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Effect of microbial community on the formation of flavor components in cigar tobacco leaves during air-curing

BMC Microbiology volume 25, Article number: 56 (2025) Cite this article

Abstract

Background

The air-curing process of cigar tobacco leaves is typically conducted in an open environment, involving the participation of various microorganisms. However, the effect of microbial communities during air-curing process on the formation of flavor components remains unclear. Therefore, this study aims to reveal the dynamics of flavor components and microbial community changes, and explore the potential role of microbial communities in flavor formation during the cigar tobacco air-curing process.

Results

High-throughput sequencing analysis showed that Pantoea, Sphingomonas and Pseudomonas were the dominant bacterial genera during air-curing process, while Aspergillus was the dominant fungal genus. Subsequently, volatile flavor analysis shows that alkaloids were the most important volatile compounds in cigar leaves, followed by esters, alcohols and aldehydes. Furthermore, 38 characteristic volatile flavor compounds at different periods of air-curing were identified based on PLS-DA in different periods of air-curing. The correlation analysis between microorganisms and flavor components showed that Pantoea and Staphylococcus might promote the flavor formation from browning to post-air-curing and were positively correlated with specific flavor components like phenylacetaldehyde and acetophenone. Phoma, Mycosphaerella, Wallemia, and Cladosporium were identified as key fungal genera influencing flavor formation, as they showed positive correlations with multiple flavor components. These information enrich our understanding of the flavor formation of cigar tobacco during air curing.

Conclusions

There is a complex correlation between the microbial community and the flavor components, which may have a great influence on the flavor formation during the air-curing process of cigar leaves. Bacterial communities have higher species diversity and richness during air-curing, and have more complex correlation characteristics with volatile flavor, which may play more roles in the flavor formation. This study revealed the potential role of microbial community on flavor formation in cigar tobacco air-curing process, and provided guidance for subsequent screening of specific functional microorganisms to improve and stabilize cigar tobacco flavor.

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Introduction

As a globally renowned premium tobacco product, cigars are known for their rich aroma, harmonious blend of sweetness and bitterness, and intoxicating charm [1]. Distinguished from conventional flue-cured tobacco processing, the processing of cigar tobacco leaves is considerably intricate, involving multiple stages such as air-curing, agricultural fermentation, industrial fermentation, rolling and aging [2]. Among these, air-curing is the initial step of cigar tobacco leaves processing, where harvested leaves undergo treatment in a well-ventilated air-curing barn with suitable temperature, humidity, and ventilation conditions. Influenced by environmental factors and microbial activity, the leaves gradually dehydrate and dry, exhibiting a gradual shift in external color from yellow to brown, while internal substances undergo chemical transformations that shape the distinctive flavor profile of cigars tobacco [3].

There are abundant microbial communities distributed on the surface of cigar tobacco leaves, which play an important role in the quality formation of cigar tobacco leaves processing [4, 5]. Previous studies have shown that microorganisms can effectively reduce the impurity, pungency and bitterness in tobacco leaves through metabolic activities, promote the accumulation of aromatic compounds, and help reduce the production of undesirable components such as ammonia and nicotine. For example, Bacillus can decompose and utilize macromolecular substances such as starch, protein, cellulose and carotene in tobacco leaves to produce aromatic components and thus promote the formation of tobacco aroma [6,7,8]. Bacillus and Aspergillus can convert sugars into organic acids, alcohols, and esters, which can give fermented tobacco a fruity, floral, or wine-like flavor that enhances the complexity and character of tobacco flavor [9]. Microbacterium pasteuri, Pseudomonas and Bacillus parabrevis have strong nicotinic degrading ability [10, 11]. These results indicate that microbial community is involved in the quality formation of tobacco leaves. Therefore, our research hypothesis is that certain dominant and characteristic microbial communities during the air-curing process of cigar tobacco leaves play crucial and specific roles in the formation of distinct flavor components, and their metabolic activities directly contribute to the generation and modification of key flavor compounds. However, due to differences in origin, fermentation technology, tobacco leaf varieties and other factors, different cigar leaves have different functional microbial communities, which also leads to differences in flavor quality of cigar leaves produced [12]. Therefore, exploring the microbial community structure and its relationship with volatile flavor compounds can deepen our understanding of the formation mechanism of flavor quality in cigar tobacco leaves, and point out the direction of subsequent improvement of tobacco quality. Unfortunately, previous studies on the air-curing process of cigar tobacco mainly focused on the characterization of the types and variation rules of microorganisms or flavor components at a single level [2, 13, 14], as well as the influence of technological conditions on the formation of tobacco quality [15, 16]. However, the internal relationship of the influence of microbial communities on the formation of flavor components during air-curing is still unclear.

