Effects of a novel microbial fermentation medium produced by Tremella aurantialba SCT-F3 on cigar filler leaf

Introduction Adding a fermentation medium is an effective way to improve the quality of cigar tobacco leaves. Methods A novel microbial fermentation medium produced by an edible medicinal fungus, Tremella aurantialba SCT-F3 (CGMCC No.23831) was used to improve the quality of cigar filler leaves (CFLs). Changes in sensory quality, chemical components, volatile flavor compounds (VFCs), and the structure and function of microbes were investigated during the fermentation process. Results The sensory quality of CFLs supplemented with the T. aurantialba SCT-F3 fermentation medium significantly improved. Adding the fermentation medium increased the total alkaloid, reducing sugar, total sugar, and 12 VFCs significantly. A total of 31 microbial genera were significantly enriched, which increased the microbial community’s richness and diversity. Microbial functions increased, including nucleotide biosynthesis, amino acid biosynthesis, fatty acid and lipid biosynthesis, nicotine degradation, and nicotinate degradation. During fermentation, the total alkaloid, reducing sugar, and total sugar content decreased. The richness and diversity of the microbial community decreased, whereas bacterial enzyme activity increased. At the end of fermentation, the sensory quality was excellent. The microbial structure gradually stabilized, and functional genes were low. The contents of the four Maillard reaction products and three nicotine degradation products increased significantly. 2-Ethyl-6-methylpyrazine, methylpyrazine, D,L-anatabine, β-nicotyrine, nicotinic degradation products, and total nitrogen were significantly and positively correlated with sensory quality. Methylpyrazine, D,L-anatabine, and β-nicotyrine were negatively correlated with Luteimonas, Mitochondria, Paracoccus, Stemphylium, and Stenotrophomonas. Conclusion This research provides not only a new microbial fermentation medium that utilizes edible and medicinal fungi to improve the quality of fermented CFLs, but also new ideas for the development and application of other edible medicinal fungi to improve the quality of cigar tobacco leaves.


Introduction
A cigar is a tobacco product made from fermented tobacco leaves and comprises three parts: a wrapper, binder, and filler.The wrapper determines the cigar's appearance, and the binder fixes the position of the filler.As the most important component accounts for approximately 75% of the cigar's weight, filler determines the style of the cigar.Fillers, rich in aroma and flavor, are essential for rolling superior-quality cigars.Fermentation is an important process to improve the quality of cigar filler leaves (CFLs).There are two fermentation methods for cigar tobacco leaves: spontaneous fermentation and artificial fermentation.When spontaneous fermentation using only microorganisms in the natural environment cannot meet the quality requirements of the product, fermentation media need to be artificially added to significantly improve the fermentation quality (China Tobacco Sichuan Industrial Co., Ltd., 2021).Traditional fermentation media include not only plant extracts, Fritillaria cirrhosa tincture, coffee tincture, cocoa tincture, holly gum, jujube juice, and cane sugar, but also fermented products, such as red rice (rice fermented by Monascus), sweet rice (rice fermented by Rhizopus), rice wine, and loquat wine (Jin, 1988;Cai et al., 2022;Hu et al., 2022;Zong et al., 2023).Novel fermentation media, strains such as Bacillus cereus, B. pumilus, Acinetobacter, and Candida, and enzymes such as proteases, amylases, cellulases, and pectinases, have been shown to improve the quality of CFLs (Li et al., 2012;Zhang et al., 2020a,b;Zheng et al., 2022a,b;Jia et al., 2023).
In this study, we added the fermentation broth of T. aurantialba into CFLs to improve the quality.After fermentation, changes in sensory quality, chemical components, volatile flavor compounds (VFCs), and the structure and function of microbes were investigated, and the contributions of chemical components and VFCs to sensory quality and correlation analysis of the predominant microbes and VFCs were analyzed.

