Molecular characterization and functional activities of bioactive peptides from panchali sheep milk fermented with Lacticaseibacillus rhamnosus MTCC 25516 and Saccharomyces cerevisiae WBS2A

This study explored the antioxidative and antidiabetic potential of bioactive peptides derived from Panchali sheep milk fermented with Lacticaseibacillus rhamnosus (M9, MTCC 25516) and Saccharomyces cerevisiae (WBS2A, MG101828). Fermentation was conducted at 30°C, and measurements were taken at 0, 12, 24, 36, and 48 h. Maximum activity was observed at 48 h, including antioxidant activity (ABTS free radical scavenging) of 28.62%, and α-glucosidase and α-amylase inhibition of 71.19% and 65.90%, respectively. Proteolysis was optimized using varying inoculum levels and incubation times, with maximal activity (6.31 mg/mL) attained at 2.5% inoculum after 48 h. Fermented sheep milk notably suppressed LPS-induced cytotoxin and ROS production in RAW 267.4 cells, suggesting anti-inflammatory effects. Structural and molecular changes in proteins were validated using CLSM and FTIR, revealing alterations in the protein networks. SDS-PAGE revealed prominent protein bands between 10-85 kDa, while no bands were detected in permeate samples. In contrast, 2D gel electrophoresis localized 35 distinct protein spots within the 15–70 kDa molecular weight range. Water-soluble extracts were further analyzed using RP-HPLC and MASCOT software, with sequences validated through the BIOPEP database for antioxidant and antidiabetic activity. Notably, molecular docking of the ITMPLW peptide showed a high binding affinity for digestive enzymes, confirming its multifunctional bioactivity. Overall, fermented Panchali sheep milk is a novel source of peptides with strong antioxidant and antidiabetic potential.