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

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Abstract

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.

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Data Availability

Data used to support the findings of this study are available from the corresponding author upon request.

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Acknowledgements

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Funding

This study was funded by the Key Science and Technology Projects of Sichuan Tobacco Company in 2019 (Grant Number SCYC201915) and the Key Science and Technology Projects of Shanghai Tobacco Group in 2019 (Grant Number 2019110004340244).

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Authors and Affiliations

  1. College of Tobacco Science/Tobacco Harm Reduction Research Center, Henan Agricultural University, No. 95 Wenhua Road, Zhengzhou, 450002, Henan, China

    Jingjing Li, Yuanyuan Zhao, Huijuan Yang, Xingyou Yang & Hongzhi Shi

  2. Deyang Branch of Sichuan Tobacco Company, Deyang, 618000, Sichuan, China

    Jun Wang

  3. Shanghai Tobacco Group, No. 717 Changyang Road, Yangpu District, Shanghai, 200082, China

    Jun Zhou

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  1. Jingjing Li
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Contributions

Conception and design of the research: JL, HS; acquisition of data: JW and JZ; analysis and interpretation of data: YZ and HY; statistical analysis: XY; obtaining funding: JW and JZ; drafting the manuscript: JL; revision of the manuscript for important intellectual content: HS. All authors have read and approved the final manuscript.

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Correspondence to Hongzhi Shi.

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284_2023_3314_MOESM1_ESM.tif

Supplementary file1 (TIF 645 KB)—Figure S1 Representative chromatograms of NNN, NAT, NAB, and NNK in the fermented tobacco sample. NNN N′-nitrosonornicotine; NAT N′-nitrosoanatabine; NAB N′-nitrosoanabasine; NNK 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone.

284_2023_3314_MOESM2_ESM.xls

Supplementary file2 (XLS 37 KB)—Table S1 The KEGG pathways enriched by microbes in the temperature gradient group. KEGG, Kyoto Encyclopedia of Genes and Genomes.

Supplementary file3 (XLS 40 KB)—Table S2 The KEGG pathways enriched by microbes in the low-temperature group.

Supplementary file4 (XLS 22 KB)—Table S3 The KEGG pathways enriched by microbes in the high-temperature group.

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Li, J., Zhao, Y., Yang, H. et al. Identification of Bacteria Associated with Tobacco Mildew and Tobacco-Specific Nitrosamines During Tobacco Fermentation. Curr Microbiol 80, 218 (2023). https://doi.org/10.1007/s00284-023-03314-z

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