双酶显色-紫外-可见分光光度法测定牛奶中组胺与腐胺的含量

Determination of Histamine and Putrescine in Milk by UV-Vis Spectrophotometry Combined with Double Enzyme Colorimetry

建立了基于二胺氧化酶-辣根过氧化物酶双酶显色的紫外-可见分光光度法测定牛奶中组胺与腐胺含量的方法。取10mL牛奶样品,加入10mL 2%(质量分数)硝酸铅标准溶液,混匀,再加入20mL 0.1%(质量分数)三氯乙酸标准溶液,混匀,离心,取上清液,将溶液pH调至7.2,得到待测样品溶液。移取1.0g·L^(-1)二胺氧化酶标准溶液100μL,加入100μL待测样品溶液,于50℃水浴中反应15min。反应结束后,依次加入1.0g·L^(-1)辣根过氧化物酶标准溶液50μL,0.5g·L^(-1)四甲基联苯胺标准溶液200μL和2mol·L^(-1)盐酸溶液50μL,采用紫外-可见分光光度计测定体系的吸光度。结果显示:样品溶液由无色变为蓝色又变为黄色;组胺、腐胺的浓度分别在2.5~150μmol·L^(-1)和5~200μmol·L^(-1)内与其对应的吸光度呈线性关系,检出限(3S/N)分别为0.6522μmol·L^(-1)和1.1341μmol·L^(-1)。对空白牛奶样品进行加标回收试验,组胺和腐胺的回收率分别为80.5%~88.3%和89.5%~92.3%,测定值的相对标准偏差(RSD,n=5)分别为2.5%~4.4%和4.4%~5.1%。

A method for determination of histamine and putrescine in milk by UV-Vis spectrophotometry based on diamine oxidase and horseradish peroxidase double enzyme colorimetry was established.2%(mass fraction)lead nitrate standard solution of 10mL was added into the milk sample of 10mL,and mixed well.Then 0.1%(mass fraction)trichloroacetic acid standard solution of 20mL was added.Atter mixing and centrifuging,the supernatant was taken and adjusted its acidity to pH 7.2.1.0g·L^(-1)diamine oxidase standard solution of 100μL was added into sample solution of 100μL in water bath at 50℃for 15 min.After the reaction,1.0g·L^(-1)horseradish peroxidase standard solution of 50μL,0.5g·L^(-1)tetramethylbenzidine standard solution of 200μL and 2mol·L^(-1)hydrochloric acid solution of 50μL were added successively.The absorbance of the system was measured by UV-Vis spectrophotometer.It was showed that the color of sample solution underwent colorless,and then changed from blue to yellow.Linear relationships between concentrations of histamine and putrescine and their absorbances were kept in the ranges of 2.5-150μmol·L^(-1)and 5-200μmol·L^(-1),with detction limits(3S/N)of 0.6522μmol·L^(-1)and 1.1341μmol·L^(-1),respectively.Recovery test was made on blank milk sample,giving results in the ranges of 80.5%-88.3%for histamine and 89.5%-92.3%for putrescine,and RSDs(n=5)of the determined values were in the ranges of 2.5%-4.4%for histamine and 4.4%-5.1%for putrescine.