高速逆流色谱法分离制备南极磷虾虾粉抗氧化肽
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摘要
采用复合蛋白酶酶解南极磷虾得到虾粉多肽,经超滤分离和高速逆流色谱技术进行分离纯化并检验其体外抗氧化活性。结果表明:经超滤分离后的多肽在分子量范围为5～10 ku时多肽抗氧化活性最好,当浓度为10 mg/m L时,其超氧阴离子自由基清除率达到21. 28%,DPPH自由基清除率为86. 52%,对羟基自由基清除率为53. 49%,Fe~(2+)螯合率为85. 35%。对分子量范围在5～10 ku的多肽进行高速逆流色谱分析,采用体积比为氯仿∶丁醇∶甲醇∶水=4∶0. 75∶3∶2作为两相溶剂体系,下相作固定相,上相作流动相;反转,转速900 r/min,恒温水浴温度为25℃,在流动相的流速1. 5 m L/min,进样浓度20 mg/m L的条件下分离得到5个组分。经测定:当浓度为5 mg/m L时,P3组分清除超氧阴离子自由基的能力和Fe~(2+)螯合能力均显著高于其余4个组分,分别为37. 18%和92. 33%,P2组分的DPPH自由基清除率最高,为90. 33%,P5组分的羟基自由基清除率最高,为70. 11%。与未经HSCCC分离的对照组相比,抗氧化活性均得到明显增强。
The compound peptide was used to digest the Euphausia superba to obtain the shrimp powder polypeptide,which was separated and purified by ultrafiltration separation and high-speed countercurrent chromatography to test its antioxidant activity in vitro. The results showed that the peptides isolated by ultrafiltration had the best antioxidant activity when the molecular weight ranged from 5 to 10 ku. When the concentration was 10 mg/m L,the superoxide anion radical scavenging rate reached 21. 28%,and the DPPH free radical scavenging rate was 86. 52%,the hydroxyl radical scavenging rate was 53. 49%,and the Fe2 +chelation rate was 85. 35%. High-speed countercurrent chromatography was carried out on peptides with a molecular weight ranging from 5 to 10 ku,using chloroform-butanol-methanol-water = 4 ∶ 0. 75 ∶ 3 ∶ 2 as the two-phase solvent system,the lower phase as the stationary phase and the upper phase as the mobile phase;As for inversion,the speed was 900 r/min,the constant temperature water bath temperature was 25 ℃,the flow rate of the mobile phase was 1. 5 m L/min,and the five components were separated under the injection concentration of 20 mg/m L. It was determined that when the concentration was 5 mg/m L,the ability of P3 component to scavenge superoxide anion radicals and Fe2 +chelation ability were significantly higher than the other four components,which were 37. 18% and 92. 33%,respectively. The free radical scavenging rate of P2 was the highest,which was 90. 33%,and the P5 component had the highest hydroxyl radical scavenging rate of 70. 11%. Antioxidant activity was significantly enhanced compared to the control group not isolated by HSCCC.
The compound peptide was used to digest the Euphausia superba to obtain the shrimp powder polypeptide,which was separated and purified by ultrafiltration separation and high-speed countercurrent chromatography to test its antioxidant activity in vitro. The results showed that the peptides isolated by ultrafiltration had the best antioxidant activity when the molecular weight ranged from 5 to 10 ku. When the concentration was 10 mg/m L,the superoxide anion radical scavenging rate reached 21. 28%,and the DPPH free radical scavenging rate was 86. 52%,the hydroxyl radical scavenging rate was 53. 49%,and the Fe2 +chelation rate was 85. 35%. High-speed countercurrent chromatography was carried out on peptides with a molecular weight ranging from 5 to 10 ku,using chloroform-butanol-methanol-water = 4 ∶ 0. 75 ∶ 3 ∶ 2 as the two-phase solvent system,the lower phase as the stationary phase and the upper phase as the mobile phase;As for inversion,the speed was 900 r/min,the constant temperature water bath temperature was 25 ℃,the flow rate of the mobile phase was 1. 5 m L/min,and the five components were separated under the injection concentration of 20 mg/m L. It was determined that when the concentration was 5 mg/m L,the ability of P3 component to scavenge superoxide anion radicals and Fe2 +chelation ability were significantly higher than the other four components,which were 37. 18% and 92. 33%,respectively. The free radical scavenging rate of P2 was the highest,which was 90. 33%,and the P5 component had the highest hydroxyl radical scavenging rate of 70. 11%. Antioxidant activity was significantly enhanced compared to the control group not isolated by HSCCC.
