Influences of tobacco straw return with lime on microbial community structure of tobacco-planting soil and tobacco leaf quality.

Soil amendment is an important strategy for improving soil quality and crop yield. From 2014 to 2019, we conducted a study to investigate the effects of tobacco straw return with lime on soil nutrients, soil microbial community structure, tobacco leaf yield, and quality in southern Anhui, China. A field experiment was conducted with four treatments: straw removed (CK), straw return (St), straw return with dolomite (St + D), and straw return with lime (St + L). Results showed that after 5 years of application, the St + L significantly increased the soil pH by 16.9%, and the contents of soil alkaline nitrogen (N) and available potassium (K) by 17.2% and 23.0%, respectively, compared with the CK. Moreover, the St + L significantly increased tobacco leaf yield (24.0%) and the appearance (9.1%) and sensory (5.9%) quality of flue-cured tobacco leaves. The addition of soil conditioners (straw, dolomite, and lime) increased both the total reads and effective sequences of soil microorganisms. Bacterial diversity was more sensitive to changes in the external environment compared to soil fungi. The application of soil amendments (lime and straw) promoted the growth of beneficial microorganisms in the soil. Additionally, bacterial species had greater competition and limited availability of resources for survival compared to fungi. The results showed that soil microorganisms were significantly influenced by the presence of AK, AN, and pH contents. These findings can provide an effective method for improving the quality of flue-cured tobacco leaves and guiding the amelioration of acidic soil in regions where tobacco-rice rotation is practiced.

Dynamic changes in fungal communities and functions in different air-curing stages of cigar tobacco leaves.

Introduction: Air curing (AC) plays a crucial role in cigar tobacco leaf production. The AC environment is relatively mild, contributing to a diverse microbiome. Fungi are important components of the tobacco and environmental microbiota. However, our understanding of the composition and function of fungal communities in AC remains limited. Methods: In this study, changes in the chemical constituents and fungal community composition of cigar tobacco leaves during AC were evaluated using flow analysis and high-throughput sequencing. Results: The moisture, water-soluble sugar, starch, total nitrogen, and protein contents of tobacco leaves exhibited decreasing trends, whereas nicotine showed an initial increase, followed by a decline. As determined by high-throughput sequencing, fungal taxa differed among all stages of AC. Functional prediction showed that saprophytic fungi were the most prevalent type during the AC process and that the chemical composition of tobacco leaves is significantly correlated with saprophytic fungi. Conclusion: This study provides a deeper understanding of the dynamic changes in fungal communities during the AC process in cigar tobacco leaves and offers theoretical guidance for the application of microorganisms during the AC process.

Unravel the Supremacy of Klebsiella variicola over Native Microbial Strains for Aroma-Enhancing Compound Production in Reconstituted Tobacco Concentrate through Metagenomic Analysis.

Sensory attributes strongly influence consumers' preferences for products. The inoculation of the Klebsiella variicola H8 strain in a reconstituted tobacco leaf concentrate (RTLC) solution increased neutral aroma-enhancing compound (NAEC) production by 45%, decreased the nicotine level by 25%, decreased the water-soluble total sugar content by ~36%, and improved the sensory quality by 5.71%. The production of NAECs such as dihydrokiwi lactone (DHKL: 192.86%), 1,2,3,4-tetrahydro-1,1,6-trimethylnaphthalene (THTMN: 177.77%), 2,4-di-tert-butylphenol (DTBP: 25%), 4-oxoisofolkone (OIFK: 116.66%,) 1,9-heptadecadiene-4,6-diyn-3-ol (HDD: 116.67%), ß-damastrone (BDS: 116.67), and megastigmatrienone A (MSTA: 116.67%) was increased. A metagenomics analysis of the microbial community in the fermented RTLC (FRTLC) was performed to elucidate the mechanism by which NAECs were produced. As a result, 24 groups of functional genes were identified, and among them, five families of carbohydrate-active enzymes, (i) glycoside hydrolase (GH), (ii) glycosyltransferase (GT), (iii) polysaccharide lyase (PL), (iv) carbohydrate esterase (CE), and (v) auxiliary active enzyme (AA), were found to be positively correlated with the production of NAECs. However, among the GHs, the GHs annotated from the H8 strain chromosome displayed the highest relative abundance and a positive correlation with the production of NAECs. Specifically, the GH13-14, GH13-20, GH13-38, GH13-25, GH13-10, GH42, and GH28 genes of the H8 strain were relatively more abundant and were key contributors to the production of NAECs. The correlation analyses revealed that the H8 strain plays a leading role among all the microorganisms in FRTLC in the production of NAECs. Our findings support the application of Klebsiella variicola in NAEC production and a reduction in nicotine content in tobacco products.

