海马水解蛋白的氨基酸组成与抗氧化能力的关系
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摘要
为了探讨大海马蛋白质的高效利用方法,本试验将脱脂大海马粉经碱性蛋白酶和胰蛋白酶联合消解后,酶解液(PH)经超滤膜分离为5组分(PH-Ⅰ、PH-Ⅱ、PH-Ⅲ、PH-Ⅳ、PH-Ⅴ),结合各组分的氨基酸组成,回归分析了各组分的自由基清除能力与氨基酸组成间的相关关系。结果表明,酶解液经超滤膜分离的5组分中PH-Ⅴ(<2 500 Da)抗氧化能力较强,清除DPPH自由基能力为34.2%±0.1%,对超氧阴离子的清除能力为29.2%±0.1%,Fe~(3+)还原力为0.28%±0.1%。海马酶解多肽各组成中疏水性氨基酸含量低于亲水性氨基酸,但疏水性氨基酸对自由基的清除能力起着决定性作用,极性氨基酸和非极性氨基酸可通过协同作用来增强海马多肽清除自由基的能力,且每个单位极性氨基酸的抗氧能力是非极性氨基酸的1.2倍,每个单位芳香族氨基酸的抗氧化能力是脂肪族氨基酸的2倍。本研究不仅制备了一类具有开发价值的抗氧化多肽,并且分析了多肽抗氧化能力与氨基酸组成的相关关系,为大海马的高值化利用提供了理论依据。
In order to study the high efficient use of Hippocampus kuda Bleeker protein, the degreased hippocampal powder was digested by alkaline protease and trypsin,and the protein hydrolysate(PH) was separated by ultrafiltration into 5 fractions(PH-Ⅰ, PH-Ⅱ, PH-Ⅲ, PH-Ⅳ, PH-Ⅴ). We analyzed and compared the amino acids composition and their antioxidative capacity of the 5 fractions of PH. The results showed that PH-Ⅴ(<2 500 Da) exhibited the highest antioxidative activity with 1, 1-diphenyl-2-picrylhyd-razyl(DPPH)(34.2%±0.1%), superoxide radicals(29.2%±0.1%) scavenging, as well as reducing power(0.28%±0.005%). Although the content of hydrophobic amino acids was lower than hydrophilic amino acids, the former played a crucial role in the scavenging efficiency to eliminate free radicals. Polar and non-polar amino acids synergistically enhanced the scavenging efficiency by increasing the effective concentration of free radical cleanup. Each unit of polar amino acids for antioxidative capacity was about 1.2 times that of nonpolar amino acids. Furthermore, each unit of aromatic amino acids for antioxidative capacity was around 2 times that of aliphatic amino acids on scavenging free radicals. This study not only prepared a class of antioxidant petides with developmental value, but also analyzed the relationship between the antioxidant capacity of those peptides and amino acid composition, which provided the theoretical basis for the high utilization of hippocampus.
In order to study the high efficient use of Hippocampus kuda Bleeker protein, the degreased hippocampal powder was digested by alkaline protease and trypsin,and the protein hydrolysate(PH) was separated by ultrafiltration into 5 fractions(PH-Ⅰ, PH-Ⅱ, PH-Ⅲ, PH-Ⅳ, PH-Ⅴ). We analyzed and compared the amino acids composition and their antioxidative capacity of the 5 fractions of PH. The results showed that PH-Ⅴ(<2 500 Da) exhibited the highest antioxidative activity with 1, 1-diphenyl-2-picrylhyd-razyl(DPPH)(34.2%±0.1%), superoxide radicals(29.2%±0.1%) scavenging, as well as reducing power(0.28%±0.005%). Although the content of hydrophobic amino acids was lower than hydrophilic amino acids, the former played a crucial role in the scavenging efficiency to eliminate free radicals. Polar and non-polar amino acids synergistically enhanced the scavenging efficiency by increasing the effective concentration of free radical cleanup. Each unit of polar amino acids for antioxidative capacity was about 1.2 times that of nonpolar amino acids. Furthermore, each unit of aromatic amino acids for antioxidative capacity was around 2 times that of aliphatic amino acids on scavenging free radicals. This study not only prepared a class of antioxidant petides with developmental value, but also analyzed the relationship between the antioxidant capacity of those peptides and amino acid composition, which provided the theoretical basis for the high utilization of hippocampus.
