Despite the booming international trade in cigar tobacco leaves (CTLs), the main characteristics of tobacco leaves from different producing areas are rarely reported. This study aimed to characterize the microbial community, volatile flavor compounds (VFCs), and flavor of CTLs from four famous cigar-producing areas, including Dominica, Brazil, Indonesia, and China. High-throughput sequencing results showed that the dominant genera in CTLs wereStaphylococcus, Pseudomonas, Aspergillus, Sampaiozyma, andAlternaria. Sensory analysis revealed that Indonesian and Chinese CTLs were characterized by leathery, peppery, and baked aroma. Brazilian CTLs were dominated by caramel and herb aroma. Dominican CTLs had aromas of milk, fruity, sour, cream, flower, nutty, and honey. Supplemented with the determination of volatile flavor compounds (VFCs), the flavor of CTLs could be scientifically quantified. Most of these VFCs were aldehydes and ketones, and 20 VFCs showed significant differences in CTLs from different regions. The microbial community, VFCs, and flavor of CTLs vary widely due to geographic differences. Network analysis revealed the microbial community was closely related to most VFCs, but the relationships between the fungal community and VFCs were less than the bacterial community, and most of them were negative. Furthermore, it also found that the bacterial community had a greater contribution to the flavor of CTLs than the fungal community. This study obtained essential information on CTLs, which laid a foundation for deeply excavating the relationship between microbes and VFCs and flavor, and establishing a tobacco information database.

Fermentation process plays an important role in the biochemical properties and quality of cigar tobacco leaves (CTLs). In industry, exogenous additive (EA) was usually adopted for improving the quality of CTLs during fermentation. However, the mechanism of enhanced quality of CTLs fermented with EA was confused. Herein, the chemical compositions and microbial communities of CTLs during fermentation with EA were analyzed. The increased contents of total nitrogen and total sugar, as well as the improved consumption rate of reducing sugar in CTLs were found with the addition of EA. Besides, fermentation with EA reduced the content of total nonvolatile organic acid, especially unsaturated fatty acid. The contents of total and several representative aroma components were improved. Additionally, the increased abundance of Staphylococcus and decreased abundance of Aspergillus were detected. Combined with the changes of chemical compositions and microbial communities, it was confirmed that the carbohydrates and alcohols originated from EA promote the enrichment of Staphylococcus and accelerate biochemical reactions, such as Maillard reaction and esterification reaction, thus improving the contents and quality of aroma components in CTLs. This study demonstrated the mechanism of enhanced quality of CTLs fermented by EA, which provides more ideas for developing novel and efficient EAs.

Metabolic activity of the microbial community greatly affects the quality of cigar tobacco leaves (CTLs). To improve the quality of CTLs, two extrinsic microbes (Acinetobacter sp. 1H8 and Acinetobacter indicus 3B2) were inoculated into CTLs. The quality of CTLs were significantly improved after fermentation. The content of solanone, 6-methyl-5-hepten-2-one, benzeneacetic acid, ethyl ester, cyclohexanone, octanal, acetophenone, and 3,5,5-trimethyl-2-cyclohexen-1-one were significantly increased after inoculated Acinetobacter sp. 1H8. The inoculation of Acinetobacter sp. 1H8 enhanced the normal evolutionary trend of bacterial community. The content of trimethyl-pyrazine, 2,6-dimethyl-pyrazine, and megastigmatrienone were significantly increased after inoculated Acinetobacter indicus 3B2. The inoculation of Acinetobacter indicus 3B2 completely changed the original bacterial community. Network analysis revealed that Acinetobacter was negatively correlated with Aquabacterium, positively correlated with Bacillus, and had significant correlations with many volatile flavor compounds. This work may be helpful for improving fermentation product quality by regulating microbial community, and gain insight into the microbial ecosystem.