CFL fermentation
The fermentation medium was potato glucose broth containing 6 g/L potato extract powder and 20 g/L glucose and was autoclaved at 121°C for 15 min.T. aurantialba SCT-F3 was inoculated by sterile loops in 250-mL flasks with 75 mL fermentation medium and cultured at 150 rpm/28°C for 5 days.The fermented potato glucose broth was centrifuged (5,000 × g for 10 min) to remove mycelium pellets, and the supernatant was used as the fermentation medium.Unfermented potato glucose broth was used as the unfermented medium.Dexue No.1 from the original Chinese tobacco drying strips was used in this study.CFLs were loosened and spread flat on the table by hand.Mixed the medium and pure water in a ratio of 1:3 (v/v), then the mixture was evenly sprayed using an electronic sprayer on the surface of the leaves with 20% inoculation amount (v/g).After 2 h, the medium and water were absorbed entirely by the CFLs.The CFLs were transferred to an oak barrel for fermentation.The oak barrels were placed in the fermentation room, the temperature of the fermentation room was controlled at 37°C, the relative humidity of the fermentation room was controlled at 75%, and the fermentation was carried out for 40 days.CFLs added with T. aurantialba SCT-F3 fermented medium were sampled at 0, 20, and 40 days and marked as J0, J20, and J40; CFLs added with unfermented potato glucose broth fermented were sampled at 0, 20, and 40 days and marked as P0, P20, and P40; uninoculated CFLs were marked as Control.Samples were taken from the four corners and central position of the oak barrel, and the five positions of the CLTs were ground and mixed for testing.All samples were mixed well, transferred into sterile bags, and stored at −20°C until further analysis.All experiments were performed in triplicates.

Sensory evaluation
The fermented CFLs were stemmed and dried to 15 ± 2% moisture content.The samples were hand-rolled to a length of 110 mm and a circumference of 47 mm, then placed in a temperature humidity chamber at a temperature of 20 ± 2°C and humidity of 62 ± 3% to balance the samples' moisture to 13 ± 1%.Five assessors from the staff trained by the China National Tobacco Quality Supervision & Test Center evaluated the sensory quality of all samples to recognize the aroma, smoke, aftertaste, combustibility, and comfort level.The samples were identified and scored according to 0-9 points, ranging from weak to strong.

Chemical components analysis
The chemical components of total alkaloids, total nitrogen, reducing sugars, and total sugars in the CFLs were determined  (Hu et al., 2022).

VFC analysis
VFCs in CFLs were assayed using headspace solid-phase microextraction coupled with gas chromatography-mass spectrometry (HS-SPME-GC-MS) as previously described (Zheng et al., 2022a,b).A 50/30 μm DVB/CAR/PMDS fiber (Supelco Inc., United States) was used for aroma extraction.The CFLs were pulverized using a grinder, and the powder (1.5 g) was placed in a 10-mL glass vial.The sample was extracted at 60°C for 30 min, and the fiber was inserted into the injection port of the GC device at 250°C for 1 min to desorb the analytes.The sample was analyzed on a DB-5MS column (60 m × 0.25 mm × 0.25 μm, Agilent Technologies, United States).The GC oven temperature was maintained at 40°C for 2 min, followed by an increase of 10°C/min to 250°C and then held for 5 min.Helium (purity: 99.999%) was used as the carrier gas at a constant flow of 1 mL/min.The mass spectrometer conditions were: EI voltage, 70 eV; ion source temperature, 300°C; mass range, m/z 35-350.The identification of volatile compounds in the extracts was based on a comparison of the mass spectra and retention times of the individual compounds with those of standard compounds deposited in the National Institute of Standards and Technology database in the MS device (SI and RSI ≥800).Peak areas were compared with those of the internal standard for quantification.

