Characterization and molecular docking analysis of multifunctional bioactive peptides from fermented paneer whey reveals promising anti-inflammatory activity in RAW 264.7 macrophages

Whey-derived bioactive peptides have gained significant scientific and commercial interest due to their unique functional properties, including antihypertensive, antioxidative, immunomodulatory, and antidiabetic effects. The present study aimed to evaluate the antidiabetic, antioxidant, and anti-inflammatory potential of paneer whey fermented with Lactobacillus helveticus GQ253959, with particular emphasis on the generation of bioactive peptides. A peptidomic approach was used for protein profiling, while HPLC–LCMS and FTIR analyses were employed for peptide separation, identification, and structural characterization. The inhibitory activities against α-glucosidase (54.53%), α-amylase (61.47%), and lipase (58.23%), along with antioxidant activity (52.06%), increased significantly during the fermentation period from 0 to 48 h using 2% culture inoculation. Maximum proteolytic activity was observed at a 2.5% inoculum level after 48 h of fermentation, compared with the 1.5% and 2% inoculum levels. SDS-PAGE revealed protein bands ranging from 10 to 120 kDa, whereas 2D-PAGE showed protein spots between 15 and 70 kDa. Among the fractionated paneer whey samples, 3 kDa permeate exhibited the highest α-glucosidase inhibition (49.82%), while 10 kDa retentate showed the highest antioxidant activity (62.89%). Fermented whey showed a maximum number of peaks compared to unfermented whey. Additionally, fermented paneer whey displayed a crystalline and irregular fractured surface morphology. FTIR analysis was also used to evaluate the presence of various functional groups. Molecular docking studies suggested that the selected peptide exhibited binding affinity toward α-glucosidase, α-amylase, and DPP-IV, indicating its potential relevance in diabetes management. A dose of 0.50 mg/mL of fermented whey significantly reduced lipopolysaccharide (LPS)-induced production of pro-inflammatory cytokines (IL-6, IL-1β, and TNF-α) in RAW 264.7 cells. These findings suggest the potential of fermented paneer whey as a source of bioactive compounds with antidiabetic, antioxidative, and anti-inflammatory properties for functional food applications.