State Key Laboratory of Tea Plant Biology and Utilization, Anhui Agricultural University, Hefei, 230036, Anhui, China.
College of Long Run Pu-erh Tea, Yunnan Agricultural University, Kunming, 650201, Yunnan, China.
BMC Microbiol. 2018 Jun 5;18(1):53. doi: 10.1186/s12866-018-1194-8.
Pu-erh tea is a traditional Chinese tea and produced by natural solid-state fermentation. Several studies show that the natural microbiota influence caffeine level in pu-erh tea. Our previous research also found that the caffeine declined significantly (p < 0.05) in the fermentation, which suggested that the caffeine level could be influenced by specific strains. The purpose of this study was to isolate and identify microorganisms for caffeine degradation, and this research explored the degradation products from caffeine and optimal condition for caffeine degradation.
11 Fungi were isolated from pu-erh tea fermentation and 7 strains could survive in caffeine solid medium. Two superior strains were identified as Aspergillus niger NCBT110A and Aspergillus sydowii NRRL250 by molecular identification. In the substrate tests with caffeine, A. niger NCBT110A could use caffeine as a potential carbon source while glucose is absent, A. sydowii NRRL250 could degrade 600 mg/L caffeine completely in a liquid medium. During the degradation product analysis of A. sydowii NRRL250, theophylline and 3-methlxanthine were detected, and the level of theophylline and 3-methlxanthine increased significantly (p < 0.05) with the degradation of caffeine. The single factor analysis showed that the optimum conditions of caffeine degradation were 1) substrate concentration of 1200 mg/L, 2) reaction temperature at 30 °C, and 3) pH of 6. In the submerged fermentation of tea infusion by A. sydowii NRRL250, 985.1 mg/L of caffeine was degraded, and 501.2 mg/L of theophylline was produced.
Results from this research indicate that Aspergillus sydowii NRRL250 was an effective strain to degrade caffeine. And theophylline and 3-methlxanthine were the main caffeine degradation products.
普洱茶是一种传统的中国茶，采用自然固态发酵制成。多项研究表明，天然微生物群会影响普洱茶中的咖啡因含量。我们之前的研究还发现，发酵过程中咖啡因显著下降（p<0.05），这表明咖啡因水平可能受到特定菌株的影响。本研究旨在分离和鉴定具有咖啡因降解能力的微生物，并探索咖啡因的降解产物和最佳降解条件。
从普洱茶发酵中分离出 11 株真菌，其中 7 株能在含有咖啡因的固体培养基中存活。通过分子鉴定，将其中两株优势菌株鉴定为黑曲霉 NCBT110A 和宛氏拟青霉 NRRL250。在含有咖啡因的底物试验中，黑曲霉 NCBT110A 能在无葡萄糖的情况下以咖啡因作为潜在碳源，宛氏拟青霉 NRRL250 能在液体培养基中完全降解 600mg/L 的咖啡因。在对宛氏拟青霉 NRRL250 降解产物的分析中，检测到了茶碱和 3-甲基黄嘌呤，并且随着咖啡因的降解，茶碱和 3-甲基黄嘌呤的水平显著增加（p<0.05）。单因素分析表明，咖啡因降解的最佳条件为：1）底物浓度 1200mg/L，2）反应温度 30°C，3）pH 值 6。在宛氏拟青霉 NRRL250 对茶浸提液的浸种发酵中，降解了 985.1mg/L 的咖啡因，产生了 501.2mg/L 的茶碱。
本研究结果表明，宛氏拟青霉 NRRL250 是一种有效的咖啡因降解菌株。茶碱和 3-甲基黄嘌呤是咖啡因的主要降解产物。
