Tobacco Fermentation Research Articles

Analysis of volatile compound metabolic profiles during the fermentation of filler tobacco leaves through integrated E-nose, GC–MS, GC-IMS, and sensory evaluation

Tobacco is an agricultural commodity with great economic significance. Producers recognize that the flavor characteristics of filler tobacco clear-out (FTLs) affect the quality of cigar tobacco items. To identify the major volatile organic compounds (VOCs) responsible for differences in FTLs, E-nose, HS-SPME-GC–MS, and HS-GC-IMS were employed for multi-dimensional analysis of nine groups of FTLs at various fermentation stages. The E-nose results indicated a progressive increase in nitrogen-oxygen compounds during fermentation. Coupled with these findings, HS-SPME-GC–MS identified 56 VOCs, and HS-GC-IMS revealed 55 VOCs in the FTLs. Principal component analysis (PCA) suggested that FTLs from various fermentation stages of the Yunxue No 39 tobacco plant exhibit significant differences. Partial least squares discriminant analysis (PLS-DA), along with the variable importance in projection (VIP) method for feature selection, pinpointed 12 VOCs as the essential unmistakable metabolites embroiled within the agrarian fermenting prepare of matured FTLs. During the fermentation process, these crucial metabolites are predominantly enriched in pathways including pyruvate metabolism, phenylalanine metabolism, as well as nicotinate and nicotinamide metabolism. The study provides insights into the differences and diversity of FTLs at agricultural fermentation stages. It offers valuable information for optimizing the quality of agricultural fermentation and guiding the directional fermentation of tobacco leaves.

Effects of Phaffia rhodozyma on microbial community dynamics and tobacco quality during tobacco fermentation.

Carotenoids are important precursors of various aroma components in tobacco and play an important role in the sensory quality of tobacco. Phaffia rhodozyma is a species of Xanthophyllomyces capable of synthesizing a highly valuable carotenoid-astaxanthin, but has not yet been used in improving tobacco quality. The dynamic changes of microbial community and metabolites during tobacco fermentation were analyzed in combination with microbiome and metabolome, and the quality of tobacco after fermentation was evaluated by sensory scores. P. rhodozyma could grow and produce carotenoids in tobacco extract, with a maximum biomass of 6.50 g/L and a maximum carotenoid production of 36.13 mg/L at 100 g/L tobacco extract. Meanwhile, the correlation analysis combined with microbiome and metabolomics showed that P. rhodozyma was significantly positively correlated with 11 metabolites such as 6-hydroxyluteolin and quercetin. Furthermore, the contents of alcohols, ketones and esters, which were important aromatic components in fermented tobacco, reached 77.57 μg/g, 58.28 μg/g and 73.51 μg/g, increasing 37.39%, 265.39% and 266.27% compared to the control group, respectively. Therefore, the aroma and flavor, and taste scores of fermented tobacco increased by 0.5 and 1.0 points respectively. This study confirmed that P. rhodozyma fermentation could effectively improve the sensory evaluation of tobacco, and provided a novel microbial fermentation method to improve tobacco quality.

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

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

Effects of different adding methods of fermentation medium on the quality of cigar.

Background: To investigate the effects of different media and media addition methods on the fermentation of tobacco. Methods: A plant extract and a bacterial agent are used for fermenting cigar tobacco leaves in a different order of addition. The chemical composition, cellulose, and pectin content, as well as changes in the microbial community, were measured. Results: The addition method of the fermentation medium affects the quality of fermented tobacco. The optimal medium formula and addition method involve first adding plant extracts and then microbial agents. The medium formula and addition method can significantly reduce cellulose in tobacco, with a reduction rate of 46%, and significantly increase the content of β-carotene, thereby enhancing the aroma of cigarettes. There is an increase in aroma components, such as alcohols, alkanes, and olefins, in tobacco. By reducing the proportion of Aspergillus, it can alter the microbial community structure of tobacco. Conclusion: Adding plant extracts before introducing microbial agents can significantly improve the quality and alter the microbial community structure of Dexue No.1 tobacco.

