Abstract:Xanthine oxidase （XOD） inhibitory peptides were prepared from skipjack tuna dark meat by enzymatic hydrolysis，and their properties and activity mechanisms were analyzed. Papain was identified as the best protease by a single-factor experiment combined with the 3-（4，5-dimethylthiazol-2-yl）-2，5-diphenyltetrazolium bromide （MTT） colorimetric assay. The optimal enzymatic conditions were determined by orthogonal test：a solid-to-liquid ratio of 1∶4 （g/mL），hydrolysis temperature of 50 ℃，pH of 7.0，hydrolysis duration of 6 h，and enzyme dosage of 600 U/g. The XOD inhibition rate of skipjack tuna dark meat hydrolysates （STDMHs） under these conditions was 70%. The molecular weight of STDMHs was lower than 3 kDa，of which 90.28% was below 1 kDa. STDMHs contained 17 amino acids，with hydrophobic and basic amino acids accounting for 14.74%and 12.07%，respectively. STDMHs demonstrated excellent activity stability under but not limited to the following conditions：temperatures ranging from -20-60 ℃，pH values between 5.0 and 7.0，in the presence of metal ions Na+，K+，Ca2+，and light conditions including darkness，ultraviolet light，and fluorescent light. STDMHs retained 79.59% of XOD inhibitory activity after simulated gastrointestinal digestion in vitro. Four potential XOD inhibitory peptides，KFLR，ERFR，KFLK，and FEKAF，were screened from STDMHs by virtual screening and molecular docking.