Thus, this study collected cigar tobacco leaves samples at five key air-curing stages (fresh leaf, withering, yellowing, browning, post-air-curing) to investigate the microbial community composition during the air-curing process of cigar tobacco leaves and their potential role in the formation of volatile flavor compounds. Initially, the structure and dynamics of microbial communities and volatile flavor components in cigar tobacco during air-curing were analyzed by high-throughput sequencing and HS-SPME-GCMS, respectively. Subsequently, the characteristic microorganisms and flavor components of cigar leaves at different air-curing periods were identified by LEfSe analysis and PLS-DA. Finally, RDA and correlation networks were employed to explore the potential associations between dominant and characteristic microbial communities and flavor component formation, respectively. This study provides scientific insights into the potential role of microbes in flavor formation during tobacco air-curing process, aiding in the selection of specific functional microbes to enhance or stabilize the flavor quality of cigar tobacco leaves in the future.

Results

Microbial community structure analysis

High-throughput sequencing of amplicons was utilized to analyze microbial communities during air-curing. Following the quality control of the original sequencing data, the merging of raw reads, and the elimination of chimeras, a total of 1,750,517 clean reads for bacteria and 1,396,212 clean reads for fungi were obtained from 15 cigar tobacco leaf samples. Moreover, each sample yielded at least 106,330 and 73,070 clean reads, with an average of 116,701 clean reads for bacteria and 93,080 clean reads for fungi. The microbial community structure of cigar tobacco during air-curing was analyzed at the microbial genus level (Fig. 1). Pantoea (3.9–34.9%), Sphingomonas (4.1–31.5%), and Pseudomonas (4.1–19.5%) were dominant bacterial genera during air-curing (Fig. 1A). Among these, Pantoea exhibited a gradual increase in relative abundance during curing, reaching a peak of 34.9% at the end of the process, becoming the overwhelmingly dominant bacterial genus, while Sphingomonas and Pseudomonas have high abundance mainly from withering stage to browning stage. Notably, Acinetobacter (31.5%) displayed high abundance only in the fresh leaf phase, suggesting a possible origin from the growing environment [17]. Regarding fungal communities, Aspergillus predominated throughout the entire air-curing process, particularly in the fresh leaf stage and withering stage with 33.3% and 63.1%, respectively (Fig. 1B). Additionally, Phoma (34.3%) and Mycosphaerella (18.8%) were dominant fungi during the yellowing phase, while Diaporthe (29.7%), Septoria (17.3%), and Alternaria (15.3%) prevailed during the browning phase, with Wallemia (60.5%) dominating in post-air-curing period.

Fig. 1
figure 1

The microbial community structure during different air-curing periods of cigar tobacco leaves. (A) Bacterial community; (B) Fungal community

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Microbial community diversity analysis and characteristic microorganism identification

The α-diversity at different air-curing periods was analyzed to understand the diversity and richness of the microbial communities in cigar tobacco leaves (Fig. 2). In terms of bacterial community, all α diversity indexes decreased with the progress of air-curing, indicating that species diversity and richness gradually decreased with the progress of air-curing (Fig. 2A). This result may be due to the gradual decrease of moisture in tobacco leaves and the poor environmental stress resistance of most microorganisms. On the contrary, the species diversity and richness of fungal communities continued to increase throughout the air-curing process (Fig. 2B), indicating that the fungal community may be continuously enriched. The β-diversity was analyzed to understand the overall variability of microbial community by PcoA (Fig. 3A and B). In essence, substantial differences were observed in the bacterial community composition from the fresh-leaf period to withering period and after the yellowing period (Fig. 3A). Conversely, the fungal community exhibited relatively minor differences before the yellowing period, but underwent notable changes thereafter. These findings suggest that the yellowing stage may be a pivotal period of microbial fermentation during the air-curing process (Fig. 3B).