Research on the Microbial Community Structure and Evolutionary Changes During the Industrial Fermentation Process of Cigar Tobacco Leaves

Cigar tobacco leaves host a diverse bacterial community on their surfaces, playing a vital role in the fermentation process. This study aimed to investigate and analyze the bacterial community dynamics during the artificial fermentation of cigar tobacco leaves, recognizing the advantages of artificial over natural fermentation. The research focused on elucidating the structural composition of the bacterial community on the leaves, exploring their metabolic activities, understanding how they influence the characteristics of the leaves during artificial fermentation, and identifying potential metabolic pathways. Significant shifts in the microbial community were observed post-fermentation, with a marked increase in diversity. Notably, genera like Oceanobacillus, Terribacillus, and Corynebacterium exhibited substantial growth during the mid-phase of fermentation, suggesting their importance in the process. The insights derived from this study shed light on the artificial fermentation process of cigar tobacco leaves, offering opportunities for enhancing safety and quality.

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.

«Алкогольная» ферментация табака в дубовых бочках. Часть 2. Количественное изменение хлорогеновой кислоты и ее определение методом ВЭЖХ

We studied a quantitative change in chlorogenic acid (CGA) content in raw tobacco during its fermentation in oak barrels in a gas-liquid medium of cognac distillate for six months. Cuban cigar tobacco of the HP variety was used as the object of research. Quantitative determination of CGA was carried out on a liquid chromatograph with a spectrophotometric detector using the external standard method in gradient elution mode. An external standard of CGA was obtained from green coffee beans by extraction with a 40% aqueous solution of methanol, followed by further treatment of the extract with Carrese solutions and evaporation of the treated extract on a rotary evaporator under vacuum. As a result of chromatography of the external standard, a chromatogram in the form of a double peak was obtained. To analyze the tobacco samples under study, their water-methanol extracts were first obtained using the Soxhlet method. HPLC results showed that the mass fraction of CGA in unfermented tobacco raw materials was 0,29%. In fermented tobacco, the content of CGA was 0,19%, which corresponds to a loss of CGA during fermentation by 42,9%. To identify the possible diffusion of CGA from tobacco raw materials into oak staves, a similar analysis was carried out on 1 mm thick oak chips cut from the inner surface of the barrel before and after the fermentation process. Chromatography results showed the absence of CGA in oak chips cut before tobacco fermentation. The chromatogram of the extract of oak chips cut after fermentation showed a peak corresponding to CGA in an amount of 0,047%. However, the peak shape does not allow to unequivocally state that this peak directly relates to CGA.

Study on the correlation between the dominant microflora and the main flavor substances in the fermentation process of cigar tobacco leaves

The flavor of cigar tobacco leaf determines the quality of finished cigar tobacco, and the enhancement of flavor generally relies on microbial fermentation. In this paper, the correlation between the dominant microorganisms and the main flavor substances of cigar tobacco leaves during fermentation and the correlation between the two were investigated to reveal the correlation between microorganisms and flavor and the metabolic pathways of microorganisms affecting the flavor substances. During the fermentation process, the main flavors of cigar tobacco leaves were sweet, light and grassy, with hexanal, 2,6-dimethylpyridine, nonanal, phenylacetaldehyde, naphthalene, and methyl benzoate as the main constituents, and the key microorganisms Haloferax mediterranei, Haloterrigena limicola, Candidatus Thorarchaeota archaeon SMTZ-45, the genera Methyloversatilis, Sphingomonas, Thauera, Pseudomonas, Penicillium, and Aspergillus. Correlation analysis revealed that fungi were negatively correlated with the main aroma and inhibited the main flavor substances, while bacteria were positively correlated with Benzoic acid, methyl ester in the main flavor substances, which was conducive to the accumulation of green aroma. Functional analysis revealed that the dominant bacterial population was producing aroma by metabolizing glycoside hydrolases and glycosyltransferases, performing amino acid metabolism, carbohydrate metabolism and film transport metabolism. The present study showed that the bacterial and fungal dominant microorganisms during the fermentation of cigar tobacco were influencing the production and degradation of the main flavor substances through the enzyme metabolism by the occurrence of the Merad reaction.

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

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

Effects of fermentation chamber temperature on microbes and quality of cigar wrapper tobacco leaves.

