Transcriptome analysis reveals the key role of overdominant expression of photosynthetic and respiration-related genes in the formation of tobacco(Nicotiana tabacum L.) biomass heterosis.

BACKGROUND: Leaves are the nutritional and economic organs of tobacco, and their biomass directly affects tobacco yield and the economic benefits of farmers. In the early stage, our research found that tobacco hybrids have more leaves and larger leaf areas, but the performance and formation reasons of biomass heterosis are not yet clear. RESULTS: This study selected 5 parents with significant differences in tobacco biomass and paired them with hybrid varieties. It was found that tobacco hybrid varieties have a common biomass heterosis, and 45 days after transplantation is the key period for the formation of tobacco biomass heterosis; By analyzing the biomass heterosis of hybrids, Va116×GDH94 and its parents were selected for transcriptome analysis. 76.69% of the differentially expressed genes between Va116×GDH94 and its parents showed overdominant expression pattern, and these overdominant expression genes were significantly enriched in the biological processes of photosynthesis and TCA cycle; During the process of photosynthesis, the overdominant up-regulation of genes such as Lhc, Psa, and rbcl promotes the progress of photosynthesis, thereby increasing the accumulation of tobacco biomass; During the respiratory process, genes such as MDH, ACO, and OGDH are overedominantly down-regulated, inhibiting the TCA cycle and reducing substrate consumption in hybrid offspring; The photosynthetic characteristics of the hybrid and its parents were measured, and the net photosynthetic capacity of the hybrid was significantly higher than that of the parents. CONCLUSION: These results indicate that the overdominant expression effect of differentially expressed genes in Va116×GDH94 and its parents plays a crucial role in the formation of tobacco biomass heterosis. The overdominant expression of genes related to photosynthesis and respiration enhances the photosynthetic ability of Va116×GDH94, reduces respiratory consumption, promotes the increase of biomass, and exhibits obvious heterosis.

Correlation study on microbial communities and volatile flavor compounds in cigar tobacco leaves of diverse origins.

To elucidate the significant influence of microorganisms on geographically dependent flavor formation by analyzing microbial communities and volatile flavor compounds (VFCs) in cigar tobacco leaves (CTLs) obtained from China, Dominica, and Indonesia. Microbiome analysis revealed that the predominant bacteria in CTLs were Staphylococcus, Aerococcus, Pseudomonas, and Lactobacillus, while the predominant fungi were Aspergillus, Wallemia, and Sampaiozyma. The microbial communities of CTLs from different origins differed to some extent, and the diversity and abundance of bacteria were greater than fungi. Metabolomic analysis revealed that 64 VFCs were identified, mainly ketones, of which 23 VFCs could be utilized to identify the geographical origins of CTLs. Sixteen VFCs with OAV greater than 1, including cedrol, phenylacetaldehyde, damascone, beta-damascone, and beta-ionone, play important roles in shaping the flavor profile of CTLs from different origins. Combined with the correlation analysis, bacterial microorganisms were more closely related to key VFCs and favored a positive correlation. Bacillus, Vibrio, and Sphingomonas were the main flavor-related bacteria. The study demonstrated that the predominant microorganisms were essential for the formation of key flavor qualities in CTLs, which provided a theoretical reference for flavor control of CTLs by microbial technology. KEY POINTS: • It is the high OAV VFCs that determine the flavor profile of CTLs. • The methylerythritol phosphate (MEP) pathway and the carotenoid synthesis pathway are key metabolic pathways for the formation of VFCs in CTLs. • Microbial interactions influence tobacco flavor, with bacterial microorganisms contributing more to the flavor formation of CTLs.

Roles of cigar microbes in flavor formation during roasted-rice leachate fermentation.