Microbial community analysis
Samples (5.0 g) were suspended in 250 mL sterile phosphate buffer saline and shaken for 2 h at 200 rpm, after which the supernatant was centrifuged (10,000 × g for 30 min).The total genomic DNA of each sample was extracted using an EZNA ® Soil DNA Kit (Omega, United States) according to the manufacturer's instructions.The bacterial genomic DNA were amplified and sequenced using primers 515F and 907R for 16S rRNA genes (Parada et al., 2016).The fungal genomic DNA was amplified using primers ITS1F and ITS2R for internal transcribed spacer (ITS) genes (Usyk et al., 2017).Amplicons were pooled in equal amounts and sequenced using a 2 × 300 paired-end configuration and an Illumina MiSeq sequencing system (Illumina, United States).Operational taxonomic units (OTUs) of qualified sequences were identified using the clustering program VSEARCH version 1.9.6 and the SILVA version 132 database (Rognes et al., 2016) with 97% similarity.Alpha diversity, including the Chao1, Shannon, and Simpon values, was analyzed using QIIME version 1.9.1 (Caporaso et al., 2010).Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt2) and Fungi Functional Guild (FUNGuild) were used to predict metagenomic functions based on normalized OTU tables (Douglas et al., 2019;Xie et al., 2021).

Statistical analysis
All experiments were repeated at least thrice.SPSS version 19 software (SPSS Inc., United States) was used to carry out a one-way analysis of variance and Duncan's multiple comparison test (p < 0.05).Heat maps and cluster analyses were performed using R version 4.0.0.Galaxy 1 was used for the linear discriminant analysis of effect size (LEfSe) to assess significant differences in CFLs with different treatments.SIMCA-P version 13.0 software (Umetrics, Sweden) enabled a partial least-squares (PLS) analysis of the contributions of conventional chemical constituents and VFCs to the sensory evaluation.Additionally, to determine the correlation between representative microbes and core VFCs based on Spearman's correlation coefficients (p < 0.05), network analysis was performed using the Gephi software.

Results of sensory evaluation
Seven CFLs were evaluated and scored according to the cigar evaluation criteria.The detailed evaluation scores for each sample are shown in Figure 1.The scores of the fermented CFLs were higher than those of the control, and as fermentation progressed, the quality of the CFLs improved gradually.Except for combustibility, other qualities were significantly improved (p < 0.05) in those with T. aurantialba SCT-F3 fermentation medium (J0, J20, and J40) compared to those in potato glucose broth (P0, P20, and P40).Specifically, aroma mellow, richness, maturity, and sweetness increased; irritation decreased, and balance sense improved.

Profiles of chemical component
During fermentation, the contents of total alkaloid, total nitrogen, total sugar and reducing sugar fluctuated from 1.8-2.8%,4.3-5.4%,0.2-1.7% and 0.2-1.4%,respectively.The total alkaloid, reducing sugar, and total sugar contents significantly increased (p < 0.05) in samples supplemented with fermentation media on day 0 (F0 and P0), as shown in Figure 2.They then decreased to their lowest levels on day 20 (J20 and P20) and gradually increased (J40 and P40).The total nitrogen content at 20 and 40 days (J20, P20, J40, and P40) was higher than that on day 0 (J0 and P0) and in control.

Profiles of VFCs
In total, 188 volatile components were detected in the CFLs, including 32 ketones, 20 aldehydes, 11 alcohols, and 10 esters.Furthermore, 34 VFCs (Figure 3), five aromatic amino acid degradation products, six plastochrome degradation products, one cembrane degradation product, 10 Maillard reaction products, and four nicotine degradation products were selected for further analysis.These compounds have been reported to have different aromas that may play important roles in the aroma profiles of cigars.The contents of aromatic amino acid degradation products, plastochrome degradation products, and cembrane degradation product, decreases gradually with fermentation, whereas four Maillard reaction products and 3 nicotine degradation products increased.

