Study on the Relationship of Metabolite Changes Driven by Bacterial Community Succession During Cigar Tobacco Leaf Fermentation

Objective This study aims to elucidate the intrinsic relationship between bacterial communities and metabolite changes during cigar tobacco leaf fermentation, and to reveal the regulatory mechanisms underlying bacterial-driven metabolite transformation.

Method Using 'Haiyan 200' cigar tobacco leaves as materials, metabolomics and 16S rDNA sequencing technologies were employed to characterize metabolite dynamics and bacterial community succession at three fermentation stages (0 d, 20 d, and 40 d). Multi-omics integration was performed through co-occurrence network analysis, procrustes analysis, and correlation analysis to uncover bacterial-driven metabolic regulation from both global association and module–metabolite interaction levels.

Result A total of 475 and 153 differential metabolites were identified in the early and late fermentation stages, respectively. These metabolites were mainly assigned to five classes: flavonoids, lipids, phenolic acids, alkaloids, and amino acids and their derivatives, with opposite accumulation patterns between the two stages. Bacterial community succession exhibited distinct stage-specific characteristics. At the phylum level, Proteobacteria dominated throughout the fermentation process, while Bacteroidota was significantly enriched in the late stage. At the genus level, Ralstonia was identified as the core bacterial genus during the whole fermentation process. The bacterial co-occurrence network showed high modularity (0.504) in the early stage, representing functionally specialized local cooperation, whereas modularity decreased to 0.290 in the late stage, indicating network integration and a shift toward global coordination. Procrustes analysis verified a strong coupling between bacterial community succession and metabolite variation, with improved fitting in stage-specific analysis. Correlation analysis revealed specific associations between bacterial modules and particular metabolite categories at different fermentation stages, and the function of the core genus Ralstonia shifted in an environment-dependent manner.

Conclusion The metabolic evolution during cigar tobacco leaf fermentation is jointly driven by orderly bacterial community succession and network structural reorganization. In the early stage, highly modularized bacterial communities achieve efficient substrate transformation through metabolic division of labor, while in the late stage, network integration and functional redundancy maintain metabolic system stability. This study advances the understanding of cigar tobacco leaf fermentation mechanisms from the perspective of bacterial ecological interactions and provides a theoretical basis for optimizing cigar tobacco leaf fermentation processes through microbial regulation.