Identification of Bacteria Associated with Tobacco Mildew and Tobacco-Specific Nitrosamines During Tobacco Fermentation.

Tobacco mildew and tobacco-specific nitrosamines (TSNAs) affect the quality of tobacco products during fermentation. Microbes are thought to play key roles in the development of specific properties of fermented tobacco; however, little is known about the bacteria involved in the fermentation process. This study aims to identify key microbes related to mildew and TSNA formation. Tobacco was fermented at 25 °C, 35 °C, and 45 °C for 2, 4, and 6 weeks, with unfermented samples used as controls. Our preliminary exploration found that TSNAs content elevated with the increase of temperature and period, and mildew was easy to occur at low temperature with short period. Hence, samples were divided into three groups: the temperature gradient group (25 °C, 35 °C, and 45 °C for 6 weeks); the low-temperature group (control, 25 °C for 2, 4, and 6 weeks); and the high-temperature group (control, 45 °C for 2, 4, and 6 weeks). After collecting fermented tobacco leaves, 16S rRNA gene sequencing was used to explore the structure and dynamic changes of bacterial community during fermentation. Methylobacterium and Deinococcus were shared between the temperature gradient and high-temperature groups and showed a linear downward trend; these might play a role in the production of TSNAs. Massilia, Ruminiclostridium, and Cellulosilyticum species increased with prolonged fermentation time in the low-temperature group; this might be associated with tobacco mildew. In summary, the microbial diversity of fermented tobacco was explored under different conditions. These findings might provide data and material support to improve the quality of fermented tobacco products; however, further omics based studies are warranted to analysis the gene and protein expression patter in the identified bacteria.