Differential salt precipitation improves recovery and biological activity of probiotic-derived bioactive peptides

Bioactive peptides derived from probiotics are gaining widespread attention for their potential as nutraceuticals for the treatment of complex metabolic disorders. However, efficient recovery of peptide fractions with enhanced biological activity remains a significant challenge. In the present study, probiotic biomass obtained from a commercially available probiotic formulation was subjected to enzymatic hydrolysis using trypsin, followed by differential precipitation using magnesium sulfate (MgSO₄) and zinc sulfate (ZnSO₄) to enrich peptide fractions. SDS-PAGE was used to characterize the recovered peptides, and reverse-phase high-performance liquid chromatography (RP-HPLC) was used to evaluate molecular weight distributions and chromatographic profiles. ZnSO₄ precipitation yielded a higher quantity of peptide fraction (160 mg/L culture) compared with MgSO₄ precipitation (140 mg/L culture). SDS-PAGE analysis revealed enrichment of low-molecular-weight peptides predominantly within the 2-10 kDa range. In contrast, RP-HPLC analysis demonstrated differences in peptide composition and relative abundance between the two fractions. The biological activities of the isolated peptides were assessed through DPPH radical scavenging, Oil Red O staining, and MTT cytotoxicity assays. Both peptide fractions exhibited dose-dependent antioxidant, anti-lipidogenic, and cytotoxic activities; however, the ZnSO₄-precipitated fraction consistently demonstrated significantly greater activity than the MgSO₄ fraction. These findings indicate that differential salt precipitation influences peptide recovery and biological activity, with ZnSO₄ being a more effective precipitating agent under the experimental conditions used. These findings suggest that probiotic-derived peptides may serve as potential bioactive molecules for future therapeutic development targeting metabolic disorders.