混菌发酵绿茶产脂肪酶及其对酯型儿茶素的水解作用分析

微生物的发酵作用导致黑茶加工过程中儿茶素的生物转化和总茶多酚含量的下降,这一转化过程的代谢途径和调控机制尚不清楚。通过添加冠突曲霉(Aspergillus cristatum)菌株PE-1和黑曲霉(A. niger)菌株PE-4混合发酵绿茶,采用鉴别培养基平板检测、酶活性测定和酶蛋白分离纯化,研究脂肪酶活性及其对酯型儿茶素的水解作用。两个菌株单菌发酵和混合菌种发酵绿茶条件下均可以检测到脂肪酶活性,混菌发酵酶活力高于两个菌株单菌发酵的酶活力。混菌发酵条件下获得一个分子量约为37 kDa的脂肪酶,对EGCG和ECG有不同的生物转化作用。UPLC检测结果表明,该脂肪酶对EGCG有较好的底物选择性,EGCG的水解率为94.46%,EGC的产率为63.33%;ECG的水解率为15.45%,EC的产率为4.15%。脂肪酶对EGCG和ECG的生物转化将为儿茶素代谢途径的研究和单体儿茶素的生产起到促进作用。

Fungal Mixed Fermentation for The Production of Lipase and Its Activity Analysis in Galloylated Catechin Hydrolysis

In the process of dark tea manufacture, microbial fermentation leads to the biotransformation of catechins and the decrease of the total polyphenol contents. Up to date, the metabolic pathways and regulatory mechanisms of this transformation process were still unclear. The green tea was fermented by mixed fermentation of Aspergillus cristatum strain PE-1 and A. niger strain PE-4. Lipase activity was detected by a differential culture medium plate test and enzyme activity assay. The results indicate that lipase activities in mixed strain fermentation were stronger than that of signal strain fermentation. In mixed fermentation, a lipase protein with a molecular weight of approximately 37βkDa was obtained by protein separation and purification, which had different biotransformation efficiency for EGCG and ECG. HPLC results show that the lipase had a better substrate selectivity for EGCG than that for ECG. The efficiency of EGCG hydrolysis and the yield of EGC were 94.46% and 63.33%. While the efficiency of ECG hydrolysis and the yield of EC were 15.45% and 4.15%. The biotransformation of EGCG and ECG by lipase would promote the study of the catechin metabolism and the production of monomeric catechins.