Production and characterization of antidiabetic peptides from kefir-fermented goat milk with anti-inflammatory activity: an in silico, in vitro and molecular docking interactions

Introduction Kefir fermentation may promote proteolysis of goat milk proteins and generate peptide-containing fractions with measurable in-vitro bioactivity. This study investigated changes in proteolysis and digestive-enzyme inhibition during fermentation, characterized peptide-containing fractions and evaluated cellular responses to fermented goat milk in an in-vitro macrophage model. Methods Goat milk was fermented with kefir culture. Proteolysis and in-vitro inhibitory activities against α-amylase, α-glucosidase and pancreatic lipase were assessed. Fermentation conditions were compared across culture concentrations and time points. FTIR and dynamic light scattering (DLS) were used to characterize spectral features and particle-size distributions. Cytotoxicity and inflammatory responses were evaluated in RAW 264.7 macrophages. Peptide-containing fractions were characterized by ultrafiltration, RP-HPLC, electrophoresis and RP-LC/MS. Candidate peptides were screened in silico and SCQDQPTTLAR was selected for exploratory molecular docking. Results and Discussion After 48 h, inhibition of α-amylase, α-glucosidase and pancreatic lipase reached 68.87%, 70.21% and 61.40%, respectively, while proteolytic activity increased to 5.523 mg/mL. Optimal fermentation was obtained with 4.8–6.8 mg kefir culture/100 mL milk for 48 h, producing 5.93 mg/mL proteolytic activity. FTIR and DLS indicated fermentation-associated structural changes. GMK was non-cytotoxic to RAW 264.7 cells and decreased TNF-α, IL-6, IL-1β and ROS at 1 mg/mL. The 3 kDa permeate exhibited the greatest α-glucosidase inhibition (75.20%), whereas the 10 kDa retentate showed the highest proteolytic activity (6.35 mg/mL). RP-LC/MS identified milk-derived peptides and docking analysis indicated interactions of SCQDQPTTLAR with hBAL, hMGA and hPAM, involving His435 and Asp300. BIOPEP-UWM, PeptideRanker and toxicity prediction further supported the potential bioactivity and safety of the identified peptides. Conclusion Kefir fermentation was associated with increased proteolysis and measurable in-vitro inhibition of digestive enzymes. Ultrafiltration produced fractions with differing inhibitory activities and fermented goat milk altered selected inflammatory and ROS-related readouts in RAW 264.7 cells. These findings support further investigation of kefir-fermented goat milk and its peptide-containing fractions as potential functional-food ingredients and further studies are required to establish clinical antidiabetic efficacy or product shelf life.