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.

Profiling the role of microorganisms in quality improvement of the aged flue-cured tobacco.

BACKGROUND: The aging process in the tobacco production, as in other food industries, is an important process for improving the quality of raw materials. In the spontaneous aging, the complex components in flue-cured tobacco (FT) improve flavor or reduce harmful compounds through chemical reactions, microbial metabolism, and enzymatic catalysis. Some believed that tobacco-microbe played a significant part in this process. However, little information is available on how microbes mediate chemical composition to improve the quality of FT, which will lay the foundation for the time-consuming spontaneous aging to seek ways to shorten the aging cycle. RESULTS: Comparing aged and unaged FT, volatile and non-volatile differential compounds (DCs) were multi-dimensionally analyzed with the non-targeted metabolomes based on UPLC-QTOP-MS (the ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry), GC-MS (gas chromatography-mass spectrometer) assisted derivatization and HP-SPME-GC/MS (headspace solid-phase micro-extraction assisted GC-MS). Products associated with the degradation pathways of terpenoids or higher fatty acids were one of the most important factors in improving FT quality. With the microbiome, the diversity and functions of microbial flora were analyzed. The high relative abundance function categories were in coincidence with DCs-related metabolic pathways. According to the correlation analysis, Acinetobacter, Sphingomonas and Aspergillus were presumed to be the important contributor, in which Aspergillus was associated with the highest number of degradation products of terpenoids and higher fatty acids. At last, the screened Aspergillus nidulans strain F4 could promote the degradation of terpenoids and higher fatty acids to enhance tobacco flavor by secreting highly active lipoxygenase and peroxidase, which verified the effect of tobacco-microbes on FT quality. CONCLUSIONS: By integrating the microbiome and metabolome, tobacco-microbe can mediate flavor-related substances to improve the quality of FT after aging, which provided a basis for identifying functional microorganisms for reforming the traditional spontaneous aging.

Revealing novel synergistic defense and acid tolerant performance of Escherichia coli in response to organic acid stimulation.

Escherichia coli is an important producer of mono- and di-acids, such as D-lactic acid, itaconic acid, and succinic acid. However, E. coli has limited acid tolerance and requires neutralizers in large-scale fermentation, which leads to increased production costs. Mutagenesis breeding has been shown to be inefficient in improving the acid tolerance of strains. Therefore, it is crucial to analyze the acid resistance mechanism of E. coli. To this end, important regulatory genes and metabolic pathways in the highly evolved acid-resistant E. coli were identified based on transcriptome sequencing. By analyzing the overlap of the genes with significantly different expression levels in the four groups, a synergistic membrane-centric defense mechanism for E. coli against organic acid stress was identified. The mechanism includes four modules: signal perception, energy countermeasures, input conditioning, and envelope reinforcement. In addition, genes related to the ABC transporter pathway, polyketide metabolism, pyrimidine metabolism, and dual-arginine translocation system pathways were found for the first time to be potentially resistant to organic acid stress after overexpression. A new antacid ingredient, RffG, increases the survival rate of E. coli by 4509.6 times. This study provides new clues for improving the performance of acid-tolerant cells and reducing the production cost of industrial organic acid fermentation. KEY POINTS: • Systematic analysis of the mechanism of membrane protein partitioning in E. coli to resist organic acids • TAT system transports correctly folded hydrogenase accessory proteins to resist D-lactic acid stress • Enhanced PG synthesis and weakened hydrolysis to reduce acid penetration into cells • Overexpression of RffG in the polyketide synthesis pathway enhances acid tolerance.

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.

Bioaugmentation of Bacillus amyloliquefaciens-Bacillus kochii co-cultivation to improve sensory quality of flue-cured tobacco.

Flue-cured tobacco (FCT) with irritating and undesirable flavor must be aged. However, the spontaneous aging usually takes a very long time for the low efficiency. Bioaugmentation with functional strains is a promising method to reduce aging time and improve sensory quality. To eliminate the adverse effect of excessive starch or protein content on the FCT quality, we used the flow cytometry to sort Bacillus amyloliquefaciens LB with high alpha-amylase and Bacillus kochii SC with high neutral protease from the FCT microflora. The mono, co-culture of strains was performed the solid-state fermentation with FCT. Bacillus amyloliquefaciens monoculture for 2 days and Bacillus kochii monoculture for 2.5 days achieved the optimum quality. B. amyloliquefaciens-B. kochii co-culture at a ratio of 3:1 for 2 days of fermentation showed a more comprehensive quality enhancement and higher functional enzyme activity than mono-cultivation. Through OPLS-DA model (orthogonal partial least-squares-discriminant analyzes), there were 38 differential compounds between bioaugmentation samples. In co-cultivation, most of Maillard reaction products and terpenoid metabolites were at a higher level than other samples, which promoted an increase in aroma, softness and a decrease in irritation. This result validated the hypothesis of quality improvement via the co-culture. In our study, we presented a promising bioaugmentation technique for changing the sensory attributes of FCT in a short aging time.

