Integrated multi-omics reveals microbial and metabolic mechanisms driving enhanced fermentation quality in cigar tobacco leaves with exogenous additives

Natural exogenous additives (EA) suitable for the tobacco fermentation need to be developed to enhance the fermentation quality and economic value of low-grade cigar tobacco leaves (CTLs). This study analyzed the impacts of three compound Chinese herbal medicine (CHM) on metabolites and microorganisms during CTLs fermentation. The results manifested that EA facilitated the degradation of total sugar, starch and protein, while enhancing the accumulation of reducing sugar in CTLs. Furthermore, EA raised contents of free amino acids (FAAs), while Asp, Glu, Ser and His were found to be key differential FAAs of CTLs. During fermentation, the total contents of volatile flavor components (VFCs) initially increased and then declined. Furthermore, EA contributed to more harmonious compositions of VFCs by promoting the formation of neophytadiene, ketones, esters and aldehydes, as well as facilitating nicotine degradation. According to variable importance in the projection (VIP) > 1 and odor activity value (OAV) > 1, 7 key differential VFCs were identified. EA enhanced positive microbial interactions and led to a more stable and coordinated symbiotic network. Linear discriminant analysis effect size (LEfSe) identified 9 genera as differentially dominant microorganisms in CTLs, which were closely associated with chemical compositions and key differential flavor metabolites. In addition, EA promoted cigar tobacco characteristics (CTCs) by altering bacterial alpha diversity and influencing the assembly of dominant microbial communities. Overall, this study offered theoretical insights into the innovative applications of CHM in CTLs fermentation, and presented new perspectives for enhancing CTLs quality and customizing flavor profiles.