Changwen Ye

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.

The combustion of excessive starch in tobacco leaves leads to more harmful substances, adversely affecting the sensory properties of tobacco and posing significant risks to human health. Therefore, there is an urgent need to develop specific amylases targeting tobacco starch to address these issues. In this study, 5 different α-amylase genes were selected for recombinant expression in Bacillus amyloliquefaciens BAX-5, and the amyA(LC) (derived from Bacillus amyloliquefaciens MK10163) was confirmed to be the optimal gene. Then, the α-amylase activity was further increased by screening host bacteria BAX-5 and signal peptides SP₀₀₃ (derived from the dacB gene of Bacillus subtilis 168). Subsequently, the α-amylase properties were characterized, such as temperature tolerance, pH tolerance and metal ion. Through replacement of culture medium, the recombinant strain BAX-5/PT-17SP₀₀₃amyA(LC) produced the maximum α-amylase activity of 904.91 IU/mL, which was about 4 times higher than that of the original culture medium. Finally, the α-amylase Amy (LC) was applied to the enzyme treatment of tobacco leaves, and the evaluation results showed that α-amylase Amy (LC) could play a positive role in reducing damage and enhancing quality of cigarettes. This research provides a novel enzymatic resource for the development of amylases, and it has enormous market potential and application value.

Due to the broad application and substantial market demand for proteases, it was vital to explore the novel and efficient protease resources. The aim of this study was to identify the novel protease for tobacco protein degradation and optimize the expression levels. Firstly, the tobacco protein was used as the sole nitrogen resource for isolation of protease-producing strains, and a strain with high protease production ability was obtained, identified as Bacillus velezensis WH-7. Then, the whole genome sequencing was conducted on the strain B. velezensis WH-7, and 7 proteases genes were mined by gene annotation analysis. By further heterologous expression of the 7 protease genes, the key protease HapR was identified with the highest protease activity (144.19 U/mL). Moreover, the catalysis mechanism of HapR was explained by amino acid sequence analysis. The expression levels of protease HapR were further improved through optimization of promoter, signal peptide and host strain, and the maximum protease activity reaced 384.27 U/mL in WX-02/pHY-P43-SP_yfkD-hapR, increased by 167% than that of initial recombinant strain HZ/pHY-P43-SP_hapR-hapR. This study identified a novel protease HapR and the expression level was significantly improved, which provided an important enzyme resource for the development of enzyme preparations in tobacco protein degradation.

Vinyl acetate is a restricted substance in food products. The quantification of the organic impurities in vinyl acetate is a major problem due to its activity, instability, and volatility. In this paper, while using the mass balance method to determine the purity of vinyl acetate, an improved method was established for the determination of the content of three impurities in vinyl acetate reference material, and the GC-FID peak area normalization for vinyl acetate was calibrated. The three trace organic impurities were identified by gas chromatography tandem high-resolution mass spectrometry to be methyl acetate, ethyl acetate, and vinyl propionate. The content and relative correction factors for the three organic impurities were measured. The purity of vinyl acetate determined by the mass balance method was 99.90% with an expanded uncertainty of 0.30%, and the total content of organic impurities was 0.08% with a relative correction factor of 1.23%. The vinyl acetate reference material has been approved as a national certified reference material in China as GBW (E) 062710.