引文
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[8]刘丽红,雷清华.茚三酮比色法与甲醛滴定法测定棉籽粕蛋白水解度的比较[J].化学工程与装备,2012(11):160-163.LIU L H,LEI Q H.Comparison of ninhydrin colorimetry and formaldehyde titration in determination of cottonseed protein hydrolytic degree[J].Chemical Engineering&Equipment,2012(11):160-163.
[9]布冠好,杨国宇,李宏基.肌肽对肉品体系中脂类氧化的抑制作用[J].河南工业大学学报(自然科学版),2011,32(4):44-48.BU G H,YANG G Y,LI H J.Inhibition effects of carnosine on lipid peroxidation in meat product system[J].Journal of Henan University of Technology(Natural Science Edition),2011,32(4):44-48.
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[15]程杰,符晓晖,王维娜.高速逆流色谱在中药分离中溶剂体系的筛选[J].中草药,2008,39(8):1272-1275.CHENG J,FU X H,WANG W N.Screening of solvent system in Chinese medicine separation by high speed countercurrent chromatography[J].Chinese Traditional and Herbal Drugs,2008,39(8):1272-1275.
[16]沈莲清,许明峰.高速逆流色谱分离芝麻菜种子中的硫代葡萄糖苷[J].食品与生物技术学报,2007,26(6):13-16.SHEN L Q,XU M F.Separation of glucosinolate from eruca sative seed by high-speed counter-current chromatography[J].Journal of Food Science and Biotechnology,2007,26(6):13-16.
[17]陆丽利.黄酒中活性肽的分离纯化的研究[D].上海:上海海洋大学,2011.LU L L.Preparation and purification of peptides with ACEihibitory activity from rice wine[D].Shanghai:Shanghai Ocean University,2011.
[18]王继宏.南极磷虾抗氧化肽制备和纯化的研究[D].上海:上海海洋大学,2015.WANG J H.Preparation and purification of antarctic krill peptides with anti-oxidant activity[D].Shanghai:Shanghai Ocean University,2015.
[19]农珍妮,赵钟兴,韦藤幼,等.具有DPPH自由基抑制活性的蚕蛹蛋白酶解液脱色工艺优化[J].食品科学,2015,36(2):12-18.NONG Z N,ZHAO Z X,WEI T Y,et al.Decolorization of silkworm pupae protein hydrolyzate with DPPH radical scavenging activity[J].Food Science,2015,36(2):12-18.
[2]林琳,李八方.鱿鱼皮胶原蛋白水解肽抗氧化活性研究[J].中国海洋药物杂志,2006,25(4):48-51.LIN L,LI B F.Studies on the antioxidation activity of squid(Dosidicus eschrichitii Steenstrup)skin gelatin hydrolysate[J].Chinese Journal of Marine Medicine,2006,25(4):48-51
[3]WANG Y C,WANG S S,WANG J F,et al.Preparation and anti-osteoporotic activities in vivo of phosphorylated peptides from Antarctic krill(Euphausia superba)[J].Peptides,2015,68:239-245.
[4]高颖,王彦超,常耀光,等.三种低氟南极磷虾肽的ACE抑制作用及抗氧化活性研究[J].食品工业科技,2016,37(2):82-87.GAO Y,WANG Y C,CHANG Y G,et al.ACE inhibition and antioxidant activities of three types of Antarctic krill(Euphausia superba)peptides with low fluorine content[J].Science and Technology of Food Industry,2016,37(2):82-87.
[5]赵敏,梁萌青,郑珂珂,等.饲料中添加南极磷虾粉对半滑舌鳎(Cynoglossus semilaevi)雄性亲鱼繁殖性能及抗氧化功能的影响[J].渔业科学进展,2016,37(6):49-55.ZHAO M,LIANG M Q,ZHENG K K,et al.Effects of dietary antarctic krill on the reproductive performance and antioxidant function in male broodstock tongue sole(Cynoglossus semilaevis)[J].Progress in Fishery Sciences,2016,37(6):49-55.
