不同方法纯化的尼罗罗非鱼下脚料蛋白酶解物锌螯合盐的理化特性
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摘要
研究罗非鱼下脚料蛋白酶解物锌螯合盐(CH-0)及乙醇纯化(CH-Ⅰ)与透析纯化组分(CH-Ⅱ)的理化特性及体外自由基的清除能力,为多肽螯合盐的纯化研究提供理论基础。结果表明,CH-0的蛋白质含量(干基计)为71.86%,CH-Ⅰ和CH-Ⅱ分别为75.94%与78.83%;CH-0的锌含量为16.08%,CH-Ⅰ和CH-Ⅱ分别为14.49%与12.20%;凝胶层析结果显示,CH-Ⅰ、CH-Ⅱ与CH-0相比,有3个相同的主吸收峰,且大分子与小分子杂峰减少;氨基酸分析结果表明CH-Ⅱ中的Asp、Glu、Gly、Lys、Arg和His的含量均高于CH-Ⅰ,且都高于CH-0,与透析纯化相比,乙醇纯化必需氨基酸与支链氨基酸损失较大;CH-0、CH-Ⅰ和CH-Ⅱ对羟基自由基与DPPH自由基的清除率随浓度增强而增强,以IC50值计,CH-Ⅰ、CH-Ⅱ比CH-0的清除能力分别提高了8.92%、24.01%与38.06%、46.13%。罗非鱼下脚料,蛋白酶解物(EH10K)有较好的超氧阴离子自由基清除能力,浓度为10.00 mg/m L时清除率为69.27%,而CH-0、CH-Ⅰ与CH-Ⅱ则显示了负清除作用。纯化方法对罗非鱼下脚料蛋白酶解物锌螯合盐的理化特性有影响。
Tilapia scraps were hydrolyzed by neutral protease and flavourzyme,and the hydrolysate was fractionated through ultrafiltration membranes with a range of molecular weight cutoffs( M WCO) of 10 ku,to yield the fraction EH10 K with M W distribution < 10 ku. The zinc chelating salts( CH-0) was produced by EH10 K chelating with Zn SO4. CH-Ⅰ and CH-Ⅱ were the purified components from CH-0 by using 95%ethanol and dialysis( FW = 500 u) respectively. The formation of Zn2 +chelating salts of hydrolysate was confirmed by the UV-VIS spectra. The physicochemical properties of CH-0,CH-Ⅰ and CH-Ⅱ were analysed and their free radical scavenging activities were evaluated also. The results showed that CH-0contained 71. 86% of protein( on dry basis),16. 08% of Zn,CH-Ⅰ and CH-Ⅱ were 75. 94% and14. 49%,78. 83% and 12. 20% respectively. The gel chromatography results showed that CH-Ⅰand CH-Ⅱhad three same main absorption peaks compared to CH-0 but had little impurity peaks. The contents of Asp,Glu,Gly,Lys,Arg and His in CH-Ⅱ were high than those of CH-Ⅰ,and all were high than those of CH-0.The content of essential amino acid and branched chain amino acid in CH-Ⅱ were high than that in CH-Ⅰ.CH-0,CH-Ⅰ and CH-Ⅱ showed evident radical scavenging activity in a dose-dependent manner with the IC50 values for hydroxyl radical and 1,1-diphenyl-2-pycrylhydrazyl( DPPH) radical being 9. 08 and 6. 07,8. 27 and 3. 76,6. 90 and 3. 27 mg / m L respectively. The superoxide radical scavenging activity of EH10 K was 69. 27% at 10. 00 mg / m L,CH-0,CH-Ⅰ and CH-Ⅱ showed no effect on superoxide radical scavenging but to promote the oxidation.
