Project description:IntroductionCigar stacking fermentation is a key step in tobacco aroma enhancement and miscellaneous gas reduction, which both have a great influence on increasing cigar flavor and improving industrial availability.MethodsTo analyze the effect of cigar tobacco leaf (CTLs) microbial community on volatiles flavor compounds (VFCs), this study used multi-omics technology to reveal the changes in microbial community structure and VFCs of different cigar varieties during stacking fermentation, in addition to exploring the interaction mechanism of microbiome and VFCs.ResultsThe results showed that the dominant microbial compositions of different CTL varieties during stacking fermentation were similar, which included Staphylococcus, Corynebacterium 1, Aerococcus, and Aspergillus. These dominant microbes mainly affected the microbial community structure and characteristic microorganisms of CTLs through microbial interactions, thereby influencing the transformation of VFCs. Characteristic microorganisms of different CTLs varieties such as Trichothecium, Trichosporon, Thioalkalicoccus and Jeotgalicoccus, were found to positively correlate with characteristic VFCs like megastigmatrienone 4, pyrazine, tetramethyl-, geranyl acetone, and 2-undecanone, 6,10-dimethyl-, respectively. This in turn affected the aroma and sensory quality of the CTLs.DiscussionThis study provides theoretical support for the analysis of the mechanism of microorganisms on VFCs and aroma, and development of microbial agents during cigar stacking fermentation.
Project description: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.
Project description:BackgroundThe 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.ResultsHigh-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.ConclusionsThere 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.
Project description:Metabolic activity of the microbial community greatly affects the quality of cigar tobacco leaves (CTLs). To improve the quality of CTLs, two extrinsic microbes (Acinetobacter sp. 1H8 and Acinetobacter indicus 3B2) were inoculated into CTLs. The quality of CTLs were significantly improved after fermentation. The content of solanone, 6-methyl-5-hepten-2-one, benzeneacetic acid, ethyl ester, cyclohexanone, octanal, acetophenone, and 3,5,5-trimethyl-2-cyclohexen-1-one were significantly increased after inoculated Acinetobacter sp. 1H8. The inoculation of Acinetobacter sp. 1H8 enhanced the normal evolutionary trend of bacterial community. The content of trimethyl-pyrazine, 2,6-dimethyl-pyrazine, and megastigmatrienone were significantly increased after inoculated Acinetobacter indicus 3B2. The inoculation of Acinetobacter indicus 3B2 completely changed the original bacterial community. Network analysis revealed that Acinetobacter was negatively correlated with Aquabacterium, positively correlated with Bacillus, and had significant correlations with many volatile flavor compounds. This work may be helpful for improving fermentation product quality by regulating microbial community, and gain insight into the microbial ecosystem.
Project description:IntroductionMicrobiomes of cigar tobacco leaves play a pivotal role during the fermentation, and fermentation temperature is a key factor in shaping the structure and function of the microbial community. This study aimed to investigate the effects of different temperatures (30°C, 35°C, 40°C, 45°C, and 50°C) on the microbiomes of cigar tobacco leaves, providing insights into the complex interactions among temperature, microbes, and physicochemical metabolites.MethodsFirstly, the physicochemical metabolites of cigar tobacco leaves under various fermentation temperatures were detected by gas chromatography-mass spectrometry. Subsequently, the impacts of different temperatures on microbial biomass and community structure were revealed by quantitative real-time PCR and amplicon sequencing, and the biomarkers at different fermentation temperatures were identified by LEfSe analysis. Finally, the functional potential of microbes was predicted by correlation analysis.ResultsThe bacterial biomass increased initially and peaked at 8.4 × 109 copies/g at 35°C, then decreased as the temperature rose. The fungal biomass exhibited a downward trend with increasing temperature, reaching a maximum of 3.9 × 106 copies/g at 30°C. When the fermentation temperature exceeded 45°C, the growth of both bacteria and fungi was significantly restricted. Amplicon sequencing results indicated that Staphylococcus and Aspergillus genera dominated the bacterial and fungal communities, respectively. As the temperature increased, the relative abundance of Staphylococcus decreased first and then increased (46.1%-98.5%), while that of Aspergillus increased first and then decreased (34.9%-77.4%). Additionally, correlation analysis suggested that microbial communities shaped by different temperatures were responsible for the differences in physicochemical metabolites of cigar leaves. The biomarkers identified in the low-temperature fermentation group, including Staphylococcus, Stemphylium, Sampaiozyma, and Filobasidium, were likely responsible for the production of flavor metabolites, the accumulation of sugars, and the elevated ratio of potassium ions to chloride ions contents. Biomarkers in medium and high-temperature fermentation groups, such as Aspergillus, Neodymella, Acinetobacter, Pelomonas, Brevundimonas, and Alkalihalobacillus, might contribute to the degradation of nitrogen-containing substances and alkaloids.DiscussionThis study revealed the unique microbial community structure shaped at different temperatures and its potential correlation with physicochemical metabolites. These findings will help to further optimize the fermentation process of cigar tobacco leaves and develop functional microorganisms suitable for different fermentation temperatures.
