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

Sep 30, 2026

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Sheep milk fermentation was used to generate bioactive peptides with antidiabetic, antioxidative, and anti-inflammatory potential, focusing on the Panchali sheep breed of India. Using Limosilactobacillus fermentum in combination with Saccharomyces cerevisiae, optimal activity occurred after 48 h at 30 °C, with antioxidant activity, α-amylase inhibition, and α-glucosidase inhibition reported. Fermented milk reduced pro-inflammatory cytotoxins and ROS in LPS-induced RAW 267.4 macrophage cells, while peptide fractions were separated by RP-HPLC and identified via BIOPEP database comparison. Molecular docking showed strong binding to human α-amylase and α-glucosidase, and further in vivo validation was recommended.

LanguageEnglish

CategoryHealthcare

FormatPDF

Pages20

Reading Time50 min

AccessFree to download

Abstract

Study Rationale

Fermentation Conditions and Bioactivity Results

Proteolytic Activity and Protein Profiling

Peptide Separation and Identification

Molecular Docking and Mechanistic Insights

Conclusions and Future Work

[www. nature.com/scientificreports](www. nature.com/scientificreports) OPEN Exploring the molecular interaction of bioactive peptides with antioxidative, antidiabetic and anti-inflammatory attributes from sheep milk fermented with lactobacillus and yeast Prashantkumar Natubhai Padhiyar1, Pooja M. Mankad2, AmarA. Sakure3, Arka Bhattacharya4, Kanthi Kiran Kondepudi4, Brij Pal Singh5, Souparno Paul6, Preetam Sarkar7, Ashish Patel8, Shaikh Adil9􀀍 & Subrota Hati1􀀍 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 theyeast 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. [www. nature.com/scientificreports/](www. nature.com/scientificreports/) Keywords Antidiabetic, Anti-inflammatory, Antioxidative, Bioactive properties, Fermentation, Sheep milk Abbreviations ABTS 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) ALA α-linolenic acid ATR Attenuated total reflection CLA Conjugated linoleic acids CLSM Confocal laser scanning microscopy DCFDA 2’,7’-dichlorofluorescin diacetate DMSO Dimethyl sulfoxide DNSA 3,5-dinitrosalicylic acid ELISA Enzyme-linked immunosorbent assay EMS Enhanced MS EPI Enhanced product ion ESI Electrospray ionization FAO Food and agriculture organization FBS Fetal bovine serum FITC Fluorescein isothiocyanate FSM Fermented sheep milk FTIR Fourier-transform infrared spectroscopy IDA Information-dependent acquisition IL-1β Interleukin 1 beta IL-6 Interleukin 6 IPG Immobilized pH gradient LAB Lactic acid bacteria LC Liquid chromatography LPS Lipopolysaccharide MPL Milk polar lipids MS Mass spectrometry MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide OPA o-phthaldialdehyde P-NPG p-nitrophenyl-α-D-glucopyranoside RP-HPLC Reverse phase high-performance liquid chromatography SDS-PAGE Sodium dodecyl sulfate–polyacrylamide gel electrophoresis TFA Trifluoroacetic acid TNF-α Tumor necrosis factor alpha WSE Water-soluble extracts 1Department of Dairy Microbiology, SMC

FAQs

Which microorganisms were used to ferment sheep milk in this study?

Fermentation used Limosilactobacillus fermentum (KGL4, MTCC 25515) combined with Saccharomyces cerevisiae (WBS2A, MG101828).

What fermentation conditions produced the best bioactivity?

Optimal bioactivity was observed after 48 hours of fermentation at 30 °C.

How were the generated peptides separated and identified?

Ultrafiltered water-soluble extracts were fractionated by reverse-phase HPLC, and identified sequences were assessed by comparison with the BIOPEP database to confirm antidiabetic and antioxidative activities.

Sep 30, 2026

Uploaded by Rizky