基于醇脱氢酶催化-动力学荧光分析法测定食品中乙醇的含量  

Determination of Ethanol in Food by Alcohol Dehydrogenase-Fluorescence Analysis

作　　者：覃建军 卢孜 吴来燕 杨安平 Qin Jian-jun;Lu Zi;Wu Lai-yan;Yang An-ping(College of Resources and Environmental Science,South-Central Minzu University,Wuhan Hubei 430074,China;Hubei Provincial Ecology and Environmental Monitoring Center Station,Wuhan Hubei 430070,China)

机构地区：[1]中南民族大学资源与环境学院,湖北武汉430074 [2]湖北省生态环境监测中心站,湖北武汉430070

出　　处：《江西化工》2023年第3期52-55,共4页Jiangxi Chemical Industry

基　　金：中央高校基本科研业务费专项资金项目平台专项项目“三维多孔少层C3N4-PMS可见光催化体系降解水体中磺胺类抗生素的机理及其应用研究”(CPT22027)。

摘　　要：醇脱氢酶(ADH)是生物体内重要的氧化还原催化剂之一,很多醇类代谢都通过醇脱氢酶催化完成。基于以还原型烟酰胺腺嘌呤二核苷酸(NADH)和乙醇为底物,在醇脱氢酶作用下催化转化为无荧光的氧化型烟酰胺腺嘌呤二核苷酸(NAD+)和乙醛,联合动力学荧光分析法测定食品基质中的乙醇含量。通过三因素四水平正交实验,考察得到最佳反应体系为20μL脱氢酶溶液(31600 U·L^(-1)),40μL NAD溶液(0.05 mol·L^(-1))和pH值8.4的Tris-HCl缓冲溶液(0.05 mol·L^(-1))。根据以上体系中还原型NAD(NADH)荧光值的变化计算酶促反应速度,结合米氏方程测定乙醇含量。金属离子、蛋白质、抗坏血酸等添加剂的干扰实验表明,酶对催化反应具有较高专一性,可以通过EDTA等有效避免,该方法在1.0~5.0 mmol·L^(-1)有较好的线性,加标回收实验回收率在96%~104%。该方法具有较高灵敏,较低检测限,能准确测定生物样品和食品样品中乙醇的含量。Alcohol dehydrogenase (ADH) is one of the most important redox catalysts in living organisms,and many alcohols metabolisms are catalyzed by alcohol dehydrogenase.The substrates of NADH and alcohol were transformed to be acetaldehyde and NADH with the enzymatic of ADH.Therefore,the rate of enzymatic reaction was calculated based on the change in the fluorescence value of reduced NAD (NADH) in a system of 20μL dehydrogenase solution (31600 U·L~(-1)),40μL NAD solution (0.05 mol·L~(-1)) and Tris-HCl buffer solution (0.05 mol·L~(-1)) at pH 8.4,and the ethanol content was determined in combination with the Mie equation.The spike test and interference experiments showed excellent recovery ranged from 96%~104%.This method is more sensitive and has lower detection limits,and can determine the content of ethanol in biological samples and food samples.

关 键 词：NADH 醇脱氢酶 酶法测定 动力学荧光法 

分 类 号：TS207.3[轻工技术与工程—食品科学]