Fig. 2
figure 2

The α-diversity of microbial communities during different air-curing periods of cigar tobacco leaves. (A) Bacterial community; (B) Fungal community

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Fig. 3
figure 3

The beta diversity of microbial communities and identification of characteristic microorganism. The PcoA score plot of bacterial community (A) and fungal community (B) during air-curing. Differential bacteria (C) and fungi (D) in different air curing periods

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Furthermore, microorganisms with LDA score greater than 2.0 were identified by LEfSe analysis and were regarded as characteristic microorganisms in different air-curing periods (Fig. 3C and D). In terms of bacterial community, the number of characteristic bacterial genera gradually decreased with the progress of air-curing (Fig. 3C). Fifteen bacterial genera, including Timonella, Empedobacter, Exiguobacterium, Solibacillus, Desemzia, Aliihoeflea and Paracoccus, have been identified as the characteristic bacteria of the fresh leaf period. Craurcoccus, Methylobacterium, Paenochrobactrum and Pseudomonas were the characteristic bacteria during withering period, and Laclobacillus, Aureimonas and Sphingomonas were characteristic of the yellowing period. Only one characteristic bacterial genus, Candidatus Methylomirabilis, was identified during the browning period. Rickettsiella, Pantoea and Serratia were the characteristic bacteria in post-air-curing. The characteristic fungi mainly concentrated in the browning stage and the post-air-curing stage (Fig. 3D). Specifically, Botryosphaeria, Septoria, Alternaria and Diaporthe were genera of fungi characteristic of the browning stage. Sphaeropsis, Cladosporium, Penicillium, Debaryomyces, Meyerozyma, Candida, Plectosphaerella, Filobasidium and Wallemia were the characteristic fungi genera in post-air-curing.

Dynamics of volatile flavor components

A total of 94 volatile components, including 21 esters, 14 aldehydes, 11 alcohols, 11 ketones, 10 acids, 8 heterocycles, 5 alkaloids, 4 terpenoids and 10 others were detected by HS-SPME-GC-MS (Supplementary material: Table S1). Among them, alkaloids are the primary volatile compounds in cigar tobacco, mainly represented by nicotine and its derivatives such as dienicotinoid (Supplementary material: Table S1). In addition, esters, alcohols and heterocyclic groups are the most abundant flavor components besides alkaloids (Fig. 4A). Notably, the content of almost all flavor components increased noticeably during the withering phase. After the yellowing stage, most flavor components including esters, heterocyclics, and ketones, excluding alkaloids, demonstrated a decline to some extent (Fig. 4A).

Fig. 4
figure 4

Analysis of volatile flavor compounds. (A) Columnar accumulations of volatile componens contens. The PLS-DA socre plot (B) and VIP values (C) of volatile flavor componens. (D) The heatmap of volatile flavor compounds with VIP more than 1

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To explore the overall changes in volatile flavor components during the air-curing process, the PLS-DA model was established. The PLS-DA score plot showed significant changes in overall flavor components during the withering and yellowing stages (Fig. 4B). After the yellowing period, the flavor score tended to stabilize, indicating that the cigar leaf flavors had essentially formed by this stage. Furthermore, 38 characteristic volatile flavor compounds with significant changes (VIP > 1) during air-curing were identified based on variable importance in the projection (Fig. 4C), and their concentration change heat maps were plotted (Fig. 4D). Specifically, the characteristic components in the fresh leaf period are mainly acids and ketones, including 2-methylbutyric acid (C44), isovaleric acid (C47), isobutyric acid (C50), 2, 3-pentanedione (C63), 3, 5-octadiene-2-one (C60), and trans, trans-3,5-octene-2-one (C59). The withering phase were characterized by isovaleraldehyde (C30), 2-methylbutanal (C35), neonicotinoid (C36), dienicotinoid (C38), 2-n-amylfuran (C70), and 2-ethylfuran (C69) (Fig. 4D). Moreover, characteristic esters such as methyl phenylacetate (C74), methyl benzoate (C76), methyl formate (C80) and methyl butyrate (C91) began to accumulate from the withering stage. The characteristic flavor components in yellow stage were 2, 3-butanedione (C61), 3-hydroxy-2-butanone (C62), ligustrazine (C66), butene ester formic acid (C46) and methyl nicotinate (C94). There were fewer characteristic flavor components in the browning stage. The content of aldehyde in phenylacetaldehyde (C23), n-glutaraldehyde (C24), 2-methylacrolein (C34) increased obviously at post-air-curing period (Fig. 4D). Most aldehydes are almond and sweet, and may contribute to enhancing the complexity and layer of the cigar aroma [18].

