仿刺参精低分子质量多肽的制备及抗氧化作用
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摘要
本实验以仿刺参精为原料,制备出多肽并考察其体外和体内抗氧化活性。以清除羟自由基(·OH)能力为指标,考察木瓜蛋白酶、复合蛋白酶和风味蛋白酶的5种酶解方式产生的酶解产物的活性,优化实验用酶。经超滤分级后,测定各级组分的分子质量分布、可溶性蛋白质含量、多肽含量和清除·OH能力。选择清除·OH能力较好的低分子质量多肽组分,测定氨基酸组成,设定低、中、高剂量组(100、200、600 mg/kg(以体质量计,下同))、模型对照组、和空白对照组进行42 d小鼠灌胃实验。实验完成后,检测小鼠血液中超氧化物歧化酶(superoxide dismutase,SOD)活力、谷胱甘肽过氧化物酶(glutathione peroxidase,GSH-Px)活力、谷胱甘肽(glutathione,GSH)含量、丙二醛(malondialdehyde,MDA)含量和蛋白质羰基含量。结果表明,由木瓜蛋白酶水解,经超滤制备的<1 000 u仿刺参精多肽具有较好的体外清除·OH能力,其半数抑制浓度为6.02 mg/m L,含量占比为58%,氨基酸组成中谷氨酸等酸性氨基酸和酪氨酸等疏水性氨基酸含量丰富。小鼠抗氧化模型实验表明,实验组生长正常,高剂量多肽组小鼠体内SOD活力显著提高(P<0.05),GSH的含量提高极显著(P<0.01)、GSH-Px活力也呈剂量依赖性升高,但未达到差异显著(P>0.05)。MDA和蛋白质羰基含量得到有效降低,后者达到差异显著(P<0.05)。研究结果显示:<1 000 u仿刺参精多肽具有提高小鼠抗氧化能力的效果,可以作为相关功能产品开发的原材料。
The chemical components and in vivo antioxidant activity of peptides prepared from Apostichopus japonicus sperm were detected. The best enzyme to prepare antioxidant peptides from Apostichopus japonicus sperm was selected by comparing the hydroxyl radical scavenging activities of five protein hydrolysates obtained with papain, protamex and flavourzyme. After ultrafiltration of the hydrolysates, the weight distribution, soluble protein and polypeptide contents and hydroxyl radical scavenging capacity of the fractions were determined. The low molecular weight component of the peptide with higher hydroxyl radical scavenging capacity was analyzed for its amino acid composition, and then it was orally administered to mice at low, middle and high dosages(100, 200, 600 mg/kg), respectively. The administration period for the three dosage groups as well as the model control and blank control groups lasted for 42 days. At the end of administration, superoxide dismutase(SOD) and glutathione peroxidase(GSH-Px) activities, GSH, malondialdehyde(MDA) and protein carbonyl contents were measured in the serum of mice. Results showed that the peptide prepared by papain hydrolysis and ultrafiltration with molecular weight cut-off of < 1 000 u had better hydroxyl radical scavenging capacity in vitro with half maximal inhibitory concentration of 6.02 mg/m L, accounting for 58% of the total peptides. The acidic amino acid glutamic acid and the hydrophobic amino acid tyrosine dominated its amino acid composition. The results of antioxidant tests with a mouse model showed that all experimental mice could normally growth, and serum SOD activity and GSH content in the mice from the high-dosage group were improved significantly(P < 0.05) and highly significantly(P < 0.01), respectively. The activity of GSH-Px also exhibited a dosage-dependent improvement, but did not show a significant difference(P > 0.05). The contents of MDA and protein carbonyl were both reduced and the latter was significant(P < 0.05). All these results demonstrated that the peptide with a molecular weight less than 1 000 u could enhance antioxidant function in mice, and therefore it has potential as an ingredient of functional foods.
