一种新型抗氧化五肽的纯化、鉴定与表征
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摘要
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 identi?ed by electrospray ionization quadrupole-time of ?ight mass(ESI Q-TOF MS), and the in vitro antioxidant activity of the puri?ed 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.
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 identi?ed by electrospray ionization quadrupole-time of ?ight mass(ESI Q-TOF MS), and the in vitro antioxidant activity of the puri?ed 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.
引文
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[2]SHARMA S,SINGH R,RANA S.Bioactive peptides:a review[J].International Journal Bioautomation,2011,15(4):223-250.
[3]CAI X X,YAN A,FU N Y,et al.In vitro antioxidant activities of enzymatic hydrolysate from Schizochytrium sp.and its hepatoprotective effects on acute alcohol-induced liver injury in vivo[J].Marine Drugs,2017,15(4):115.
[4]STADTMAN E R.Role of oxidant species in aging[J].Current Medicinal Chemistry,2004,11(9):1105-1112.
[5]VALKO M,LEIBFRITZ D,MONCOL J,et al.Free radicals and antioxidants in normal physiological functions and human disease[J].The International Journal of Biochemistry and Cell Biology,2007,39(1):44-84.DOI:10.1016/j.biocel.2006.07.001.
[6]VALKO M,RHODES C J,MONCOL J,et al.Free radicals,metals and antioxidants in oxidative stress-induced cancer[J].Chemico-Biological Interactions,2006,160(1):1-40.DOI:10.1016/j.cbi.2005.12.009.
[7]TRETTER L,SIPOS I,ADAM-VIZI V.Initiation of neuronal damage by complex I deficiency and oxidative stress in Parkinson’s disease[J].Neurochemical Research,2004,29(3):569-577.DOI:10.1023/B:NERE.0000014827.94562.4b.
[8]江勇,汪少芸,赵珺,等.鲨鱼皮明胶多肽的纯化与生物活性研究[J].中国食品学报,2013(5):53-61.
[9]方菲,陈惠敏,汪少芸.鲷鱼鳞多肽-木糖美拉德反应产物的制备、结构与功能[J].食品科学,2018,39(8):182-190.DOI:10.7506/spkx1002-6630-201808029.
[10]李灵,徐梁棕,汪少芸.罗非鱼鱼鳞胶原多肽的优化制备及其生物活性研究[J].福州大学学报(自然科学版),2018,46(1):120-127.
[11]李茹,韦玉白,高永利,等.牡蛎(Crassostrea gigas)酶解物中的新型抗氧化肽[J].食品科技,2017,42(8):19-25.
[12]WANG B,LI L,CHI C F,et al.Purification and characterisation of a novel antioxidant peptide derived from blue mussel(Mytilus edulis)protein hydrolysate[J].Food Chemistry,2013,138(2/3):1713-1719.DOI:10.1016/j.foodchem.2012.12.002.
[13]王卫东,顾增权,孙月娥,等.响应面法优化虾加工下脚料抗氧化肽酶解工艺[J].食品工业,2013,34(9):25-27.
[14]罗春艳,吴杨阳,孙海燕,等.响应面优化鱿鱼须脱皮液胶原肽酶解工艺及抗氧化活性[J].食品科学,2016,37(21):176-182.DOI:10.7506/spkx1002-6630-201621030.
[15]蒋哲,王勤,邱凌,等.鲨鱼皮胶原蛋白肽成分分析[J].厦门大学学报(自然科学版),2006(A1):169-171.
[16]江勇.鲨鱼皮胶原多肽的制备、纯化及生物活性研究[D].福州:福州大学,2011.
[17]UMAYAPARVATHI S,MEENAKSHI S,VIMALRAJ V,et al.Isolation and structural elucidation of antioxidant peptides from oyster(Saccostrea cucullata)protein hydrolysate[J].Protein and Peptide Letters,2014,21(10):1073-1083.
[18]LI Y W,LI B.Characterization of structure-antioxidant activity relationship of peptides in free radical systems using QSAR models:key sequence positions and their amino acid properties[J].Journal of Theoretical Biology,2013,318(3):29-43.
[19]孙颖,徐红华,张艳杰.乳源生物活性肽构效关系的研究进展[J].中国乳品工业,2008,36(9):42-46.
[20]GUO H,KOUZUMA Y,YONEKURA M.Structures and properties of antioxidative peptides derived from royal jelly protein[J].Food Chemistry,2009,113(1):238-245.
[21]ADLER N.Enzymatic hydrolysis of food proteins[M].Elsevier Applied Science Publishers,1989.
[22]WU H C,CHEN H M,SHIAU C Y.Free amino acids and peptides as related to antioxidant properties in protein hydrolysates of mackerel(Scomber austriasicus)[J].Food Research International,2003,36(9/10):949-957.DOI:10.1016/S0963-9969(03)00104-2.
[23]YOU J,LUO Y K,WU J P.Conjugation of ovotransferrin with catechin shows improved antioxidant activity[J].Journal of Agricultural and Food Chemistry,2014,62(12):2581-2587.DOI:10.1021/jf405635q.
[24]胡新颖.山核桃蛋白多肽制备方法的研究[D].长春:吉林农业大学,2002.
[25]任婷婷.玉米蛋白的提取、功能特性及应用研究[D].石家庄:河北科技大学,2014.
[26]王春艳.杏仁短肽制备及其降血压活性研究[D].北京:中国农业科学院,2011.
[27]张少敏,钱杨鹏,刘慧燕,等.底物浓度对酸性蛋白酶水解面筋流变特性的影响[J].食品工业科技,2017,38(9):66-69.
[28]MALAYPALLY S P.Invasive silver carp(Hypophthalmichthys molitrix)protein hydrolysates-A potential source of natural antioxidant[D].West Lafayette:Purdue University,2013.
[29]LI X C,LIN J,HAN W J,et al.Antioxidant ability and mechanism of rhizoma Atractylodes macrocephala[J].Molecules,2012,17(11):13457-13472.DOI:10.3390/molecules171113457.
[30]LEVINE R L,MOSONI L,BERLETT B S,et al.Methionine residues as endogenous antioxidants in proteins[J].Proceedings of the National Academy of Sciences of the United States of America,1996,93(26):15036-15040.DOI:10.1073/pnas.93.26.15036.