在恒温恒湿箱内对海南光村茄衣发酵42 d，并对整个过程中烟叶表面微生物进行分离、纯化和鉴定，研究探讨了茄衣人工发酵过程中叶面微生物区系的变化。结果显示：在茄衣人工发酵过程中细菌为优势菌群，霉菌所占比例很小，没有检测出放线菌和酵母菌;所有细菌均为芽孢杆菌，数量由高到低依次是：巨大芽孢杆菌>枯草芽孢杆菌>蜡状芽孢杆菌>环状芽孢杆菌>蕈状芽孢杆菌>嗜热脂肪芽孢杆菌>短小芽孢杆菌>凝结芽孢杆菌;茄衣表面各菌种数量在发酵过程前24 d内呈急剧下降趋势；巨大芽孢杆菌和枯草芽孢杆菌为雪茄茄衣人工发酵中的优势菌种，分别占芽孢杆菌数量的50%以上和19%左右。

该研究通过恒温发酵明确不同温度对烟叶品质的影响规律，在此基础上进行三段式变温发酵，根据理化指标、致香成分和感官评吸确定最佳组合发酵温度。结果表明，恒温发酵实验组中低温发酵（35 ℃）组的总糖、还原糖和致香成分含量较高，分别为0.57%、0.25%和3.11 g/kg，可改善香韵丰富度，而高温发酵组的蛋白质和生物碱含量较低，分别为5.65%和3.22%，对刺激性具有改善作用。三段式变温发酵可显著提升香韵丰富度，降低刺激性。相较恒温35 ℃发酵，35 ℃-40 ℃-45 ℃和35 ℃-45 ℃-40 ℃发酵组的总氮含量分别降低了18.6%和21.7%，生物碱含量分别降低了5.7%和28.8%，总糖含量分别提高了28.1%和56.1%，致香成分总含量分别提高了21.9%和4.1%。低-高-中（35 ℃-45 ℃-40 ℃）和低-中-高（35 ℃-40 ℃-45 ℃）三段式发酵可有效弥补恒温发酵的不足，提高国产雪茄烟叶的品质。

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.© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.

Fermentation process plays an important role in the biochemical properties and quality of cigar tobacco leaves (CTLs). In industry, exogenous additive (EA) was usually adopted for improving the quality of CTLs during fermentation. However, the mechanism of enhanced quality of CTLs fermented with EA was confused. Herein, the chemical compositions and microbial communities of CTLs during fermentation with EA were analyzed. The increased contents of total nitrogen and total sugar, as well as the improved consumption rate of reducing sugar in CTLs were found with the addition of EA. Besides, fermentation with EA reduced the content of total nonvolatile organic acid, especially unsaturated fatty acid. The contents of total and several representative aroma components were improved. Additionally, the increased abundance of Staphylococcus and decreased abundance of Aspergillus were detected. Combined with the changes of chemical compositions and microbial communities, it was confirmed that the carbohydrates and alcohols originated from EA promote the enrichment of Staphylococcus and accelerate biochemical reactions, such as Maillard reaction and esterification reaction, thus improving the contents and quality of aroma components in CTLs. This study demonstrated the mechanism of enhanced quality of CTLs fermented by EA, which provides more ideas for developing novel and efficient EAs.© 2022. The Author(s).

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.© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.

为有效降低烟叶中的淀粉含量、提高烟叶品质，从不同产地原烟表面初步分离具有降解淀粉能力的菌株，通过测定菌株的淀粉酶活性筛选高效产淀粉酶菌株，并对筛选菌株基因组DNA进行16S rDNA测序，同时结合生理生化试验对其进行鉴定。结果表明：在分离得到的22株菌株中，XC-3菌株产淀粉酶活性最高（116 U/mL）；根据其16S rDNA测序结果以及生理生化试验结果推断，XC-3为苏云金芽孢杆菌（Bacillus thuringiensis）。进一步采用正交试验优化XC-3菌株在烟叶中的发酵条件，并考察其对烟叶中淀粉的降解效果。结果显示，将培养后的XC-3菌液（OD600=2.0）按照3%的烟叶质量比例接种于抽梗后的烟叶表面，在37 ℃条件下发酵48 h后，烟叶中的淀粉降解率可达29.84%，烟叶的总糖、还原糖含量上升，有效提升了烟叶的整体质量。

