Influence of aging methods on the volatile aroma compounds of tobacco leaves and the structure of surface microbial communities

Tobacco aging is a critical process for developing desirable flavor profiles, which is largely driven by microbial and enzymatic activities. This study systematically investigated the effects of bacterial inoculation ( Bacillus clausii ) and bacterial-enzyme co-treatment (with cellulase) on the surface microbial communities and flavor compounds of Yunyan 87 tobacco leaves during a 9-month aging period. High-throughput 16S rRNA sequencing revealed that the microbial-enzyme co-treatment (JM) significantly enhanced microbial diversity and community stability compared to the control (CK) and bacterial-only (FJ) treatments. PICRUSt functional prediction indicated a significant enrichment in metabolic pathways, particularly carbohydrate metabolism, amino acid metabolism, and metabolism of cofactors and vitamins. Gas chromatography–mass spectrometry (GC–MS) analysis identified 29 key aroma compounds, demonstrating that the JM treatment effectively promoted the accumulation of esters and ketones—such as ethyl palmitate, 4,7,9-megastigmatrien-3-one, and damascenone—which contribute desirable fruity, floral, and sweet notes. Correlation analysis further linked dominant bacterial genera (e.g., Pseudomonas , Bacillus , Acinetobacter ) with the formation of these characteristic volatiles. These findings demonstrate that microbial-enzyme co-fermentation is a promising strategy to accelerate the aging process and improve the flavor quality of tobacco, offering significant potential for industrial application.