引文
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[21] 李婷婷, 张晖, 吴彩娥, 范龚建. 油茶籽糖蛋白提取工艺优化及抗氧化性[J]. 农业机械学报, 2012, 43(4):148-155
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[23] 杨健, 李芳菲, 武淑影, 潘男, 夏秀芳. 大豆蛋白美拉德反应产物的抗氧化特性及其机理[J]. 中国食品学报, 2017, 17(2):44-51
[24] Yoshii H, Tachi N, Ohba R, Sakamura O,Takeyaama H. Antihypertensive effect of ACE inhibitory oligopeptides from chicken egg yolks[J]. Comparative Biochemistry & Physiology Part C Toxicology & Pharmacology, 2001, 128(1):27
[25] 李莹. 泥鳅蛋白源ACE抑制肽的酶法制备及其降压活性研究[D]. 南京: 南京农业大学, 2012
[26] Sun J, He H, Xie B J. Novel antioxidant peptides from fermented mushroom Ganoderma lucidum[J]. Journal of Agricultural & Food Chemistry, 2004, 52(21):6646-6652
[27] Chen H M, Muramoto K, Yamauchi F, Nokihara K. Antioxidant activity of designed peptides based on the antioxidative peptide isolated from digests of a soybean protein[J]. Journal of Agricultural & Food Chemistry, 2014, 44(9):2619-2623
[28] 宋佳天, 赵新淮. 大豆蛋白的酶水解及类蛋白反应对其抗氧化活性的影响[J]. 食品科学, 2012, 33(1):115-119
[29] 黄湛媛, 李丽, 熊宇飞, 方旭波, 陈小娥, 余辉, 傅鹏程.超声辅助竹节虾头酶解及抗氧化肽分离研究[J]. 核农学报, 2017, 31(8):1556-1566
[30] Mendis E, Rajapakse N, Kim S K. Antioxidant properties of a radical-scavenging peptide purified from enzymatically prepared fish skin gelatin hydrolysate[J]. Journal of Agricultural & Food Chemistry, 2005, 53(3):581-587
[31] Park P J, Jung W K, Nam K S, Shahidi F, Kim S K. Purification and characterization of antioxidative peptides from protein hydrolysate of lecithin-free egg yolk[J]. Journal of the American Oil Chemists Society, 2001, 78(6):651-656
[32] Saiga A, Tanabe S, Nishimura T. Antioxidant activity of peptides obtained from porcine myofibrillar proteins by protease treatment[J]. Journal of Agricultural & Food Chemistry, 2003, 51(12):3661-7
[33] Rajapakse N, Mendis E, Jung W K, Je J Y. Purification of a radical scavenging peptide from fermented mussel sauce and its antioxidant properties[J]. Food Research International, 2005, 38(2):175-182
[34] 向枭, 周兴华, 曾本和, 萝莉, 文华. 蛋氨酸水平对吉富罗非鱼肌肉氨基酸组成及血清抗氧化能力的影响[J]. 水产学报, 2016, 40(9):1359-1367
[35] 徐力, 李相鲁, 吴晓霞, 王华, 侯瑞珍. 一种新的玉米抗氧化肽的制备与结构表征[J]. 高等学校化学学报, 2004, 25(3):466-469
[36] Uchida K, Kawakishi S. Sequence-dependent reactivity of histidine-containing peptides with copper(Ⅱ)/ascorbate[J]. Journal of Agricultural and Food Chemistry, 1992, 40(1):13-16
[37] Schwab S, Shearer J, Conklin S E, Alies B, Hass K L. Sequence proximity between Cu(Ⅱ) and Cu(Ⅰ) binding sites of human copper transporter 1 model peptides defines reactivity with ascorbate and O2[J]. Journal of Inorganic Biochemistry, 2015, 158:70-76
[2] Horton J W. Free radicals and lipid peroxidation mediated injury in burn trauma: the role of antioxidant therapy[J]. Toxicology, 2003, 189(1/2):75-88