Project description: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.
Project description:Metabolic enzyme activity and microbial composition of the air-curing and fermentation processes determine the quality of cigar tobacco leaves (CTLs). In this study, we reveal the evolution of the dominant microorganisms and microbial community structure at different stages of the air-curing and fermentation processes of CTLs. The results showed that the changes in metabolic enzymes occurred mainly during the air-curing phase, with polyphenol oxidase (PPO) being the most active at the browning phase. Pseudomonas, Bacteroides, Vibrio, Monographella, Bipolaris, and Aspergillus were the key microorganisms in the air-curing and fermentation processes. Principal coordinate analysis revealed significant separation of microbial communities between the air-curing and fermentation phases. Redundancy analysis showed that bacteria such as Proteobacteria, Firmicutes, Bacteroidota, and Acidobacteriota and fungi such as Ascomycota and Basidiomycota were correlated with enzyme activity and temperature and humidity. Bacteria mainly act in sugar metabolism, lipid metabolism, and amino acid metabolism, while fungi mainly degrade lignin, cellulose, and pectin through saprophytic action. Spearman correlation network analysis showed that Firmicutes, Proteobacteria, and Actinobacteria were the key bacterial taxa, while Dothideomycetes, Sordariomycetes, and Eurotiomycetes were the key fungal taxa. This research provides the basis for improving the quality of cigars by improving the air-curing and fermentation processes. KEY POINTS: • Changes in POD and PPO activity control the color change of CTLs at the air-curing stage. • Monographella, Aspergillus, Pseudomonas, and Vibrio play an important role in air-curing and fermentation. • Environmental temperature and humidity mainly affect the fermentation process, whereas bacteria such as Proteobacteria, Firmicutes, Bacteroidota, and Acidobacteriota and fungi such as Ascomycota and Basidiomycota are associated with enzyme activity and temperature and humidity.
Project description: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.
Project description:IntroductionIn order to enhance the quality of cigar tobacco leaves (CTLs), a gradient variable temperature fermentation approach was employed.MethodsThe temperature gradient demonstrated a gradual increase from low temperature (35 ± 2°C) to moderate temperature (45 ± 2°C), and then to high temperature (55 ± 2°C). Each temperature gradient underwent a 10-day fermentation process, resulting in a total duration of 30 days. Changes in sensory evaluation, chemical composition, and bacterial absolute quantitative structure and function were examined throughout the process of gradient variable temperature fermentation.ResultsCompared to constant temperature fermentation, gradient variable temperature fermentation improved the sensory quality of CTLs, reduced total sugar and cembrane degradation products, and increased the amino acid contents. It resulted in significant changes in bacterial quantity and function of CTLs, but had no significant effects on the richness and diversity of bacterial communities. The results of correlation analyses showed that sensory quality had significant correlation with chemical composition, which effected by predominant microbes. The gradient variable temperature fermentation process underwent a three-phase model to characterize the alterations of CTLs. Phase I (35°C) was the microbial stage, during which there was a significant decrease in both the total number and function of microorganisms. The dominant genera shifted from Acinetobacter to Staphylococcus and Corynebacterium, and with high reducing sugar, polyphenol compound and low sensory score. Phase II (45°C) marked a chemical stage, with an enhancement in sensory evaluation. A total of 17 chemicals significantly decreased and six increased, and the decline of microbial populations persisted. The enhanced relative abundances of four microecological hubs, namely Staphylococcus, Corynebacterium, Oceanobacillus, and Bacillus, had the potential to produce protease and lipase to the production of peptides, amino acids, and organic acid, catabolizing sugars and polyphenol compounds, through carbohydrate metabolism and amino acid metabolism, resulted an increase in sensory quality of CTLs. Phase III (55°C) indicated a relative mature stage with the highest score of sensory evaluation. Eight compositions from plamochromic pigments and polyphenol compounds exhibited gradual decreases, while relative contents of carbohydrate metabolism and amino acid metabolism increased.ConclusionThe gradient variable temperature fermentation had demonstrated a significant positive influence on the quality of CTL by providing optimal fermentation temperature for microbial growth, metabolism, and the generation of quality-related chemical compositions.