Abstract: In this study, antioxidant peptides derived from sheep hemoglobin were prepared from fresh sheep blood. Sephadex G-25 chromatography, DEAE Sephadex A-50 chromatography, reverse phase high performance liquid chromatography (RP-HPLC) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) were used to separate, purify, and identify peptides from the hydrolysate of sheep hemoglobin. The antioxidant activity of the obtained peptides was evaluated by their free radical scavenging capacity, stability against simulated gastrointestinal digestion and protective effect on H2O2-induced oxidative injury in Caco-2 cells. The results indicated that three novel antioxidant peptides, Ala-Tyr-Glu-Val-Asp (AYEVD), Phe-His-Thr-Met-Glu (FHTME) and Ser-Phe-Met-Tyr-Glu-Lys (SFMYEK), were purified and identified from the hydrolysate, with molecular masses of 595.60, 663.74 and 803.92 Da, respectively. Among them, AYEVD had the strongest scavenging capacity to scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, and AYEVD showed stronger antioxidant activity after simulated gastrointestinal digestion. In addition, AYEVD inhibited H2O2-induced oxidative injury in Caco-2 cells, significantly reduced the accumulation of reactive oxygen species (ROS), suppressed early cell apoptosis and the formation of malondialdehyde (MDA), and increased the activity of intracellular antioxidant enzymes. Therefore, this study could provide a theoretical basis for the application of novel antioxidant peptides from sheep hemoglobin in functional foods.

Key words: sheep hemoglobin; antioxidant peptide; purification; identification; Caco-2 cells; cytoprotection