Roasted-rice leachate fermentation, a distinctive local tobacco fermentation method in Sichuan, imparts a mellow flavor and glossy texture to tobacco leaves, along with a roasted rice aroma. In order to find out the impact of roasted-rice leachate on cigar tobacco leaves, the physicochemical properties, volatile flavor profile, and microbial community were investigated. The content of protein significantly decreased after fermentation. The volatile flavor compounds increased following roasted-rice leachate fermentation, including aldehydes, alcohols, acids, and esters. High-throughput sequencing identified Staphylococcus, Pseudomonas, Pantoea, Oceanobacillus, Delftia, Corynebacterium, Sphingomonas, Aspergillus, Weissella, and Debaryomyces as the primary genera. Network and correlation analysis showed Debaryomyces played a crucial role in roasted-rice leachate fermentation, due to its numerous connections with other microbes and positive relationships with linoelaidic acid, aromandendrene, and benzaldehyde. This study is useful for gaining insight into the relationship between flavor compounds and microorganisms and provides references regarding the effect of extra nutrients on traditional fermentation products. KEY POINTS: • Volatile flavor compounds increased following roasted-rice leachate fermentation • Staphylococcus was the primary genera in fermented cigar • Debaryomyces may improve the quality of tobacco leaves.

Analyzing microbial community and volatile compound profiles in the fermentation of cigar tobacco leaves.

Variations in industrial fermentation techniques have a significant impact on the fermentation of cigar tobacco leaves (CTLs), consequently influencing the aromatic attributes of the resulting cigars. The entire fermentation process of CTLs can be categorized into three distinct phases: phase 1 (CTLs prior to moisture regain), phase 2 (CTLs post-moisture regain and pile fermentation), and phase 3 (CTLs after fermentation and drying). These phases were determined based on the dynamic changes in microbial community diversity. During phase 2, there was a rapid increase in moisture and total acid content, which facilitated the proliferation of Aerococcus, a bacterial genus capable of utilizing reducing sugars, malic acid, and citric acid present in tobacco leaves. In contrast, fungal microorganisms exhibited a relatively stable response to changes in moisture and total acid, with Aspergillus, Alternaria, and Cladosporium being the dominant fungal groups throughout the fermentation stages. Bacterial genera were found to be more closely associated with variations in volatile compounds during fermentation compared to fungal microorganisms. This association ultimately resulted in higher levels of aroma components in CTLs, thereby improving the overall quality of the cigars. These findings reinforce the significance of industrial fermentation in shaping CTL quality and provide valuable insights for future efforts in the artificial regulation of secondary fermentation in CTLs. KEY POINTS: • Industrial fermentation processes impact CTLs microbial communities. • Moisture and total acid content influence microbial community succession in fermentation. • Bacterial microorganisms strongly influence CTLs' aldehyde and ketone flavors over fungi.

Sesquiterpenoids from cigar tobacco leaves.

Phytochemical investigation on cigar tobacco leaves led to four unknown sesquiterpenoids as well as nine reported ones. Among of them, 3-acetoxy-ß-damascone was first found in tobacco leaves. All the structures were elucidated by intensive spectroscopic analyses and X-ray diffraction. The relationship between the newly isolates and known ones was tried to describe.

Ionones from cigar tobacco leaves.

Phytochemical studies on cigar tobacco leaves led to the isolation of 18 ionone-type compounds, including previously undescribed cigatobanes E (1) and F (2). Additionally, compounds vomifoliol acetate (3), dehydrovomifoliol (4), 8,9-dihydromegastigmane-4,6-diene-3-one (5), 7α,8α-epoxyblumenol B (6), 3-oxoactinidol (12), and loliolide acetate (15), 4ß-hydroxy-dihydroactinidiolide (17), were found in tobacco leaves for the first time. The structural elucidation of all compounds was accomplished through rigorous spectral analysis.

Metabolite-based cell sorting workflow for identifying microbes producing carbonyls in tobacco leaves.