[3] 郑萍. 天然抗氧化剂的应用与发展[J]. 科技展望, 2016, 26(1): 123-124
[4] Zhao J, Xiong Y L. Interfacial peptide partitioning and undiminished antioxidative and emulsifying activity of oxidatively stressed soy protein hydrolysate in an O/W emulsion[J]. LWT - Food Science and Technology, 2015, 61(2):322-329
[5] Mamilla R K, Mishra Ⅴ K. Effect of germination on antioxidant and ACE inhibitory activities of legumes[J]. LWT - Food Science and Technology, 2017, 75:51-58
[6] 李杰, 闫鸣艳, 刘均洪, 秦松. 鱼胶原蛋白的研究进展[J]. 食品安全质量检测学报, 2015, 6(10):3941-3946
[7] Rhee K S, Ziprin Y A, Rhee K C. Water soluble antioxidant activity of oilseed protein derivatives in model lipid peroxidation system of meat [J]. Journal of Food Science, 1979, 44(4): 1132-1135
[8] Zhang J, Zhang H, Li W, Guo X N, Wang X G, Yao H Y. Isolation and identification of antioxidative peptides from rice endosperm protein enzymatic hydrolysate by consecutive chromatography and MALDI-TOF/TOF MS/MS[J]. Food Chemistry, 2010, 119(1):226-234
[9] 刘文颖, 徐姗姗, 谷瑞增, 鲁军, 潘兴昌. 海洋胶原低聚肽中抗氧化肽的分离及鉴定[J]. 食品工业科技, 2017, 38(6):101-105
[10] Qian Z J, Jung W K, Byun H G. Protective effect of an antioxidative peptide purified from gastrointestinal digests of oyster, Crassostrea gigas against free radical induced DNA damage[J]. Bioresource Technology, 2008, 99(9): 3365-3371
[11] Dávalos A, Miguel M, Bartolomé B. Antioxidant activity of peptides derived from egg white proteins by enzymatic hydrolysis[J]. Journal of Food Protection, 2004, 67(9):1939-1944
[12] Liu P, Zhao M, Cao Y, Lu Y, Liang M, Huang Z, Ren J. Purification and identification of anti-oxidant soybean peptides by consecutive chromatography and electrospray ionization-mass spectrometry[J]. Food Chemistry, 2014, 17(2):209 -211
[13] 伊鹏, 李英新, 张萌萌,王颖,叶波平. 海洋多肽类化合物研究进展[J]. 药物生物技术, 2014, 21(3):264-269
[14] Jiang Z, Xu Y, Su Y. Preparation process of active enzymolysis polypeptides from seahorse bone meal[J]. Food Science Nutrition, 2014, 2(5):490-499
[15] 袁学会, 陈成波, 陈政, 易美华. 三斑海马酶解工艺的优化及其液化蛋白对DPPH·的清除作用[J]. 食品工业科技, 2011, 32(9):291-293
[16] Sanaye S Ⅴ, Pise N M, Pawar A P. Evaluation of antioxidant activities in captive-bred cultured yellow seahorse, Hippocampus kuda, (Bleeker, 1852)[J]. Aquaculture, 2014, 434:100-107
[17] 姜展志. 海马骨粉多肽制备及其抗氧化活性研究[D]. 宁波: 宁波大学, 2014
[18] You L, Zhao M, Joem R, Ren J. Purification and identification of antioxidative peptides from loach (Misgurnus anguillicaudatus) protein hydrolysate by consecutive chromatography and electrospray ionization-mass spectrometry[J]. Food Research International, 2010, 43(4):1167-1173
[19] 成静, 周国仪, 陈栋梁, 王阿敬,杨国燕. 白蛋白多肽清除DPPH·测定方法的研究[J]. 食品工业科技, 2009, 30(8):121-122
