酶联免疫吸附法与二喹啉甲酸法检测核桃蛋白浓度的比较研究
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摘要
目的探究间接酶联免疫吸附法(enzyme-linked immunosorbent assay,ELISA)和二喹啉甲酸(bicinchoninic acid,BCA)试剂盒2种方法在核桃蛋白浓度检测中的优缺点和适用范围。方法对本实验室提取的核桃蛋白进行烘烤、高温高压以及微波加热处理后,用ELISA法和BCA法2种方法分别检测其蛋白质浓度。结果在不同的热加工处理条件下, ELISA对蛋白浓度测定结果存在显著性差异,高度依赖热处理的类型。与之相比,使用BCA法测定蛋白浓度时,受到热加工处理影响较小。结果提示,在食物加工中的一些剧烈条件下,蛋白质的高级结构很容易遭到破坏,蛋白质结构的改变会干扰其与抗体结合部位的作用,导致ELISA法测定的结果不准确。BCA法测定蛋白质浓度的准确性较高,且受加热影响较小。结论 在剧烈的食品加工条件下,尤其当这种条件引起蛋白结构发生改变后,对其蛋白质浓度进行测定时,可选用BCA法进行测定。
Objective To investigate the advantages, disadvantages and application range of indirect enzyme-linked immunosorbent assay(ELISA) and bicinchoninic acid(BCA) kits in determination of walnut protein concentration. Methods After baking, high temperature, high pressure and microwave heating, the walnut protein extracted in our laboratory was tested by ELISA method and BCA method. Results There were significant differences in the results of protein concentration detected by ELISA under different thermal processing conditions and it highly dependents on the type of heat treatment. In contrast, the BCA method used to determine the protein concentration had less affected by thermal processing. The results suggested that the high-level structure of the protein could easily be destroyed under severe conditions in food processing. The change of protein structure could interfere with the binding site of antibody, resulting in the inaccurate result of ELISA. It was of high accuracy of utilizing BCA to determine the protein concentration and it was less affected by the heating. Conclusion In the severe food processing conditions, especially when the conditions resulted in changes in the protein structure, the protein concentration can be measured by BCA method.
Objective To investigate the advantages, disadvantages and application range of indirect enzyme-linked immunosorbent assay(ELISA) and bicinchoninic acid(BCA) kits in determination of walnut protein concentration. Methods After baking, high temperature, high pressure and microwave heating, the walnut protein extracted in our laboratory was tested by ELISA method and BCA method. Results There were significant differences in the results of protein concentration detected by ELISA under different thermal processing conditions and it highly dependents on the type of heat treatment. In contrast, the BCA method used to determine the protein concentration had less affected by thermal processing. The results suggested that the high-level structure of the protein could easily be destroyed under severe conditions in food processing. The change of protein structure could interfere with the binding site of antibody, resulting in the inaccurate result of ELISA. It was of high accuracy of utilizing BCA to determine the protein concentration and it was less affected by the heating. Conclusion In the severe food processing conditions, especially when the conditions resulted in changes in the protein structure, the protein concentration can be measured by BCA method.
引文
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[12]Venkatachalam,Roux K,Sathe S,et al.Antigenic stability of pecan(Carya illinoinensis(Wangenh.)K.Koch)proteins:effects of thermal treatments and in vitro digestion[J].J Agric Food Chem,2006,(54):1449-1458.
[13]王振华,张波,张影全,等.面条干燥过程的湿热传递机理研究进展[J].农业工程学报,2016,32(13):310-314.Wang ZH,Zhang B,Zhang YQ,et al.Research progress on the mechanism of moist heat transfer in noodle drying process[J].Trans Chin Soc Agr Eng,2016,32(13):310-314.
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[15]解冝华,陈红兵.加工对牛乳过敏原的影响[J].食品科学,2006,27(11):598-601.Xie GH,Chen HB.Effects of processing on allergens in milk[J].Food Sci,2006,27(11):598-601.
[16]Nowak-Wegrzyn A,Fiocchi A.The effect of heating and food matrix on food protein allergenicity[J].Curr Opin Allergy Cl,2009,9(3):234-237.
[17]涂宗财,迟海霞,刘成梅,等.动态超高压微射流对卵清蛋白功能性质的影响[J].食品研究与开发,2010,31(3):4-6.Tu ZC,Chi HX,Liu CM,et al.Effects of dynamic ultra high pressure micro jets on functional properties of ovalbumin[J].Food Res Dev,2003,31(3):4-6.
[18]谢丹丹,胡志和,薛璐,等.超高压结合酶法消减南美白对虾虾肉中的过敏原[J].食品科学,2012,33(3):68-71.Xie DD,Hu ZH,Xue L,et al.Ultra-high pressure combined with enzymatic method to reduce allergens in shrimp and shrimp of P.vannamei[J].Food Sci,2012,33(3):68-71.
[19]Gustavo AP,Dorcas W,Michael A,et al.Effect of processing on the detectability of pecan proteins assessed by immunological and proteomic tools[J].Food Anal Method,2012,(5):216-225.
[2]方凤.过敏原与过敏性疾病[J].人民军医,2004,47(12):730-732.Fang F.Allergens and allergic diseases[J].People Mil Surg,2004,47(12):730-732.
