| In order to prepare highly active antioxidant peptide, Papain, Alcalase, Neutral protease, and Trypsin were used to hydrolyze Oviductus Ranae in the optimal conditions of each enzyme. The ability of scavenging·OH, O2-· and DPPH· free radicals were used to measure the anti-oxidation of the enzymes hydrolysates. At the same time, the anti-oxidation of the enzymic hydrolysates also being measured separately by three different compound-enzyme, the Papain and Neutral protease, Alcalase and Trypsin, Neutral and Trypsin. The results show that the pepitide hydrolyzed by Neutral protease have highest anti-oxidative activity.Four factors (E/S、temperature, pH of solvent and time) were screened by single factor experiment depending on the anti-oxidation of the enzymic hydrolysates. Subsequently, four factors plus their best levels were chosen to optimize the hydrolysis using a Box-Behnken design experiment with the Design Expert8.05. It showed that the optimal extraction conditions were E/S2.0%, temperature50℃, pH7.5, hydrolyse10h through the response surface methodology when the rate of scavenging hydroxyl radicals of the peptide reach42.75%, which have only0.07%difference with the theoretical value. By comparing the oxidation resistance of the peptides, it found that the ability of scavenging free radicals of the peptide after optimization improved. The IC50of scavenged the·OH fell to27.35mg/mL while the IC50of scavenged O2-fell from22.32mg/mL to19.17mg/mL. The ability of scavenging DPPH changed not significant, with IC50only fell from12.39mg/mL to11.68mg/mL.Enzymic hydrolysates was preliminary separated by ultrafiltration, and four components with different molecular weight<5k,5-10k,10k-30k,>30k were collected to test the oxidation resistance. The result showed that the components of molecular weight is5-10k have the highest oxidation resistance which contained four components separated further by SephadexG-50gel column with peak Ⅰ,Ⅲ showed higher oxidation resistance. |
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