从14种烟叶表面筛选出一株能够利用烟末发酵产香的菌株GXY35，通过生理生化实验和16S rDNA序列对其进行分析，并用GC/MS对发酵液的香气成分进行分析。结果表明：GXY35分离株属于克雷伯氏杆菌，发酵液中特征香味物质4乙烯基愈创木酚的含量最高。在单因素试验的基础上通过正交试验，对菌株的发酵条件进行优化。结果表明，最适宜的产香发酵条件为：料液比1∶20，接种量12%，发酵温度37 ℃，发酵时间72 h，pH值7.0。在此条件下，特征香味物质4乙烯基愈创木酚的含量最高可达0.725 mg·mL-1。

雪茄烟叶中的半纤维素在叶脉及叶片韧性等特性中发挥重要作用,烟叶叶脉较粗,半纤维素含量过高,导致雪茄烟叶韧性较差,可用性变差。本实验室前期筛选到一株产木聚糖酶的蜡样芽孢杆菌,该菌株对烟碱有较好的耐受性,利用液态发酵方法研究菌株以雪茄烟叶为营养源产木聚糖酶的最佳发酵条件,并对最佳发酵条件下菌株降解烟叶中半纤维素的效果进行测定,最后分析比较了菌株对雪茄烟叶香气物质成分及含量的影响。结果表明,菌株在含有不高于1.0 g/L烟碱的培养基中生长受抑制较小,菌株对烟碱有良好的耐受性;当培养基中初始 pH 6.7,接种量为5%,雪茄烟叶底物浓度为20 g/L,发酵24 h,菌株产木聚糖酶活性最高,可达（4.04±0.18）U/mL;在最佳发酵条件下,雪茄烟叶中半纤维素含量为3.38%,降低了6.63%,纤维素含量为10.54%,降低了8.19%,利用菌株发酵同时降低了雪茄烟叶中纤维素和半纤维素含量;通过对香气物质影响的分析比较,发现经菌株发酵后的雪茄烟叶能够提升叶绿醇（2.85%）、油酸（0.62%）、正二十六烷（1.75%）和正三十一烷（14.47%）等物质的含量。蜡样芽孢杆菌 B. cereus 对雪茄烟叶中半纤维素及纤维素降解具有一定作用,后期可应用于雪茄外包皮烟叶的固态发酵,进一步改善雪茄外包皮烟叶的物理特性,为该菌株的开发利用奠定基础。

该研究从雪茄烟叶表面筛选具有解磷作用的菌株，结合形态观察和16S rDNA序列分析对筛选出的菌株进行鉴定。以烟叶自然发酵为对照，分别以添加筛选菌株、添加筛选菌株和营养剂的方式进行发酵，分析发酵过程中生物量、无机磷、还原糖和游离总氨基酸含量及致香成分的变化，并对接种量进行优化。结果表明，获得的7株具有解磷作用的菌株，其中菌株Q627解磷作用最好，经鉴定为高地芽孢杆菌（Bacillus altitudinis）。与自然发酵及接种发酵方式相比，接种菌株Q627及添加营养剂的发酵方式解磷效果较好，发酵结束时，其无机磷含量为1.60 μg/g，比自然发酵（1.28 μg/g）和接种菌株Q627发酵（1.18 μg/g）分别提高29%和37%，且生物量、还原糖、总游离氨基酸含量分别为1.20×107 CFU/g、15.01 mg/g、1.44 mg/g；发酵6 d时，其美拉德反应产物、类胡萝卜素降解产物和西柏烷类降解产物含量最高，分别为20.31 μg/g、47.57 μg/g、47.83 μg/g，香气成分总含量为156.18 μg/g；确定雪茄烟叶发酵最佳接种量为7.5×108 CFU/g。