The natural fermentation of cigar tobacco leaves usually utilizes natural temperature and humidity for fermentation. Cigars produced in China are often fermented in winter, and the low environmental temperatures can lead to slow heating of the tobacco stack, affecting the cigar tobacco leaves quality. This study aimed to determine the minimum chamber temperature required to initiate the process of fermentation for cigar tobacco leaves and to explore the impact of temperature on the microbial community of tobacco leaves. Here, the cigar variety "Dexue 1" were subjected to stacking fermentation under three temperature parameters (20 ℃, 27 ℃, 34 ℃). With an increase in environmental temperature, the temperature inside the stack of cigar leaves increased significantly, the protein, total sugar, starch, and total alkaloid content in fermented tobacco leaves decreased, and the aroma components and amino acid content increased. Microbial richness and community diversity associated with fermented tobacco were highest at chamber temperatures of above 27 ℃. The relative abundance of Chryseobacterium and Rhodococcus was significantly negatively correlated with protein, alkaloids, total sugar, and starch, and positively correlated with amino acids and aroma components. Chryseobacterium and Rhodococcus may be responsible for the degradation of macromolecular substances and the conversion of favorable aromatic substances, thus improving the tobacco leaves quality. This study demonstrated that increasing the fermentation chamber temperature above 27 ℃ was conductive to raising the inner-stack temperature, increased microbial diversity and aromatic quality, reduced the strength and irritation, and extremely enhanced the overall quality of fermented cigar tobacco leaves. KEY POINTS: • The environmental temperature of the fermentation chamber has a significant impact on the quality of tobacco • Temperature > 27 ℃ can initiate the process of cigar tobacco leaves fermentation and increase inner-stack temperature and microbial diversity and abundance • Chryseobacterium and Rhodococcus may be related to the degradation of macromolecular substances and the transformation of aromatic substances, thereby improving the quality of tobacco leaves.

Effects of adding cocoa fermentation medium on cigar leaves in agricultural fermentation stage.

Background and Objective: With the development of the world economy and the integration of cultures, the Chinese cigar market has shown a significant upward trend. However, high-quality cigar leaves are mostly produced in Dominica, Cuba, Nicaragua and other places. In contrast, Chinese cigar leaves have problems such as insufficient aroma, which has become one of the main factors restricting the development of Chinese cigars. Adding medium to ferment is a traditional method in the cigar industry. At present, it mostly relies on manual experience, and lacks systematic and scientific research. At the same time, the addition of medium fermentation is mainly concentrated in the industrial fermentation process, and has not yet begun to be applied in the agricultural fermentation process. In this study, the medium was added to the agricultural fermentation process for the first time to explore the possibility of the application. The effects of adding cocoa medium to ferment on the chemical composition, sensory quality and surface microbial diversity of eggplant core cigar leaves were investigated.wrapper. Method: With Dexue 7' as the experimental material, the changes of main chemical components of wrapper fermented with water and cocoa medium were determined, and microbial community structure on the surface and relative abundance of cigar leaves at different turning periods were analyzed, and the functional genes were predicted. The results of the study were as follows: 1) The results of sensory evaluation showed that the addition of cocoa medium could highlight the aroma of bean, cocoa and coffee, improve the sweetness and fluency and the combustibility of cigar leaves. 2) The addition of cocoa medium increased the contents of proline and malic acid which were positively correlated with sensory quality, and decreased the contents of citric acid, linoleic acid, basic amino acids and aromatic amino acids which were negatively correlated with sensory quality. 3) The addition of cocoa medium increased the total amount of aroma components in cigar leaves, especially carotenoid degradation products, and changed the structural composition of some aroma substances in wrappercigar leaves. 4) The similarity of species composition between the water-added group and the cocoa-added group was higher, but the dominant microorganisms were more concentrated. Staphylococcus and Arthrobacter maintained a high relative abundance throughout the fermentation process, which may be the key microorganisms in the agricultural fermentation stage. 5) The addition of cocoa medium increased the expression abundance of related functional genes in cigar leaves, accelerated the fermentation process of cigar leaves, and bacteria played a major role in the fermentation process. Conclusion: Adding cocoa medium in the agricultural fermentation stage, the changes of bacterial community and dominant flora on the surface of cigar leaves are the main factors affecting their internal chemical components, and the addition of media has a positive effect on tobacco fermentation.

Study on the effect of enzymatic treatment of tobacco on HnB cigarettes and microbial succession during fermentation.