Contributions of chemical components and VFCs to sensory
PLS was performed on scaled values of chemical constituents and VFCs as X-variables and on scaled values of total sensory evaluation scores as Y-variables for correlations.Two significant principal components of the total variance in the data matrix were extracted.The R2X (cum), R2Y (cum), and Q2 (cum) were 0.522, 0.808, and 0.701, respectively, which meant that 52.2% variation was due to these two components, with a total of 80.8% dummy Y-variables per class and 70.1% overall cross-validated R 2 for these two components.The data indicated that the PLS model was suitable for this study.The distributions of the samples in the first and second components of the statistical analysis are shown in Figure 4. Groups J0 and P0 are located on the right side of the plot, J20 and P20 on the lower left, and J40 and P40 on the upper left.Table 1 shows that 14 variables with a VIP higher than 1.0 were important in the first and second most significant principal components.2-ethyl-6-methylpyrazine, methylpyrazine, D,L-anatabine, β-nicotyrine, nicotinic degradation products, and total nitrogen were significantly positively correlated with the total sensory evaluation scores.In contrast, 1-phenylethanol, geranylacetone, megastigmatonone, D-solanone, 6-methyl-3,5-heptadiene-2-one, Maillard reaction products, 1,2-dimethoxybenzene, and γ-caprolactone were significantly negatively correlated with the total sensory evaluation scores.

Overview of microbial community
Figure 5A shows alpha diversity indices, including the Chao1, Shannon, and Simpson indices, of bacterial and fungal communities in CFLs.The Chao1, Shannon, and Simpson values of the bacterial communities were higher than those of the fungal communities, indicating that the richness and diversity of the bacterial communities were generally higher than those of the fungal communities.The Chao1 values of J0 were higher than those of others, indicating that the richness of the bacterial and fungal communities increased after adding the T. aurantialba SCT-F3 fermentation medium.The Shannon and Simpson values of microbes in CFLs with medium without fermentation (P0 and J0) were higher than those in the other samples, indicating that adding fermentation medium increased the diversity of the microbial community.As fermentation progressed, fungal alpha diversity indices decreased.
Unconstrained principal coordinate analysis of weighted UniFrac distance revealed that the microbiota of CFLs with different treatments explained 77.4% of the bacterial variance (Figure 5B) and 98.1% of the fungal variance (Figure 5C).The addition of the fermentation medium altered the structure of the bacterial and fungal communities.The fungal community structure of sample J20 significantly differed from the other samples.The abundance of bacterial and fungal taxa is shown in Figure 6.The dominant bacterial phyla were Bacillota and Pseudomonadota, and the dominant fungal phyla were Ascomycota and Basidiomycota (Figures 6A,B).Pseudomonadota and Ascomycota of unfermented samples (control, P0, and J0) were significantly higher than those of the fermented samples.The dominant bacterial genera, with relative abundances higher than 5.0% in at least one sample, were Staphylococcus, Burkholderia-Caballeronia-Paraburkholderia, unclassified Enterobacteriaceae, Pantoea, Aerococcus, Pseudomonas, and unclassified Burkholderiaceae, which represented between 44.6 and 96.1% of the total abundance in each CFL sample (Figure 6C).The relative abundance of Staphylococcus decreased significantly at P0 and J0.The dominant fungal genera were Aspergillus and Alternari (Figure 6D).The relative abundance of Aspergillus and Alternari significantly decreased in P0 and J0, whereas the relative abundance of Stemphylium showed the opposite trend.
LEfSe analyses revealed significant differences below the phylum level to explore the different microbes among the CFLs under different treatments (Figure 7).The circles from innermost to the outermost represent bacterial and fungal classifications from the phylum to genus levels, and the corresponding colors in every group denote bacterial and fungal taxa with a significant difference.Notably, 85 different bacteria appeared at the LDA threshold of 2, judging by statistically significant differences (p < 0.05), which consisted of two phyla, four classes, 10 orders, 22 families, and 47 genera (Figure 7A).In detail, 30 genera were significantly enriched in inoculated T. aurantialba SCT-F3 CFLs without fermented (J0), such as Escherichia-Shigella, Luteimonas, and Brachybacterium.Four genera were significantly enriched in P0: Brevundimonas, Clostridium sensu stricto1, Variovorax, and Moraxellaaceae.Two genera were significantly enriched in J20, including Oceanobacillus and Curvibacter.Eight genera were significantly enriched in J40, such as Aquabacterium, Burkholderia-Caballeronia-Paraburkholderia, and Prauserella.There were 12 fungi of two classes, one order, four families, and five genera (Figure 7B).Colletotrichum and Symmetrospora were significantly enriched in J0 and P0, Sagenomella was enriched in J20, and Aspergillus in J40.