Carbonyl compounds represented by aldehydes and ketones make an important contribution to the flavor of tobacco. Since most carbonyl compounds are produced by microbes during tobacco fermentation, identifying their producers is important to improve the quality of tobacco. Here, we created an efficient workflow that combines metabolite labeling with fluorescence-activated cell sorting (ML-FACS), 16S rRNA gene sequencing, and microbial culture to identify the microbes that produce aldehydes or ketones in fermented cigar tobacco leaves (FCTL). Microbes were labeled with a specific fluorescent dye (cyanine5 hydrazide) and separated by flow cytometry. Subsequently, the sorted microbes were identified and cultured under laboratory conditions. Four genera, Acinetobacter, Sphingomonas, Solibacillus, and Lysinibacillus, were identified as the main carbonyl compound-producing microbes in FCTL. In addition, these microorganisms could produce flavor-related aldehydes and ketones in a simple synthetic medium, such as benzaldehyde, phenylacetaldehyde, 4-hydroxy-3-ethoxy-benzaldehyde, and 3,5,5-trimethyl-2-cyclohexene-1-one. On the whole, this research has developed a new method to quickly isolate and identify microorganisms that produce aldehydes or ketones from complex microbial communities. ML-FACS would also be used to identify other compound-producing microorganisms in other systems. KEY POINTS: • An approach was developed to identify target microbes in complex communities. • Microbes that produce aldehyde/ketone flavor compounds in fermented cigar tobacco leaves were identified. • Functional microbes that produce aldehyde/ketone flavor compounds from the native environment were captured in pure cultures.

The Consequence of Aqueous Extract of Tobacco Leaves (Nicotiana tabacum. L) on Feed Intake, Body Mass, and Hematological Indices of Male Wistar Rats fed under Equal Environmental Conditions.

OBJECTIVE: While the dangers of consuming tobacco by smoking has been of concern, the hazardous effect of other forms of tobacco consumption (in smokeless form) on health indices are less well explored. In this paper, we explored the effect of different doses of aqueous extract of tobacco leaves on feed intake, body mass, and hematological indices of male Wistar rats under equal environmental conditions. METHOD: Using an oral route of administration, the rats (n = 24; w = 65-85 g; 2-3 weeks old) were administered at different doses of 100, 200, 400, 0 mg/kg body weight (b.w.) per day to group A, B, C, and D for 42 days, after phytochemical and acute toxicity testing of the tobacco leaves. RESULTS: Compared to the negative control group (D), packed cell volume, hemoglobin concentration, red blood cells, and lymphocytes reduced dose-dependently in contrast to the white blood cells, neutrophils, mean corpuscular hemoglobin concentration, mean corpuscular hemoglobin, and mean corpuscular volume. As body mass and feed intake in relation to the different doses of aqueous extract of tobacco leaves reduced significantly (p < 0.05), the reverse was observed for body mass and feed intake in relation to room temperature. CONCLUSIONS: By influencing hematological indices, feed intake, and body mass, the extract of tobacco leaves can be hazardous to health. However, to assess safety and to come up with a conscious conclusion, future studies should explore the effect of the extract on organs histopathology, biochemical parameters, and lipid profile of the body.

Sub-acute oral toxicity study of aqueous extract of tobacco leaves (Nicotiana tabacum L.) on lipid profile, the tissue, and serum of the liver and kidney of male Wistar rats.

CONTEXT: Tobacco consumption may pose a very serious threat to the physiological state of the body; yet, fewer records have been documented in that regard. OBJECTIVE: We investigated the impact of aqueous extract of tobacco leaves on the lipid profile, the tissue, and serum levels of the liver and kidney of male Wister rats. MATERIALS AND METHODS: Rats (n = 52; weight = 33 - 47 g; ∼ 2½ weeks old) were acclimatised for 7 days and administered aqueous extract of tobacco leaves at 100, 200, 400, 0 mg/kg of body weight (to group A, B, C, D) for 30 days. RESULTS: Compared with the control group, the kidney tissue and serum (i.e., urea and creatinine) were not influenced, in contrast, indices of the liver such as AST, ALT, and ALP, dose-dependently increased. Changes such as coagulative necrosis resulted in the infiltration of mononuclear inflammatory cells and the vacuolar degeneration of the liver. Beside the reduction in the high-density lipoprotein of the rats, there was an increase in the concentration of triglycerides, very low-density lipoprotein, low-density lipoprotein, and the total cholesterol. CONCLUSION: Thus, extract of tobacco leaves can greatly influence the body lipid profile, beside the serum and tissues of the liver.

