蛹虫草TTC-脱氢酶测定方法的优化

The optimization of TTC-dehydrogenase assay in Cordyceps militaris

TTC-脱氢酶还原法可用于检测和鉴别优良及退化蛹虫草菌株。这一方法的应用依赖测定参数的优化。本文优化了蛹虫草TTC-脱氢酶的测定方法,结果显示:测定蛹虫草TTC-脱氢酶活性时,培养6d的蛹虫草菌液先以生理盐水洗涤4次后配成0.15g/mL的待用菌液;反应缓冲液为Tris-HCl,最适pH值为8.4;最适反应温度为37℃、最适反应时间为3h。酶促反应完成后无需加终止剂,立即将样品放置冰上,4℃离心再以无菌水洗2次,用无水乙醇萃取便可测定吸光度得到脱氢酶活性。本文优化的蛹虫草TTC-脱氢酶活性检测方法具有测定结果稳定可靠、重现性好、准确度高、操作方便等优点。

2, 3, 5 -triphenyl tetrazolium chloride （Trc） -dehydrogenase assay can be applied in screening and detecting the degenerate strains of Cordyceps rrtilitaris. The effective utilization of this assay heavily depends on the optimum experimental parameters. TFC - dehydrogenase assay of C. militaris was optimized in this study. The results showed that the mycelia sample （0. 15 g/mL, age 6 d） washed with physiological saline for four times before test, the buffer solution （ Tris - HC1, pH8.4）, the incubation condition （37~C for 3 h） and extractant （absolute ethyl alcohol） were optimum parameters for the effec- tive dehydrogenase activity. Ice, instead of adding terminating agent was found to terminate reaction. The optimized method in this study were reliable, reproducible, easy and efficient for TI＇C - dehydrogen- ase assay of C. militaris.