The chemical components and in vivo antioxidant activity of peptides prepared from Apostichopus japonicus sperm were detected. The best enzyme to prepare antioxidant peptides from Apostichopus japonicus sperm was selected by comparing the hydroxyl radical scavenging activities of five protein hydrolysates obtained with papain, protamex and flavourzyme. After ultrafiltration of the hydrolysates, the weight distribution, soluble protein and polypeptide contents and hydroxyl radical scavenging capacity of the fractions were determined. The low molecular weight component of the peptide with higher hydroxyl radical scavenging capacity was analyzed for its amino acid composition, and then it was orally administered to mice at low, middle and high dosages(100, 200, 600 mg/kg), respectively. The administration period for the three dosage groups as well as the model control and blank control groups lasted for 42 days. At the end of administration, superoxide dismutase(SOD) and glutathione peroxidase(GSH-Px) activities, GSH, malondialdehyde(MDA) and protein carbonyl contents were measured in the serum of mice. Results showed that the peptide prepared by papain hydrolysis and ultrafiltration with molecular weight cut-off of < 1 000 u had better hydroxyl radical scavenging capacity in vitro with half maximal inhibitory concentration of 6.02 mg/m L, accounting for 58% of the total peptides. The acidic amino acid glutamic acid and the hydrophobic amino acid tyrosine dominated its amino acid composition. The results of antioxidant tests with a mouse model showed that all experimental mice could normally growth, and serum SOD activity and GSH content in the mice from the high-dosage group were improved significantly(P < 0.05) and highly significantly(P < 0.01), respectively. The activity of GSH-Px also exhibited a dosage-dependent improvement, but did not show a significant difference(P > 0.05). The contents of MDA and protein carbonyl were both reduced and the latter was significant(P < 0.05). All these results demonstrated that the peptide with a molecular weight less than 1 000 u could enhance antioxidant function in mice, and therefore it has potential as an ingredient of functional foods.
引文
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[8]向怡卉,苏秀榕,董明敏,等.复合蛋白酶水解海参生殖腺工艺的研究[J].食品工业科技,2007,28(3):143-146.DOI:10.3969/j.issn.1002-0306.2007.03.042.
[9]赵鸿霞,周大勇,秦磊,等.响应面法优化海参卵酶解工艺[J].食品与机械,2010,26(5):114-117.DOI:10.3969/j.issn.1003-5788.2010.05.033.
[10]曹荣,刘淇,殷邦忠.响应面法优化海参性腺酶解工艺的研究[J].食品科学,2012,33(2):29-33.
[11]陈卉卉,于平,励建荣.东海海参胶原蛋白多肽的制备及清除自由基功能研究[J].中国食品学报,2010,10(1):19-25.DOI:10.3969/j.issn.1009-7848.2010.01.003.
[12]王颖,张健,王茂剑,等.仿刺参肠道组织酶解工艺优化及肽段分析[J].食品科学,2014,35(23):182-187.DOI:10.7506/spkx1002-6630-201423036.
[13]许长成,邹琦,程炳嵩.硫代巴比妥酸(TBA)法检测脂质过氧化水平的探讨[J].植物生理学报,1989(6):58-60.
[14]孟雪莲,陈长兰,孔维娟,等.虫草素抑制脂多糖诱导的小胶质细胞活化及神经保护作用[J].食品科学,2014,35(19):224-230.DOI:10.7506/spkx1002-6630-201419045.
[15]YAO Ping,LI Ke,JIN You,et al.Oxidative damage after chronic ethanolin take in rattissues:prophylaxis of Ginkgobiloba extract[J].Food Chemistry,2006,99(2):305-314.DOI:10.1016/j.foodchem.2005.07.047.
[16]ZHOU Xiaoqiu,WANG Changhai,JIANG Aili.Antioxidant peptides isolated from sea cucumber Stichopus Japonicus[J].European Food Research and Technology,2012,234(3):441-447.DOI:10.1007/s00217-011-1610-x.
[17]赵强忠,刘丹.秋刀鱼抗氧化肽制备及其抗氧化活性的研究[J].现代食品科技,2014,30(10):165-171.
