Discovery and characterization of xanthine oxidase inhibitory peptides from milk protein hydrolysates: in vitro screening, molecular docking and QSAR modeling

Milk proteins were hydrolyzed to release xanthine oxidase inhibitory peptides. • Peptides smaller than 3 kDa showed the strongest inhibitory activities. • Six candidate peptides were synthesized and validated with in vitro assays. • Molecular docking confirmed stable binding of peptides to XO active sites. • A QSAR model accurately predicted peptide inhibitory activities against XO. This study aimed to identify and validate xanthine oxidase (XO) inhibitory peptides derived from milk proteins through an integrated experimental and computational strategy. Whey protein and casein were hydrolyzed using different proteases, and the hydrolysates were evaluated for XO inhibitory activity. Low-molecular-weight fractions (<3 kDa) exhibited the strongest inhibition, and LC-MS/MS analysis identified numerous short peptides. Six candidate peptides were selected by bioinformatics screening and molecular docking, synthesized, and subsequently verified in vitro, with TTMPLW showing the most potent inhibition (IC 50  = 3.44 mM). To further support peptide discovery, a quantitative structure-activity relationship (QSAR) model was established using 81 reported peptides and descriptor sets. The optimized model (AESNN3-RF) showed good predictive performance (R 2  = 0.87, RMSEP = 2.33 mM) and reliably reproduced experimental outcomes. This work suggests a combined workflow of enzymatic hydrolysis, peptide identification, and computational modeling, providing a framework for the discovery of natural XO inhibitors from food proteins.