Fermentation-Driven Generation of α-Glucosidase Inhibitory Whey Peptides by Marine-Derived Probiotic Lacticaseibacillus casei DS31: Activity Enrichment and Peptidomics

This study investigated the generation of α-glucosidase inhibitory peptides from whey protein fermented by the marine-derived probiotic Lacticaseibacillus casei DS31 (isolated from the intestinal microbiota of the large yellow croaker, Larimichthys crocea) and assessed their potential for practical glycemic management. Fermentation markedly increased inhibitory activity, with the freeze-dried crude supernatant exhibiting an IC50 of 2.115 mg/mL. Activity was further enriched through stepwise purification: ultrafiltration (<3 kDa) improved potency (IC50 = 1.206 mg/mL), and subsequent Sephadex (crosslinked dextran) G-15 gel filtration yielded a more active E fraction (IC50 = 1.145 mg/mL). LC–MS/MS characterized 19 peptides, and integrated in silico screening (PeptideRanker combined with molecular docking) highlighted GEPGPEGPAG as a leading candidate, showing a more favorable predicted binding energy (−82.50 kcal/mol) than the positive control acarbose (−69.31 kcal/mol). Docking analysis suggests that GEPGPEGPAG may inhibit α-glucosidase by forming a stable network of hydrogen bonds, salt bridges, and hydrophobic interactions within the catalytic pocket. Overall, DS31-fermented whey and its enriched fractions show promise as functional ingredients for postprandial glycemic control.