[18]王天明,苏意钢,马永钧,等.海地瓜多肽分离及抗氧化活性研究[J].现代食品科技,2014,30(5):75-81.
[19]WANG Jingfeng,WANG Yuming,TANG Qingjuan,etal.Antioxidation activities of low-molecular-weight gelatin hydrolysate isolated from the sea cucumber Stichopus japonicus[J].Journal of Ocean University of China,2010,9(1):94-98.DOI:10.1007/s11802-010-0094-9.
[20]RANATHUNGA S,RAJAPAKSE N,KIM S K.Purification and characterization of antioxidative peptide derived from muscle of conger eel(Conger myriaster)[J].European Food Research&Technology,2006,222(3):310-315.DOI:10.1007/s00217-005-0079-x.
[21]BAMDAD F,CHEN Lingyun.Antioxidant capacities of fractionated barley hordein hydrolysates in relation to peptide structures[J].Molecular Nutrition&Food Research,2013,57(3):493-503.DOI:10.1002/mnfr.201200252.
[22]BAMDAD F,AHMED S,CHEN L Y.Specifically designed peptide structures effectively suppressed oxidative reactions in chemical and cellular systems[J].Journal of Functional Foods,2015,18(Part A):35-46.DOI:10.1016/j.jff.2015.06.055.
[23]UDENIGWE C C,ALUKO R E.Chemometric analysis of the amino acid requirements of antioxidant food protein hydrolysates[J].International Journal of Molecular Sciences,2011,12(5):3148-3161.DOI:10.3390/ijms12053148.
[24]SAIGA A,TANABE S,NISHIMURA T.Antioxidant activity of peptides obtained from porcine myofibrillar proteins by protease treatment[J].Journal of Agricultural and Food Chemistry,2003,51(12):3661-3667.DOI:10.1021/jf021156g.
[25]常飞,杨雪果,肖士成,等.脱脂羊脑蛋白水解多肽的分离纯化及抗氧化活性[J].食品科学,2016,37(1):33-39.DOI:10.7506/spkx1002-6630-201601007.
[26]HARMAN D.Free-radical theory of aging:an update:increasing the functional life span[J].Annals of the New York Academy of Sciences,1994,1067:10-21.DOI:10.1111/j.1749-6632.1994.tb12069.x.
[27]MARITIM A C,SANDERS R A,WATKINS J B,et al.Diabetes,oxidative stress,and antioxidants:a review[J].Journal of Biochemical&Molecular Toxicology,2003,17(1):24-38.DOI:10.1002/jbt.10058.
[28]HALLIWELL B,WHITEMAN M.Measuring reactive species and oxidative damage in vivo and in cell culture:how should you do it and what do the results mean?[J].British Journal of Pharmacology,2004,142(2):231-255.DOI:10.1038/sj.bjp.0705776.
[29]KUMAR N S S,NAZEER R A.In vivo antioxidant activity of peptide purified from viscera protein hydrolysate of horse mackerel(Magalaspis cordyla)[J].International Journal of Food Science&Technology,2012,47(7):1558-1562.DOI:10.1111/j.1365-2621.2012.03002.x.
[30]RILEY P A.Free radicals in biology:oxidative stress and the effects of ionizing radiation[J].International Journal of Radiation Biology,1994,65(1):27-33.DOI:10.1080/09553009414550041.
[31]DAS I,SAHA T.Effect of garlic on lipid peroxidation and antioxidation enzymes in DMBA-induced skin carcinoma[J].Nutrition,2009,25(4):459-471.DOI:10.1016/j.nut.2008.10.014.
[32]文镜,张春华,董雨,等.蛋白质羰基含量与蛋白质氧化损伤[J].食品科学,2003,24(10):153-157.
[33]REZNICK A Z,PACKER L.Oxidative damage to proteins:spectrophotometric method for carbonyl assay[J].Methods in Enzymology,1994,233:357-363.DOI:10.1016/S0076-6879(94)33041-7.