Changes in physicochemical properties and microbial community succession during leaf stacking fermentation.

Leaf stacking fermentation involves enzymatic actions of many microorganisms and is an efficient and environmentally benign process for degrading macromolecular organic compounds. We investigated the dynamics of metabolite profiles, bacterial and fungal communities and their interactions during fermentation using cigar leaves from three geographic regions. The results showed that the contents of total sugar, reducing sugar, starch, cellulose, lignin, pectin, polyphenol and protein in cigar tobacco leaves was significantly decreased during fermentation. Notably, the furfural, neophytadiene, pyridine, benzyl alcohol, geranylacetone, 3-hydroxy-2-butanone, N-hexanal, 3-Methyl-1-butanol and 2,3-pentanedione were important features volatile aroma compounds during fermentation. The α-diversity of fungi and bacteria initially increased and then decreased during fermentation. An analysis of variance showed that microbial diversity was influenced by fermentation stages and growing locations, in which the all stages had greater impacts on α- and ß-diversity than all regions. Microbiome profiling had identified several core bacteria including Sphingomonas, Bacillus, Staphylococcus, Pseudomonas, Ralstonia, Massilia and Fibrobacter. Fungal biomarkers included Aspergillus, Penicillium, Fusarium, Cladosporium and Trichomonascus. Interestingly, the molecular ecological networks showed that the core taxa had significant correlations with metabolic enzymes and physicochemical properties; bacteria and fungi jointly participated in the carbohydrate and nitrogen compound degrading and volatile aroma compound chemosynthesis processes during fermentation. These studies provide insights into the coupling of material conversion and microbial community succession during leaf fermentation.

Hormonal Regulation and Stimulation Response of Jatropha curcas L. Homolog Overexpression on Tobacco Leaf Growth by Transcriptome Analysis.

The Flowering locus T (FT) gene encodes the florigen protein, which primarily regulates the flowering time in plants. Recent studies have shown that FT genes also significantly affect plant growth and development. The FT gene overexpression in plants promotes flowering and suppresses leaf and stem development. This study aimed to conduct a transcriptome analysis to investigate the multiple effects of Jatropha curcas L. homolog (JcFT) overexpression on leaf growth in tobacco plants. The findings revealed that JcFT overexpression affected various biological processes during leaf development, including plant hormone levels and signal transduction, lipid oxidation metabolism, terpenoid metabolism, and the jasmonic-acid-mediated signaling pathway. These results suggested that the effects of FT overexpression in plants were complex and multifaceted, and the combination of these factors might contribute to a reduction in the leaf size. This study comprehensively analyzed the effects of JcFT on leaf development at the transcriptome level and provided new insights into the function of FT and its homologous genes.

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.

Microbial and enzymatic changes in cigar tobacco leaves during air-curing and fermentation.

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.

Efficient heterogeneous Fenton-like degradation of methylene blue using green synthesized yeast supported iron nanoparticles.

To reduce the consumption of oxidant and catalyst in Fenton-like reaction and to realize the reuse of catalyst, yeast supported iron nanoparticles (nZVI@SCM) was synthesized by tobacco leaf extract and applied in the heterogeneous Fenton-like degradation of aqueous methylene blue (MB) at ambient conditions. The performance of the composite was exploited in terms of catalytic activity and factors influencing MB degradation. The surface changes of nZVI@SCM before and after reaction were characterized by XPS, SEM, FT-IR and XRD. Iron leaching, primary reactive oxidizing species, and the storage stability and reusability of catalyst were also investigated. Typically, 99.7% removal of 50 mg/L MB, with a TOC removal of 97.2%, could be achieved within 10 h by 0.1 g/L nZVI@SCM coupled with 1.0 mM H2O2. The MB degradation is in good agreement with the pseudo-first-order model, and hydroxyl radicals in the bulk solution is the main reactive oxidizing species responsible for MB degradation. Based on the identified intermediates by liquid chromatography/mass spectrometry, the possible MB degradation mechanism in the nZVI@SCM/H2O2 system is discussed. The developed high-performance nZVI@SCM catalyst strategy can provide a new route in enhancing the Fenton-like degradation of organic contaminants with less consumption of catalyst and oxidant.