Tilapia scraps were hydrolyzed by neutral protease and flavourzyme,and the hydrolysate was fractionated through ultrafiltration membranes with a range of molecular weight cutoffs( M WCO) of 10 ku,to yield the fraction EH10 K with M W distribution < 10 ku. The zinc chelating salts( CH-0) was produced by EH10 K chelating with Zn SO4. CH-Ⅰ and CH-Ⅱ were the purified components from CH-0 by using 95%ethanol and dialysis( FW = 500 u) respectively. The formation of Zn2 +chelating salts of hydrolysate was confirmed by the UV-VIS spectra. The physicochemical properties of CH-0,CH-Ⅰ and CH-Ⅱ were analysed and their free radical scavenging activities were evaluated also. The results showed that CH-0contained 71. 86% of protein( on dry basis),16. 08% of Zn,CH-Ⅰ and CH-Ⅱ were 75. 94% and14. 49%,78. 83% and 12. 20% respectively. The gel chromatography results showed that CH-Ⅰand CH-Ⅱhad three same main absorption peaks compared to CH-0 but had little impurity peaks. The contents of Asp,Glu,Gly,Lys,Arg and His in CH-Ⅱ were high than those of CH-Ⅰ,and all were high than those of CH-0.The content of essential amino acid and branched chain amino acid in CH-Ⅱ were high than that in CH-Ⅰ.CH-0,CH-Ⅰ and CH-Ⅱ showed evident radical scavenging activity in a dose-dependent manner with the IC50 values for hydroxyl radical and 1,1-diphenyl-2-pycrylhydrazyl( DPPH) radical being 9. 08 and 6. 07,8. 27 and 3. 76,6. 90 and 3. 27 mg / m L respectively. The superoxide radical scavenging activity of EH10 K was 69. 27% at 10. 00 mg / m L,CH-0,CH-Ⅰ and CH-Ⅱ showed no effect on superoxide radical scavenging but to promote the oxidation.
引文
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[15]Chen Y F.Study on preparation of soy oligopeptide,function of ACE inhibitory activity and metal-chelate[D].Beijing:China academy of agricultural science,2009.[陈亚房.大豆低聚肽制备及其ACE抑制活性和螯合特性的研究.北京:中国农业科学院,2009.]
[16]Wang Z T.Methods of food hygienic analysis(physical and chemical sectionⅠ)[M].Beijing:Standards Press of China,2012.[王竹天.食品卫生检验方法(理化部分1).北京:中国标准出版社,2012.]
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[18]Halliwell B,Gutteridge J M,Aruoma O I.The deoxyribose method:A simple of“test-tube”assay for determination of rate constants for reactions hydroxyl radicals[J].Analytical Biochemistry,1987,165(1):215-219.
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[20]Marklund S,Marklund G.Involvement of the superoxide anion radical in the autoxidation of pyrogallal and a convenient assay for superoxide dismutase[J].European Journal of Biochemistry,1974,47(3):469-474.
[21]Guan H Y.Study on synthesis and characterization of chelate of glycine with trace element[D].Hangzhou:Zhejiang Uiniversity,2007.[管海跃.甘氨酸微量元素螯合物的合成与表征研究.杭州:浙江大学,2007.]
[22]Lu Y,Liu Q,Bao X,et al.Identification and characteristics of iron-chelating peptides from soybean protein hydrolysates using IM AC-Fe3+[J].Journal of Agricultural and Food Chemistry,2009,57(11):4593-4597.
[23]Torres F C,Alaiz M,Vioque J.Affinity purification and characterisation of chelating peptides from chickpea protein hydrolysates[J].Food Chemistry,2011,129(2):485-490.
[24]Wang X R,Peng X W,Kan J Q.Study on antioxidant activity of chelated pig blood polypeptide ferrite in vitro[J].Science and Technology of Food Industry,2011,32(10):135-138.[汪学荣,彭祥伟,阚建全.猪血多肽亚铁螯合盐的体外抗氧化作用研究.食品工业科技,2011,32(10):135-138.]
[25]Guo Y X,Lin Q L,Wu W.Study on antioxidant properties of defatted rice bran protein by enzymatic hydrolysis[J].Cereals&Oils,2012(10):15-19.[郭延熙,林亲录,吴伟.酶法水解脱脂米糠蛋白抗氧化性质研究.粮食与油脂,2012(10):15-19.]