Correlation analysis between dominant microorganisms and characteristic volatile flavor components

Because of the high species abundance, the dominant microbial community may exert more metabolic activities to lead the flavor transformation. Therefore, analyzing the internal relationship between the dominant microbial community and the formation of flavor components may help to explain the flavor formation mechanism of cigar tobacco during the air-curing process. The relationship between the top 10 microbial genera and the overall flavor and characteristic flavor components of samples in different periods was explored by redundancy analysis (Fig. 5). In terms of bacteria (Fig. 5A), Pantoea and Staphylococcus may promote flavor formation between browning and post-air-curing period, showing positive correlations with specific flavor components like 2-methylacrolein (C34),) 1-hexadecanol (C3, 2-methylfuran (C71), phenylacetic acid (C41), phenylacetaldehyde (C23), N-glutaraldehyde (C24), acetophenone (C55), damadone (C58), methyl phenylacetate (C74). Acinetobacter, Comamonas, Chryseomicrobium seemed to influence the characteristic flavor components of the fresh-leaf stages, showing positive correlations with butene ester formic acid (C46), isobutyric acid (C50), geranyl acetone (C53), trans, trans-3,5-octene-2-one (C59), 2,3-butanedione (C61). Furthermore, bacterial genera including Brevundimonas, Pseudomonas, Methylobacterium, Sphingomonas, Aureimonas may dominate the overall flavor formation in withering and yellowing stages, with positive correlation with cis-2-pentenol (C2), trans-3-hexene-1-ol (C4), isovaleraldehyde (C30), 2-methylbutanal (C35), neonicotinoid (C36), 6-methyl-5-hepten-2-one (C56).

Fig. 5
figure 5

Redundancy analysis of the top 10 dominant microorganisms in relative abundance with characteristic volatile flavor components. (A) Bacteria; (B) Fungi

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The results of RDA indicate that Phoma, Mycosphaerella, Wallemia and Cladosporium might be key fungi genera affecting the formation of characteristic flavor components during the air-curing process of cigar tobacco leaves (Fig. 5B). Among them, Phoma, Mycosphaerella show a positive correlation with the characteristic flavor in the yellowing stage, including ligustrazine (C66), 3-hydroxy-2-butanone (C62), butene ester formic acid (C46), 2,3-butanedione (C61), methyl benzoate (C76), benzaldehyde (C22). Moreover, Wallemia and Cladosporium were positively correlated at the post-air-curing with characteristic substances such as methyl phenylacetate (C74), phenylacetic acid (C41), phenylacetaldehyde (C23), N-glutaraldehyde (C24), acetophenone (C55), 2-methylfuran (C71), 1-hexadecanol (C3), 2-methylacrolein (C34). In addition, Aspergillus and Lycoperdon are closely related to the various flavor components of organic acids and ketones.

Correlation analysis between characteristic microorganisms and characteristic volatile flavor components

Microbial communities contribute to the improvement of sensory properties such as aroma and taste through metabolic activity [12]. Differences in microbial communities may be a potential factor contributing to changes in volatile flavor components [17]. Therefore, investigating the potential associations between characteristic microbes and significantly altered flavor components could provide insights into the flavor formation mechanisms of cigar tobacco during air-curing. The Spearman correlation coefficients between characteristic microbes and flavor components were calculated, and visualized using correlation networks (Fig. 6). In comparison to characteristic fungi, the relationships between characteristic bacterial communities and volatile flavors appear more complex, suggesting that bacteria may play a more significant role in flavor formation (Fig. 6A). Within the bacterial community, Serratia and Rickettsiella showed significant positive correlations with 1-hexadecanol (C3), phenylacetic acid (C41), phenylacetaldehyde (C23), 2-methylfuran (C71), and 2-methylacrolein (C34). Additionally, trans-2-hexenol (C7), guaiacol (C17), isobutyric acid (C50), 2,3-pentanedione (C63), (1′S,2′S)-Nicotine-1’-oxide (C39), isovaleric acid (C47), β-cyclocitral (C29), 2-methylbutyric acid (C44) and heptanal (C25) showed significant positive correlations with most characteristic bacteria, but exhibited significant negative correlations with Pantoea and Paracoccus. Interestingly, Pseudomonas displayed negative correlations with 2-methylacrolein (C34), 2-methylfuran (C71), phenylacetaldehyde (C23), 1-hexadecanol (C3), and phenylacetic acid (C41), which is consistent with the RDA results in the previous section.