[34]SOHAL R S,AGARWAL S,DUBEY A,et al.Protein oxidative damage is associated with life expectancy of houseflies[J].Proceedings of the National Academy of Sciences,1993,90(15):7255-7259.DOI:10.1073/pnas.90.15.7255.
[2]苏秀榕,娄永江,常亚青,等.海参的营养成分及海参多糖的抗肿瘤活性的研究[J].营养学报,2003,25(2):181-182.DOI:10.3321/j.issn:0512-7955.2003.02.025.
[3]盛文静,薛长湖,赵庆喜,等.不同海参多糖的化学组成分析比较[J].中国海洋药物杂志,2007,26(1):44-49.DOI:10.3969/j.issn.1002-3461.2007.01.012.
[4]王静,张京楼,王铎喜,等.海参多肽的抗氧化性能研究[J].食品与机械,2010,26(2):67-71.
[5]孙鹏,易杨华,李玲,等.海参皂苷的生源分类和化学结构特征(楯手目)[J].中国天然药物,2007,5(6):463-469.
[6]樊廷俊,袁文鹏,丛日山,等.仿刺参水溶性海参皂苷的分离纯化及其抑瘤活性研究[J].药学学报,2009,44(1):25-31.DOI:10.3321/j.issn:0513-4870.2009.01.004.
[7]张健,王茂剑,马晶晶,等.仿刺参生殖腺营养成分分析[J].食品科学,2013,34(14):232-236.DOI:10.7506/spkx1002-6630-201314047.
[8]向怡卉,苏秀榕,董明敏,等.复合蛋白酶水解海参生殖腺工艺的研究[J].食品工业科技,2007,28(3):143-146.DOI:10.3969/j.issn.1002-0306.2007.03.042.
[9]赵鸿霞,周大勇,秦磊,等.响应面法优化海参卵酶解工艺[J].食品与机械,2010,26(5):114-117.DOI:10.3969/j.issn.1003-5788.2010.05.033.
[10]曹荣,刘淇,殷邦忠.响应面法优化海参性腺酶解工艺的研究[J].食品科学,2012,33(2):29-33.
[11]陈卉卉,于平,励建荣.东海海参胶原蛋白多肽的制备及清除自由基功能研究[J].中国食品学报,2010,10(1):19-25.DOI:10.3969/j.issn.1009-7848.2010.01.003.
[12]王颖,张健,王茂剑,等.仿刺参肠道组织酶解工艺优化及肽段分析[J].食品科学,2014,35(23):182-187.DOI:10.7506/spkx1002-6630-201423036.
[13]许长成,邹琦,程炳嵩.硫代巴比妥酸(TBA)法检测脂质过氧化水平的探讨[J].植物生理学报,1989(6):58-60.
[14]孟雪莲,陈长兰,孔维娟,等.虫草素抑制脂多糖诱导的小胶质细胞活化及神经保护作用[J].食品科学,2014,35(19):224-230.DOI:10.7506/spkx1002-6630-201419045.
[15]YAO Ping,LI Ke,JIN You,et al.Oxidative damage after chronic ethanolin take in rattissues:prophylaxis of Ginkgobiloba extract[J].Food Chemistry,2006,99(2):305-314.DOI:10.1016/j.foodchem.2005.07.047.
[16]ZHOU Xiaoqiu,WANG Changhai,JIANG Aili.Antioxidant peptides isolated from sea cucumber Stichopus Japonicus[J].European Food Research and Technology,2012,234(3):441-447.DOI:10.1007/s00217-011-1610-x.
[17]赵强忠,刘丹.秋刀鱼抗氧化肽制备及其抗氧化活性的研究[J].现代食品科技,2014,30(10):165-171.
[18]王天明,苏意钢,马永钧,等.海地瓜多肽分离及抗氧化活性研究[J].现代食品科技,2014,30(5):75-81.