[6]王继宏,汪之和,田鑫,等.南极磷虾抗氧化多肽制备的研究[J].食品工业科技,2014,35(3):109-112.WANG J H,WANG Z H,TIAN X,et al.Preparation of Antarctic krill peptides with anti-oxidant activity[J].Science and Technology of Food Industry,2014,35(3):109-112.
[7]曹学丽.高速逆流色谱分离技术及应用[M].北京:化学工业出版社,2005:11-13.CAO X L.High-speed countercurrent chromatography separation technology and its application[M].Beijing:Chemical Industry Press,2005:11-13.
[8]刘丽红,雷清华.茚三酮比色法与甲醛滴定法测定棉籽粕蛋白水解度的比较[J].化学工程与装备,2012(11):160-163.LIU L H,LEI Q H.Comparison of ninhydrin colorimetry and formaldehyde titration in determination of cottonseed protein hydrolytic degree[J].Chemical Engineering&Equipment,2012(11):160-163.
[9]布冠好,杨国宇,李宏基.肌肽对肉品体系中脂类氧化的抑制作用[J].河南工业大学学报(自然科学版),2011,32(4):44-48.BU G H,YANG G Y,LI H J.Inhibition effects of carnosine on lipid peroxidation in meat product system[J].Journal of Henan University of Technology(Natural Science Edition),2011,32(4):44-48.
[10]FERNANDES J M O,SMITH V J.A novel antimicrobial function for a ribosomal peptide from rainbow trout skin[J].Biochemical and Biophysical Research Communications,2002,296(1):167-71.
[11]邹晓莉,黎源倩,曾红燕,等.反相高效液相色谱法测定人肌腱中的胶原蛋白[J].色谱,2006,24(3):263-266.ZOU X L,LI Y Q,ZENG H Y,et al.Determination of collagen in tendon by reversed-phase high performance liquid chromatography[J].Chinese Journal of Chromatography,2006,24(3):263-266.
[12]胡菊华,冷欣夫.反相高效液相色谱法测定某些有机化合物的分配系数[J].色谱,1992,10(6):344-346.HU J H,LENG X F.Determination of partition coefficients for some pesticides by using reversed-phase high performance liquid chromatography(HPLC)[J].Chinese Journal of Chromatography,1992,10(6):344-346.
[13]张元元,汪之和,陈敏,等.南极磷虾肽制备工艺优化及体外抗氧化研究[J].上海海洋大学学报,2016,25(5):786-793.ZHANG Y Y,WANG Z H,CHEN M,et al.Technology optimization of the peptide preparation for Euphausia superba and study on the antioxidant activity in vitro[J].Journal of Shanghai Ocean University,2016,25(5):786-793.
[14]WU H C,CHEN H M,SHIAN 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.
[15]程杰,符晓晖,王维娜.高速逆流色谱在中药分离中溶剂体系的筛选[J].中草药,2008,39(8):1272-1275.CHENG J,FU X H,WANG W N.Screening of solvent system in Chinese medicine separation by high speed countercurrent chromatography[J].Chinese Traditional and Herbal Drugs,2008,39(8):1272-1275.
[16]沈莲清,许明峰.高速逆流色谱分离芝麻菜种子中的硫代葡萄糖苷[J].食品与生物技术学报,2007,26(6):13-16.SHEN L Q,XU M F.Separation of glucosinolate from eruca sative seed by high-speed counter-current chromatography[J].Journal of Food Science and Biotechnology,2007,26(6):13-16.
[17]陆丽利.黄酒中活性肽的分离纯化的研究[D].上海:上海海洋大学,2011.LU L L.Preparation and purification of peptides with ACEihibitory activity from rice wine[D].Shanghai:Shanghai Ocean University,2011.
[18]王继宏.南极磷虾抗氧化肽制备和纯化的研究[D].上海:上海海洋大学,2015.WANG J H.Preparation and purification of antarctic krill peptides with anti-oxidant activity[D].Shanghai:Shanghai Ocean University,2015.
[19]农珍妮,赵钟兴,韦藤幼,等.具有DPPH自由基抑制活性的蚕蛹蛋白酶解液脱色工艺优化[J].食品科学,2015,36(2):12-18.NONG Z N,ZHAO Z X,WEI T Y,et al.Decolorization of silkworm pupae protein hydrolyzate with DPPH radical scavenging activity[J].Food Science,2015,36(2):12-18.