Starch and cellulose are the fundamental components of tobacco, while their excessive content will affect the quality of tobacco. Enzymatic treatment with different enzymes is a promising method to modulate the chemical composition and improve the sensory quality of tobacco leaves. In this study, enzymatic treatments, such as amylase, cellulase, and their mixed enzymes, were used to improve tobacco quality, which could alter the content of total sugar, reducing sugar, starch, and cellulose in tobacco leaves. The amylase treatment changed surface structure of tobacco leaves, increased the content of neophytadiene in tobacco by 16.48%, and improved the total smoking score of heat-not-burn (HnB) cigarette products by 5.0 points compared with the control. The Bacillus, Rubrobacter, Brevundimonas, Methylobacterium, Stenotrophomonas, Acinetobacter, Pseudosagedia-chlorotica, and Sclerophora-peronella were found to be significant biomarkers in the fermentation process by LEfSe analysis. The Basidiomycota and Agaricomycetes were significantly correlated with aroma and flavor, taste, and total score of HnB. The results showed that microbial community succession occurred due to amylase treatment, which promoted the formation of aroma compounds, and regulated the chemical composition of tobacco, and improved tobacco quality during tobacco fermentation. This study provides a method for enzymatic treatment to upgrade the quality of tobacco raw materials, thereby improving the quality of HnB cigarettes, and the potential mechanism is also revealed by chemical composition and microbial community analysis. KEY POINTS: Enzymatic treatment can change the chemical composition of tobacco leaves. The microbial community was significantly affected by enzymatic treatment. The quality of HnB cigarettes was significantly improved by amylase treatment.

Identification of Bacteria Associated with Tobacco Mildew and Tobacco-Specific Nitrosamines During Tobacco Fermentation.

Tobacco mildew and tobacco-specific nitrosamines (TSNAs) affect the quality of tobacco products during fermentation. Microbes are thought to play key roles in the development of specific properties of fermented tobacco; however, little is known about the bacteria involved in the fermentation process. This study aims to identify key microbes related to mildew and TSNA formation. Tobacco was fermented at 25°C, 35°C, and 45°C for 2, 4, and 6weeks, with unfermented samples used as controls. Our preliminary exploration found that TSNAs content elevated with the increase of temperature and period, and mildew was easy to occur at low temperature with short period. Hence, samples were divided into three groups: the temperature gradient group (25°C, 35°C, and 45°C for 6weeks); the low-temperature group (control, 25°C for 2, 4, and 6weeks); and the high-temperature group (control, 45°C for 2, 4, and 6weeks). After collecting fermented tobacco leaves, 16S rRNA gene sequencing was used to explore the structure and dynamic changes of bacterial community during fermentation. Methylobacterium and Deinococcus were shared between the temperature gradient and high-temperature groups and showed a linear downward trend; these might play a role in the production of TSNAs. Massilia, Ruminiclostridium, and Cellulosilyticum species increased with prolonged fermentation time in the low-temperature group; this might be associated with tobacco mildew. In summary, the microbial diversity of fermented tobacco was explored under different conditions. These findings might provide data and material support to improve the quality of fermented tobacco products; however, further omics based studies are warranted to analysis the gene and protein expression patter in the identified bacteria.

Tobacco microbial screening and application in improving the quality of tobacco in different physical states

The first-cured tobacco contains macromolecular substances with negative impacts on tobacco products quality, and must be aged and fermented to mitigate their effects on the tobacco products quality. However, the natural fermentation takes a longer cycle with large coverage area and low economic efficiency. Microbial fermentation is a method to improve tobacco quality. The change of chemical composition of tobacco during the fermentation is often correlated with shapes of tobacco. This study aimed to investigate the effects of tobacco microorganisms on the quality of different shapes of tobacco. Specifically, Bacillus subtilis B1 and Cytobacillus oceanisediminis C4 with high protease, amylase, and cellulase were isolated from the first-cured tobacco, followed by using them for solid-state fermentation of tobacco powder (TP) and tobacco leaves (TL). Results showed that strains B1 and C4 could significantly improve the sensory quality of TP, enabling it to outperform TL in overall texture and skeleton of tobacco products during cigarette smoking. Compared with the control, microbial fermentation could increase reducing sugar; regulate protein, starch, and cellulose, reduce nicotine, improve total aroma substances, and enable the surface of fermented TP and TL to be more loose, wrinkled, and porous. Microbial community analysis indicated that strains B1 and C4 could change the native structure of microbial community in TP and TL. LEfSe analysis revealed that the potential key biomarkers in TP and TL were Bacilli, Pseudonocardia, Pantoea, and Jeotgalicoccus, which may have cooperative effects with other microbial taxa in improving tobacco quality. This study provides a theoretical basis for improving tobacco fermentation process for better cigarettes quality.Graphical

Development of Candida autochthonous starter for cigar fermentation via dissecting the microbiome.

