从上部低次烟叶中筛选出耐高温淀粉酶产生菌GW 2和耐高温蛋白酶产生菌GW 3，通过对菌落的形态观察、16S rRNA的鉴定和同源性分析，初步确定GW 2为解淀粉芽胞杆菌，GW 3为枯草芽胞杆菌。对上部低次烟叶进行双菌种固态发酵，并优化发酵条件，最佳发酵条件：GW 2菌悬液（ OD600＝1）接种量为7�5 mL／g，GW 3菌悬液（ OD600＝1）接种量为10 mL／g，物料填充度为500 mL三角瓶中装50 g烟叶干基，当烟叶发酵水分质量分数为45％时，48℃发酵5 d。在该发酵条件下，上部低次烟叶的淀粉降解率为22�33％，蛋白质降解率为16�74％。烟叶淀粉和蛋白质的质量分数较大幅度下降，其含量指标接近优等烟叶的含量。该发酵方式比传统的贮存式烟叶发酵大大缩短了发酵时间。%In this study,two thermotolerant strains were isolated from tobacoo leaves�One is an amylase producing bacterial strain named GW⁃2 and another is a protease producing bacterial strain named GW⁃3�By morphological observation of colonies and 16S rRNA sequence homology analysis, they were identified as Bacillus subtilis and Bacillus amyloliquefaciens� Then the two strains were used for tobacco solid⁃state fermentation,and the condition was optimized by monofactorial experiments�The experimental results show that the best conditions for solid⁃state fermentation were that:inoculum of bacterial suspension( OD600=1) of GW⁃2 and GW⁃3 were 7�5 mL/g and 10 mL/g,respectively,charge amount of tobacoo was 50 g ( 500 mL flask ) , fermentation temperature was 48 ℃, moisture content of the tobacco was 45% and fermentation time was 5 d�Under these conditions of solid⁃state fermentation,the starch and protein decomposition rates in the upper leaves of flue⁃cured tobacco were 22�33% and 16�74%, respectively�The content of starch and protein in the upper leaves of flue⁃cured tobacco was close to the standard of the top quality tobacco�In this way, the fermentation time was greatly shortened than traditional storage fermentation.
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