Peptidomic profiling and molecular dynamics study of Bioactive peptides from fermented camel milk: Considering the fermentation time dependent proteolysis by Lactobacillus and Saccharomyces with antidiabetic, antioxidative and anti-inflammatory activities

Prashantkumar Natubhai Padhiyar¹Pooja M. Mankad¹Krupali Ramanuj²AMAR ASHOK SAKURE³ARKA BHATTACHARYA⁴Kanthi Kiran Kondepudi⁴Bipransh Kumar Tiwary⁵Zhenbin Liu⁶Ashish Patel¹Subrota Hati^1*

• ¹Kamdhenu University, Gandhinagar, India
• ²Gujarat Natural Farming Science University, Hallol, India
• ³Anand Agricultural University, Anand, India
• ⁴BRIC National Agri-Food and Biomanufacturing Institute, Sahibzada Ajit Singh Nagar, India
• ⁵University of North Bengal, Darjeeling, India
• ⁶Shaanxi University of Science and Technology, Xi'an, China

This study addresses the growing demand for natural functional foods with therapeutic potential, particularly for managing diabetes, oxidative stress, and inflammation. The aim of this research was to investigate the biofunctional attributes of camel milk fermented with Limosilactobacillus fermentum (KGL4) and Saccharomyces cerevisiae (WBS2A), with a focus on its antidiabetic, antioxidative, proteolytic, anti-inflammatory, and cytoprotective activities. Fermentation was performed for 0, 12, 36 and 48 h at 30 °C (2% inoculum rate). Fermented camel milk exhibited a significant enhancement insignificantly enhanced α-amylase (81.33%) and α-glucosidase (68.37%) inhibition, demonstrating strong antidiabetic potential. Antioxidant activity, as assessed through the ABTS assay, progressively increased with incubation time, reaching a peak of 65.87% at 48 h. Proteolytic activity also rose significantly, attaining a maximum of 6.41 mg/mL (2.5% inoculum concentration forat 30 °C/48 h). Chromatographic profiling via RP-HPLC revealed increased bioactivity in 3KDa3 kDa permeate (antidiabetic) and 10KDa10 kDa retentate (antioxidant) samples, suggesting the role of low -molecular -weight peptides. 2D gel electrophoresis and SDS-PAGESDS‒PAGE confirmed proteolytic cleavage, revealing the presence of smaller peptide fragments in the fermented samples compared tothan in the unfermented controls. Further structural analysis usingvia FTIR and Confocal Laser Scanning Microscopyconfocal laser scanning microscopy (CLSM) demonstrated secondary structure modifications, including increased β-sheet formation and reduced aggregate size. Molecular docking studies showedrevealed that the identified peptide sequence CCFSSCAMR effectively bound to the human digestive enzymes, hBAL, hPAM, and hMGA via hydrogen bonding and hydrophobic interactions, supporting its potential inhibitory function. Additionally, fermented camel milkCM displayed strong anti-inflammatory and cytoprotective properties ineffects on LPS-stimulated RAW 264.7 macrophage cellsmacrophages. This study highlights the potential of fermented camel milk as a value-added functional food with significant antidiabetic, antioxidant, anti-inflammatory, and cytoprotective properties. The generation of low -molecular -weight bioactive peptides through targeted microbial fermentation provides a scientific foundation for developing natural, non-pharmacologicalnonpharmacological interventions for metabolic and inflammation-related disorders. These findings support the application of fermented camel milk in functional food and nutraceutical development.