Discussion
Tobacco leaves are the most widely planted nonfood crops worldwide.The quality of tobacco leaves is affected by the breed, climate, soil, and culture conditions.Due to the poor quality of cigar tobacco grown in China at present, developing a new measure to improve their quality would have important economic benefits for both tobacco farmers and manufacturers.In the present study, the addition of an edible medicinal fungus, T. aurantialba SCT-F3 fermented broth as the fermentation medium improved the quality of CFLs.According to the results of sensory evaluation, the mellow aroma, richness, maturity, and sweetness of fermented CFLs increased, irritation decreased, and the sense of balance improved.The addition of fermentation media significantly changed the total alkaloid, total nitrogen, reduced sugar, and total sugar contents.The CFLs' total sugar and reduced sugar contents significantly increased at the beginning of fermentation.The fermented broth of T. aurantialba contains not only a large amount of monosaccharides, such as mannose, glucose, and galactose (Deng et al., 2017), but also amylase and cellulase, which can decompose starch and cellulose (Chen et al., 2021), and might be the cause of this phenomenon.At the end of fermentation, there were no significant difference in the reducing sugar and total sugar content of all samples, and there was no significant difference in the total alkaloid content of the samples with added culture medium.Microbes consume nicotine and aniline compounds, and alkaloid compounds produced by microbial metabolism or protein decomposition, which may cause the total alkaloid content first decreased and then increased during fermentation.The fluctuation of total sugar and reducing sugar content during the fermentation process is due to the degradation of glycogen, the utilization of microorganisms, and the Maillard reaction (Ye et al., 2019).Total nitrogen and nicotine are related to smoke concentration and pungency, and sugar is related to sweetness (Hu et al., 2022).However, within a certain concentration range, the contents of chemical components, total alkaloids, total nitrogen, reducing sugar, and total sugar may not directly reflect the sensory qualities of CFLs.Therefore, we measured VFCs to explore the effects of The relative abundance of bacterial metabolic pathways in CFLs.Function prediction of the bacteria (A) and fungi (B) in CFLs.Volcano plot of the numbers of differentially expressed genes between the two groups (C).Red represents the up-regulated, and green represents the down-regulated genes in group A. Blue represents that these genes had no differential expression between the two groups.T. aurantialba SCT-F3 fermentation medium on the quality of CFLs.Aromatic amino acid degradation products, plastochrome degradation products, cembrane degradation products, Maillard reaction products, and nicotine degradation products are especially important for forming characteristic tobacco flavors and aromas (Xie, 2009;Yin et al., 2012).Four VFCs from the Maillard reaction, 2,3-butanedione with creamy fragrance, methylpyrazine with sweet fragrance, 2,6-dimethylpyrazine and 2-ethyl-6-methylpyrazine with baked potato fragrance (Yan and Zhao, 2008) increased in CFLs fermented with T. aurantialba SCT-F3 fermentation medium.Fermentation led to the increase in nicotine degradation products, including β-nicotyrine, D,L-anatabine, and cotinine.VFCs from T. aurantialba SCT-F3 broth increased in unfermented CFLs added T. aurantialba SCT-F3 fermentation medium.However, the increase in VFCs did not significantly improve the sensory quality scores.Some VFCs in CFLs gradually decreased or increased as fermentation progressed; however, the sensory quality continuously increased.Additionally, through PLS analysis of conventional chemical constituents, VFCs, and sensory evaluations, it was found that in the complex aroma system by our study, 2-ethyl-6-methylpyrazine, methylpyrazine, D,L-anatabine, β-nicotyrine, nicotinic degradation products and total nitrogen had a significantly positive contribution to the sensory quality.In contrast, 1-phenylethanol with rose flavor, geranylacetone with green flavor, megastigmatrienone with flowery and woody flavor, D-solanone enhancing the fragrance of tobacco, "-"meant that functional genes were not detected in samples inoculated with potato glucose broth (P0, P20, and P40).Functional genes were up-regulated in samples inoculated with T. aurantialba SCT-F3 (J0, J20, and J40) compared to P0, P20, and P40.Functional genes were down-regulated in samples inoculated with fermented medium (J0, J20, and J40) compared to those inoculated with unfermented medium (P0, P20, and P40). 10.3389/fmicb.2023.1267916 Frontiers in Microbiology 13 frontiersin.org6-methyl-3,5-heptadiene-2-one with cinnamon aroma, 1,2-dimethoxybenzene with pleasant odor, and γ-caprolactone with herbal and sweet flavor showed the opposite trend.Although higher content of VFCs would endow CFLs with more fragrant flavor, the coordination and balance of VFCs in tobacco leaves determine the sensory quality of tobacco leaves.A significant increase or decrease in a VFC might break the balance (Zhang et al., 2021).However, there is no criteria for evaluating coordination and balance to measure the quality of cigar tobacco yet.Further study is needed to establish the criteria for evaluating coordination and balance of chemical composition and VFCs to reduce sensory evaluators workload and provide objective criterion.
