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. 2025 Jul 16:16:1623279.
doi: 10.3389/fmicb.2025.1623279. eCollection 2025.

Isolation of indigenous Bacillus velezensis from aging tobacco leaves for improving the flavor of flue-cured tobacco

Xiao-Jie Shan  1 , Lifeng Jin  2 , Feng Li  2 , Sheng-Bing Yang  1 , Xiao-Juan Zhang  1   3 , Li-Juan Chai  1   3 , Jin-Song Gong  4 , Jin-Song Shi  4 , Zhen-Ming Lu  1   3 , Zong-Yu Hu  5 , Zheng-Hong Xu  1   6
Affiliations

Isolation of indigenous Bacillus velezensis from aging tobacco leaves for improving the flavor of flue-cured tobacco

Xiao-Jie Shan et al. Front Microbiol. .

Abstract

Introduction: Aging of flue-cured tobacco is a slow microbial fermentation process that usually lasts for 2-3 years, which plays an important role in improving the quality of final products.

Methods: Re-dried tobacco leaves from seven Chinese regions were subjected to a controlled aging environment for 12 months. The bacterial community succession and volatile compounds dynamics in tobacco leaves throughout aging process were monitored. The main functional microorganisms were isolated from the tobacco leaves and inoculated into lower-grade tobacco leaves to evaluate their metabolic functions.

Results: Spearman's rank correlation analysis revealed that Bacillus was a key genus driving aroma production. By using the pure culture method, strain Bacillus velezensis TB-1 with the best enzyme-producing capacity was screened from high-grade tobacco leaves. Furthermore, Bacillus velezensis TB-1 was applied to low-grade tobacco leaves for solid-state fermentation. GC-MS results showed a significant increase in volatile compounds in fermented tobacco leaves compared to unfermented controls. The contents of 2-methoxy-4-vinylphenol (clove and wood flavors), megastigmatrienone (tobacco and spicy aromas), 2-methyl-hexanoic acid (cream flavor) increased significantly. Sensory evaluation confirmed that fermentation with Bacillus velezensis TB-1 markedly enhanced tobacco leaf flavor quality.

Discussion: This study identified Bacillus velezensis as a core aroma-producing microorganism in tobacco aging, demonstrating its application for fermentation to enhance leaf quality, thus establishing a novel strategy for tobacco improvement.

Keywords: Bacillus velezensis; aging; bacterial community; bioaugmentation; flue-cured tobacco.

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Conflict of interest statement

Z-YH was employed by the China Tobacco Jiangsu Industrial Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Two bar graphs depict microbial community changes with aging. Graph A shows relative abundance over 12 months, with various bacteria genera represented by colored bars. Graph B highlights differences across several samples over the same period, using similar color coding. A legend identifies multiple bacterial genera and their specific strains.
FIGURE 1
Succession of bacterial communities during the accelerated aging process of tobacco leaves under controlled environmental condition (temperature, 25–30°C; humidity, 60–65%). Species with an unannotated classification and a relative abundance below 0.5% in the sample were grouped into the “others” category. (A) Succession of bacterial communities within all tobacco leaf samples. (B) Succession of bacterial communities during the aging process of tobacco leaves originated from seven tobacco-producing regions. YNLJ, Lijiang in Yunnan province; GZZY, Zunyi in Guizhou province; HNCZ, Chenzhou in Hunan province; SCLS, Liangshan in Sichuan province; FJSM, Sanming in Fujian province; HASMX, Sanmenxia in Henan province; SDLY, Linyi in Shandong province.
The image features five panels displaying box plots and a scatter plot related to PCoA (Principal Coordinate Analysis). The top and bottom panels on the right and center show box plots with various data points and categories labeled with letters such as a, b, c, etc. The central scatter plot displays data points distributed in two dimensions, highlighting clusters with different colors. The panels indicate multiple data categories and groupings with distinct labels, showing variation and distribution in the datasets.
FIGURE 2
PCoA analysis of bacterial communities of different aged tobacco samples according to the classification of aging time, tobacco origin, tobacco grade and tobacco leaf collection site.
Heatmap and graphs depict the concentration of various compounds over time, categorized as terpenoids, ketones, esters, alcohols, aldehydes, heterocyclics, and phenols. The heatmap (left) shows concentration levels using a red-to-blue color scale. Graphs (right) provide detailed trends for specific compounds like solanone and acetoin with concentration on the y-axis and time on the x-axis.
FIGURE 3
(A) Heatmap showing the content of neutral flavor components in tobacco leaves during 1 year of accelerated aging. Red color representing higher content and blue color representing lower content. (B) Line plot of the relative contents of the six volatile compounds that changed significantly by one-way ANOVA.
Heatmap and bar chart showing average Spearman correlations of various compounds and bacterial genera. The top bar chart displays positive correlations in red and negative correlations in blue. The heatmap below details individual correlation values with a color gradient from red (positive) to blue (negative). Compounds are listed vertically, and bacterial genera are listed horizontally.
FIGURE 4
Spearman correlation heatmap illustrating associations between neutral flavor constituents of tobacco leaves undergoing accelerated aging and bacterial taxa (abundance > 0.1%). The heatmap is ordered by average correlation coefficient. Red indicates positive correlations, blue negative correlations, and * denotes P < 0.05.
A four-part image showcases bacterial analysis. Part A is a table comparing ASV sequences with strains, showing identity and similarity percentages. Part B is a bubble chart depicting enzyme activity in micrograms per milliliter per minute for various isolates. Part C is a phylogenetic tree illustrating relationships among different bacterial strains. Part D shows a petri dish with bacterial colonies grown on an agar plate.
FIGURE 5
Isolation and identification of Bacillus species from aged tobacco leaves. (A) Matching results between the 16S rRNA sequences of isolated strains and the ASV sequences in 16S rRNA gene amplicon sequencing of tobacco leaves. (B) Amylase activity, protease activity, cellulase activity, and pectinase of isolated Bacillus species. (C) Evolutionary tree relationship between strain TB-1 and Bacillus ASV of aged tobacco leaves. (D) Plate colony morphology of Bacillus velezensis TB-1.
Heatmap, bar chart, and stacked bar chart illustrating volatile compounds analysis. Panel A: Heatmap shows compound concentration in log scale across samples. Panel B: Green bar chart represents VIP scores for various compounds. Panel C: Stacked bar chart depicts concentration of compound classes like alcohols, acids, and others across control and TB samples.
FIGURE 6
(A) Heatmap of volatile compound composition in Bacillus velezensis TB-1 fermented tobacco leaves. (B) Volatile compounds with VIP value of > 1 after PLS-DA analysis. (C) Relative content of various compounds (μg/kg).
Radar charts labeled A and B compare sensory attributes. Chart A shows attributes like herb, caramel sweet, and burnt with red (TB-1) and blue (Control) lines. Chart B measures aroma quality, fineness, aftertaste, and other attributes. Red indicates TB-1 and blue indicates Control. Both charts use a star rating system.
FIGURE 7
Sensory evaluation of Bacillus velezensis TB-1 fermented tobacco leaves. (A) Sensory odor radar chart of flavor characteristics in different treatment groups,“*,” significant (P ≤ 0.05); “**,” highly significant (P ≤ 0.01); “***,” very highly significant (P ≤ 0.001). (B) Radar chart of smoking evaluation scores of different treatment groups.
See this image and copyright information in PMC

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