Based on the theoretical basis of bioinformatics, this paper used the computer to simulate the enzymatic digestion of Tibetan sheep hemoglobin, using online activity scoring, activity prediction, and molecular docking techniques to screen their antioxidant peptides and verify their antioxidant properties by DPPH free radical and ABTS cationic free radical scavenging rate experiments.The amino acid sequences of Tibetan sheep hemoglobin were screened using the UniProt database to evaluate its potential bioactivities, ExPASy Peptide Cutter online tool was applied to enzymatically digest the screened peptide chains, Peptide Ranker was applied to score the bioactivity prediction, and the online tools Innovagen, ToxinPred, iBitter-SCM, A11erTOP, and AdmetSAR for solubility, toxicity, bitterness, sensitization and ADMET, molecular docking using Keap1 receptor for activity prediction, and finally validation of the screened antioxidant peptides of Tibetan sheep hemoglobin using DPPH free radical and ABTS cationic free radical scavenging rate assay.Results showed that five Tibetan sheep hemoglobin chains contained 20 amino acids, and a total of 258 kinds of peptide (485 peptides) were obtained by enzymatic digestion, and 110 kinds of peptide (194 peptides) with ratings greater than 0.5 were screened out by the bioactivity scoring system, and 18 kinds of peptide with good solubility, non-toxicity, non-bitterness, and non-allergenicity were obtained through the prediction of their activities, and the molecular docking was carried out for the 10 peptides that had a better intestinal absorption, and the top three peptides with molecular docking scores, KRM, FPL, and FL, were subjected to in vitro antioxidant activity assay, and the results showed that the free radical scavenging ability of FPL and KRM exceeded the positive control, with KRM exceeding the positive control for DPPH radicals and ABTS cationic radicals with IC50 of (0.125 0±0.012 3) and (0.339 1±0.031 4) mg/mL, respectively, and FPL exceeding the positive control for DPPH radicals and ABTS cationic radicals with IC50 of (0.256 5±0.014 1) and (0.374 6±0.047 1) mg/mL, respectively. and the scavenging ability of FL for DPPH radicals and ABTS cation radicals reached 88.90% and 94.40% of that of the positive control, with IC50 of (1.106 1±0.279) and (1.601±0.031 4) mg/mL, respectively, both of which showed strong antioxidant activity. This method could screen antioxidant peptides quickly and economically compared with the traditional enzyme digestion experiments, and provide a research basis for the high-value utilization and development of Tibetan sheep blood resources.
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