Fig. 6
figure 6

Correlation network analysis of characteristic bacteria and characteristic volatile flavor components. (A) Bacteria; (B) Fungi

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In the terms of fungi (Fig. 6B), Penicillium, Alternaria, Septoria, and Botryosphaeria exhibit positive correlations with various flavor components, suggesting which may be characteristic fungi contributing positively to the flavor formation of cigar tobacco. Among these, it is worth noting that Penicillium shows significant positive correlations with 3-hydroxy-2-butanone (C62), isovaleraldehyde (C30), 2,3-butanedione (C61) and ligustrazine (C66). Additionally, Cladosporium, Alternaria, and Septoria show positive correlations with 6-methyl-5-hepten-2-one (C56), 2-methylbutanal (C35), and 2-n-amylfuran (C70), potentially contributing to enhancing the baking and sweet aroma of cigar tobacco leaves.

Discussion

Tobacco leaves have a long history of cultivation and processing worldwide and are one of the most widely cultivated non-food crops in the world [19]. Cigar tobacco leaves require air-curing after picking for subsequent fermentation, which is crucial for their unique flavor [20]. This process occurs in an open environment where environmental conditions and tobacco’s organic matter influence microorganism accumulation and metabolism, thus affecting flavor quality. Hence, this study analyzed the microbial community dynamics and flavor composition during the air-curing process and explored their relationship to explain the impact of the microbial community on flavor formation.

The air-curing of tobacco leaves is typically conducted in an open environment, where suitable temperature, humidity, and organic matter in the tobacco provide an optimal environment and substrate for the proliferation and metabolism of microorganisms [21]. Exploring the structure and dynamics of microbial communities is instrumental in deepening our understanding of the biochemical transformations involved in tobacco leaves during air-curing. In this study, amplicon-based high-throughput sequencing was conducted during the air-curing of cigar leaves. It was found that the number of bacterial genera was much higher than that of fungal genera. This might imply that the bacterial community is more diverse and complex compared to the fungal community and dominates the air-curing process, thus having a greater impact [13]. Further, the microbial community composition of cigar tobacco leaves was analyzed. Pantoea, Sphingomonas and Pseudomonas were the dominant bacterial genera, which was basically consistent with our previous study [22]. Previous studies have indicated that the Pantoea is widely present in the brewing environment of traditional fermented food such as rice wine [23], Baijiu [24], and vinegar [25] due to its good environmental stress resistance and closely related to the formation of various flavor compounds. Additionally, Pseudomonas has been reported to possess significant nicotine-degrading capabilities [26], potentially leading to a decrease in alkaloids concentration during the yellowing period. Aspergillus, a dominant fungal genus throughout the entire air curing process, exhibits a gradual decline in relative abundance as air curing progresses, consistent with a previous finding [14]. Additionally, a previous study has indicated that Aspergillus is also a leading fungal genus in heap fermentation processes, capable of producing various enzymes such as proteases and amylases to enhance the generation of aromatic compounds, thus improving the quality of cigars [18]. The dominance of Phoma and Mycosphaerella during the yellowing phase indicates their potential importance in this particular stage of cigar tobacco air-curing. Although there is relatively less prior research specifically focusing on their roles in cigar processing compared to Aspergillus, it can be hypothesized that they also contribute to the flavor transformation during this period. Therefore, this study also conducted a follow-up analysis of their association with characteristic flavor components to reveal their potential role. Furthermore, the characteristic microorganisms of different air curing periods were also identified by LEfSe analysis. The number of characteristic bacteria was significantly greater than that of fungi. And they were mainly concentrated in the fresh leaf stage. The possible reason is that during this stage, tobacco leaves undergo gradual dehydration. As a result, most of the characteristic microorganisms in the fresh leaf period, which had poor stress resistance, could not survive [27]. This finding was consistent with the results of microbial community diversity analysis.

