Study on tobacco quality improvement and bacterial community succession during microbial co-fermentation

The high levels of starch, cellulose and protein in tobacco can produce a strong irritating smoke and diminish its flavor and overall quality, but the tobacco quality can be improved by microorganisms abundant in tobacco during fermentation. In this study, we screened and identified three strains from tobacco with high amylase, cellulase, and protease activities. The co-fermentation of Bacillus velezensis A2 and Bacillus endophyticus A4 could effectively reduce the content of starch, cellulose and protein in tobacco, leading to an increase in the content of reducing sugar and alterations in the surface of fermented tobacco, making it looser, more wrinkled, and porous, coupled with the potential to mitigate the presence of the harmful substance nicotine. Additionally, compared with the control, A2 +A4 co-fermentation increased the content of aroma components by 108.33%, and remarkably improved the sensory quality of Heat-not-Burn (HnB) cigarettes. Moreover, Bacillus, Pseudomonas and Klebsiella were genera with high abundance before and after A2 +A4 co-fermentation, but Bacillus gradually became the dominant genus, with its relative abundance increased from 0.49% to 32.65%. LEfSe analysis identified Bacillus as a crucial biomarker in tobacco after microbial co-fermentation. PICRUSt analysis showed that microbial co-fermentation could promote the metabolic pathways associated with substrate utilization. This study provided new strains and a theoretical basis for optimizing the fermentation process to improve the quality of tobacco for HnB cigarettes.