[26]Han H,Zhan S M,Shan L G,et al.Studies on the free radical scavenging activity of bioactive substances from Sargassum fusif orme[J].China Journal M arine Drugs,2008,27(8):31-34.[韩华,战松梅,单联刚,等.羊栖菜中清除自由基活性物质的研究.中国海洋药物,2008,27(8):31-34.]
[2]Yang P,Xia Y J,Fan W Q.Enzymatic hydrolysis of protein from tilapia scraps using papain[J].Fisheries Science,2008,27(6):290-292.[杨萍,夏永军,范伟群.木瓜蛋白酶对罗非鱼下脚料的水解作用[J].水产科学,2008,27(6):290-292.]
[3]Zhang Z,Yang X Q,Ma H X.Optimization of Enzymatic Hydrolysis of Tilapia Waste by PlackettBurman Design and Central Composite Design[J].Food Science,2011,32(18):1-5.[张桢,杨贤庆,马海霞.Plackett-Burman法和中心组合法优化罗非鱼下脚料酶解工艺[J].食品科学,2011,32(18):1-5.]
[4]Miquel E,Farre R.Effects and future trends of casein phosphopeptides on zinc bioavailability[J].Trends in Food Science and Technology,2007,18(3):139-143.
[5]Xie J,Li Z J.Theomtical innovation on calcium reinforcement-combination of collagen polypeptide and calcium[J].International Journal of Traditional Chinese M edicine,,2008,30(1):69-71.[谢静,李宗军.补钙理论创新—胶原多肽与钙结合物.国际中医中药杂志,2008,30(1):69-71.]
[6]Lu J F,Meng C W,Li J,et al.Preparation and characterization of collagen polypeptide chelated calcium from fish bone powder of channel catfish(Ictalurus punctatus)[J].Journal of Fisheries of China,2012,36(2):314-320.[陆剑锋,孟昌伟,李进,等.斑点叉尾鱼骨胶原多肽螯合钙的制备及其特征.水产学报,2012,36(2):314-320.]
[7]Bao X L,Lv Y,Yang B C,et al.A study of the soluble complexes formed during calcium binding by soybean protein hydrolysates[J].Journal of Food Science,2008,73(3):117-121.
[8]Huo J C,Deng S G,Xie C,et al.Study on compound enzymic peptides iron from hairtaill offchuts and its activity of antioxidation[J].Science and Technology of Food Industry,2009,30(4):267-270.[霍健聪,邓尚贵,谢超.带鱼下脚料蛋白多肽亚铁螯合物的制备及抗氧化活性研究.食品工业科技,2009,30(4):267-270.]
[9]Zhang L,Li J,Zhou K.Chelating and radical scavenging activities of soy protein hydrolysates prepared from microbial proteases and their effect on meat lipid peroxidation[J].Bioresource Technology,2010,101(7):2084-2089.
[10]Yang J,Zhao H L,Xu S F,et al.Antioxidant and zinc supplementation effect of zinc chelating small peptides from fish protein in mice[J].Journal of Huazhong Agricultural University,2011,30(4):516-520.[杨杰,赵洪雷,徐淑芬,等.鱼蛋白小肽螯合锌对小鼠的补锌效果及抗氧化作用.华中农业大学学报,2011,30(4):516-520.]
[11]Carlson M S,Boren C A,Wu C,et al.Evaluation of various inelusion rate of organic zinc either as polysaccharide or proteinate complex on the growth performance,plasma,and excretion of nursery pigs[J].Journal of Animal Science,2004,82(5):1359-1366.
[12]Yang S,Deng S G,Qin X M.Study on synthesis of peptides calcium chelation from low-value fish and antioxidation and antibacterial activities[J].Food Science,2008,29(1):202-206.[杨燊,邓尚贵,秦小明.低值鱼蛋白多肽-钙螯合物的制备和抗氧化、抗菌活性研究.食品科学.2008,29(1):202-206.]
