Identification, characterization, in vitro and vivo evaluation of dual-protein peptides based on virtual screening

Functional food-derived peptides with α-glucosidase inhibitory activity exhibit great potential for dietary management of type 2 diabetes (T2DM), especially given the growing demand for non-pharmaceutical interventions against this global epidemic. Dual-protein composite peptides were prepared from soy protein and casein under optimized enzymatic hydrolysis conditions, yielding <5 kDa fractions (fractionated soy-casein composite peptides, FSCPs) with α-glucosidase inhibitory activity (IC₅₀ = 0.78 ± 0.15 mg/mL). Enzyme kinetic analyses verified competitive binding to the α-glucosidase active site. Molecular docking demonstrated that peptide binding to α-glucosidase was mediated by hydrogen bonds and salt bridges with key catalytic residues. Furthermore, the inhibitory activity of these peptides was validated via the synthesis of IH-11 and IV-9. In the Caco-2 cell model, FSCPs significantly inhibited glucose transport while enhancing α-glucosidase inhibitory activity. Furthermore, this dual bioactivity was corroborated by in vivo experiments, in which oral administration of FSCPs to mice reduced postprandial glycemia in a dose-dependent manner. Efficacy was further validated via randomized, double-blind human trials, which exhibited a marked reduction in postprandial blood glucose levels. This study established an integrated approach for generating dual-protein composite peptides with enhanced α-glucosidase inhibitory activity. This study employed a comprehensive validation framework, integrating in vitro assays to elucidate functional mechanisms, primarily α-glucosidase inhibition and glucose transport suppression, and in vivo assessments, including animal experiments and human trials, to verify physiological efficacy, with a focus on dose-dependent regulation of postprandial glycemia. The framework offers promising potential for non-pharmaceutical management of T2DM through targeted nutritional interventions.