Genotoxic effect induced by dried nicotiana tabacum leaves from tobacco barns (kiln-houses) in chinese hamster lung fibroblast cells (V79).

Nicotiana tabacum is the most cultivated tobacco species in the state of Rio Grande do Sul, Brazil. Workers who handle the plant are exposed to the leaf components during the harvesting process and when separating and classifying the dried leaves. In addition to nicotine, after the drying process, other components may be found including tobacco-specific nitrosamines, polycyclic aromatic hydrocarbons, as well as pesticides residues. The objective of this study was to examine the genotoxicity attributed to the aqueous extract of dried tobacco leaves obtained from tobacco barns using Chinese hamster lung fibroblast cells (V79) as a model system by employing alkaline comet assay, micronucleus (MN) and Ames test. MTT assay was used to assess cytotoxicity and establish concentrations for this study. Data demonstrated cell viability > 85% for concentrations of 0.625-5 mg/ml while the comet assay indicated a significant increase in DNA damage at all concentrations tested. A significant elevation of MN and nuclear buds (NBUD) was found for 5 mg/ml compared to control and other dry tobacco leaves concentrations (0.625-2.5 mg/ml). Mutagenicity was not found using the Salmonella/Microsome test (TA98, TA100, and TA102 strains) with and without metabolic activation. The concentration of inorganic elements was determined employing the PIXE technique, and 13 inorganic elements were detected. Using CG/MS nicotine amounts present were 1.56 mg/g dry tobacco leaf powder. Due to the observed genotoxicity in V79 cells, more investigations are needed to protect the health of tobacco workers exposed daily to this complex mixture of toxic substances present in dry tobacco leaves.

Quantitative Relationship Between Paddy Soil Properties and Cadmium Content in Tobacco leaves.

Tobacco readily accumulates cadmium (Cd), an unnecessary and poisonous element. A total of 107 soil and tobacco leaf samples were collected from South China, to clarify the quantitative relationship between soil properties and Cd content in tobacco leaves. The results showed that 86.9% of the total sampling points had soil cadmium in excess of standard value, and the ratio of active Cd content to total soil Cd content was 24.0%. The enrichment factor of tobacco Cd was 3.43. There was a significant positive correlation between Cd concentration in tobacco leaves and soil Cd content. Soil pH, organic matter and cation exchange amount were negatively correlated with the Cd enrichment factor of tobacco. This present study has provided a regression model of tobacco Cd content based on soil factors, which could accurately predict Cd content in different parts of tobacco.

The effects of threshing and redrying on bacterial communities that inhabit the surface of tobacco leaves.

Before being subjected to the aging process, raw tobacco leaves (TLs) must be threshed and redried. We propose that threshing and redrying affect the bacterial communities that inhabit the TL surface, thereby influencing the aging process. However, these effects remain unclear. In this study, Illumina sequencing was applied to analyze the bacterial communities on both raw and redried TLs. Shannon's diversity value decreased from 3.38 to 2.52 after the threshing and redrying processes, indicating a large reduction in TL bacterial diversity. The bacterial communities also largely differed between raw TLs and redried TLs. On unaged raw TLs, Proteobacteria was the most dominant phylum (56.15%), followed by Firmicutes (38.99%). In contrast, on unaged redried TLs, Firmicutes (76.49%) was the most dominant phylum, followed by Proteobacteria (21.30%). Thus, the dominant genus Proteobacteria, which includes Sphingomonas, Stenotrophomonas, and Pantoea, decreased after the threshing and redrying processes, while the dominant genus Firmicutes, which includes Bacillus and Lactococcus, increased. Changes in the bacterial communities between raw and redried TLs were also noted after 1 year of aging. The relative abundance of dominant Proteobacteria taxa on raw TLs decreased from 56.15 to 16.92%, while the relative abundance of Firmicutes taxa increased from 38.99 to 79.10%. However, small changes were observed on redried TLs after 1 year of aging, with a slight decrease in Proteobacteria (21.30 to 17.64%) and a small increase in Firmicutes (76.49 to 79.10%). Based on these results, Firmicutes taxa may have a higher tolerance for extreme environments (such as high temperature or low moisture) than Proteobacteria bacteria. This study is the first report to examine the effects of threshing and redrying on bacterial communities that inhabit TLs.