A total of 94 volatile compounds were identified during the air-curing process of cigar tobacco leaves using HS-SPME-GC-MS. Notably, the content of almost all flavor components increased noticeably during the withering phase. This increase may be attributed to the breakdown and utilization of large molecular substances like cellulose by microbes, resulting in the formation of flavor components, or could be due to the relative increase in compound concentration from substantial dehydration of the leaves. Alkaloids are the predominant volatile compounds in cigar tobacco leaves, primarily nicotine. Ensuring an appropriate alkaloid content is crucial for both the sensory experience of cigars and the health of smokers. Smokers perceive varying levels of bitterness, smoke intensity, and complexity of tobacco based on the nicotine content. Insufficient alkaloid content results in a faint aroma and lack of power, while excessive levels lead to strong, irritating flavors and unpleasant experiences [28]. Additionally, nicotine, a highly addictive and hazardous substance, poses numerous health risks with prolonged and excessive consumption, potentially increasing the risks of cardiovascular diseases, respiratory disorders, and certain cancers [29]. It is noteworthy that the nicotine content in cigar tobacco leaves is relatively lower compared to other tobacco products [18]. Apart from alkaloids, cigar tobacco leaves also contain a rich array of esters, alcohols, aldehydes, ketones, and heterocyclic compounds, providing a richer aroma experience and softening the harshness of the tobacco leaves [4]. For instance, esters and alcohols often impart floral, fruity and sweet aromas; aldehydes and ketones contribute to nutty, creamy, and toasted aromas; furan compounds offer a distinctive caramel aroma [32,33,34]. Forexample, 3-hydroxy-2-butanone (also known as acetoin), a volatile flavor compound with a nutty and creamy aroma, is widely utilized in food additives and chemical flavor production and has the potential to convert into ligustrazine (2,3,5,6-tetramethylpyrazine) during the aging process to offer certain health benefits. The combined effects of these flavor components shape the unique and captivating aroma characteristics of cigar tobacco leaves.

Finally, the potential associations between dominant and characteristic microbial communities and characteristic flavor components were investigated, respectively. A previous study showed that Pseudomonas and Sphingomonas were significantly positively correlated with the formation of flavor compounds such as acetophenone, decyl aldehyde, and β-cyclocitral during the industrial fermentation of cigar tobacco leaves, which may have played an important contribution to the formation of tobacco flavor [1]. Similarly, in this study, the dominant bacterial genera Pseudomonas and Sphingomonas were positively correlated with a variety of ketones, aldehydes, and alcohols, which may be responsible for the overall flavor formation during the withering and yellowing periods. On the other hand, it is worth mentioning that there are more significant correlation characteristics between characteristic bacteria and flavor characteristics, suggesting that bacterial communities may play more roles in flavor formation during the air-curing process, which is consistent with previous research results [5, 22]. Previous studies have reported that Acinetobacter, Sphingomonas, Solibacillus, and Lysinibacillus are the major carbonyl compound producing microorganisms in cigar tobacco leaves [22]. Similarly, Solibacillus has been identified in this study as one of the characteristic microorganisms in the air-curing process and has also been analyzed to be significantly positively correlated with the formation of various carbonyl compounds such as 3,5-octadiene-2-one, geranyl acetone and 2,3-pentanedione. There have been few studies on the function of fungal communities during cigar leaf processing, which may be related to the low biomass of fungi [5]. In this study, the dominant fungus Aspergillus showed close correlation between various organic acids and ketone components. As is commonly known, Aspergillus, which widely exists in numerous fermented food environments, can secrete abundant and varied enzymes to convert carbohydrates into organic acids, alcohols, and esters, thus promoting the formation of food flavor [33]. In addition, the characteristic fungi Penicillum, Alternaia, Septoria, Botryosphaeria also showed positive correlation with a variety of flavor components, which were considered to have a positive contribution to the formation of cigar tobacco flavor. These results provide us with some insights into the role of fungi in the flavor formation during tobacco air-curing. It should be noted that due to the amplification bias of primer pairs selected for amplicons towards different species, they cannot accurately represent the fine structure of the real microbial flora, which might affect the accurate judgment of the functions of microbial communities in subsequent analyses. Meanwhile, the sequencing methods based on amplicons are unable to obtain the genomic information of microorganisms, thereby limiting a comprehensive understanding of the specific metabolic functions of microbial communities. Consequently, it remains necessary to further elaborate on the roles of microbial communities in the flavor formation of cigar tobacco leaves from the perspective of functional genes through metagenomic or metatranscriptomic sequencing in furture works. Additionally, it is essential to conduct strain screening based on the revealed functions of specific strains and carry out subsequent laboratory or industrial fermentation verification.