[3]Rahaman T,Vasiljevic T,Ramchandran L.Effect of processing on conformational changes of food proteins related to allergenicity[J].Trend Food Sci Technol,2016,(49):24-34.
[4]王海艳,袁飞,吴亚君,等.食品中过敏原胡桃蛋白间接竞争ELISA检测方法研究[J].中国食品学报,2010,10(5):217-222.Wang HY,Yuan F,Wu YJ,et al.Indirect competitive ELISA assay for allergen walnut protein in foods[J].J Chin Inst Food Sci Technol,2010,10(5):217-222.
[5]叶玫,吴成业,刘海新,等.酶联免疫吸附法在水产品安全检测中的应用[J].上海海洋大学学报,2002,11(2):171-175.Ye M,Wu CY,Liu HX,et al.Application of enzyme-linked immunosorbent assay in the safety detection of aquatic products[J].JShanghai Ocean Univ,2002,11(2):171-175.
[6]孟庆威,郭磊,申睿,等.一种基于二喹啉甲酸-Cu+显色反应的毛细管甴泳检测蛋白质的新方法[J].色谱,2010,28(7):682-687.Meng QW,Guo L,Shen R,et al.A new method for the determination of proteins by capillary electrophoresis based on bisquinolinecarboxylic acid-Cu+color reaction[J].Chin J Chromatogr,2010,28(7):682-687.
[7]韩富亮,袁春龙,郭安鹊,等.二喹啉甲酸法(BCA)分析蛋白多肽的原理、影响因素和优点[J].食品与发酵工业,2014,40(11):202-207.Han FL,Yuan CL,Guo AQ,et al.Principles,influencing factors and advantages of bisquinolinecarboxylic acid(BCA)analysis of protein peptides[J].Food Ferm Ind,2014,40(11):202-207.
[8]邱文英.小反刍兽疫病毒抗体、抗原免疫学检测方法和纳米金核酸检测方法的建立[D].北京:中国农业科学院,2011.Qiu WY.Establishment of antibodies against small ruminant virus,antigen immunoassay and nanogold nucleic acid detection method[D].Beijing:Chinese Academy of Agricultural Sciences,2011.
[9]陈朝银,方娟,赵声兰,等.用于核桃蛋白定性定量检测的ELISA试剂盒及其检测方法:中国,103760362A[P].2014.Chen CY,Fang J,Zhao SL,et al.ELISA kit for qualitative and quantitative detection of walnut protein and its detection method:CN,103760362A[P].2014.
[10]李晓玺,陈玱,邹芳建,等.微波对高链玉米淀粉颗粒抗消化性能的影响[J].食品科学,2007,28(10):105-108.Li XX,Chen L,Zou FJ,et al.Effects of microwave on anti-digestibility of high-chain corn starch granules[J].Food Sci,2007,28(10):105-108.
[11]姜松松,赵博,毕源,等.核桃过敏原分子特征与检测方法研究进展[J].农业生物技术学报,2016,(12):1935-1944.Jiang SS,Zhao B,Bi Y,et al.Research progress on molecular characteristics and detection methods of walnut allergens[J].J Agric Biotechnol,2016,(12):1935-1944.
[12]Venkatachalam,Roux K,Sathe S,et al.Antigenic stability of pecan(Carya illinoinensis(Wangenh.)K.Koch)proteins:effects of thermal treatments and in vitro digestion[J].J Agric Food Chem,2006,(54):1449-1458.
[13]王振华,张波,张影全,等.面条干燥过程的湿热传递机理研究进展[J].农业工程学报,2016,32(13):310-314.Wang ZH,Zhang B,Zhang YQ,et al.Research progress on the mechanism of moist heat transfer in noodle drying process[J].Trans Chin Soc Agr Eng,2016,32(13):310-314.
[14]Kato A,Takagi T.Formation of intermolecular beta-sheet structure during heat denaturation of ovalbumin[J].Agric Biol Chem,1988,36(6):1156-1159.
[15]解冝华,陈红兵.加工对牛乳过敏原的影响[J].食品科学,2006,27(11):598-601.Xie GH,Chen HB.Effects of processing on allergens in milk[J].Food Sci,2006,27(11):598-601.
[16]Nowak-Wegrzyn A,Fiocchi A.The effect of heating and food matrix on food protein allergenicity[J].Curr Opin Allergy Cl,2009,9(3):234-237.
[17]涂宗财,迟海霞,刘成梅,等.动态超高压微射流对卵清蛋白功能性质的影响[J].食品研究与开发,2010,31(3):4-6.Tu ZC,Chi HX,Liu CM,et al.Effects of dynamic ultra high pressure micro jets on functional properties of ovalbumin[J].Food Res Dev,2003,31(3):4-6.
[18]谢丹丹,胡志和,薛璐,等.超高压结合酶法消减南美白对虾虾肉中的过敏原[J].食品科学,2012,33(3):68-71.Xie DD,Hu ZH,Xue L,et al.Ultra-high pressure combined with enzymatic method to reduce allergens in shrimp and shrimp of P.vannamei[J].Food Sci,2012,33(3):68-71.
[19]Gustavo AP,Dorcas W,Michael A,et al.Effect of processing on the detectability of pecan proteins assessed by immunological and proteomic tools[J].Food Anal Method,2012,(5):216-225.