The main goal of tobacco fermentation technology is to minimize the alkaloid content while improving flavor substance content. This study revealed the microbial community structure and their metabolic functions during cigar leaf fermentation by high-throughput sequencing and correlation analysis, and evaluated the fermentation performance of functional microbes based on in vitro isolation and bioaugmentation fermentation. The relative abundance of Staphylococcus and Aspergillus increased first but then decreased during the fermentation, and would occupy the dominant position of bacterial and fungal communities, respectively, on the 21st day. Correlation analysis predicted that Aspergillus, Staphylococcus and Filobasidium could contribute to the formation of saccharide compounds, Bacillus might have degradation effects on nitrogenous substances. In particular, Candida, as a co-occurring taxa and biomarker in the later stage of fermentation, could not only degrade nitrogenous substrates and synthesize flavor substances, but also contribute to maintaining the stability of microbial community. Moreover, based on in vitro isolation and bioaugmentation inoculation, it was found that Candida parapsilosis and Candida metapsilosis could significantly reduce the alkaloids content and increase the content of flavor components in tobacco leaves. This study found and validated the critical role of Candida in the fermentation of cigar tobacco leaves through high-throughput sequencing and bioaugmentation inoculation, which would help guide the development of microbial starters and directional regulation of cigar tobacco quality.

High throughput screening of key functional strains based on improving tobacco quality and mixed fermentation.

Background: Tobacco alcoholization is an important step in increasing the quality of tobacco leaf, which may convert a portion of low-grade tobacco leaves into useable product, however this may take to 2-3years. The addition of exogenous microorganisms to tobacco leaves and treating them by biological fermentation can shorten the maturation time of tobacco leaves, and improve the quality and applicability of low-grade tobacco leaves Methods: Several strains were screened from low-grade tobacco by flow cytometry, including the bacteria Bacillus amyloliticus, with starch degradation ability and Bacillus kochii, with protein degradation ability, and the fungus Filobasidium magnum with lipid oxidase ability, and were inoculated onto tobacco leaves, both individually and in combination, for solid-state fermentation Results: The greatest improvement in tobacco quality was observed when strains 4# and 3# were applied at a ratio of 3:1. The Maillard reaction products, such as 2-amyl furan, 1-(2-furanmethyl) -1h-pyrrole, furfural and 2, 5-dimethylpyrazine, were significantly increased, by up to more than 2times. When strains F7# and 3# were mixed at a ratio of 3:1, the improvement of sensory evaluation index was better than that of pure cultures. The increase of 3-(3, 4-dihydro-2h-pyrro-5-yl) pyridine, β -damasone and benzyl alcohol was more than 1times. The increase of 2-amyl-furan was particularly significant, up to 20times Conclusion: The functional strains screened from tobacco leaves were utilized for the biological fermentation of tobacco leaves, resulting in the reduction of irritation and an improvement in quality of final product, showing a good potential for application.

Abstract 2398: The tobacco cembranoid β-cembrenediol as promising prostate cancer recurrence suppressor lead