[13]Li T G,Chen K J,Hong P Z,et al.Study on preparation of multiple-enzyme hydrolysate of tilapia scraps chelating with zinc[J].Packaging and Food M achinery 2013,31(3):13-17.[李同刚,陈康健,洪鹏志,等.罗非鱼下脚料复合酶水解物锌螯合盐的制备.包装与食品机械,2013,31(3):13-17.]
[14]Li T G,Hong P Z,Yang P,et al.Optimization of echnological conditions of ilapia scraps enzymatic hydrolysates chelated with zinc by quadratic regression orthogonal rotary[J].Science and Technology of Food Industry,2013,34(2):234-237.[李同刚,洪鹏志,杨萍,等.二次回归正交旋转组合优化罗非鱼下脚料酶解液螯合锌的工艺条件.食品工业科技,2013,34(2):234-237.]
[15]Chen Y F.Study on preparation of soy oligopeptide,function of ACE inhibitory activity and metal-chelate[D].Beijing:China academy of agricultural science,2009.[陈亚房.大豆低聚肽制备及其ACE抑制活性和螯合特性的研究.北京:中国农业科学院,2009.]
[16]Wang Z T.Methods of food hygienic analysis(physical and chemical sectionⅠ)[M].Beijing:Standards Press of China,2012.[王竹天.食品卫生检验方法(理化部分1).北京:中国标准出版社,2012.]
[17]Wang Z T.Methods of food hygienic analysis(physical and chemical sectionⅡ)[M].Beijing:Standards Press of China,2012.[王竹天.食品卫生检验方法(理化部分2).北京:中国标准出版社,2012.]
[18]Halliwell B,Gutteridge J M,Aruoma O I.The deoxyribose method:A simple of“test-tube”assay for determination of rate constants for reactions hydroxyl radicals[J].Analytical Biochemistry,1987,165(1):215-219.
[19]Shimada K,Fujikawa K,Tahara K,et al.Antioxidative properties of xanthan on the autoxidation of soybean oil in cyclodextrin emulsion[J].Journal of Agricultural and Food Chemistry,1992,40(6):945-948.
[20]Marklund S,Marklund G.Involvement of the superoxide anion radical in the autoxidation of pyrogallal and a convenient assay for superoxide dismutase[J].European Journal of Biochemistry,1974,47(3):469-474.
[21]Guan H Y.Study on synthesis and characterization of chelate of glycine with trace element[D].Hangzhou:Zhejiang Uiniversity,2007.[管海跃.甘氨酸微量元素螯合物的合成与表征研究.杭州:浙江大学,2007.]
[22]Lu Y,Liu Q,Bao X,et al.Identification and characteristics of iron-chelating peptides from soybean protein hydrolysates using IM AC-Fe3+[J].Journal of Agricultural and Food Chemistry,2009,57(11):4593-4597.
[23]Torres F C,Alaiz M,Vioque J.Affinity purification and characterisation of chelating peptides from chickpea protein hydrolysates[J].Food Chemistry,2011,129(2):485-490.
[24]Wang X R,Peng X W,Kan J Q.Study on antioxidant activity of chelated pig blood polypeptide ferrite in vitro[J].Science and Technology of Food Industry,2011,32(10):135-138.[汪学荣,彭祥伟,阚建全.猪血多肽亚铁螯合盐的体外抗氧化作用研究.食品工业科技,2011,32(10):135-138.]
[25]Guo Y X,Lin Q L,Wu W.Study on antioxidant properties of defatted rice bran protein by enzymatic hydrolysis[J].Cereals&Oils,2012(10):15-19.[郭延熙,林亲录,吴伟.酶法水解脱脂米糠蛋白抗氧化性质研究.粮食与油脂,2012(10):15-19.]
[26]Han H,Zhan S M,Shan L G,et al.Studies on the free radical scavenging activity of bioactive substances from Sargassum fusif orme[J].China Journal M arine Drugs,2008,27(8):31-34.[韩华,战松梅,单联刚,等.羊栖菜中清除自由基活性物质的研究.中国海洋药物,2008,27(8):31-34.]