Effects of drying pretreatment and particle size adjustment on the composting process of discarded flue-cured tobacco leaves.

The main characteristic of discarded flue-cured tobacco leaves is their high nicotine content. Aerobic composting is an effective method to decrease the nicotine level in tobacco leaves and stabilize tobacco wastes. However, high levels of nicotine in discarded flue-cured tobacco leaves complicate tobacco waste composting. This work proposes a drying pretreatment process to reduce the nicotine content in discarded flue-cured tobacco leaves and thus enhance its carbon-to-nitrogen ratio to a suitable level for composting. The effect of another pretreatment method, particle size adjustment, on composting efficiency was also tested in this work. The results indicated that the air-dried (nicotine content: 1.35%) and relatively long discarded flue-cured tobacco leaves (25 mm) had a higher composting efficiency than damp (nicotine content: 1.57%) and short discarded flue-cured tobacco leaves (15 mm). When dry/25 mm discarded flue-cured tobacco leaves mixed with tobacco stems in an 8:2 ratio was composted at a temperature above 55 °C for 9 days, the nicotine content dropped from 1.29% to 0.28%. Since the discarded flue-cured tobacco leaves was successfully composted to a fertile and harmless material, the germination index values increased to 85.2%. The drying pretreatment and particle size adjustment offered ideal physical and chemical conditions to support microbial growth and bioactivity during the composting process, resulting in efficient conversion of discarded flue-cured tobacco leaves into a high quality and mature compost.

Metabolic Profiling with Gas Chromatography-Mass Spectrometry and Capillary Electrophoresis-Mass Spectrometry Reveals the Carbon-Nitrogen Status of Tobacco Leaves Across Different Planting Areas.

The interaction between carbon (C) and nitrogen (N) metabolism can reflect plant growth status and environmental factors. Little is known regarding the connections between C-N metabolism and growing regions under field conditions. To comprehensively investigate the relationship in mature tobacco leaves, we established metabolomics approaches based on gas chromatography-mass spectrometry (GC-MS) and capillary electrophoresis-time-of-flight-mass spectrometry (CE-TOF-MS). Approximately 240 polar metabolites were determined. Multivariate statistical analysis revealed that the growing region greatly influenced the metabolic profiles of tobacco leaves. A metabolic correlation network and related pathway maps were used to reveal the global overview of the alteration of C-N metabolism across three typical regions. In Yunnan, sugars and tricarboxylic acid (TCA) cycle intermediates were closely correlated with amino acid pools. Henan tobacco leaves showed positive correlation between the pentose phosphate pathway (PPP) intermediates and C-rich secondary metabolism. In Guizhou, the proline and asparagine had significant links with TCA cycle intermediates and urea cycle, and antioxidant accumulation was observed in response to drought. These results demonstrate that combined analytical approaches have great potential to detect polar metabolites and provide information on C-N metabolism related to planting regional characteristics.

Quantitative analysis of 17 amino acids in tobacco leaves using an amino acid analyzer and chemometric resolution.

A method was developed for quantifying 17 amino acids in tobacco leaves by using an A300 amino acid analyzer and chemometric resolution. In the method, amino acids were eluted by the buffer solution on an ion-exchange column. After reacting with ninhydrin, the derivatives of amino acids were detected by ultraviolet detection. Most amino acids are separated by the elution program. However, five peaks of the derivatives are still overlapping. A non-negative immune algorithm was employed to extract the profiles of the derivatives from the overlapping signals, and then peak areas were adopted for quantitative analysis of the amino acids. The method was validated by the determination of amino acids in tobacco leaves. The relative standard deviations (n = 5) are all less than 2.54% and the recoveries of the spiked samples are in a range of 94.62-108.21%. The feasibility of the method was proved by analyzing the 17 amino acids in 30 tobacco leaf samples.

