Isolation of enzyme-producing microbial strains and their effects on the color and quality of tobacco stems.

China is one of the world's major tobacco producers, yielding more than 2.2 million tons of tobacco leaves annually, among which approximately 25-30% constitute by-products such as tobacco stems. Dark-colored stems are favored for their high contents of reducing and total sugars, porous structure, thermal stability, and rich aroma precursors; however, their limited availability constrains large-scale industrial utilization. In contrast, light-colored stems are frequently discarded because of inadequate natural aging, resulting in significant resource waste. To enhance stem utilization and improve cigarette quality, enzyme-producing strains were isolated from dark-colored stems (cellulase: C8, Z16; polyphenol oxidase: A59, Z1), and a synthetic microbial community (C8-Z1) was established to ferment light-colored stems. Amplicon-based high-throughput sequencing and UPLC-QTOF-MS non-targeted metabolomics were utilized to comprehensively characterize microbial succession, the dynamics of volatile aroma compounds, and their correlations with dominant genera during fermentation. The results demonstrated that microorganisms facilitated stem darkening and aroma enhancement through macromolecule degradation, enzymatic browning, and modulation of pigment metabolism. Key genera, including Bacillus, Aspergillus, and Filobasidium were identified as major contributors to aroma formation and color regulation. This study elucidates the microbial enzymatic mechanisms underlying the improvement of stem quality and offers theoretical as well as technical support for reducing the aging period and enhancing the industrial value of tobacco stems.