Study On Antioxidative Peptide From Controllable Enzymatic Hydrolyzed Laver

In this dissertation, the mixture of neutral protease and some aminopeptidases were used to produce antioxidant peptides by hydrolyzing laver. The hydrolyzation conditions were optimized, and the obtained antioxidative peptides were purified and their characteristics were studied.First of all, acid protease, alkaline protease, neutral protease, trypsin and pepsin were used to hydrolyze defatted laver powder respectively. Based on the yield of antioxidative peptide and their ability to eliminate DPPH, neutral protease was chosen as hydrolyzation enzyme. By performing orthogonal test, the optimal hydrolysis conditions were set as: 30g/L laver in pH 7.5 Na2HPO4-NaH2PO4 buffer, 1.5×104U/g of neutral protease, hydrolyzed for 6 hours at 50℃. After incubation, the degree of hydrolysis is 36.43%, the content of peptide in hydrolytic products reaches 12.01%, and DPPH radical elimination rate of the products is 20.17%.Then the laver was further hydrolyzed by the mixture of neutral protease and aminopeptidase from Bacillus subtilis prepared in our laboratory. The optimal hydrolysis conditions are: 1.48×104U/g of neutral protease and 1.30×104U/g of aminopeptidase were added into pH8.5 Na2HPO4-KH2PO4 buffer containing 30g/L of lever. The hydrolyzation was carried out for 6 hours at 50℃. The results showed that the degree of hydrolysis is 47.51%, the content of peptide in hydrolytic products is 19.01%, and the DPPH scavenging rate of the peptides is 42.17%.The laver was hydrolyzed with neutral protease and aminopeptidase under the optimal conditions. The obtained antioxidative peptides mixture was further purified through Sephadex G-10 gel filtration, DEAE-52 anion-exchange and SOURCE 3RPC and a main peptide was obtained. According to the result of analytical reverse-phase high performance liquid chromatography (RP-HPLC), the purity of this purified peptide is up to 87.44%. Furthermore, the peptide was identified as a hexapeptide AGVGTG (Asp-Gly-Val-Gly-Tyr-Gly) through ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (Q-TOF-MS) analysis.By using synthetized hexapeptide DGVGYG (purity≥98%) as the standard, the analysis showed that the content of antioxidant peptide in the crude enzymatic products of the laver was about 20%. At the concentration of 100μg/mL, the purified antioxidative peptide was at the scavenging activity of superoxide-radical, DPPH radical and hydroxyl-radical, and reducing ability were of 79%, 87%, 91% and 123% of the same concentration of vitamin C. While in the case of the synthetized peptide, was at 100μg/mL, the scavenging activity of superoxide-radical, DPPH radical and hydroxyl-radical, and reducing ability were of 151%, 68%, 114% and 168% of the same concentration of vitamin C. Both of the peptides exhibit strong antioxidant activity.Compared with the synthetized hexapeptide, the laver enzymatic peptide shows better thermal stability. After incubation under the temperature ranged from 30℃to 90℃, the purified enzymatic laver antioxidative peptide remained more than 80% of its scavenging activities of superoxide radical, DPPH radical, hydroxyl radical and reducing ability, while the synthetized hexapeptide kept only 60~70% of its activities. The laver enzymatic antioxidant peptide showed a good stability in the buffer with pH range of 6~10, its antioxidant activity maintained more than 90% after incubation. While the synthetized hexapeptide was more sensitive to the change of pH. The highest antioxidant activity emerges in pH7.5 buffer. In pH6~8 buffer, its antioxidant activity maintained above 90% of its activities. These results may attribute to certain compounds existed in the laver enzymatic products, which have not high antioxidant activity, but can make help to maintain the stability of the antioxidant activity.The laver enzymatic antioxidant peptide (DGVGYG) in our study has high antioxidant activity as well as good stability under the certain conditions, therefore, it can be used as a quality indicators in controlled enzymatic hydrolysis of lever, and to establish the basis of laver comprehensive utilization technology.