Solid-State Fermentation Improves Tobacco Leaves Quality via the Screened Bacillus subtilis of Simultaneously Degrading Starch and Protein Ability.

The process of tobacco aging plays a significant role in enhancing the smoking experience by improving the flavor and quality of tobacco leaves. During natural aging, the metabolic activity of the microbes on the surface of tobacco leaves will be greatly changed. Besides, starch and protein are two of the main macromolecular compounds causing the poor smoking quality of tobacco leaves which to be degraded for better tobacco quality. In this study, a bacterium with the simultaneously degrading ability of starch (degradation rate of 33.87%) and protein (degradation rate of 20%) has been screened out from high-class tobacco leaf and then inoculated into low-class tobacco leaf by solid-state fermentation for quality improvement. The changes in components related to carbon and nitrogen showed that the strain had an obvious effect on the quality improvement of tobacco leaves. After that, GC-MS analyses displayed the volatile flavor compounds which become rich and the flavor has been improved. It has been proved that inoculation solid-state fermentation by dominant strain could improve tobacco quality, as well as instead of the traditional natural aging process which greatly shortens the aging process. The work also offers a helpful strategy for solid-state products for deep fermentation.

Integrated characterization of filler tobacco leaves: HS-SPME-GC-MS, E-nose, and microbiome analysis across different origins.

This study delves into the aroma characteristics and microbial composition of filler tobacco leaves (FTLs) sourced from six distinct cigar-growing regions within Yunnan, China, following standardized fermentation. An integrated approach using gas chromatography-mass spectrometry (GC-MS), electronic nose (E-nose), and microbiome analysis was employed for comprehensive profiling. Results derived from Linear Discriminant Analysis (LDA) using E-nose data confirmed the presence of notable variability in flavor substance profiles among the FTLs from six regions. Additionally, GC-MS was used to discern disparities in volatile organic compound (VOC) distribution across FTLs from these regions, identifying 92, 81, 79, 58, 69, and 92 VOCs within each respective sample set. Significantly, 24 VOCs emerged as pivotal determinants contributing to the heterogeneity of flavor profiles among FTLs from diverse origins, as indicated by Variable Importance for the Projection (VIP) analysis. Furthermore, distinctions in free amino acid content and chemical constituents were observed across FTLs. Of noteworthy significance, solanone, isophorone, durene, (-)-alpha-terpineol, and 2,3'-bipyridine exhibited the strongest correlations with microbiome data, with fungal microorganisms exerting a more pronounced influence on metabolites, as elucidated through two-way orthogonal partial least-squares (O2PLS) modeling. These findings provide a theoretical and technical basis for accurately evaluating the synchronization of FTLs in aromas and fermentation processes, and they will enhance the quality of fermented FTLs and foster the growth of the domestic cigar tobacco industry ultimately.

Genotoxicity of Nicotiana tabacum leaves on Helix aspersa.

Tobacco farmers are routinely exposed to complex mixtures of inorganic and organic chemicals present in tobacco leaves. In this study, we examined the genotoxicity of tobacco leaves in the snail Helix aspersa as a measure of the risk to human health. DNA damage was evaluated using the micronucleus test and the Comet assay and the concentration of cytochrome P450 enzymes was estimated. Two groups of snails were studied: one fed on tobacco leaves and one fed on lettuce (Lactuca sativa L) leaves (control group). All of the snails received leaves (tobacco and lettuce leaves were the only food provided) and water ad libitum. Hemolymph cells were collected after 0, 24, 48 and 72 h. The Comet assay and micronucleus test showed that exposure to tobacco leaves for different periods of time caused significant DNA damage. Inhibition of cytochrome P450 enzymes occurred only in the tobacco group. Chemical analysis indicated the presence of the alkaloid nicotine, coumarins, saponins, flavonoids and various metals. These results show that tobacco leaves are genotoxic in H. aspersa and inhibit cytochrome P450 activity, probably through the action of the complex chemical mixture present in the plant.

Widely targeted metabolomic analysis reveals that volatile metabolites in cigar tobacco leaves dynamically change during fermentation.

