Traditional tobacco-leaf fermentation relies heavily on the spontaneous activity of microorganisms naturally present in the environment. These microorganisms participate in the transformation of sugars, proteins and other chemical components and can influence the aroma and sensory characteristics of tobacco leaves. However, natural fermentation can be lengthy and strongly affected by variations in environmental conditions and raw materials, making consistent fermentation outcomes difficult to achieve.
Seeking a more controllable microbial approach, researchers from Guizhou University, China Tobacco Guizhou Industrial Co., Ltd., and Guizhou Tobacco Company Zunyi Branch turned to a natural microbial source outside the tobacco environment: grapes.
The team isolated three yeast strains and four bacterial strains from the surface of commercially available Kyoho grapes. When the cultures were compared by sensory evaluation, only one yeast strain, designated PT-1, produced a pronounced fruity aroma accompanied by a mild wine-like note. Morphological characterization and rDNA-ITS sequence analysis subsequently identified PT-1 as Debaryomyces hansenii .
D. hansenii is a non-Saccharomyces yeast capable of adapting to a range of fermentation environments. It has previously attracted attention in fermented foods including cheese, fermented meat products and fruit wines. In the new study, the researchers investigated whether a D. hansenii strain obtained from fruit could also be used for directed tobacco-leaf fermentation.
The researchers prepared a PT-1 yeast suspension at 1×10 8 CFU/mL and sprayed it evenly onto redried tobacco leaves at an application rate equivalent to 4% of the leaf mass. The leaves were then fermented for three days at 30 °C and a relative humidity of 70±2%. The team subsequently measured chemical composition and sensory quality and used high-throughput sequencing to characterize changes in bacterial and fungal communities.
The results showed that PT-1 rapidly established itself on the tobacco-leaf surface.
Following fermentation, Debaryomyces accounted for 99.66% of the fungal community in the treatment group, making it overwhelmingly dominant. Fungal Shannon diversity was significantly lower than in untreated controls, indicating a pronounced simplification of the fungal community. According to the researchers, this result suggests that PT-1 has a strong ability to establish itself on the leaf surface and may influence resident fungi through resource competition or other microbial interactions.
The bacterial community was more stable. PT-1 treatment did not significantly change bacterial Shannon diversity, although analyses of overall community composition revealed separation between treated and control samples. The findings indicate that the intervention primarily affected the fungal community while potentially producing more moderate changes in bacterial community structure.
The microbial changes were accompanied by shifts in tobacco-leaf chemistry and sensory scores. Total sugar increased significantly from 18.27% in the control group to 19.12% after PT-1 treatment, an increase of 0.85 percentage points. Reducing sugar rose from 15.27% to 15.73%. Total alkaloids and total nitrogen, meanwhile, showed modest decreases, from 3.83% to 3.76% and from 2.44% to 2.39%, respectively.
Professional sensory evaluation also indicated improvements in several characteristics. PT-1-treated samples received higher scores for aroma quality, sweetness and aftertaste, while scores reflecting off-notes and irritation also improved. The overall sensory score increased significantly from 33.93 in the control group to 36.03 after PT-1 treatment.
“One distinctive aspect of this study is that it expands the search for functional microorganisms beyond tobacco leaves and tobacco-processing environments to the surface of fruit,” said corresponding author Xiao Zou of the College of Life Sciences at Guizhou University. “PT-1, isolated from Kyoho grapes, not only showed a clear fruity aroma but also rapidly established itself during short-term tobacco fermentation. This gives us another direction for exploring new aroma-producing microbial resources.”
Correlation analysis provided further clues to the relationship between the fungal community and tobacco quality. Debaryomyces was positively correlated with total sugar, reducing sugar, aroma quality and sweetness, while showing negative correlations with total nitrogen, total alkaloids and sensory strength. The researchers caution that these correlations do not establish direct causality, and functional experiments will be needed to determine the specific metabolic mechanisms involved.
The team suggests that the potential value of PT-1 extends beyond its aroma-producing properties. A defined functional strain could provide a more controllable microbial intervention during fermentation. Compared with fermentation that depends entirely on naturally occurring microbial communities, directed fermentation with a selected and characterized strain may ultimately help reduce environmental variability and improve process consistency.
“An important next question is whether the advantages observed during this short fermentation period can be maintained through longer aging periods and reproduced consistently at a larger scale,” said corresponding author Jianyu Gou of Guizhou Tobacco Company Zunyi Branch. “Pilot-scale studies, longer-term aging experiments and further characterization of key metabolites will be important for evaluating the practical potential of PT-1.”
The researchers emphasize that the present study is an initial evaluation of a functional microbial inoculant during a short tobacco-aging stage. The findings cannot yet be extrapolated to long-term aging or the stability of final products, and longer-term and larger-scale studies are still required. The study evaluated leaf chemistry, microbial communities and sensory characteristics; it did not investigate combustion products or health risks associated with smoking.
Overall, the study identifies a new candidate microbial resource for tobacco fermentation from the surface of grapes and shows how D. hansenii PT-1 can influence microbial community structure and quality-related indicators during short-term fermentation. The findings provide an experimental basis for further development of efficient and controllable functional microbial fermentation systems.
ACTA MICROBIOLOGICA SINICA
Screening of fruit-flavored yeast strains and their fermentation effects on the microbial community of tobacco leaves
4-Oct-2026