The effects of T. aurantialba SCT-F3 fermentation medium and fermentation on the microbial community of CFLs were amplified using Illumina high-throughput sequencing.The addition of the fermentation medium significantly changed the microbes' richness, diversity, and structure.Fungal communities have more stable structures than bacterial communities.Aspergillus was the dominant fungus, mainly clustered in CFLs, and may play an important role in maintaining the stability of fungal communities.Aspergillus species, filamentous fungi with GRAS status, are widely used in the food industry to produce multiple enzymes such as amylases, proteases, lipases, cellulases, and aroma compounds (Raveendran et al., 2018;Kumar et al., 2021;Chilakamarry et al., 2022).It is worth noting that in the fermentation process of CFLs, avoiding Aspergillus sp.mycelium formation affects the appearance quality of CFLs, while applying their properties of enzyme and aroma production is very important.
Changes in the microbial communities of the CFLs may be separated into three distinct phases.Phase I is the initial disturbance period.In this phase, the endogenous microbial community and its functions changed dramatically with the addition of fermentation media.Nutrients, including sugars, proteins, ketones, aldehydes, alcohols, and esters from T. aurantialba SCT-F3 fermentation medium facilitated the growth of 31 microbial genera, which led to an increase in diversity and species richness.Furthermore, the activity of 20 microbial functional genes increased, including nucleotide biosynthesis, amino acid biosynthesis, fatty acid and lipid biosynthesis, fermentation, nicotine degradation, and nicotinate degradation.This indicates that adding T. aurantialba SCT-F3 accelerated the VFCs of CFLs and shortened the fermentation time (Cai et al., 2022).Phase II was the transitional period.The loss of diversity and species richness likely resulted from the interactions between microorganisms, such as competition and antagonism (Tao et al., 2014).Functional genes related to nucleotide biosynthesis, amino acid biosynthesis, fatty acid, and lipid biosynthesis, and fermentation further increased, indicating that substance consumption and new substance synthesis occurred in CFLs during this period.Phase III was relatively stable.The microbial community gradually stabilized owing to the complete suppression of external disturbances (Wu et al., 2016).The abundance of functional genes was low, indicating that the metabolic activity of microorganisms has also become less active.
VFCs are affected by microbial metabolism (Yang et al., 2021).Network analysis has shown that microbes in CFLs are closely related to VFCs, consistent with previous study findings (Zhang et al., 2020a,b;Zheng et al., 2022a,b).Aspergillus sp.produces aromatic compounds (Chilakamarry et al., 2022); this was positively related to 2,3-butanedione in our study.Alternaria, a cosmopolitan fungal genus, is one of the dominant resident fungi in tobacco leaves Metabolic pathways of nicotine degradation and nicotinate degradation and the expression levels of the genes encoding related enzymes in CFLs according to Encyclopedia of Genes and Genomes (KEGG) (Jimenez et al., 2008;Qiu et al., 2012 (Chattopadhyay et al., 2021) and is positively related to benzene acetaldehyde with a fruity and sweet aroma (Koan Sik et al., 2006), and geranylacetone.Pseudomonas was positively related to 1-octen-3-one with a mushroom flavor (Bauer et al., 2022), γ-undecalactone with a sweet peach flavor (Chen et al., 2022), and benzeneacetaldehyde with a hyacinth aroma.Achromobacter, Cellulomonas, Enterobacter, Arthrobacter, Alcaligene s, Pseudomonas, and Aspergillus have been reported to degrade nicotine (Meng et al., 2010).In our study, Burkholderia-Caballeronia-Paraburkholderia and Prauserella were positively correlated with cotinine and Mycosphaerella and Stemphylium were positively correlated with 2,4,6-collidine.Although microbial enzymes that catabolize nicotine to 6-hydroxynicotine (EC:1.5.99.4) or N-methylmyosmine (EC: 1.4.2.2) were not detected, enzymes associated with nicotine degradation and the nicotinate degradation pathway increased after the addition of T. aurantialba SCT-F3 fermentation medium.Microbial enzymes mainly participate in succinate semi-aldehyde-transfer and fumarate-transfer pathways.Hence, it is speculated that microbial enzymes in CFLs catalyze the increase of β-nicotyrine, D,L-anatabine, and cotinine during fermentation; some intermediates of these four strains participate in the transformation process of nitrogen-containing substances and some strains use nitrogen-containing substances in the metabolic process.