[19]WANG Jingfeng,WANG Yuming,TANG Qingjuan,etal.Antioxidation activities of low-molecular-weight gelatin hydrolysate isolated from the sea cucumber Stichopus japonicus[J].Journal of Ocean University of China,2010,9(1):94-98.DOI:10.1007/s11802-010-0094-9.
[20]RANATHUNGA S,RAJAPAKSE N,KIM S K.Purification and characterization of antioxidative peptide derived from muscle of conger eel(Conger myriaster)[J].European Food Research&Technology,2006,222(3):310-315.DOI:10.1007/s00217-005-0079-x.
[21]BAMDAD F,CHEN Lingyun.Antioxidant capacities of fractionated barley hordein hydrolysates in relation to peptide structures[J].Molecular Nutrition&Food Research,2013,57(3):493-503.DOI:10.1002/mnfr.201200252.
[22]BAMDAD F,AHMED S,CHEN L Y.Specifically designed peptide structures effectively suppressed oxidative reactions in chemical and cellular systems[J].Journal of Functional Foods,2015,18(Part A):35-46.DOI:10.1016/j.jff.2015.06.055.
[23]UDENIGWE C C,ALUKO R E.Chemometric analysis of the amino acid requirements of antioxidant food protein hydrolysates[J].International Journal of Molecular Sciences,2011,12(5):3148-3161.DOI:10.3390/ijms12053148.
[24]SAIGA A,TANABE S,NISHIMURA T.Antioxidant activity of peptides obtained from porcine myofibrillar proteins by protease treatment[J].Journal of Agricultural and Food Chemistry,2003,51(12):3661-3667.DOI:10.1021/jf021156g.
[25]常飞,杨雪果,肖士成,等.脱脂羊脑蛋白水解多肽的分离纯化及抗氧化活性[J].食品科学,2016,37(1):33-39.DOI:10.7506/spkx1002-6630-201601007.
[26]HARMAN D.Free-radical theory of aging:an update:increasing the functional life span[J].Annals of the New York Academy of Sciences,1994,1067:10-21.DOI:10.1111/j.1749-6632.1994.tb12069.x.
[27]MARITIM A C,SANDERS R A,WATKINS J B,et al.Diabetes,oxidative stress,and antioxidants:a review[J].Journal of Biochemical&Molecular Toxicology,2003,17(1):24-38.DOI:10.1002/jbt.10058.
[28]HALLIWELL B,WHITEMAN M.Measuring reactive species and oxidative damage in vivo and in cell culture:how should you do it and what do the results mean?[J].British Journal of Pharmacology,2004,142(2):231-255.DOI:10.1038/sj.bjp.0705776.
[29]KUMAR N S S,NAZEER R A.In vivo antioxidant activity of peptide purified from viscera protein hydrolysate of horse mackerel(Magalaspis cordyla)[J].International Journal of Food Science&Technology,2012,47(7):1558-1562.DOI:10.1111/j.1365-2621.2012.03002.x.
[30]RILEY P A.Free radicals in biology:oxidative stress and the effects of ionizing radiation[J].International Journal of Radiation Biology,1994,65(1):27-33.DOI:10.1080/09553009414550041.
[31]DAS I,SAHA T.Effect of garlic on lipid peroxidation and antioxidation enzymes in DMBA-induced skin carcinoma[J].Nutrition,2009,25(4):459-471.DOI:10.1016/j.nut.2008.10.014.
[32]文镜,张春华,董雨,等.蛋白质羰基含量与蛋白质氧化损伤[J].食品科学,2003,24(10):153-157.
[33]REZNICK A Z,PACKER L.Oxidative damage to proteins:spectrophotometric method for carbonyl assay[J].Methods in Enzymology,1994,233:357-363.DOI:10.1016/S0076-6879(94)33041-7.
[34]SOHAL R S,AGARWAL S,DUBEY A,et al.Protein oxidative damage is associated with life expectancy of houseflies[J].Proceedings of the National Academy of Sciences,1993,90(15):7255-7259.DOI:10.1073/pnas.90.15.7255.
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