The first-cured tobacco contains macromolecular substances with negative impacts on tobacco products quality, and must be aged and fermented to mitigate their effects on the tobacco products quality. However, the natural fermentation takes a longer cycle with large coverage area and low economic efficiency. Microbial fermentation is a method to improve tobacco quality. The change of chemical composition of tobacco during the fermentation is often correlated with shapes of tobacco. This study aimed to investigate the effects of tobacco microorganisms on the quality of different shapes of tobacco. Specifically,Bacillus subtilisB1 andCytobacillus oceanisediminisC4 with high protease, amylase, and cellulase were isolated from the first-cured tobacco, followed by using them for solid-state fermentation of tobacco powder (TP) and tobacco leaves (TL). Results showed that strains B1 and C4 could significantly improve the sensory quality of TP, enabling it to outperform TL in overall texture and skeleton of tobacco products during cigarette smoking. Compared with the control, microbial fermentation could increase reducing sugar; regulate protein, starch, and cellulose, reduce nicotine, improve total aroma substances, and enable the surface of fermented TP and TL to be more loose, wrinkled, and porous. Microbial community analysis indicated that strains B1 and C4 could change the native structure of microbial community in TP and TL. LEfSe analysis revealed that the potential key biomarkers in TP and TL wereBacilli,Pseudonocardia, Pantoea, andJeotgalicoccus, which may have cooperative effects with other microbial taxa in improving tobacco quality. This study provides a theoretical basis for improving tobacco fermentation process for better cigarettes quality.Graphical Abstract

The composition and content of aroma substances in flue-cured tobacco (Nicotiana tabacum L.) will affect the quality of tobacco. To investigate the correlation between various aroma substances of K326 before and after flue-curing, and their impact on tobacco quality and diversity, this study employed the middle leaves of K326 and KRK26 as test materials. Samples were collected both before and after flue-curing for untargeted metabolomics analysis. The results of K326 showed that 584 metabolites were significantly different and there were 44 aroma-related metabolites, including alcohols, aldehydes, phenols, organic acids, etc. The analyzed aroma compounds consist of 37 known tobacco aroma substances, while 7 metabolites, previously not associated with tobacco aroma, have been identified as aroma substances in other food products. These findings suggest that these seven metabolites might may be potential tobacco aroma compounds. Further analysis showed that the content of phenols, alcohols, and aldehydes increased significantly after flue-curing, but the content of organic acids decreased. Furthermore, the analysis of KRK26 revealed a correlation between the quantity of aroma substances and the type of tobacco. These findings serve as a reference for enhancing the flue-curing process of K326 and optimizing the industrial production of cigarettes that use cured K326 tobacco leaves.

Soil sickness a severe problem in tobacco production, leading to soil-borne diseases and reduce in tobacco yield. This occurs as a result of the interaction between root exudates and rhizosphere microorganisms, which is however, little studied until now. By combining the field investigation and pot experiment, we found the output yield consistently decreased during the first 10 years of continuous cropping in a tobacco field, but increased at the 15th year (15Y). The root exudate and rhizosphere bacterial community was further analyzed to reveal the underlying mechanism of the suppressive soil formation. Root exudate of 15Y tobacco enriched in amino acids and derivatives, while depleted in the typical autotoxins including phenolic acids and alkaloids. This was correlated to the low microbial diversity in 15Y, but also the changes in community composition and topological properties of the co-occurrence network. Especially, the reduced autotoxins were associated with low Actinobacteria abundance, low network complexity and high network modularity, which significantly correlated with the recovered output yield in 15Y. This study revealed the coevolution of rhizosphere microbiota and root exudate as the soil domesticated by continuous cropping of tobacco, and indicated a potential role of the autotoxins and theirs effect on the microbial community in the formation of suppressive soil.

Introduction: Carotenoids are important precursors of various aroma components in tobacco and play an important role in the sensory quality of tobacco. Phaffia rhodozyma is a species of Xanthophyllomyces capable of synthesizing a highly valuable carotenoid-astaxanthin, but has not yet been used in improving tobacco quality.Methods: The dynamic changes of microbial community and metabolites during tobacco fermentation were analyzed in combination with microbiome and metabolome, and the quality of tobacco after fermentation was evaluated by sensory scores.Results: P. rhodozyma could grow and produce carotenoids in tobacco extract, with a maximum biomass of 6.50 g/L and a maximum carotenoid production of 36.13 mg/L at 100 g/L tobacco extract. Meanwhile, the correlation analysis combined with microbiome and metabolomics showed that P. rhodozyma was significantly positively correlated with 11 metabolites such as 6-hydroxyluteolin and quercetin. Furthermore, the contents of alcohols, ketones and esters, which were important aromatic components in fermented tobacco, reached 77.57 μg/g, 58.28 μg/g and 73.51 μg/g, increasing 37.39%, 265.39% and 266.27% compared to the control group, respectively. Therefore, the aroma and flavor, and taste scores of fermented tobacco increased by 0.5 and 1.0 points respectively.Discussion: This study confirmed that P. rhodozyma fermentation could effectively improve the sensory evaluation of tobacco, and provided a novel microbial fermentation method to improve tobacco quality.

Introduction: During tobacco growth and development, variations in environmental conditions and cultivation practices can lead to the emergence of low-quality tobacco leaves (multi fertilizer and reviving tobacco leaves). Although most experiments have focused on the late roasting process of tobacco and related metabolites, few have reported the physiological and biochemical changes in the internal development stage of tobacco. Methods: Comparative analyses of physiological, colorimetric, pigment, and routine chemical parameters across normal mature tobacco leaves and low-quality tobacco leaves revealed significant disparities in growth vigor, chromatic evolution, pigment accumulation, and primary metabolite profiles. At the same time, the ultrastructural changes of leaf cells were observed by electron microscope to reveal the dysfunction at the cellular level. Results: This study investigated physiological and biochemical differences between normal mature tobacco leaves and low-quality tobacco leaves during their growth stages. Results showed that normal mature tobacco leaves exhibited significantly lower a* values but higher L* and b* values compared to low-quality tobacco leaves. Additionally, the contents of β-carotene, lutein, and chlorophylls a/b in three tobacco types decreased through developmental stages. Normal mature tobacco leaves exhibited lower plastid pigments than low-quality tobacco leaves at all stages. The contents of starch, reducing sugar, and total sugar in normal mature tobacco leaves reached the highest levels of 45.98%, 25%, and 27.65%, respectively, which are generally higher than those in low-quality tobacco leaves. At the same time, the contents of protein, total phytoalkaloid, and total nitrogen in most parts of normal mature tobacco leaves were the lowest, reaching as low as 2.7%, 0.07%, and 0.97%. Chlorine and potassium levels were more balanced in normal mature tobacco leaves than in low-quality ones. Discussion: Different quality tobacco leaves show different growth characteristics and chemical composition changes in the development process. Future research should investigate cultivation practices (e.g., fertilization timing, topping protocols) to optimize leaf phenotype and quality, thereby informing sustainable tobacco production strategies.