Exploration of seasonal fermentation differences and the possibility of flavor substances as regulatory factors in Daqu.

Medium-high temperature Daqu is a characteristic starter for Chinese strong-flavor Baijiu fermentation, and its final quality determines the character and type of Baijiu. Nonetheless, its formation is affected by the interaction of physical and chemical, environmental and microbial interaction, and the differences in seasonal fermentation performance emerge. Here, the differences in the two seasons' Daqu fermentation properties were revealed by the detection of the enzyme activity. The respective dominant enzyme in summer Daqu (SUD) was protease and amylase, while cellulase and glucoamylase in spring Daqu (SPD). The underlying causes of this phenomenon were then investigated through an evaluation of nonbiological variables and microbial community structure. A greater absolute number of microorganisms, particularly Thermoactinomyces, were created in the SPD as a result of the superior growth environment (higher water activity). Additionally, the correlation network and discriminant analysis hypothesized that the volatile organic compound (VOC) guaiacol, which had a different content between SUD and SPD, may be a contributing element to the microbial composition. In contrast to SUD, the enzyme system activity related to guaiacol production in SPD was significantly higher. To support this notion that the volatile flavor chemicals mediate microbial interactions in Daqu, the growth effect of guaiacol on several bacteria isolated from the Daqu was examined in both a contact and non-contact manner. This study emphasized that VOCs not only have the basic characteristics of flavor compounds but also have ecological significance. Because the strains' varied structures and enzyme activities affected how the microorganisms interacted, the VOCs produced in this way ultimately had a synergistic effect on the various effects of Daqu fermentation.

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, ß-nicotyrine, nicotinic degradation products, and total nitrogen were significantly and positively correlated with sensory quality. Methylpyrazine, D,L-anatabine, and ß-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.

Analysis of the structure and metabolic function of microbial community in cigar tobacco leaves in agricultural processing stage.

The agricultural fermentation processing of cigar tobacco leaves (CTLs), including air-curing and agricultural fermentation, carried out by tobacco farmers has rarely been studied. In this study, we have investigated the microbial community in the CTLs during air-curing and agricultural fermentation by 16S rRNA and ITS gene high-throughput sequencing. The results showed that the richness of microbial communities gradually increased with the development of agricultural fermentation, which means that not all microorganisms in CTLs come from the fields where tobacco grows, but gradually accumulate into CTLs during the fermentation process. Enterobacteriaceae, Chloroplast, and Alternaria were the dominant genera in the air-cured CTLs. Aquabacterium, unclassified Burkholderiaceae, Caulobacter, Brevundimonas, and Aspergillus were the dominant genera in the agriculturally fermented CTLs. Acinetobacter, Methylobacterium, Sampaiozyma, and Plectosphaerella first significantly increased, and then significantly decreased during agricultural processing. The changes in microbial communities are mainly related to their different functions during fermentation. This means that when the fermentation effect of the original microbial community in cigar tobacco leaves is not ideal, we can optimize or design the microbial community based on the fermentation function that the microbial community needs to achieve. These results may help adjust and optimize the agricultural fermentation process of CTLs, and help develop the quality of CTLs and increase the income of tobacco farmers.

Effects of Inoculation With Acinetobacter on Fermentation of Cigar Tobacco Leaves.

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.

Function-driven design of Bacillus kochii and Filobasidium magnum co-culture to improve quality of flue-cured tobacco.

Flue-cured tobacco (FCT) is an economical raw material whose quality affects the quality and cost of the derived product. However, the time-consuming and inefficient spontaneous aging is the primary process for improving the FCT quality in the industry. In this study, a function-driven co-culture with functional microorganisms was built in response to the quality-driven need for less irritation and more aroma in FCT. The previous study has found that Bacillus kochii SC could degrade starch and protein to reduce tobacco irritation and off-flavors. The Filobasidium magnum F7 with high lipoxygenase activity was screened out for degrading higher fatty acid esters and terpenoids to promote the aroma and flavor of FCT. Co-cultivation with strain SC and F7 obtained better quality improvement than mono-culture at an initial inoculation ratio of 1:3 for 2 days, representing a significant breakthrough in efficiency and a reduction in production costs compared to the more than 2 years required for the spontaneous aging process. Through the analysis of microbial diversity, predicted flora functions, enzyme activities and volatile compositions within the mono- and co-cultivation, our study showed the formation of a function-driven co-culture between two strains through functional division of labor and nutritional feeding. Herein, the function-driven co-culture via bioaugmentation will become an increasingly implemented approach for the tobacco industry.

Analysis of Microbial Community, Volatile Flavor Compounds, and Flavor of Cigar Tobacco Leaves From Different Regions.

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