Antioxidant, Anti-Inflammatory and Attenuating Intracellular Reactive Oxygen Species Activities of Nicotiana tabacum var. Virginia Leaf Extract Phytosomes and Shape Memory Gel Formulation.

Oxidative stress is one of the major causes of skin aging. In this study, the shape memory gels containing phytosomes were developed as a delivery system for Nicotiana tabacum var. Virginia fresh (VFL) and dry (VDL) leaf extracts. The extracts were loaded in the phytosomes by a solvent displacement method. The physical and chemical characteristics and stability of phytosomes were evaluated by dynamic light scattering and phytochemistry, respectively. The in vitro antioxidant activity and intracellular reactive oxygen species reduction of phytosomes and/or extracts were investigated by the DPPH and ABTS radical scavenging assays, FRAP assay, and DCFH-DA fluorescent probe. The cytotoxicity and anti-inflammatory activity of VDL and VFL phytosomes were studied by an MTT and a nitric oxide assay, respectively. Here, we first reported the total phenolic content in the dry leaf extract of N. tabacum var. Virginia was significantly greater than that of the fresh leaf extract. The HPLC analysis results revealed that VDL and VFL extracts contained 4.94 ± 0.04 and 3.13 ± 0.01 µg/mL of chlorogenic acid and 0.89 ± 0.00 and 0.24 ± 0.00 µg/mL of rutin, respectively. The phytosomes of the VDL and VFL extracts displayed stable size, polydispersity index, zeta potential values, and good chemical stability. VDL and VDL phytosomes showed higher phenolic and flavonoid contents which showed stronger DPPH and ABTS radical scavenging effects and reduced the intracellular ROS. The results suggested that the phenolic compounds are the main factor in their antioxidant activity. Both VDL and VFL phytosomes inhibited nitric oxide production induced by LPS, suggesting the anti-inflammatory activity of the phytosomes. The shape memory gel containing VDL and VFL phytosomes had good physical stability in terms of pH and viscosity. The VDL and VFL phytosomes dispersed in the shape memory gels can be considered as a promising therapeutic delivery system for protecting the skin from oxidation and reactive oxygen species.

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.

Investigation of the growth performance, blood status, gut microbiome and metabolites of rabbit fed with low-nicotine tobacco.

Tobacco contains a large amount of bioactive ingredients which can be used as source of feed. The objective of this study was to evaluate the effects of dietary addition of low-nicotine tobacco (LNT) on the growth performance, blood status, cecum microbiota and metabolite composition of meat rabbits. A total of 80 Kangda meat rabbits of similar weight were assigned randomly as four groups, and three of them were supplemented with 5%, 10%, and 20% LNT, respectively, with the other one fed with basal diet as control group. Each experiment group with 20 rabbits was raised in a single cage. The experiments lasted for 40 days with a predictive period of 7 days. The results revealed that LNT supplementation had no significant effect on the growth performance, but increased the half carcass weight compared with control group. Dietary supplemention of LNT decreased the triglycerides and cholesterol content in rabbit serum, and significantly increased the plasma concentration of lymphocytes (LYM), monocytes, eosinophils, hemoglobin HGB and red blood cells. In addition, LNT supplementation significantly changed the microbial diversity and richness, and metagenomic analysis showed that LNT supplementation significantly increased Eubacterium_siraeum_group, Alistipes, Monoglobus and Marvinbryantia at genus level. Moreover, LC-MS data analysis identified a total of 308 metabolites that markedly differed after LNT addition, with 190 significantly upregulated metabolites and 118 significantly downregulated metabolites. Furthermore, the correlation analysis showed that there was a significant correlation between the microbial difference and the rabbit growth performance. Overall, these findings provide theoretical basis and data support for the application of LNT in rabbits.

Effects of fermentation medium on cigar filler.

