Production, Characterization, and Molecular Dynamic Study of Antidiabetic and Antioxidative Peptides of Fermented Cheese Whey with Anti-inflammatory Properties using Limosilactobacillus fermentum

Introduction: This study aims to valorise cheese whey waste by converting it into bioactive peptides that have several health benefits, potentially leading to the development of nutraceuticals and functional foods and also used in pharmaceutical industry. Methods: The study evaluates the antidiabetic, antioxidative, and anti-inflammatory properties of fermented cheese whey with Limosilactobacillus fermentum (M4), along with the production of antioxidative and antidiabetic peptides. SDS PAGE and 2D PAGE were also performed to identify proteins by molecular weight and isoelectric point, while RP-HPLC distinguished peptide fractions. Peptide sequences from 2D gel spots were identified using RPLC/MS, and RP-HPLC analyzed 3 kDa and 10 kDa permeates. Peakview software characterized the LC/MS results, and FTIR analysis measured structural changes in bioactive peptides. Results: The antioxidative and antidiabetic properties in cheese whey fermented with M4 showed a progressive growth over extended incubation periods, higher effects were observed after fermentation for 48 hours. Inhibitory activities in α-glucosidase, α-amylase & lipase were found to be 65.39%, 66.09%, and 56.74% respectively. ABTS assay was performed to measure antioxidant activity (63.39%) and the highest proteolytic activity (7.62 mg/ml) was measured at 2.5% inoculation rate for 48 hours. In SDS-PAGE, protein bands between 10 & 30 kDa were observed, whereas peptide spots within the range of 10 to 70 kDa were also visualized on the 2D PAGE. RP-HPLC was used to distinguish different fractions of a peptide. Peptide sequences from 2D gel spots were identified using RP-HPLC & RPLC/MS. Peakview software was utilized to characterize the LC/MS results. Sequences of peptides generated from α-lactalbumin and β-lactoglobulin were searched in the BIOPEP database to validate the antidiabetic and antioxidative activities of fermented cheese whey peptides. 0.50 mg/mL of fermented cheese whey significantly LPS suppressed the production of proinflammatory cytokines as well as the mediators that govern them including IL-6, IL-1β, NO, and TNF-α in RAW 264.7 cells. FTIR was used to analysis of protein secondary structure and conformational changes. Conclusion: This study aims to the production of antidiabetic and antioxidative peptides from dairy waste, and cheese whey.