Comprehensive evaluation and mechanisms of tobacco quality improvement via microbial fermentation

Microbial fermentation is an effective strategy to enhance the quality of conventional tobacco, and exploring its application in Heat-not-Burn (HnB) tobacco is of great industrial significance. This study investigated the fermentation process of tobacco using the highly enzyme-active microorganisms Bacillus velezensis A2 and Bacillus endophyticus A4. Principal component analysis combined with the entropy weight method was employed to comprehensively evaluate tobacco quality. The optimized fermentation conditions were determined as follows: a compound ratio of A2 to A4 at 1:2, an inoculum amount of 8 × 107 CFU/g, a fermentation time of 14 days, and a moisture content of 32 %. Fermentation significantly modified the chemical composition, microbial communities, and metabolite profiles of tobacco. In particular, the enrichment of Bacillus was positively associated with increased relative abundances of key aroma-active metabolites, including butanoic acid, butyl ester, acetic acid, 4-methylphenyl ester, and p-cresol. These compounds exhibited higher relative odor activity values (rOAV), contributing fruity, sweet, and phenolic notes that enhanced the overall aroma and smoke smoothness of tobacco. This integrative approach not only elucidates the mechanistic role of microbial fermentation but also provides practical guidance for the process optimization of HnB tobacco products.