Metabolomics-based study of chemical compositions in cellulase additives derived from a tobacco-origin Bacillus subtilis and their impact on tobacco sensory attributes.

To enhance the quality of tobacco leaves and optimize the smoking experience, diverse strains of functional bacteria and their associated metabolites have been used in tobacco aging. Exogenous cellulase additives are frequently employed to facilitate the degradation of cellulose and other macromolecular matrices and enhance the quality of the tobacco product. However, little is known about how microbial metabolites present in exogenous enzyme additives affect tobacco quality. In this study, crude cellulase solutions, produced by a tobacco-originating bacterium Bacillus subtilis FX-1 were employed on flue-cured tobacco. The incorporation of cellulase solutions resulted in the reduction of cellulose crystallinity in tobacco and the enhancement of the overall sensory quality of tobacco. Notably, tobacco treated with cellulase obtained from laboratory flask fermentation demonstrated superior scent and flavor attributes in comparison to tobacco treated with enzymes derived from industrial bioreactor fermentation. The targeted and untargeted metabolomic analysis revealed the presence of diverse flavor-related precursors and components in the cellulase additives, encompassing sugars, alcohols, amino acids, organic acids, and others. The majority of these metabolites exhibited significantly higher levels in the flask group compared to the bioreactor group, probably contributing to a pronounced enhancement in the sensory quality of tobacco. Our findings suggest that the utilization of metabolic products derived from B. subtilis FX-1 as additives in flue-cured tobacco holds promise as a viable approach for enhancing sensory attributes, establishing a solid theoretical foundation for the potential development of innovative tobacco aging additives.

Application of starch degrading bacteria from tobacco leaves in improving the flavor of flue-cured tobacco.

Starch is an essential factor affecting the quality of flue-cured tobacco, and high starch content can affect the sensory quality and safety. Recently, the degradation of macromolecules in tobacco raw materials by using additional microorganisms to improve their intrinsic quality and safety has become a new research hotspot in the tobacco industry. However, the technical maturity and application scale are limited. Our study analyzed the correlation between microbial community composition and volatile components on the surface of tobacco leaves from 14 different grades in Fujian tobacco-producing areas. The PICRUSt software was utilized to predict the function of the microbial community present in tobacco leaves. Furthermore, dominant strains that produced amylase were screened out, and an enzyme solution was prepared to enhance the flue-cured tobacco flavor. Changes in the content of macromolecules and volatile components were determined, and sensory evaluations were conducted to assess the overall quality of the tobacco leaves. The results showed that the dominant bacterial genera on the surface of Fujian tobacco leaves were Variovorax, Sphingomonas, Bacillus, etc. Bacillus was positively correlated with various volatile components, which contributed to the sweet and aromatic flavors of Fujian flue-cured tobacco. The main genetic functions of Fujian flue-cured tobacco surface bacteria were carbohydrate metabolism and amino acid metabolism. After treating flue-cured tobacco with an enzyme preparation prepared by the fermentation of Paenibacillus amylolyticus A17 #, the content of starch, pectin, and cellulose in flue-cured tobacco decreased significantly compared with the control group. Meanwhile, the content of total soluble sugar and reducing sugar was significantly increased, and the volatile aroma components, such as 3-hydroxy--damascone, 2,3-dihydro-3,5-dihydroxy-6-methyl-4 H-Pyran-4-one, ethyl palmitate, ethyl linolenic acid, etc., were significantly increased. The aroma quality and quantity of flue-cured tobacco were enhanced, while impurities were reduced. The smoke characteristics were improved, with increased fineness, concentration, and moderate strength. The taste characteristics were also improved, with reduced irritation and a better aftertaste. In conclusion, Bacillus, as the dominant genus in the abundance of bacterial communities on tobacco surfaces in Fujian, had an essential impact on the flavor of tobacco leaves by participating in carbohydrate metabolism and finally forming the unique flavor style of flue-cured tobacco in Fujian tobacco-producing areas. Paenibacillus amylolyticus A17 #, a target strain with amylase-producing ability, was screened from the surface of Fujian flue-cured tobacco. The enzyme preparation, produced by the fermentation of Paenibacillus amylolyticus A17 #, was utilized to reduce the content of macromolecules, increase the content of water-soluble total sugar and reducing sugar, and produce a variety of crucial volatile aroma components, which had a significant improvement on the quality of tobacco leaves.