The addition of medium during industrial fermentation can improve the quality of cigar tobacco leaves after agricultural fermentation. In this study, the cigar filler tobacco "Brazilian Frogstrips YA14" was used as the test material to determine the contents of main chemical components in cigar tobacco leaves after fermentations with the additions of water (control group) and a medium (test group), and the changes in the community structure and abundances of bacteria on tobacco leaves during the fermentation process were analyzed. The results of the study were as follows: 1) During the fermentation process, the protein content of tobacco leaves fluctuated slightly, basically stabilized at 19%-20%. 2) Under the impact of the medium, the total content of main amino acids in tobacco leaves showed a downward trend, and the difference of which between the control group and the test group was the most obvious on the fourth day of fermentation. 3) The change trend of the content of petroleum ether extract in cigar leaves for the control group was not obvious, and the content of petroleum ether extract in the tobacco leaves for the test group decreased by 12.4% under the impact of the medium. 4) After fermentation, the relative content of saturated fatty acids for the control group and the test group all increased, while the relative content of unsaturated fatty acids all decreased. 5) After the addition of the medium, the diversity of bacteria on tobacco leaves changed significantly, the number of OTUs in tobacco leaves increased, and the bacterial community structure changed. This research indicates that after adding the medium to ferment cigar filler, the changes of bacterial community and dominant bacterial group on cigar tobacco leaves have impacts on the contents of chemical components in tobacco leaves, and the fermentation with the addition of medium has a positive effect on improving the quality of tobacco leaves.

Anxiolytic Effect of Two Tobacco Essential Oils (Nicotiana tabacum Linn.) on Mice.

Tobacco (Nicotiana tabacum Linn.) is a famous traditional herb used in folk medicine. The essential oils of tobacco have been demonstrated in modern studies to possess antioxidant, anti-inflammatory, and neuroprotective properties, while its anxiolytic effect has not been reported. The purpose of this study was to evaluate the anxiolytic effect of Yunnan tobacco essential oil (YTO) and Zimbabwe tobacco essential oil (ZTO) on mice. The constituents of YTO and ZTO were analyzed by GC/MS. The anxiolytic effect of YTO and ZTO (0.1%, 1%, and 10%, v/v) on male ICR mice was evaluated in the light-dark box test (LDB) and the elevated plus maze test (EPM) test via inhalation and transdermal administration. After the behavioral tests, salivary corticosterone levels in mice were measured. The behavioral analysis showed that the administration of both YTO and ZTO elevated the time that the mice spent in the light chamber in the LDB test compared to the untreated control. In the EPM test, YTO and ZTO increased the time spent in open arms and the number of entries into the open arms. In addition, both YTO and ZTO significantly decreased salivary corticosterone levels in mice (p ≤ 0.001). In summary, our results demonstrated that inhalation and transdermal administration of both YTO and ZTO showed anxiolytic effect on male ICR mice.

Exogenous application of brassinosteroids regulates tobacco leaf size and expansion via modulation of endogenous hormones content and gene expression.

Brassinosteroids (BR) play diverse roles in the regulation of plant growth and development. BR promotes plant growth by triggering cell division and expansion. However, the effect of exogenous BR application on the leaf size and expansion of tobacco is unknown. Tobacco seedlings are treated with different concentrations of exogenous 2,4-epibrassinolide (EBL) [control (CK, 0 mol L-1), T1 (0.5 × 10-7 mol L-1), and T2 (0.5 × 10-4 mol L-1)]. The results show that T1 has 17.29% and T2 has 25.99% more leaf area than control. The epidermal cell area is increased by 24.40% and 17.13% while the number of epidermal cells is 7.06% and 21.06% higher in T1 and T2, respectively, relative to control. So the exogenous EBL application improves the leaf area by increasing cell numbers and cell area. The endogenous BR (7.5 times and 68.4 times), auxin (IAA) (4.03% and 25.29%), and gibberellin (GA3) contents (84.42% and 91.76%) are higher in T1 and T2, respectively, in comparison with control. Additionally, NtBRI1, NtBIN2, and NtBES1 are upregulated showing that the brassinosteroid signaling pathway is activated. Furthermore, the expression of the key biosynthesis-related genes of BR (NtDWF4), IAA (NtYUCCA6), and GA3 (NtGA3ox-2) are all upregulated under EBL application. Finally, the exogenous EBL application also upregulated the expression of cell growth-related genes (NtCYCD3;1, NtARGOS, NtGRF5, NtGRF8, and NtXTH). The results reveal that the EBL application increases the leaf size and expansion by promoting the cell expansion and division through higher BR, IAA, and GA3 contents along with the upregulation of cell growth-related genes. The results of the study provide a scientific basis for the effect of EBL on tobacco leaf growth at morphological, anatomical, biochemical, and molecular levels. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12298-021-00971-x.

