胶原纤维的酶解液中羟脯氨酸含量测定方法的优化
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摘要
针对文献报道的测定蛋白水解液中羟脯氨酸含量的氯胺T显色法(比色法)中的操作条件不一致的问题,对该方法的关键条件进行了优化。用优化后的方法和氨基酸分析仪法(AA法)对系列皮粉和皮块蛋白酶水解液中的羟脯氨酸的含量进行了测定和比较。结果表明:优化后比色法的最适测定条件是:氧化时间5 min、显色时间15 min、消解时间4 h、样品pH 6.0～6.5;优化后的比色法的测定结果具有较好的重复性(RSD小于1.17%);比色法与AA法测定相同样品的结果比值在0.97～1.09之间,测定结果基本一致。与AA法相比,比色法具有快速、简便、成本低、易实现等优点,可以用于表征酶对胶原纤维的作用性能。
In order to solve the problem of inconsistent operating conditions in the chloramine T colorimetric method(colorimetric method) for the determination of hydroxyproline(Hypro) in protein hydrolysate reported in literatures, the key conditions of the method were optimized. Then the contents of Hypro in hydrolysis solutions of different proteases toward hide powder and skin were measured with the optimized method, and compared with amino acid analyzer method(AA).The results showed that the optimal conditions of the optimized colorimetric method were oxidation time of 5 min, color development time of 15 min, digestion time of 4 h, and pH of 6.0-6.5 after digestion. The results of the colorimetric method showed good repeatability(RSD less than 1.17%). The ratio of measured values from the two methods for the same sample was between 0.97 and 1.09. Compared with AA method, colorimetric method has the advantages of rapid, simple, low cost and easy realization in ordinary laboratory, which can be used to characterize the function of enzyme on collagen fiber.
In order to solve the problem of inconsistent operating conditions in the chloramine T colorimetric method(colorimetric method) for the determination of hydroxyproline(Hypro) in protein hydrolysate reported in literatures, the key conditions of the method were optimized. Then the contents of Hypro in hydrolysis solutions of different proteases toward hide powder and skin were measured with the optimized method, and compared with amino acid analyzer method(AA).The results showed that the optimal conditions of the optimized colorimetric method were oxidation time of 5 min, color development time of 15 min, digestion time of 4 h, and pH of 6.0-6.5 after digestion. The results of the colorimetric method showed good repeatability(RSD less than 1.17%). The ratio of measured values from the two methods for the same sample was between 0.97 and 1.09. Compared with AA method, colorimetric method has the advantages of rapid, simple, low cost and easy realization in ordinary laboratory, which can be used to characterize the function of enzyme on collagen fiber.
引文
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[8]曾暖茜,王洪健,周兴起,等.氨基酸自动分析仪对乳制品中羟脯氨酸的测定方法研究[J].现代食品科技,2008,(07):719-721.
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[12]Fayad S,Morin P,NehméR.Use of chromatographic and electrophoretic tools for assaying elastase,col()lagenase,hyaluronidase,and tyrosinase activity[J].Journal of Chromatography A.2017,1529:1-28.
[13]赵一晖,刘思洁,张培军,等.高效液相色谱法测定乳与乳制品中L-羟脯氨酸的含量[J].食品安全质量检测学报,2016,7(01):55-58.
[14]刘芳,李德富,林炜,等.羟脯氨酸含量的测定方法与应用[J].中国皮革,2007,(15):51-54.
[15]郭恒斌,曾庆祝.分光光度法测定鱼皮中羟脯氨酸含量[J].食品研究与开发,2007,(10):145-148.
[16]迟原龙,文振华,崔敏,等.胶原水解度的测定[J].中国皮革,2010,(19):13-16.
[17]金博,李玉彬,王援朝,等.氯胺T法测定经脯氨酸含量的方法改进[J].陕西医学检验,1994,9(3):176.
[18]张笑,刘杰,陈翠岚.比色法测定明胶中的L-羟脯氨酸[J].食品安全质量检测学报,2010,27(3):124-127.
[19]王海轩,谷瑞增,金振涛,等.胶原蛋白水解物中L-羟脯氨酸两种检测法对比分析[J].食品研究与开发,2016,37(22):119-122.
[20]赵天珍,袁秀金,谭贵良,等.比色法快速测定奶粉和含乳饮料中游离L-羟脯氨酸[J].食品研究与开发,2008,29(12):111-113.
[2]RLR,IZD,MA.Essential modification of the Sircol Collagen assay for the accurate quantification of collagen content in complex protein solutions[J].Acta Biomaterialia.2010,6(8):3146-3151.
[3]S U.Formation of Hydroxyproline in Collagen[J].Science.1966,152(3727):1335.
[4]李艳红.蛋白酶水解弹性蛋白和胶原蛋白性能对酶脱毛效果和皮粒面作用程度的影响[D].四川大学,2016.
[5]潘红英,迟原龙,廖学品,等.蛋白酶种类及使用方法对胶原水解程度的影响[J].西部皮革,2013,(06):10-14.
[6]李景红,杨再山,孟祥晨.比色法检测乳中掺加的动物胶原水解蛋白[J].中国乳品工业,2007,(09):50-52.
[7]江明芳,陈敏,吴金键,等.基于氯胺T法的胶原酶活力测定[J].皮革科学与工程,2017,(06):14-18.
[8]曾暖茜,王洪健,周兴起,等.氨基酸自动分析仪对乳制品中羟脯氨酸的测定方法研究[J].现代食品科技,2008,(07):719-721.
[9]蔡梅,吉文亮,刘华良,等.氨基酸自动分析仪对牛乳中羟脯氨酸快速测定方法研究[J].中国食品卫生杂志,2012,24(06):542-545.
[10]高建华,宁正祥.禽畜皮和鱼鳞的基本成分及氨基酸组成分析[J].现代食品科技,2007,(12):77-79.
[11]金苏英,林笑容,赵志红,等.高效液相色谱法测定奶粉及其他乳制品中的L-羟脯氨酸[J].饮料工业,2009,(07):28-30.
[12]Fayad S,Morin P,NehméR.Use of chromatographic and electrophoretic tools for assaying elastase,col()lagenase,hyaluronidase,and tyrosinase activity[J].Journal of Chromatography A.2017,1529:1-28.
[13]赵一晖,刘思洁,张培军,等.高效液相色谱法测定乳与乳制品中L-羟脯氨酸的含量[J].食品安全质量检测学报,2016,7(01):55-58.
[14]刘芳,李德富,林炜,等.羟脯氨酸含量的测定方法与应用[J].中国皮革,2007,(15):51-54.
[15]郭恒斌,曾庆祝.分光光度法测定鱼皮中羟脯氨酸含量[J].食品研究与开发,2007,(10):145-148.
[16]迟原龙,文振华,崔敏,等.胶原水解度的测定[J].中国皮革,2010,(19):13-16.
[17]金博,李玉彬,王援朝,等.氯胺T法测定经脯氨酸含量的方法改进[J].陕西医学检验,1994,9(3):176.
[18]张笑,刘杰,陈翠岚.比色法测定明胶中的L-羟脯氨酸[J].食品安全质量检测学报,2010,27(3):124-127.
[19]王海轩,谷瑞增,金振涛,等.胶原蛋白水解物中L-羟脯氨酸两种检测法对比分析[J].食品研究与开发,2016,37(22):119-122.
[20]赵天珍,袁秀金,谭贵良,等.比色法快速测定奶粉和含乳饮料中游离L-羟脯氨酸[J].食品研究与开发,2008,29(12):111-113.