skim milk (SM), to determine how representative the EV content of IMF is to SM. Contaminant casein micelles, due to their abundance and overlapping size, were removed followed by either differential ultracentrifugation (DUC) or gradient ultracentrifugation (GUC). Characterisation included BCA, SDS-PAGE, nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), immunoblotting, and imaging flow cytometry (IFCM). NTA reported significantly reduced concentrations of EVs/particles in IMF versus SM; TEM showed intact SM-derived EVs to sparse and disrupted EV-like structures in IMF. Compared to IMF, noticeably stronger bands for EV biomarkers were observed by immunoblotting in SM, indicating compromised EV proteins in IMF; also supported by IFCM. Altogether, we established that EVs are substantially compromised during IMF processing. Furthermore, an optimised method combining acid pre-treatment and GUC for EV separation from milk products has been established.