Ultrasound-shear-assisted enzymatic preparation of α-glucosidase inhibitory peptides from Juglans sigillata D. and elucidation of their molecular inhibitory mechanism.

This study aimed to efficiently valorize protein resources from Juglans sigillata D. by employing ultrasound-shear-assisted enzymatic hydrolysis to generate α-glucosidase-inhibitory peptides. The process parameters were optimized using response surface methodology, and the structural basis underlying this enhanced inhibitory activity was further elucidated through multiscale structural characterization and molecular docking analysis. The optimal conditions were determined as follows: ultrasound treatment time of 21 min, ultrasonic power of 400 W, shear speed of 12000 r/min, and enzyme loading of 12 KU/g. Under these conditions, the α-glucosidase inhibition rate of the hydrolysate reached 54.38 ± 0.77 %, which was significantly higher than that obtained by enzymatic hydrolysis alone, with the latter showing an inhibition rate of 45.82 ± 3.28 % (p < 0.05). Structural analyses indicated that the combined ultrasound-shear treatment modified the conformational structure and increased the surface hydrophobicity of walnut protein, potentially enhancing protease accessibility and facilitating the release of α-glucosidase-inhibitory peptides. In total, 4492 peptides were identified by LC-MS/MS, from which 20 candidate peptides were prioritized using a BIOPEP-aided, two-stage in silico workflow. Molecular docking was subsequently used to compare the predicted binding modes and docking scores of selected peptides with α-glucosidase. The peptides FFPGSP, FGPSQPF, and APSKDAPMF were synthesized and experimentally validated. Their IC50 values were 1745, 5393, and 4090 μM, respectively. These findings show that combining peptide identification, bioinformatics screening, molecular docking, and in vitro validation can help facilitate the preliminary discovery and selection of α-glucosidase-inhibitory peptides. This study provides a basis for the high-value utilization of J. sigillata protein and the development of natural peptides with potential α-glucosidase-inhibitory activity.