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• The 0.05% rennet enzyme treatment minimizes casein interference during mEVs separation. • Compared with vacuum heating concentration (VHC) and vacuum freeze concentration (VFC), membrane concentration (MC) better protects the morphological characteristics and functional components of mEVs. • Rennet enzyme precipitation + MC + ultracentrifugation can rapidly and nondestructively enrich mEVs in cow and goat milk. • Sequential protocol for the mass separation of EVs from liquid milk developed. The large-scale and efficient production of high quality milk-derived extracellular vesicles (mEVs) is essential to study their function and properties. The low concentrations of mEVs in milk, coupled with interference from casein, makes the enrichment of EV from large quantities of milk a major challenge. Therefore, the effects of casein removal via enzyme precipitation (EP) and acid precipitation (AP) on mEVs separation were compared in this study. Then, three concentration methods, vacuum heating concentration (VHC), vacuum freeze concentration (VFC) and membrane concentration (MC), were employed to concentrate mEVs-rich whey to improve the efficiency before ultracentrifugation. Subsequently, the morphology, particle size and marker proteins of mEVs were characterized by nanoparticle tracking analysis, transmission electron microscopy and western blotting. Finally, the function of mEVs obtained was verified by RAW264.7 cells. The results indicated that mEVs with intact morphology and higher purity could be obtained quickly through MC concentration combined with ultracentrifugation after casein removal from milk via EP, thereby providing a promising pathway for the large-scale production of milk-derived mEVs.
Ye et al. (Mon,) studied this question.