黑藻抗氧化肽制备及其活性表征

Preparation of antioxidative peptides from Hydrilla verticillata(Linn.f.)Royle and its activity

采用胰蛋白酶酶解黑藻蛋白制备抗氧化肽,以酶解物清除2,2'-联氮-双-3-乙基苯并噻唑啉-6-磺酸(ABTS)自由基的百分率为响应值,考察酶底比(酶与底物的比例,以质量百分比表示)、酶解pH和酶解时间对酶解物清除自由基的影响.响应面法优化黑藻蛋白的最佳酶解条件为:酶底比8.6%、时间5.9 h、酶解pH值7.3.所得黑藻蛋白酶解物(1000μg/mL)对ABTS、DPPH自由基、超氧阴离子、羟自由基的清除率分别为67.9%、40.1%、50.1%、25.3%.酶解物经截留分子量3 kDa和10 kDa的滤膜超滤和Sephadex G-25凝胶柱层析分离纯化后,所得黑藻抗氧化肽P-1和P-1-A(1000μg/mL)对ABTS、DPPH自由基、超氧阴离子、羟自由基的清除率分别显著提升到80.6%、43.1%、52.1%、31.4%和88.2%、48.2%、54.4%、35.5%.研究结果为黑藻抗氧化肽应用于功能性食品或天然抗氧化剂提供理论依据.

Protein from Hydrilla verticillata(Linn.f.)Royle(HVR)was hydrolyzed with trypsin to prepare antioxidant peptides.The effects of enzyme-to-substrate ratio(the rate of enzyme to substrate,expressed by mass percentage),pH and time on the scavenging radical performance of hydrolysates were investigated and the process conditions were optimized using response surface methodology(RSM)based on scavenging activity to 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)(ABTS)radical.The optimal hydrolysis conditions were determined as follows:enzyme-to-substrate ratio,8.6%;pH,7.3;time,5.9 h.The hydrolysate prepared under these optimal conditions could scavenge 67.9%,40.1%,50.1%,25.3%of ABTS,DHHP radical,superoxide anion,hydroxyl radical,respectively.Subsequently,the hydrolysate is treated by ultrafiltration and separating on G-25 Sephadex column,the scavenging percentage of achieved antioxidative peptides P-1and P-1-A to above-mentioned four radicals significantly promoted to 80.6%,43.1%,52.1%,31.4%and 88.2%,48.2%,54.4%,35.5%,respectively.The results of this study provide a theoretical basis for the application of Hydrilla verticillata(Linn.f.)Royle antioxidant peptides in functional foods or natural antioxidants.