Abstract Prostate cancer (PC) is the second leading cause of men death in the US, annually killing >34,000 men. Despite progress in early PC diagnosis and treatment, many patients have locally and distant disease recurrence. Therapeutic androgens deprivation have limited curative potential and associated with severe side effects and rapid resistance development justifying the dire need for discovery of novel recurrence inhibitors. (1S,2E,4R,6R,-7E,11E)-2,7,11-cembratriene-4,6-diol (β-CBT) and its 4-epi-analog (α-CBT) are the precursors to key flavor ingredients in leaves of most Nicotiana species. Nearly 40-60% of β- and α-CBT are purposely degraded during the commercial tobacco fermentation. Earlier, we reported the lack of β-CBT cytotoxicity below 50 µM while at 50 nM it reversed the calcitonin-stimulated decrease in transepithelial resistance and increased paracellular permeability in PC-3M PC cells. β-CBT also proven to stimulate tight junction formation between PC-3M cells, producing a tighter intercellular barrier. β-CBT 10-50 nM potently inhibited the calcitonin-stimulated PC-3M cells invasion in Matrigel assay. This study demonstrates the in vitro β-CBT anti-migratory (wound-healing assay) and anti-clonogenicity (colony-formation assay) activities against five PC cell lines, including the metastatic castration-recurrent human prostate cancer (mCRPC) CWR-R1ca. Meanwhile, β-CBT potently suppressed the in vivo locoregional and distant recurrences after the primary tumor surgical excision of the tumor formed by engrafting the human metastatic prostate PC-3M-Luc cells at the suprascupular region in male nude mice model. β-CBT treatments prevented organ and significantly suppressed bone metastasis and lacked any major toxicity over the 60-day study course. Therefore, β-CBT emerges as an effective promising PC recurrence and metastasis suppressive lead appropriate for near future use as a prospective nutraceutical. Citation Format: Ethar A. Mudhish, Abu Bakar Siddique, Khaldoun S. Abdelwahed, Judy A. King, Khalid A. El Sayed. The tobacco cembranoid β-cembrenediol as promising prostate cancer recurrence suppressor lead [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2398.

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

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

The Tobacco β-Cembrenediol: A Prostate Cancer Recurrence Suppressor Lead and Prospective Scaffold via Modulation of Indoleamine 2,3-Dioxygenase and Tryptophan Dioxygenase.

Prostate cancer (PC) is the second leading cause of death in men in the US. PC has a high recurrence rate, and limited therapeutic options are available to prevent disease recurrence. The tryptophan-degrading enzymes 2,3-indoleamine dioxygenase (IDO1) and tryptophan dioxygenase (TDO2) are upregulated in invasive PC. (1S,2E,4R,6R,7E,11E)-2,7,11-cembratriene-4,6-diol (β-CBT) and its C-4 epimer α-CBT are the precursors to key flavor ingredients in tobacco leaves. Nearly 40–60% of β- and α-CBT are purposely degraded during commercial tobacco fermentation. Earlier, β-CBT inhibited invasion, reversed calcitonin-stimulated transepithelial resistance decrease, and induced tighter intercellular barriers in PC-3M cells. This study demonstrates the in vitro β-CBT anti-migratory (wound-healing assay) and anti-clonogenicity (colony-formation assay) activities against five diverse human PC cell lines, including the androgen-independent PC-3, PC-3M, and DU-145, the castration-recurrent CWR-R1ca, and the androgen-dependent CWR-22rv1. Meanwhile, β-CBT potently suppressed in vivo locoregional and distant recurrences after the primary tumor surgical excision of PC-3M-Luc cell tumor engrafted in male nude mice. β-CBT treatments suppressed organ and bone metastasis and lacked any major toxicity over the 60-day study course. β-CBT treatments significantly suppressed IDO1, TDO2, and their final metabolite kynurenine levels in PC-3M cells. β-CBT treatments significantly suppressed the tumor recurrence marker PSA and kynurenine levels in treated animals’ plasma. β-CBT emerges as a promising PC recurrence suppressive lead.

Exogenous additives facilitate the fermentation of cigar tobacco leaves: improving sensory quality and contents of aroma components

The cigar market is growing rapidly in China. However, insufficient aroma value and quality, as well as confused subject fragrance were found in cigar tobacco leaves (CTLs), which has greatly restricted the development of Chinese cigar. In this study, several new and effective additives, including coffee, cocoa, ginger, cumin, and rhodiola rosea, were screened out for facilitating the fermentation process. Significant improvements on the aroma and sensory quality of CTLs were detected. The above-mentioned additives can increase the contents of total sugar, alcohols, alkenes, ketonic and acid aroma components in tobacco leaves. Besides, the content of representative aroma component, such as neophytadiene, increased by 1~2 times. Moreover, abundant new aroma components were introduced into CTLs. As a result, the performances on aroma richness, aroma and smoke characteristics, as well as aftertaste and combustion of CTLs were enhanced. The findings provide a promising strategy for enhancing sensory quality of domestic CTLs and the flourishment of Chinese cigar.