Microbial-enzyme synergistic treatment stabilizes surface microbial communities and enhances flavor quality during tobacco leaf aging

Abstract

Tobacco aging is a critical for developing desirable flavor profiles, driven primarily by microbial and enzymatic activities. This study systematically evaluated the effects of Bacillus clausii inoculation (FJ) and B. clausii-cellulase co-treatment (JM) on the surface microbial communities and aroma compounds of Yunyan 87 tobacco leaves during 9 months of aging, with natural aging as the control (CK). High-throughput 16S rRNA sequencing revealed that the JM treatment significantly increased microbial diversity and enhanced the structural similarity of microbial communities across different aging stages compared with the CK and FJ treatments, while also promoting the proliferation of beneficial taxa (e.g., Bacteroidota, Cyanobacteria). PICRUSt functional prediction revealed that JM enriched metabolic pathways related to carbohydrate metabolism (18.2–20.6%), amino acid metabolism (15.1–16.7%), and metabolism of cofactors and vitamins (16.5–17.7%)–key pathways for flavor precursor conversion. A total of 29 key aroma compounds (odor activity value > 1) were significantly influenced the quality of tobacco leaves, with JM significantly increasing total volatile content (317.1–388.8 μg/g vs. 124.7–143.0 μg/g in CK and 283.2–300.8 μg/g in FJ). Notably, JM promoted the accumulation of esters (ethyl palmitate, ethyl linoleate) and ketones (4,7,9-megastigmatrien-3-one, damascenone), contributing desirable fruity, floral, and sweet notes. Correlation analysis linked Pseudomonas, Bacillus, and Acinetobacter to the formation of key volatiles. These findings demonstrate that microbial-enzyme co-fermentation enriched desirable aromas, and stabilized microbial communities, providing a practical and efficient strategy for industrial production of high-quality aged tobacco.

Graphical abstract

Keywords

Tobacco aging / Microbial community / Volatile compounds / Microbial-enzyme co-fermentation

References

[1] Cai W, Zhang QY, Zhu PC, Hu WR, An HY, Huo DQ, Li DL. Study on dynamic changes and correlations of microbial diversity, key enzyme activity and conventional components in tobacco leaves during the aging process. Bioresour Bioprocess, 2025, 12(1): 130

[2] Di Bella JM, Bao Y, Gloor GB, Burton JP, Reid G. High throughput sequencing methods and analysis for microbiome research. J Microbiol Methods, 2013, 95(3): 401-414

[3] Fang X, Qin YQ, Liu TT, Guo SP, Wu CD, Zhang RN, Zhong Q, Lei YK, Shi HZ, Zhou RQ, Zeng SH, Jin Y. Roles of cigar microbes in flavor formation during roasted-rice leachate fermentation. Appl Microbiol Biotechnol, 2024, 108(1): 457

[4] Hao J, Wang XS, Li LQ, Sun ZK, Li XW, Ji Q, Huang XH, Qin L. A multi-omics fusion of metabolomics, lipidomics, and flavoromics to analyze the effect of enzyme treatment on tobacco quality. Ind Crop Prod, 2025, 226 120721

[5] Hu HY, Liu YL, Huang YQ, Zhang Z, Tang HZ. The leaf microbiome of tobacco plants across eight Chinese provinces. Microorganisms, 2022, 10(2): 450

[6] Jiang CD, Kong DC, Li YY, Sun JG, Chen ZG, Yang MF, Cao ST, Yu CF, Wang ZY, Jiang JZ, Zhu CG, Zhang N, Sun GW, Zhang Q. Degradation and mechanism analysis of protein macromolecules by functional bacteria in tobacco leaves. Front Microbiol, 2024, 15: 1416734

[7] Li JJ, Zhao YY, Qin YQ, Shi HZ. Influence of microbiota and metabolites on the quality of tobacco during fermentation. BMC Microbiol, 2020, 20(1): 356

[8] Li WL, Yu J, Li H, Yang CL, Peng Z, Zhang J. The dynamics of microbial community structure and metabolic function in different parts of cigar tobacco leaves during air-curing. Front Microbiol, 2024, 15: 1438566

[9] Liu F, Wu ZY, Zhang XP, Xi GL, Zhao Z, Lai M, Zhao MQ. Microbial community and metabolic function analysis of cigar tobacco leaves during fermentation. MicrobiologyOpen, 2021, 10(2): e1171

[10] Liu Z, Xu GX, Song CH, Zuo W, Li SY, Gui C, Liu N, Hu P, Yang J, Zhou GF, Zhang W, Chen YB. Effect of steam explosion on the structure, chemical composition and pyrolytic volatile composition of tobacco waste. J Biobased Mater Bioenergy, 2024, 18(1): 106-114

