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

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.