Study of tobacco-derived proteins in paper coatings.

Replacing synthetic polymers with renewable alternatives is a critical challenge for the packaging industry. This research investigated the use of leaf-based proteins as a sustainable co-binder in the coating formulations for paper-based packaging and other applications. Protein isolates from tobacco leaf and alfalfa concentrates were characterized using the Pierce protein assay, Kjeldahl nitrogen, and gel electrophoresis. The proteins were tested as co-binders in a typical latex-based paper coating formulation. The rheology and water retention properties of the wet coating and the surface, optical, structural, and strength properties of coated papers were measured. The coating performance was affected by the purity, solubility, and molecular weight of the tobacco protein and exhibited a shear-thinning behavior with lower water retention than soy protein. Analysis by scanning electron microscopy and time of flight secondary ion mass spectroscopy on the dried coating layer containing tobacco protein showed enhanced porosity (advantageous for package glueability) relative to the control latex coating. The tobacco protein offers adequate coverage and coating pigment distribution, indicating that this protein can be a suitable option in coatings for packaging applications.

Characterization and discrimination of microbial community and co-occurrence patterns in fresh and strong flavor style flue-cured tobacco leaves.

Fermentation, also known as aging, is vital for enhancing the quality of flue-cured tobacco leaves (FTLs). Aged FTLs demonstrate high-quality sensory characteristics, while unaged FTLs do not. Microbes play important roles in the FTL fermentation process. However, the eukaryotic microbial community diversity is poorly understood, as are microbial associations within FTLs. We aimed to characterize and compare the microbiota associated with two important categories, fresh and strong flavor style FTLs, and to reveal correlations between the microbial taxa within them. Based on 16S and 18S rRNA Illumina MiSeq sequencing, the community richness and diversity of prokaryotes were almost as high as that of eukaryotes. The dominant microbes of FTLs belonged to seven genera, including Pseudomonas, Bacillus, Methylobacterium, Acinetobacter, Sphingomonas, Neophaeosphaeria, and Cladosporium, of the Proteobacteria, Firmicutes, and Ascomycota phyla. According to partial least square discriminant analysis (PLS-DA), Xanthomonas, Franconibacter, Massilia, Quadrisphaera, Staphylococcus, Cladosporium, Lodderomyces, Symmetrospora, Golovinomyces, and Dioszegia were significantly positively correlated with fresh flavor style FTLs, while Xenophilus, Fusarium, unclassified Ustilaginaceae, Tilletiopsis, Cryphonectria, Colletotrichum, and Cyanodermella were significantly positively correlated with strong flavor style FTLs. Network analysis identified seven hubs, Aureimonas, Kocuria, Massilia, Brachybacterium, Clostridium, Dietzia, and Vishniacozyma, that may play important roles in FTL ecosystem stability, which may be destroyed by Myrmecridium. FTL microbiota was found to be correlated with flavor style. Species present in lower numbers than the dominant microbes might be used as microbial markers to discriminate different flavor style samples and to stabilize FTL microbial communities. This research advances our understanding of FTL microbiota and describes a means of discriminating between fresh and strong flavor FTLs based on their respective stable microbiota.

Enantiomeric composition of nicotine in tobacco leaf, cigarette, smokeless tobacco, and e-liquid by normal phase high-performance liquid chromatography.

Evaluating the source of nicotine in e-liquid is a problem. Tobacco-derived nicotine contains predominantly (S)-(-)-nicotine, whereas tobacco-free nicotine products may not. Thus, we developed a new normal phase high-performance liquid chromatography method to determinate the enantiomeric composition of nicotine in 10 kinds of flue-cured tobacco, 3 kinds of burley, 1 kind of cigar tobacco, 2 kinds of oriental tobacco, 5 kinds of Virginia cigarette, 5 kinds of blend cigarette, 10 kinds of e-liquid, and 4 kinds of smokeless tobacco. The amount of (R)-(+)-nicotine ranged from ~0.02% to ~0.76% of total nicotine. An e-liquid sample had the highest level of (R)-(+)-nicotine. The extraction and purification processes used to obtain commercial (S)-(-)-nicotine from the tobacco do not decrease the amount of (R)-(+)-nicotine in tobacco. So the amount of (R)-(+)-nicotine in samples in our work were the same as tobacco samples.