[20] 郭雪峰, 岳永德, 汤锋, 王进, 姚曦. 用清除超氧阴离子自由基法评价竹叶提取物抗氧化能力[J]. 光谱学与光谱分析, 2008, 28(8):1823-1826
[21] 李婷婷, 张晖, 吴彩娥, 范龚建. 油茶籽糖蛋白提取工艺优化及抗氧化性[J]. 农业机械学报, 2012, 43(4):148-155
[22] 中华人民共和国卫生部, 中国国家标准化管理委员会. GB/T 5009.124-2003食品中氨基酸的测定[J]. 北京:中国标准出版社,2003
[23] 杨健, 李芳菲, 武淑影, 潘男, 夏秀芳. 大豆蛋白美拉德反应产物的抗氧化特性及其机理[J]. 中国食品学报, 2017, 17(2):44-51
[24] Yoshii H, Tachi N, Ohba R, Sakamura O,Takeyaama H. Antihypertensive effect of ACE inhibitory oligopeptides from chicken egg yolks[J]. Comparative Biochemistry & Physiology Part C Toxicology & Pharmacology, 2001, 128(1):27
[25] 李莹. 泥鳅蛋白源ACE抑制肽的酶法制备及其降压活性研究[D]. 南京: 南京农业大学, 2012
[26] Sun J, He H, Xie B J. Novel antioxidant peptides from fermented mushroom Ganoderma lucidum[J]. Journal of Agricultural & Food Chemistry, 2004, 52(21):6646-6652
[27] Chen H M, Muramoto K, Yamauchi F, Nokihara K. Antioxidant activity of designed peptides based on the antioxidative peptide isolated from digests of a soybean protein[J]. Journal of Agricultural & Food Chemistry, 2014, 44(9):2619-2623
[28] 宋佳天, 赵新淮. 大豆蛋白的酶水解及类蛋白反应对其抗氧化活性的影响[J]. 食品科学, 2012, 33(1):115-119
[29] 黄湛媛, 李丽, 熊宇飞, 方旭波, 陈小娥, 余辉, 傅鹏程.超声辅助竹节虾头酶解及抗氧化肽分离研究[J]. 核农学报, 2017, 31(8):1556-1566
[30] Mendis E, Rajapakse N, Kim S K. Antioxidant properties of a radical-scavenging peptide purified from enzymatically prepared fish skin gelatin hydrolysate[J]. Journal of Agricultural & Food Chemistry, 2005, 53(3):581-587
[31] Park P J, Jung W K, Nam K S, Shahidi F, Kim S K. Purification and characterization of antioxidative peptides from protein hydrolysate of lecithin-free egg yolk[J]. Journal of the American Oil Chemists Society, 2001, 78(6):651-656
[32] Saiga A, Tanabe S, Nishimura T. Antioxidant activity of peptides obtained from porcine myofibrillar proteins by protease treatment[J]. Journal of Agricultural & Food Chemistry, 2003, 51(12):3661-7
[33] Rajapakse N, Mendis E, Jung W K, Je J Y. Purification of a radical scavenging peptide from fermented mussel sauce and its antioxidant properties[J]. Food Research International, 2005, 38(2):175-182
[34] 向枭, 周兴华, 曾本和, 萝莉, 文华. 蛋氨酸水平对吉富罗非鱼肌肉氨基酸组成及血清抗氧化能力的影响[J]. 水产学报, 2016, 40(9):1359-1367
[35] 徐力, 李相鲁, 吴晓霞, 王华, 侯瑞珍. 一种新的玉米抗氧化肽的制备与结构表征[J]. 高等学校化学学报, 2004, 25(3):466-469
[36] Uchida K, Kawakishi S. Sequence-dependent reactivity of histidine-containing peptides with copper(Ⅱ)/ascorbate[J]. Journal of Agricultural and Food Chemistry, 1992, 40(1):13-16
[37] Schwab S, Shearer J, Conklin S E, Alies B, Hass K L. Sequence proximity between Cu(Ⅱ) and Cu(Ⅰ) binding sites of human copper transporter 1 model peptides defines reactivity with ascorbate and O2[J]. Journal of Inorganic Biochemistry, 2015, 158:70-76