Selection of optimal primer pairs in 16S rRNA gene sequencing is a pivotal issue in microorganism diversity analysis. However, limited effort has been put into investigation of specific primer sets for analysis of the bacterial diversity of aging flue-cured tobaccos (AFTs), as well as prediction of the function of the bacterial community. In this study, the performance of four primer pairs in determining bacterial community structure based on 16S rRNA gene sequences in AFTs was assessed, and the functions of genes were predicted using Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt). Results revealed that the primer set 799F–1193R covering the amplification region V5V6V7 gave a more accurate picture of the bacterial community structure of AFTs, with lower co-amplification levels of chloroplast and mitochondrial genes, and more genera covered than when using the other primers. In addition, functional gene prediction suggested that the microbiome of AFTs was involved in kinds of interested pathways. A high abundance of functional genes involved in nitrogen metabolism was detected in AFTs, reflecting a high level of bacteria involved in degrading harmful nitrogen compounds and generating nitrogenous nutrients for others. Additionally, the functional genes involved in biosynthesis of valuable metabolites and degradation of toxic compounds provided information that the AFTs possess a huge library of microorganisms and genes that could be applied to further studies. All of these findings provide a significance reference for researchers working on the bacterial diversity assessment of tobacco-related samples.

Tobacco stalk (TS), a major agricultural waste abundant in pectin, has resulted in concerns about the need for its reuse. The nicotine in TS is considered a chemical that is to\xic and hazardous to the environment. In this study, Bacillus tequilensis CAS-MEI-2-33 was isolated from cigar wrappers to produce alkaline pectinase using TS. Subsequently, the medium and fermentation conditions for the production of pectinase by B. tequilensis CAS-MEI-2-33 were optimized. The optimal fermentation period, pH of the initial fermentation medium, concentration of TS, and inoculum amount for B. tequilensis CAS-MEI-2-33 were 40 h, 40 g/L, 7.0, and 3%, respectively. Under optimal conditions, the pectinase activity was 1370 U/mL. Then, the enzymatic properties, such as the optimum pH, reaction temperature, temperature stability, and effects of metal ions, were studied. The optimal pH was determined to be 10.0, indicating that the enzyme was an alkaline pectinase. The optimal temperature was 40 °C, and pectinase activity was stable at 40 °C. The Ag+ metal ions were shown to remarkably promote enzyme activity. The pectinase was partly purified by ammonium sulfate precipitation, ion exchange chromatography, and Sephacryl S-100 chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and LC-MS/MS analyses were utilized to analyze the pectinase. This study provided a new alkaline pectinase candidate and a new strategy for the use of TS.

Introduction: Cigar tobacco leaves (CTLs) are nutrient-rich and susceptible to mold contamination during fermentation and storage. This study aims to investigate the specific volatile profiles of dominant culturable molds recovered under the applied isolation conditions in CTLs and to establish a robust volatile organic compounds (VOCs)-based targeted monitoring strategy for preventing early-stage mold outbreaks. Methods: Eight dominant culturable molds were isolated from natural mold CTLs, and their spores were inoculated onto sterile CTLs. A combined visual scoring and mold counting system was utilized to evaluate the mold-inducing potential of the isolates. Gas chromatography-ion mobility spectrometry (GC-IMS) was employed to detect VOCs at the initial stage of visible mold onset. Furthermore, multivariate chemometric analyses, including principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA), were performed to process the data and screen for differential markers. Results: Based on the evaluation system, the isolates were quantitatively classified into high-moldogenicity ( Aspergillus montevidensis , Penicillium chrysogenum , Aspergillus pseudoglaucus , and Aspergillus versicolor ) and low-moldogenicity ( Talaromyces funiculosus , Trichoderma longibrachiatum , Aspergillus chevalieri , and Aspergillus sydowii ) strains. A total of 66 VOCs were identified by GC-IMS. PCA and PLS-DA not only clearly differentiated mold-contaminated samples from uninoculated controls but also clustered the strains according to their defined mold-inducing potential. Through strict variable screening (VIP > 1, P < 0.05), three VOCs—3-octanone (M), 2-pentanol, and 2-methyl-1-butanol—were identified as broad-spectrum early-warning markers for mold contamination. Additionally, 2-pentanol exhibited a distinctly higher relative abundance in high-moldogenicity strains, highlighting its potential as a quantitative indicator of infection severity. Conclusion: GC-IMS serves as a rapid and effective analytical tool for profiling dynamic VOC changes during fungal infection. The newly identified broad-spectrum markers and the quantitative classification model offer a promising targeted monitoring strategy to ensure safe cigar tobacco production.