The Preraration, Identification And Activity Of Ginkgo Antioxidant Peptides

In this dissertation, taking ginkgo seed as material, the optimum process for ginkgo antioxidant peptide preparation was studied. Further, ginkgo antioxidant peptides were identified and its antioxidant activities were validated. The research would provide a basis for the comprehensive utilization of ginkgo seed. The main findings were as follows:(1) Uniform design method was used for the optimization of alkali extraction process of ginkgo seed proteins. The results showed the optimum technological conditions:extraction time 12 h, so lid-liquid ratio 1:26, sodium hydroxide solution concentration 0.2%. The extraction rate was 89.14% in this condition.(2) Test with reducing capacity as index, ginkgo seed proteins were hydrolyzed by alkaline protease 2709 and pepsin. The response surface experiments and orthogonal experimental design were used to optimize the conditions of enzymatic hydrolysis processing, results showed that the enzyme concentration was 6976 U per gram of substrate; the hydrolysis process was performed under 55℃, pH 8.9 for 5 h. And then pepsin was added, the optimal conditions were:the enzyme concentration was 9000 U per gram of substrate; pH 3.0, the hydrolysis process was performed under 50℃for 2 h. The antioxidant value was 1.636.(3) In order to research the functional properties of ginkgo seed proteins hydrolysates, water-holding capacity, oil-holding capacity, solubility, foamability and emulsifying properties were studied. The result showed that the functional properties of enzymatic hydrolysate was improved, solubility, foamability and emulsifying properties were improved significantly.(4) The antioxidant activities of ginkgo seed proteins hydrolysates were evaluated using nine different antioxidant assays in vitro. The concentration of ginkgo seed proteins hydrolysates showed a certain dose-effect relationship with the antioxidant activities. The results showed that Two-step enzymatic hydrolysis could improve the antioxidant activity of Ginkgo seed proteins hydrolysates.(5) Ginkgo seed proteins hydrolysates were separated by Sephadex G-25, Sephadex G-10 and RP-HPLC. The result of radical scavenging activity showed that fraction C8 and C9 had stronger antioxidant activity than others.(6) The amino acids composition analysis showed that the major amino acids of the fraction C8 were Asp, Gly, Val and Tyr, and the fraction C9 were Asp, Thr, Gly, Ala, Val, Leu, Tyr, His and Asn. The fraction C8 and C9 were then identified by MALDI SYNAPT Q-TOF MS. The molecular weights of the fraction C8 was 452.21 Da, and the amino acid sequence of C8 was YVGD(Tyr-Val-Gly-Asp); The molecular weights of the fraction C9 was 988.49 Da, and the amino acid sequence of C9 was LGNTDYAVH (Leu-Gly-Asn-Thr-Asp-Tyr-Ala-Val-His). The sequence Alignment by bioactive peptide database showed that this two antioxidant peptide were new active peptide sequence.(7) YVGD and LGNTDYAVH were synthesized by Fmoc method. The molecular weights of the synthesized YVGD was 452.16 Da, the molecular weights of the synthesized LGNTDYAVH was 988.54 Da. The antioxidant activities were evaluated by different antioxidant assays, the results showed that YVGD and LGNTDYAVH had good antioxidant activity. The scavenging activity against·OH and chelating metal ion capacity of LGNTDYAVH were better than YVGD. And YVGD showed better antioxidant activities than LGNTDYAVH in other antioxidant assays. We deduced that the amino acid such as Tyr, Gly, Asp, Val, Leu and His in YVGD and LGNTDYAVH might play an important role in their activities. The amino acid compositions, sequences of peptides and the stability of peptides structure might play an important role in antioxidant activities of peptides, and showed different antioxidant activities in different antioxidant assays.