Exploring the molecular interaction of bioactive peptides with antioxidative, antidiabetic and anti-inflammatory attributes from sheep milk fermented with lactobacillus and yeast.

Abstract

Sheep milk is a rich source of proteins with potential to generate bioactive peptides through microbial fermentation. However, limited studies have focused on the Panchali sheep breed of India and the multifunctional health properties of its fermented products. This study investigated the generation of bioactive peptides with antidiabetic, antioxidative, and anti-inflammatory properties through the fermentation of sheep milk using a potent LAB, Limosilactobacillus fermentum (KGL4, MTCC 25515), in combination with the yeast strain Saccharomyces cerevisiae (WBS2A, MG101828). Optimal bioactivity was observed after 48 h of fermentation at 30 °C, with antioxidant activity reaching 40.08%, α-amylase inhibition at 72.48%, and α-glucosidase inhibition at 75.96%. Proteolytic activity was assessed at varying inoculum concentrations (1.5, 2 and 2.5%) and incubation time (0, 12, 24, 36 and 48 h), with the highest activity (6.74 mg/mL) observed at a 2.5% inoculum after 48 h. Additionally, fermented milk effectively lowered the production of pro-inflammatory cytotoxins and ROS in LPS-induced RAW 267.4 macrophage cells, indicating its anti-inflammatory properties. Protein profiling through SDS-PAGE revealed bands between 15 and 70 kDa, while 2D electrophoresis identified 39 distinct protein spots associated with peptide production. Reverse-phase HPLC of ultrafiltered water-soluble extracts (WSEs) was used to separate peptide fractions, and identified sequences were assessed through comparison with BIOPEP database to confirm antidiabetic and antioxidative activities. The most potent selected peptide showed strong binding affinity and stable interaction within the active pockets of human α-amylase and α-glucosidase, as well as high HADDOCK and HPEPDOCK scores in molecular docking analyses. These findings indicate that fermented sheep milk is a promising source of multifunctional bioactive peptides with potential applications in functional foods targeting diabetes and inflammation. Further in vivo studies are warranted to validate physiological relevance, bioavailability, and health benefits.

Supplementary Information: The online version contains supplementary material available at 10.1038/s41598-025-30657-3.

Keywords: Anti-inflammatory; Antidiabetic; Antioxidative; Bioactive properties; Fermentation; Sheep milk.