Conclusion
When spontaneous fermentation cannot meet the demands of consumers, medium fermentation is an important processing method for improving the sensory quality of cigar tobacco leaves.Our present study demonstrates a new microbial medium fermentation method for improving the quality of CFLs.The addition of the microbial medium produced by Tremella aurantialba SCT-F3 significantly affected the chemical components and VFCs, mainly increasing the content of Maillard reaction products and nicotine degradation products.The fermentation medium changed the microbial community's structure and improved CFL microbial activity.We also revealed the contributions of chemical components and VFCs to sensory evaluation and the association between predominant microbes and VFCs.

FIGURE 3
FIGURE 3Hierarchical clustering of volatile flavor compounds in CFLs.

FIGURE 4
FIGURE 4Score scatter three-dimensional plots of the partial least-squares regression (PLS).PLS of various CFLs is represented as a twodimensional representation of the scores (t[1] and t[2]) on the first and second PLS components.

FIGURE 5
FIGURE 5 Diversity of microbial communities in CFLs.Alpha diversity was determined based on the Chao1, Shannon, and Simpson indices (A).Bacterial beta diversity (B) and fungal beta diversity (C) were measured by weighted UniFrac distance.**means significance at the level of 0.01.

FIGURE 7
FIGURE 7Evolutionary branch map of the bacteria (A) and fungi (B) significantly differs in CFLs.

TABLE 1
VIP values and correlation coefficients between variables and total score of sensory evaluation † .
† Means significance at the level of 0.05.The coefficient is significant (p < 0.05).VIP[1] and VIP[2] represent the VIP of the first and second principal components, respectively, and the coefficient values [1] and [2] represent the coefficient values of the first and second principal components, respectively.

TABLE 2
Significant differential functional genes.

TABLE 3
Spearman's correlation coefficients between representative microbes and core volatile flavor compounds.