Conclusion

This study shows that bacterial communities possess higher species diversity and richness during air-curing. They exhibit a more intricate correlation with volatile flavor characteristics, which implies their potential to play a more significant role in the formation of flavor quality. Alkaloids, esters, and alcohols are the primary flavor components of cigar tobacco leaves, all exhibiting a trend of initial increase followed by a decrease during air curing. Furthermore, the potential roles played by dominant and characteristic microbes such as Pseudomonas, Sphingomonas, Acinetobacter, Aspergillus, and Penicillium in the tobacco leaf curing process were elucidated through correlation analysis. These research findings deepen our understanding of the mechanisms underlying flavor changes during cigar tobacco leaf curing, offering insights for selecting functional microbes to enhance or stabilize the quality of cigar tobacco leaves in subsequent studies.

Materials and methods

Sample collection

All the tested samples were obtained from the same batch of cigar tobacco leaves during air-curing, provided by Hubei China Tobacco Industry Co., Ltd. Samples were randomly and evenly collected during five key periods of the air-curing process: fresh leaf, withering, yellowing, browning, and post-air-curing, with each stage sampled three times for biological replicates. All samples were stored at -20 ℃ for subsequent testing.

DNA extraction and high-throughput sequencing

The tobacco leaf samples (5 g) were ground into powder in a mortar pre-cooled with liquid nitrogen, then mixed with 100 mL NaCl solution (0.9%, w/v) in a 250 mL conical bottle and elution in a shaker at 10 ℃ and 220 r/min for 3 h. After the tobacco residue was filtered by four layers of gauze, the microbial cells were collected after centrifugation at 4 ℃ and 7000 r/min for 15 min. Total microbial DNA was extracted from tobacco leaf using DNeasy PowerSoil Pro Kit (QIAGEN, Hilden, Germany) according to the instructions. The DNA concentration was determined using a NanoDrop 2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA), and the quality of the DNA samples was verified through agarose gel electrophoresis. Each sample was performed for three biological replicates, and the extracted DNA samples were stored at -80 ℃ for subsequent amplicon-sequencing analysis.

Using the extracted DNA as template, bacterial primers 515 F (5’-GTGCCAGCMGCCGCGGTAA-3’) and 907R (5’-CCGTCAATTCMTTTRAGTTT-3’) were used to amplify the genes in the V4-V5 region of bacterial 16 S rRNA gene. The gene in ITS1 region of the fungus was amplified using the fungal prims ITS1F (5ʹ-CTTGGTTCATTTAGAGGAAGTAA-3ʹ) and ITS2R (5ʹ-GCTGCGTTCTTCATCGATGC-3ʹ). The PCR amplification and library construction were performed in accordance with the methods described by a previous study [34]. Subsequently, the amplicon library was sequenced using the high-throughput Illumina HiSeq 2500 platform (Shanghai Personalbio Technology Co. Ltd., Shanghai, China). QIIME2 software was used to process the original sequence [35], including cutting low-quality parts, splitting sample data according to Barcode, and intercepting Barcode and primer sequences to obtain raw data after initial quality control. UCHIME Algorithm was used to detect and remove chimera sequences to obtain valid data. The valid data were aggregated into amplicon sequence variants (ASVs) at 99% similarity level, and the species classification was annotated by q2-feature-classifier plugin. All raw sequences were deposited in the NCBI Sequence Read Archive with accession number PRJNA1165000.

