茶叶微生物固态发酵中咖啡碱降解途径初探

为探究微生物作用下咖啡碱的降解产物与途径,将普洱茶发酵中筛选鉴定的Aspergillus sydowii NRRL250（聚多曲霉）、Aspergillus pallidofulvus NRRL4789、Aspergillus sesamicola CBS137324和Penicillium mangini CBS253.31等优势菌株分别接种至晒青毛茶进行单菌种固态发酵,并采用高效液相色谱（HPLC）测定咖啡碱、可可碱、茶碱的含量,探究微生物对咖啡碱代谢的影响;另外,基于UHPLC-QTOF-MS代谢组学技术,以灭菌处理组（ST组）和原料组（RM组）为对照,对聚多曲霉接种发酵样进行代谢组学分析。结果表明,A. pallidofulvus NRRL4789、A. sesamicola CBS137324和Penicillium mangini CBS253.31等优势菌株对咖啡碱等嘌呤类碱代谢均无显著影响,而在聚多曲霉接种发酵中,咖啡碱含量显著下降（P<0.05）,降幅达83.89%;茶碱含量显著增加（P<0.05）,发酵末期含量为(25.03±1.17) mg·g^-1;而可可碱保持基本稳定。由此可知,聚多曲霉对咖啡碱降解代谢有显著影响。采用UHPLC-QTOF-MS方法检出茶碱、3-甲基黄嘌呤、1,7-二甲基黄嘌呤等9种与咖啡碱降解相关的代谢物。在聚多曲霉作用下,茶碱、3-甲基黄嘌呤、1,7-二甲基黄嘌呤、7-甲基黄嘌呤含量显著提高（P<0.05）。茶碱、3-甲基黄嘌呤、1,7-二甲基黄嘌呤和1-甲基黄嘌呤与咖啡碱及其相关代谢物的N-脱甲基化途径相关。1,7-二甲基尿酸、1-甲基尿酸与咖啡碱相关代谢物的氧化途径相关。由此可知,聚多曲霉为降解普洱茶咖啡碱的优势菌株,且具有将咖啡碱转化为茶碱的潜在能力;在咖啡碱降解代谢过程中,存在聚多曲霉作用下的N-脱甲基化和氧化,并以N-脱甲基化为主。

A Preliminary Study on the Degradation Pathway of Caffeine in Tea Microbial Solid-state Fermentation

In order to explore caffeine degradation products and pathways under the action of microorganisms, the dominant strains including Aspergillus sydowii NRRL250, Aspergillus pallidofulvus NRRL4789, Aspergillus sesamicola CBS137324 and Penicillium mangini CBS253.31 were screened and identified during pu-erh tea fermentation. Strains were inoculated into sun-dried green tea leaves for solid-state fermentation. High performance liquid chromatography (HPLC) was used to determine caffeine, theobromine and theophylline contents to explore the effect of microorganisms on caffeine metabolism. UHPLC-QTOF-MS was used for the metabonomic analysis of Aspergillus sydowii inoculated fermentation with sterilization treatment group (ST group) and raw material group (RM group). The results show that the dominant strains such as A. pallidofulvus NRRL4789, A. sesamicola CBS137324 and Penicillium mangini CBS253.31 had no significant effects on the metabolism of caffeine and other purine alkaloids. However, caffeine content was decreased significantly (P<0.05) with a great reduction about 83.89% during the inoculated fermentation of Aspergillus sydowii. Additionally, theophylline content was increased significantly (P<0.05) and arrived to (25.03±1.17) mg·g^-1 at the end of fermentation. While theobromine content remained stable. Therefore, Aspergillus sydowii has a profound effect on caffeine degradation metabolism. Nine metabolites related to caffeine degradation were detected by UHPLC-QTOF-MS during the inoculated fermentation, Among them, theophylline, 3-methylxanthine, 1,7-dimethylxanthine and 7-methylxanthine contents were significantly increased (P<0.05) under the action of Aspergillus sydowii which were related to N-demethylation pathway of caffeine and its related metabolites. 1,7-dimethyluric acid and 1-methyluric acid were related to the oxidation pathway of caffeine-related metabolites. It can be seen that Aspergillus sydowii is the dominant strain that can degrade caffeine and has the potential ability to convert caffeine into theophylline. Under the action of spergillus sydowii, both N-demethylation and oxidation were found in caffeine degradation metabolism, and the former was the dominant.