Exosomes separation with aqueous two-phase systems from bovine milk

The exosomes hold significant potential in disease diagnosis and therapeutic interventions. The objective of this study was to investigate the potential of aqueous two-phase systems (ATPSs) for the separation of bovine milk exosomes. The milk exosome partition behaviors and bovine milk separation were investigated, and the ATPSs and bovine milk whey addition was optimized. The optimal separation conditions were identified as 16% (mass) polyethylene glycol 4000, 10% (mass) dipotassium phosphate, and 1% (mass) enzymatic hydrolysis bovine milk whey. During the separation process, bovine milk exosomes were predominantly enriched in the interphase, while protein impurities were primarily found in the bottom phase. The process yielded bovine milk exosomes of 2.0 × 10¹¹ particles per ml whey with high purity (staining rate>90%, 7.01 × 10¹⁰ particles per mg protein) and high uniformity (polydispersity index <0.03). The isolated exosomes were characterized and identified by transmission electron microscopy, zeta potential and size distribution. The results demonstrated aqueous two-phase extraction possesses a robust capability for the enrichment and separation of exosomes directly from bovine milk whey, presenting a novel approach for the large-scale isolation of exosomes.