[11] Mai J, Zhu MJ, Hu BB, Zhang H, Liu ZH, Sun JF, Hu Y, Zhao L. Effects of Phaffia rhodozyma on microbial community dynamics and tobacco quality during tobacco fermentation. Front Microbiol, 2024, 15: 1451582

[12] Mai J, Ning Y, Lin ZL, Zhang H, Sun JF, Chen Y, Jiang YL, Ma YF, Zhu MJ, Hu BB. Diversity of adapted tobacco microbial community and its application in improving tobacco quality. Ind Crops Prod, 2025, 234: 121580

[13] Pan J, Huang MD, Wang J, Zhao JX, Yang B, Yang HH, Huang JS, Su YL, Song XR, Wang WG, Bu LD. Unraveling the microbiome–aroma nexus: a metagenomic and volatile compound analysis of Yunnan cigars. Front Microbiol, 2025, 16: 1597501

[14] Pei Q, Jiang X, Li ZQ, Xu H, Xie MY, Xiong T, Liu ZG. Study on quality enhancement during cigar tobacco fermentation by Staphylococcus nepalensis: insights into microbial community, volatile substances and sensory evaluation. Front Microbiol, 2025, 16: 1526178

[15] Ren MJ, Zhao YY, Zhou T, Zhang RN, Li JY, Zhang LY, Qin YQ, Shi HZ. Integrated 16S rDNA, UPLC-MS, and GC-MS analyses to investigate the effects of roasted-rice leachate fermentation on flavor formation in cigar tobacco leaves. Food Biosci, 2025, 73: 107723

[16] Ruan YA, Xu SG, Tang ZX, Liu XL, Zhang QR, Chen ZB. Microbial diversity in tobacco rhizosphere soil at different growth stages. J Biobased Mater Bioenergy, 2021, 15(5): 606-614

[17] Shan XJ, Jin LF, Li F, Yang SB, Zhang XJ, Chai LJ, Gong JS, Shi JS, Lu ZM, Hu ZY, Xu ZH. Isolation of indigenous Bacillus velezensis from aging tobacco leaves for improving the flavor of flue-cured tobacco. Front Microbiol, 2025, 16: 1623279

[18] Shu M, Yang Y, Pan FD, Bian TF, Li Q, Liao F, He WM, Li ST, Xu J, Hu T, Qiao P, Zhong WH. Effects of the multi-stress-resistant strain Zygosaccharomyces parabailii MC-5K3 bioaugmentation on microbial communities and metabolomics in tobacco waste extract. Arch Microbiol, 2023, 205(8): 299

[19] Shu M, Xue HJ, Yang Y, Zhang X, Li ST, Bian TF, Yuan KL, Xu CP. Microbial-enzyme co-fermentation of low-grade tobacco: metagenomics and metabolomic insights into flavor formation. Enzyme Microb Technol, 2026, 194: 110803

[20] Van Gemert LJ. Odour thresholds, compilations of odour threshold values in air, water and other media, 2011, 2Zeist. Published by Oliemans Punter & Partners BV2

[21] Wang F, Jin YM, Chen XN, Zhang Y, Jiang XL, Zhang G, Chen GQ, Yang MJ, Leng FF, Li HT, Wu LJ, Zhang HB. The diversity, structure and function of microbial communities changes across aging process of tobacco leaves. Environ Res Commun, 2022, 4(9): 095012

[22] Wang HQ, Guo DF, Zhang MZ, Wu GL, Shi YQ, Zhou JL, Ding NH, Chen XS, Li XJ. Correlation study on microbial communities and volatile flavor compounds in cigar tobacco leaves of diverse origins. Appl Microbiol Biotechnol, 2024, 108(1): 236

[23] Wang YQ, Fan JQ, Chen YQ, Chen SY, Deng XH, Huang HM, Wu LX, Ni L, Long T, He W, Jiang ZK, Cui ZW, Huang YJ, Li JJ, Zhang W. Correlation between microbial diversity and flavor substances on the surface of flue-cured tobacco. Anal Lett, 2024, 57(8): 1197-1214

[24] Wei JB, Song K, Zang ZP, Yang HJ, Gao YZ, Zhang JD, Wang Z, Liu C. Influence of specific tobacco endophytic Bacillus on tobacco leaf quality enhancement during fermentation. Front Microbiol, 2024, 15: 1468492

[25] Weng SN, Deng MZ, Chen SY, Yang RQ, Li JJ, Zhao XB, Ji SH, Wu LX, Ni L, Zhang ER, Wang CC, Qi LF, Liao KQ, Chen YQ, Zhang W. Application of pectin hydrolyzing bacteria in tobacco to improve flue-cured tobacco quality. Front Bioeng Biotechnol, 2024, 12: 1340160

