基于水-甲醇体系的ONPG法测定调制乳粉中乳糖酶活力研究

Determination ofβ-galactosidase activity in special modified milk powder by ONPG method based on water-methanol system

建立了基于水-甲醇体系的ONPG法测定调制乳粉中乳糖酶活力的测定方法。调制乳粉中的乳糖酶经水溶解提取,在特定温度、pH条件下将ONPG水解生成ONP和半乳糖,以碳酸钠作为反应终止剂,使ONP显色,加入甲醇去除杂质,分光光度法定量,计算酶活力。首次提出的基于水-甲醇体系的ONPG分光光度法,较好地去除杂质,并提高了响应,在0.005~0.15 mmol/L范围内线性相关系数0.9997,加标回收率95.0%~98.8%,相对标准偏差0.78%~0.98%,定量限1.0 U/g。该方法线性良好,结果可靠,可以作为常规实验室测定调制乳粉中乳糖酶活力的经济、可靠的测定方法。

A method for the determination of lactase activity in modulated milk powder by ONPG method based on water-methanol system was established.The lactase in the modulated milk powder is dissolved and extracted in water,and ONPG is hydrolyzed to ONP and galactose under specific temperature and pH conditions by lactase.Sodium carbonate is used as a reaction terminator,and it made ONP show color,and methanol is added to remove impurities,and the ONP is quantified by spectrophotometry,and calculate the enzyme activity.This study proposes an innovative ONPG spectrophotometric method based on water-methanol system,it can remove impurities well and improve the response.The linear correlation coeficient is 0.9997 in the range of 0.005-0.15 mmol/L,and the recovery rate of standard adition is 95.0%~98.8%,which is relatively standard Deviation 0.78%-0.98%,limit of quantitation 1.0 U/g.The method has good linearity and reliable results,and can be used as an economical and reliable determination method for the determination of lactase activity in powdered milk for the conventional laboratory.