Volatile compound analysis

The tobacco leaves samples underwent drying in an oven at 40 ℃. Subsequently, 1.5 g of the desiccated tobacco leaves was finely powdered and deposited in a headspace vial for the quantification of volatile compound levels through Headspace Solid-Phase Microextraction coupled with Gas Chromatography-Mass Spectrometry (HS-SPME-GC-MS). An internal standard of 2-Octanol was introduced at a concentration of 100 µg/µL. Extraction of flavor components was carried out using the SPME fiber assembly (50/30 µm DVB/CAR/PDMS, Supelco, USA) at 60 ℃ for 30 min.

The volatile compounds were scrutinized using a Gas Chromatography-Mass Spectrometer (7890 A/5975 C, Agilent Technologies, USA) under specific parameters: desorption time was fixed at 5 min; DB-5MS column (30 m × 250 μm × 0.25 μm, Agilent Technologies, USA) was utilized with a column flow rate of 1 mL/min; the injection temperature was set at 250 ℃, beginning at 40 ℃ for 2 min, ascending at a rate of 10 ℃/min to 250 ℃, then maintained for 6 min. Electron Impact (EI) was employed for ionization at -70 eV; transfer line and ion source temperatures were maintained at 280 ℃ and 210 ℃, respectively. Mass spectral data were captured in full-scan mode, ranging from m/z 33 to 400, with a solvent delay time of 3 min and a scan rate of 10 spectra per second. Data processing encompassed tasks such as raw peak extraction, baseline filtering and calibration, peak alignment, deconvolution analysis, peak identification, and integration, facilitated by Chroma TOF 4.3X software and the LECO-Fiehn Rtx5 database. Qualitative examination of volatile flavor compounds involved matching against the WILEY 8.0 and NIST14 database. A semi-quantitative analysis of volatile flavor compounds in cigar tobacco was carried out by internal standard method. Each sample was added with 100 µg/µL 2-Octanol before HS-SPME extraction. The concentration of each volatile flavor compound was calculated by the ratio of the peak area of each substance to the internal standard substance.

Statistical analysis and data visualization

The microorganisms with LDA score > 2 were identified as characteristic microorganisms in different curing periods of cigar tobacco leaves based on Linear discriminant analysis effect size (LEfSe) analysis [36]. RDA was used to explore the relationship between dominant microbial genera and characteristic flavor components in cigar tobacco leaves. The Spearman correlation coefficients between characteristic microorganisms and characteristic flavor components were analyzed. p < 0.05 and r > 0.7 were defined as significant positive correlation, and p < 0.05 and r < -0.7 were defined as significant negative correlation. The heat map of the concentration change of volatile flavor compounds and the correlation network with characteristic microorganisms were mapped on the OmicStudio analysis platform [37].

Data availability

All raw sequences datasets analysed during the current study were deposited in the NCBI Sequence Read Archive with accession number PRJNA1165000.

Abbreviations

HS-SPME-GCMS:

Headspace-Solid Phase Microextraction Gas Chromatography-Mass Spectrometry

LefSe:

Linear discriminant analysis effect size

PLS-DA:

Partial Least Squares Discriminant Analysis

RDA:

Redundancy Analysis

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Acknowledgements

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Funding

This work was supported by the Hubei Tobacco Company Science and Technology project (027Y2022-008).

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Authors and Affiliations

  1. Key Laboratory of Industrial Biotechnology, School of Biotechnology, Ministry of Education, Jiangnan University, 1800 Lihu Road, Wuxi, 214122, China

    Lin Zhang, Wenlong Li, Zheng Peng & Juan Zhang

  2. Science Center for Future Foods, Jiangnan University, 1800 Lihu Road, Wuxi, 214122, China

    Lin Zhang, Wenlong Li, Zheng Peng & Juan Zhang

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  2. Wenlong Li
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  4. Juan Zhang
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LZ and WL: investigation, writing-original drafting, formal analysis, methodology. ZP and JZ: funding acquisition, supervision, and writing-reviewing and editing. All authors have read and agreed to the published version of the manuscript.

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12866_2025_3774_MOESM1_ESM.xlsx

Supplementary Material 1: Supplementary material Table S1: The concentration of volatile components identified in cigar tobacco leaves during air curing.

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Zhang, L., Li, W., Peng, Z. et al. Effect of microbial community on the formation of flavor components in cigar tobacco leaves during air-curing. BMC Microbiol 25, 56 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12866-025-03774-2

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Keywords

BMC Microbiology

ISSN: 1471-2180

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