普洱茶中微生物的筛选及其安全性初步评价

目的 在对普洱茶中微生物筛选和鉴定基础上对蜡样芽胞杆菌毒素基因的分布、普洱茶下调毒素基因的表达和改善肠上皮细胞的损伤进行研究。方法 分别采用无菌水和沸水泡制普洱茶，获得分离株，通过16S rDNA测序以及生理生化试验确定其归属；对所筛选的菌株进行耐模拟胃肠液能力评价和毒力基因的检测；采用细胞实验和荧光定量PCR技术，研究普洱茶对蜡样芽胞杆菌毒素的抑制作用。结果 无菌水浸泡普洱茶获得的45株菌中44株为芽胞杆菌属，沸水浸泡获得的7株菌均为蜡样芽胞杆菌（FBCE01、FBCE06、FBCE10、FBCE14、FBCE20、FBCE26和FBCE29），多重PCR技术结果表明其分别含有毒力基因cytK、nheA和hblD的2种或3种。耐模拟胃肠液实验表明，7株菌均具有很强的耐模拟胃肠液消化能力；细胞实验结果发现，普洱茶汤能显著降低蜡样芽胞杆菌对Caco-2细胞的粘附（P<0.05）；MTT实验结果显示，普洱茶能有效降低蜡样芽胞杆菌对细胞的损伤；荧光定量PCR技术结果进一步说明，普洱茶使蜡样芽胞杆菌肠毒素的mRNA表达水平下调。结论 普洱茶具有抑制蜡样芽胞杆菌毒素的作用。

Microbiota isolated from Pu-erh tea and preliminary evaluation on its safety

Abstract: Objective To screen and identify the microorganisms in Pu-erh tea, and explore the distribution of toxin genes of Bacillus cereus, the down-regulation of toxin gene expression and the improvement of intestinal epithelial cell damage by Pu-erh tea. Methods The tea was prepared using sterile water and boiling water respectively to isolate microbial strains which were then identified with 16S rDNA sequencing; The species of the strains were determined through physiological and biochemical tests. The tolerance of the strains to simulated gastrointestinal fluids was evaluated, and the virulence genes of the isolated strains were detected. Cellular assay and fluorescent quantitative PCR technique were used to study the inhibitory effect of Pu''er tea on Bacillus cereus. Results 44 strains out of the 45 strains obtained by immersing the tea in sterile water were Bacilli, and the 7 strains (FBCE01, FBCE06, FBCE10, FBCE14, FBCE20, FBCE26 and FBCE29) obtained through boiling water immersion were identified as Bacillus cereus. The 7 strains of Bacillus cereus were found to contain 2 or all the 3 virulence genes cytK, nheA, and hblD, and they were highly resistant to simulated gastrointestinal fluids. The results of cellular assay showed that the adhesion of Bacillus cereus to Caco-2 cells was significantly reduced by Pu-erh tea solution (P<0.05). MTT test showed that Pu-erh tea effectively reduced the damage of cells by Bacillus cereus and enhanced the cell viability. RT-qPCR further demonstrated that Pu-erh tea reduced the mRNA expression of Bacillus cereus enterotoxin. Conclusion Pu-erh tea has the effect of inhibiting the toxin of Bacillus cereus.