【作者】 贾韶千；

【导师】 吴彩娥；

【作者基本信息】 南京林业大学 ， 森林培育， 2011， 博士

【摘要】 本论文以银杏种仁为原料,研究了银杏抗氧化肽的制备工艺,并对其进行了结构鉴定,检验了其抗氧化活性,为银杏种仁的综合利用提供了依据。主要研究结果如下：(1)通过均匀设计对碱法制备银杏种仁蛋白的工艺进行了优化,得到的最佳提取条件为：提取时间12h、料液比1：26、NaOH溶液质量分数0.2%,在此条件下提取率达到了89.14%。(2)采用2709碱性蛋白酶和胃蛋白酶为双酶法的酶源,对银杏种仁蛋白进行酶解,以还原能力为指标考察酶解效果。通过单因素试验和响应面分析优化得到双酶法酶解银杏种仁蛋白制备抗氧化肽的最佳工艺条件,首先添加2709碱性蛋白酶,酶解条件为：底物浓度2%、酶用量为6976U.g1、酶解温度为55℃、pH值为8.9、酶解时间为5h；然后添加胃蛋白酶,酶解条件为：酶用量为9000U.g-1、pH值为3.0、酶解温度为50℃、酶解时间为2h。在此条件下得到的银杏种仁蛋白酶解液的还原能力A700为1.636。(3)通过考察持水性、吸油性、溶解性、起泡性及泡沫稳定性、乳化性及乳化稳定性等指标,对银杏种仁蛋白及其酶解产物的功能特性进行研究。结果发现,银杏种仁蛋白酶解后,其酶解产物的功能特性得到了提高,溶解性、起泡性和乳化性得到了显著的改善。(4)采用不同的体外抗氧化指标对不同浓度的银杏种仁蛋白酶解产物的抗氧化活性进行了测定。随着银杏种仁蛋白酶解产物浓度的增加,其抗氧化活性呈现不断上升的趋势,表现出一定的量效关系。双酶分步水解可以提高银杏种仁蛋白酶解产物的抗氧化活性。(5)采用葡聚糖凝胶Sephadex G-25、Sephadex G-10和半制备RP-HPLC对银杏种仁蛋白酶解产物进行分离纯化,通过对自由基清除能力的测定,得到了抗氧化活性较强的组分C8和C9。(6)采用MALDI-SYNAPT-Q-TOF-MS质谱分析法测定组分C8和C9的相对分子量分别为452.21和988.49。结合TOF-MS/MS串联质谱和氨基酸组成分析得知C8的氨基酸序列为：YVGD(Tyr-Val-Gly-Asp); C9的氨基酸序列为：LGNTDYAVH (Leu-Gly-Asn-Thr-Asp-Tyr-Ala-Val-His)。(7)采用固相合成法中的快速Fmoc法合成了抗氧化活性肽YVGD和LGNTDYAVH,并采用不同的体外抗氧化试验对其抗氧化活性进行验证。结果表明银杏抗氧化肽YVGD和LGNTDYAVH具有较高的抗氧化活性,且抗氧化活性随浓度的升高而增强,呈现出较强的量效关系。更多还原

【Abstract】 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.更多还原