[26] Wu XY, Cai W, Zhu PC, Peng Z, Zheng TF, Li DL, Li JH, Zhou GY, Du GC, Zhang J. Profiling the role of microorganisms in quality improvement of the aged flue-cured tobacco. BMC Microbiol, 2022, 22(1): 197

[27] Wu QY, Peng Z, Pan Y, Liu LP, Li LL, Zhang J, Wang J. Interaction analysis of tobacco leaf microbial community structure and volatiles flavor compounds during cigar stacking fermentation. Front Microbiol, 2023, 14: 1168122

[28] Wu GL, Zhang MZ, Han P, Guo DF, Shi YQ, Mu DD, Li XJ, Wu XF. Microbial community succession patterns and metabolite profiles in cigar tobacco during different mildew stages. Ind Crop Prod, 2024, 222: 120005

[29] Wu P, Zong DY, Yang L, Jia XW, Qu LL, Wu YH, Xu CP. Volatile compounds and characteristic aroma perception in blue lotus (Nymphaea nouchali var. caerulea) three processed products: insights from flavoromics and molecular docking. LWT, 2025, 229: 118193

[30] Yin YM, Song XR, Cui YH, Zhang JL, Fu KJ, Zhou Q, Feng YX, Huang JK, Hu CL, Deng YS, Su YB. Application of starch-degradation bacteria in cigar tobacco leaf fermentation: effects on starch degradation, microbial communities and metabolic pathways. Front Microbiol, 2025, 16: 1632731

[31] Zhang QY, Geng ZZ, Li DL, Ding ZY. Characterization and discrimination of microbial community and co-occurrence patterns in fresh and strong flavor style flue-cured tobacco leaves. MicrobiologyOpen, 2020, 9(2): e965

[32] Zhang Q, Kong GH, Zhao GK, Liu J, Jin HG, Li ZH, Zhang GH, Liu T. Microbial and enzymatic changes in cigar tobacco leaves during air-curing and fermentation. Appl Microbiol Biotechnol, 2023, 107(18): 5789-5801

[33] Zhang LY, Mai J, Shi JF, Ai KB, He L, Zhu MJ, Hu BB. Study on tobacco quality improvement and bacterial community succession during microbial co-fermentation. Ind Crop Prod, 2024, 208: 117889

[34] Zhang MZ, Guo DF, Wang HQ, Wu GL, Shi YQ, Zhou JL, Zhao E, Zheng TF, Li XJ. Analyzing microbial community and volatile compound profiles in the fermentation of cigar tobacco leaves. Appl Microbiol Biotechnol, 2024, 108(1): 243

[35] Zhang MZ, Guo DF, Wang HQ, Wu GL, Shi YQ, Zhou JL, Zheng TF, Zhao EY, Wu XL, Li XJ. Comparative profiling of microbial communities and volatile organic compounds in fermented wrapper, binder, and filler cigar tobaccos. Chem Biol Technol Agric, 2024, 11(1): 68

[36] Zhang L, Li WL, Peng Z, Zhang J. Effect of microbial community on the formation of flavor components in cigar tobacco leaves during air-curing. BMC Microbiol, 2025, 25(1): 56

[37] Zhang W, Deng Q, Zhu BK, Xiao D, Chen QM, Pan HY, Chen J. Improving the quality of low-grade tobacco by enzymatic treatment and co-fermentation with yeast and lactic acid bacteria. Appl Biochem Biotechnol, 2025, 197(1): 613-630

[38] Zhang X, Li HY, Nie JG, Wu D, Huang QL. Unveiling flavor formation and variation in fermented vinasse grass carp based on the dynamic correlation of microbiota with metabolites by multi-omics and bioinformatics approaches. Food Chem, 2025, 487: 144730

[39] Zheng TF, Zhang QY, Li PH, Wu XY, Liu Y, Yang Z, Li DL, Zhang J, Du GC. Analysis of microbial community, volatile flavor compounds, and flavor of cigar tobacco leaves from different regions. Front Microbiol, 2022, 13 907270

[40] Zhu JJ, Wang JT, An ZC, Shen C, Dong HX, Wang HJ, Peng ZX, Yang B, Liu JJ, Wang XJ, Fang ZM. Microbial community succession during tobacco fermentation reveals a flavor-improving mechanism. Front Bioeng Biotechnol, 2025, 13: 1627842

Funding

Major Science and Technology Project of China National Tobacco Corporation(110202201005(JY-05))

Major Science and Technology Project of Henan Province(231100310200)

RIGHTS & PERMISSIONS

The Author(s)