一种新型抗氧化五肽的纯化、鉴定与表征

Puri?cation, Identi?cation and Characterization of a Novel Antioxidant Pentapeptide

以黑鲨鱼皮为原料,利用蛋白酶酶解制备和分离纯化抗氧化多肽,并探究其抗氧化机理。以1,1-二苯基-2-苦肼基(1,1-diphenyl-2-picrylhydrazyl,DPPH)自由基清除率为主要指标,以水解度为辅助指标,优化酶解工艺条件。通过反相高效液相色谱和电喷雾四极杆飞行时间质谱分离纯化和鉴定氨基酸序列并探究其体外抗氧化活性。结果显示,最佳水解蛋白酶为碱性蛋白酶,最优酶解工艺为pH 8.0、酶添加量311 U/mL、底物质量分数3%、酶解温度45℃、酶解时间4.9 h,所得的酶解液DPPH自由基清除率为77.61%,水解度为14.21%。经反相高效液相色谱分离,得到组分F19的DPPH自由基清除能力最强。经鉴定,选择抗氧化活性高的总离子流色谱峰(保留时间为10.02 min左右)进行一级质谱和二级质谱分析,获得纯化的新型抗氧化五肽,其分子质量为461 Da,氨基酸序列结构为Pro-Gly-Gly-Thr-Met,其DPPH自由基清除率为75.01%(1.0 mg/mL),氧自由基吸收能力(以Trolox当量计)为2 490.01μmol/g。新型五肽的Pro和Met在抗氧化中起到关键性作用。本研究为黑鲨鱼皮抗氧化五肽的抗氧化机理及其构效关系提供了一定理论依据。

Black shark skin was enzymatically hydrolyzed by protease to produce antioxidant peptides. The mechanism of action of the peptides was explored. The hydrolysis process was optimized based on 1,1-diphenyl-2-picrylhydrazyl(DPPH)radical scavenging capacity and also by taking into account degree of hydrolysis. The antioxidant peptides were separated and purified by reversed-phase high performance liquid chromatography(RP-HPLC). The amino acid sequences were identified by electrospray ionization quadrupole-time of flight mass(ESI Q-TOF MS), and the in vitro antioxidant activity of the purifled peptides was analyzed. The optimal hydrolysis parameters were determined as follows: use of alkaline protease as the best enzyme, enzyme dosage 311 U/mL, substrate concentration 3%, temperature 45 ℃, pH 8.0 and time 4.9 h. The hydrolysate obtained with a degree of hydrolysis of 14.21% under the optimized conditions scavenged 77.61% of DPPH radical. The hydrolysate was separated into 26 fractions. F19 was found to possess the strongest DPPH radical scavenging activity. The total ion current peak with higher antioxidant activity(at 10.02 min retention time) was selected for mass spectrometric(MS) and tandem mass spectrometric(MS/MS) analysis. The amino acid sequence of F19 was determined as Pro-Gly-Gly-Thr-Met. F19 at 1.0 mg/mL scavenged 75.01% of DPPH radical, and its oxygen radical absorption capacity(ORAC, calculated as Trolox equivalent) was 2 490.01 μmol/g. Pro and Met residues played a leading role in the antioxidant activity of F19. This study provides a theoretical basis for understanding the antioxidant mechanism and the structureactivity relationship of the pentapeptide derived from black shark skin.