多酚氧化酶的提取工艺与活性测定研究

Study on Extraction Process and Activity Determination of Polyphenol Oxidase

多酚氧化酶活性测定常使用分光光度法。本试验对目前常用的茶叶多酚氧化酶(Polyphenol oxidase,PPO)的提取工艺和活性测定条件进行了改进,考察了4℃浸提时间、离心处理、反应pH值及温度、底物浓度和反应终止剂对茶叶PPO酶活的影响,并对不同植物来源的PPO酶活进行了比较。结果表明:茶叶多酚氧化酶活性的最佳测定条件为鲜叶匀浆后4℃浸提12 h,8000 r/min离心15 min,取上清酶液于pH值6.0和37℃条件下,添加质量浓度为1.5%的邻苯二酚作为底物,反应10 min后使用3 mL 1 mol/L的偏磷酸终止反应,于460 nm处测定吸光度值,改进后的方法测定标准差和相对标准偏差均小于5%。使用改进方法测定了福鼎大白(春)、福鼎大白(夏)、香蕉、苹果、皇冠梨和葡萄等植物的PPO酶活大小,发现福鼎大白夏季茶叶PPO酶活性高于春季茶叶,而香蕉PPO的酶活高于其他植物,这为使用茶鲜叶以外的材料制备茶黄素提供了一定的科学依据。

The determination of polyphenol oxidase activity often uses spectrophotometry.In this paper,the current extraction process and measurement conditions for measuring tea polyphenol oxidase activity are improved.The effects of extraction time,centrifugation,pH and temperature,substrate concentration and reaction termination method on enzyme activity determination were investigated,and the enzyme activities of polyphenol oxidase from different plants were compared.The results showed that the best conditions for the determination of polyphenol oxidase activity were extraction at 4℃for 12 h after homogenization of fresh leaves,centrifugation at 8000 rpm for 15 min,then the supernatant enzyme solution was added with 1.5%catechol as a substrate at pH 6.0 and 37℃,after 10 minutes the reaction was terminated with 3 mL•1mol/L metaphosphoric acid and the absorbance was measured at 460 nm.The standard deviation and relative standard deviation of the improved method are both less than 5%.The PPO activities of FudingDabai(spring),FudingDabai(summer),banana,apple,crown pear and grape were measured by the improved method.It was found that the PPO activity of FudingDabai tea in summer was higher than in spring,while the PPO activity of banana was higher than that of other plants,which provided a scientific basis for the preparation of theaflavin from materials other than fresh tea leaves.