Changes in volatile metabolites during cigar tobacco leaves fermentation as well as the metabolic pathways of metabolites with significant differences were investigated to determine the influence of cigar tobacco leaves fermentation on its flavor. The volatile substances in cigar tobacco leaves at different stages were detected by headspace-solid phase microextraction gas chromatography-mass spectrometry (HS-SPME-GC-MS), and the main differences in volatile substances in cigar tobacco leaves at different fermentation stages of Yunxue1 in Yuxi production area were analyzed by principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA). The results show that in the process of cigar tobacco leaves fermentation (YXF0, YXF1, YXF2, YXF3, YXF4, YXF5), a total of 613 volatile metabolites were detected, and a significant difference was found in 263 kinds of metabolites. Among them, the main upregulated differential metabolites were 1,3,6,10-Cyclotetradecatetraene, 3,7,11-trimethyl-14-(1-methylethyl)-, [S-(E,Z,E,E)]-, Benzoic acid, Benzaldehyde, etc. While the main downregulated differential metabolites included beta.-Myrcene, trans-Farnesol, etc. The metabolites with significant differences are mainly concentrated in the biosynthesis of monoterpenes, diterpenes, sesquiterpenes and triterpenes, the degradation metabolism of amino acids, such as valine, leucine and isoleucine, and the biosynthesis of phenylpropyl. There were 8 different metabolites in 5 groups, including 4- (1-methylethyl) -1-cyclohexene-1-formaldehyde、2, 4-dihydroxyacetophenone、2-methylbutyl 3-methylbutyrate and methylpyrazine, all of which showed upregulation trend during fermentation. In the fermentation process, volatile metabolites participate in various synthesis and degradation pathways. The biosynthesis pathway of terpenes and amino acid synthesis and degradation pathway are connected to produce various terpenes, aldehydes and other substances, such as 1,3,6,10-Cyclotetradecatetraene, 3,7,11-trimethyl-14-(1-methylethyl)-, [S-(E,Z,E,E)]-、benzaldehyde and 4-hydroxybenzaldehyde, which are conducive to the overall flavor and quality of cigar tobacco leaves.

The bacterial succession and its role in flavor compounds formation during the fermentation of cigar tobacco leaves.

Fermentation is the key process required for developing the characteristic properties of cigar tobacco leaves, complex microorganisms are involved in this process. However, the microbial fermentation mechanisms during the fermentation process have not been well-characterized. This study investigated the dynamic changes in conventional chemical composition, flavor compounds, and bacterial community during the fermentation of cigar tobacco leaves from Hainan and Sichuan provinces in China, as well as the potential roles of bacteria. Fermentation resulted in a reduction of conventional chemical components in tobacco leaves, with the exception of a noteworthy increase in insoluble protein content. Furthermore, the levels of 10 organic acids and 19 amino acids showed a significant decrease, whereas the concentration of 30 aromatic substances exhibited a unimodal trend. Before fermentation, the bacterial community structures and dominant bacteria in Hainan and Sichuan tobacco leaves differed significantly. As fermentation progressed, the community structures in the two regions became relatively similar, with Delftia, Ochrobactrum, Rhodococcus, and Stenotrophomonas being dominant. Furthermore, a total of 12 functional bacterial genera were identified in Hainan and Sichuan tobacco leaves using bidirectional orthogonal partial least squares (O2PLS) analysis. Delftia, Ochrobactrum, and Rhodococcus demonstrated a significant negative correlation with oleic acid and linoleic acid, while Stenotrophomonas and Delftia showed a significant negative correlation with undesirable amino acids, such as Ala and Glu. In addition, Bacillus showed a positive correlation with benzaldehyde, while Kocuria displayed a positive correlation with 2-acetylfuran, isophorone, 2, 6-nonadienal, and ß-damascenone. The co-occurrence network analysis of microorganisms revealed a prevalence of positive correlations within the bacterial network, with non-abundant bacteria potentially contributing to the stabilization of the bacterial community. These findings can improve the overall tobacco quality and provide a novel perspective on the utilization of microorganisms in the fermentation of cigar tobacco leaves.