Introduction: The main goal of tobacco fermentation technology is to minimize the alkaloid content while improving flavor substance content.Methods: This study revealed the microbial community structure and their metabolic functions during cigar leaf fermentation by high-throughput sequencing and correlation analysis, and evaluated the fermentation performance of functional microbes based on in vitro isolation and bioaugmentation fermentation.Results: The relative abundance of Staphylococcus and Aspergillus increased first but then decreased during the fermentation, and would occupy the dominant position of bacterial and fungal communities, respectively, on the 21st day. Correlation analysis predicted that Aspergillus, Staphylococcus and Filobasidium could contribute to the formation of saccharide compounds, Bacillus might have degradation effects on nitrogenous substances. In particular, Candida, as a co-occurring taxa and biomarker in the later stage of fermentation, could not only degrade nitrogenous substrates and synthesize flavor substances, but also contribute to maintaining the stability of microbial community. Moreover, based on in vitro isolation and bioaugmentation inoculation, it was found that Candida parapsilosis and Candida metapsilosis could significantly reduce the alkaloids content and increase the content of flavor components in tobacco leaves.Discussion: This study found and validated the critical role of Candida in the fermentation of cigar tobacco leaves through high-throughput sequencing and bioaugmentation inoculation, which would help guide the development of microbial starters and directional regulation of cigar tobacco quality.

Background: Tobacco alcoholization is an important step in increasing the quality of tobacco leaf, which may convert a portion of low-grade tobacco leaves into useable product, however this may take to 2–3 years. The addition of exogenous microorganisms to tobacco leaves and treating them by biological fermentation can shorten the maturation time of tobacco leaves, and improve the quality and applicability of low-grade tobacco leavesMethods: Several strains were screened from low-grade tobacco by flow cytometry, including the bacteria Bacillus amyloliticus, with starch degradation ability and Bacillus kochii, with protein degradation ability, and the fungus Filobasidium magnum with lipid oxidase ability, and were inoculated onto tobacco leaves, both individually and in combination, for solid-state fermentationResults: The greatest improvement in tobacco quality was observed when strains 4# and 3# were applied at a ratio of 3:1. The Maillard reaction products, such as 2-amyl furan, 1-(2-furanmethyl) -1 h-pyrrole, furfural and 2, 5-dimethylpyrazine, were significantly increased, by up to more than 2 times. When strains F7# and 3# were mixed at a ratio of 3:1, the improvement of sensory evaluation index was better than that of pure cultures. The increase of 3-(3, 4-dihydro-2h-pyrro-5-yl) pyridine, β -damasone and benzyl alcohol was more than 1 times. The increase of 2-amyl-furan was particularly significant, up to 20 timesConclusion: The functional strains screened from tobacco leaves were utilized for the biological fermentation of tobacco leaves, resulting in the reduction of irritation and an improvement in quality of final product, showing a good potential for application.

Fermentation, also known as aging, is vital for enhancing the quality of flue‐cured tobacco leaves (FTLs). Aged FTLs demonstrate high‐quality sensory characteristics, while unaged FTLs do not. Microbes play important roles in the FTL fermentation process. However, the eukaryotic microbial community diversity is poorly understood, as are microbial associations within FTLs. We aimed to characterize and compare the microbiota associated with two important categories, fresh and strong flavor style FTLs, and to reveal correlations between the microbial taxa within them. Based on 16S and 18S rRNA Illumina MiSeq sequencing, the community richness and diversity of prokaryotes were almost as high as that of eukaryotes. The dominant microbes of FTLs belonged to seven genera, including Pseudomonas, Bacillus, Methylobacterium, Acinetobacter, Sphingomonas, Neophaeosphaeria, and Cladosporium, of the Proteobacteria, Firmicutes, and Ascomycota phyla. According to partial least square discriminant analysis (PLS‐DA), Xanthomonas, Franconibacter, Massilia, Quadrisphaera, Staphylococcus, Cladosporium, Lodderomyces, Symmetrospora, Golovinomyces, and Dioszegia were significantly positively correlated with fresh flavor style FTLs, while Xenophilus, Fusarium, unclassified Ustilaginaceae, Tilletiopsis, Cryphonectria, Colletotrichum, and Cyanodermella were significantly positively correlated with strong flavor style FTLs. Network analysis identified seven hubs, Aureimonas, Kocuria, Massilia, Brachybacterium, Clostridium, Dietzia, and Vishniacozyma, that may play important roles in FTL ecosystem stability, which may be destroyed by Myrmecridium. FTL microbiota was found to be correlated with flavor style. Species present in lower numbers than the dominant microbes might be used as microbial markers to discriminate different flavor style samples and to stabilize FTL microbial communities. This research advances our understanding of FTL microbiota and describes a means of discriminating between fresh and strong flavor FTLs based on their respective stable microbiota.