Analyzing multiple pesticides in tobacco leaf using gas chromatography with quadrupole time-of-flight mass spectrometry.

A method combining gas chromatography with quadrupole time-of-flight mass spectrometry has been developed for the simultaneous analysis of multiple pesticide residues in tobacco leaf. The retention index and high accurate masses of ions from the first-stage and the second-stage mass spectra of each pesticide were collected for qualitation and quantification. A total of 115 pesticides were evaluated. The extract from organic tobacco leaf was used as a model matrix. The limit of detection was <10 ng/mL, and the limit of quantification was in the range of 1-20 ng/mL for 95% of the tested pesticides. The correlation coefficients were >0.9900 for all tested pesticides. At three concentrations (10, 50, and 100 ng/mL), most compounds presented satisfactory recoveries ranging from 70 to 120% and good precision <20%. Finally, three tobacco leaf samples collected from a local market were analyzed. A total of three pesticides were found, including dimethachlon, triadimenol, and flumetralin. Each pesticide was confirmed by the presence of three ions at the expected retention index and mass. In conclusion, gas chromatography with quadrupole time-of-flight mass spectrometry appears to be one of the most efficient tools for the analysis of pesticide residues in tobacco leaf.

Metabolic changes in primary, secondary, and lipid metabolism in tobacco leaf in response to topping.

As an important cultivation practice used for flue-cured tobacco, topping affects diverse biological processes in the later stages of development and growth. Some studies have focused on using tobacco genes to reflect the physiological changes caused by topping. However, the complex metabolic shifts in the leaf resulting from topping have not yet been investigated in detail. In this study, a comprehensive metabolic profile of primary, secondary, and lipid metabolism in flue-cured tobacco leaf was generated with use of a multiple platform consisting of gas chromatography-mass spectrometry, capillary electrophoresis-mass spectrometry, and liquid chromatography-mass spectrometry/ultraviolet spectroscopy. A total of 367 metabolites were identified and determined. Both principal component analysis and the number of significantly different metabolites indicated that topping had the greatest influence on the upper leaves. During the early stage of topping, great lipid level variations in the upper leaves were observed, and antioxidant defense metabolites were accumulated. This indicated that the topping activated lipid turnover and the antioxidant defense system. At the mature stage, lower levels of senescence-related metabolites and higher levels of secondary metabolites were found in the topped mature leaves. This implied that topping delayed leaf senescence and promoted secondary metabolite accumulation. This study provides a global view of the metabolic perturbation in response to topping. Graphical abstract Metabolic alterations in tobacco leaf in response to topping using a multiplatform metabolomics.

Prevalence, pattern and sociodemographic differentials in smokeless tobacco consumption in Bangladesh: evidence from a population-based cross-sectional study in Chakaria.

BACKGROUND: The health hazards associated with the use of smokeless tobacco (SLT) are similar to those of smoking. However, unlike smoking, limited initiatives have been taken to control the use of SLT, despite its widespread use in South and Southeast Asian countries including Bangladesh. It is therefore important to examine the prevalence of SLT use and its social determinants for designing appropriate strategies and programmes to control its use. OBJECTIVE: To investigate the use of SLT in terms of prevalence, pattern and sociodemographic differentials in a rural area of Bangladesh. DESIGN: Population-based cross-sectional household survey. SETTING AND PARTICIPANTS: A total of 6178 individuals aged ≥13 years from 1753 households under the Chakaria HDSS area were interviewed during October-November 2011. METHODS: The current use of SLT, namely sadapatha (dried tobacco leaves) and zarda (industrially processed leaves), was used as the outcome variable. The crude and net associations between the sociodemographic characteristics of respondents and the outcome variables were examined using cross-tabular and multivariable logistic regression analysis, respectively. RESULTS: 23% of the total respondents (men: 27.0%, women: 19.3%) used any form of SLT. Of the respondents, 10.4% used only sadapatha,13.6% used only zarda and 2.2% used both. SLT use was significantly higher among men, older people, illiterate, ever married, day labourers and relatively poorer respondents. The odds of being a sadapatha user were 3.5-fold greater for women than for men and the odds of being a zarda user were 3.6-fold greater for men than for women. CONCLUSIONS: The prevalence of SLT use was high in the study area and was higher among socioeconomically disadvantaged groups. The limitation of the existing regulatory measures for controlling the use of non-industrial SLT products should be understood and discussion for developing new strategies should be a priority.