枯草芽孢杆菌分离鉴定及其对烟叶化学成分和吸味品质的影响

为研究枯草芽孢杆菌对烟叶化学成分和吸味品质的影响，从初烤后烟叶中筛选出一株微生物（H11），结合菌株形态和16S rDNA序列分析，初步鉴定为枯草芽孢杆菌枯草亚种（Bacillus subtilis subsp. subtilis）；用H11对20 kg河南平顶山低等级烟叶（2017 DCFB）发酵30 h，对比分析发酵前后烟叶感官质量、常规化学成分、中性香味成分的变化，并采用宏基因组测序技术，分析了发酵前后烟叶表面微生物群落变化，探讨了酶在烟叶发酵中的作用。与发酵前相比，烟气透发性好、甜感增强、香气质提升、香气量增加；水溶性总糖和还原糖含量（质量分数）分别增加了2.52%和1.92%；（E）-β-大马酮、二氢猕猴桃内酯和巨豆三烯酮C等香味成分含量（质量分数）分别增加了25.92%、10.57%和12.15%；烟叶表面微生物数量明显减少，芽孢杆菌属（Bacillus）微生物丰度增加至微生物总量的76%。枯草芽孢杆菌（Bacillus subtilis）对烟叶化学成分和吸味品质有重要影响，且具有发酵时间短和处理量大等特点，可为微生物发酵低等级烟叶提供参考。 

Isolation and identification of a Bacillus subtilis strain and its effects on chemical composition and sensory quality of tobacco leaves

In order to study the effects of Bacillus subtilis on the chemical composition and sensory quality of tobacco leaves, a microorganism (H11) was screened from the cured tobacco leaves. Through strain morphology and 16S rDNA sequence analyses, H11 was preliminarily identified as Bacillus subtilis subsp. subtilis. It was used to ferment 20 kg low-grade tobacco leaves(2017 DCFB) from Pingdingshan County, Henan Province for 30 h, and the variations in sensory quality, routine chemical components and neutral aroma components of tobacco leaves before and after the fermentation were comparatively analyzed. Metagenomic sequencing technology was adopted to analyze the changes in microbial population on the tobacco leaves before and after fermentation, and the roles of enzymes in the leaf fermentation were discussed. Compared with no fermentation, the fermentation promoted the volatility, sweet taste, aroma quality and aroma quantity of tobacco smoke, increased the contents(mass fraction)of water-soluble total sugar and reducing sugar by 2.52% and 1.92% respectively, raised the contents(mass fractions) of neutral aroma components such as (E)-β-damascenone, dihydroactinidiolide and megastigmatrienone C by 25.92%, 10.57% and 12.15% respectively. The amounts of microorganisms on the tobacco leaves decreased significantly after fermentation, and the relative abundance of Bacillus increased to 76% of the total amount of microorganisms. Bacillus subtilis has important influences on the chemical composition and sensory quality of tobacco leaves, and it is characterized by a short fermentation time and high throughput, which can provide references for the microbiological treatment of low-grade tobacco leaves.