蔡 文¹，吴鑫颖²，张倩颖¹，李东亮¹，张 娟²，张宝玉²，朱鹏程^1*

（1.四川中烟工业有限责任公司，四川成都 610000；

2.江南大学，江苏无锡 214000）

摘要：烟叶发酵是改善烟叶品质的重要手段之一，烟叶中蛋白质含量偏高会对烟叶品质产生不良影响，所以筛选一株具有产蛋白酶功能的微生物，对烟叶品质提升具有重要意义。本研究从烟叶表面筛选出一株产蛋白酶的菌株，采用分子生物学方法进行菌株鉴定。对菌株发酵条件进行优化，并将优化后的菌株发酵液喷洒于烟叶表面进行固态发酵，利用近红外光谱仪和顶空固相微萃取-气相色谱-飞行时间-质谱联用技术分别检测了发酵前后烟叶中六项常规化学指标及挥发性成分。经鉴定该菌株为高斯芽孢杆菌（Bacillus kochii），优化后培养基组成为：1%（w/v）碳源（小麦粉∶葡萄糖＝2∶1）、2.5%（w/v）玉米浆、30%（v/v）烟叶提取物，最佳发酵温度为40 ℃，发酵24 h蛋白酶活力达到51.05 u/g，发酵后烟叶中总氮和总植物碱下降，两糖差上升。β-紫罗兰酮、β-环柠檬醛、异戊酸香叶酯等类胡萝卜素的降解产物含量明显提高，在减轻了烟叶的刺激性、杂气的同时，促进烟叶致香物质的形成，增香提质。

关键词：高斯芽孢杆菌；蛋白酶；发酵优化；烤烟；提质增香

中图分类号：TS414    文献标识码：A   文章编号：1674-506X（2022）03-0092-0008

Optimization of Neutral Protease Production by Bacillus kochii and its Effect on Fermentation of Tobacco Leaf

CAI Wen¹, WU Xinying², ZHANG Qianying¹, LI Dongliang¹

, ZHANG Juan², ZHANG Baoyu², ZHU Pengcheng^1*

（1.China Tobacco Sichuan Industrial Co., Ltd., Chengdu Sichuan 610000, China;

2.Jiangnan University, Wuxi Jiangsu 214000, China）

Abstract：The fermentation is one of the important means to improve the quality of flue-cured tobacco (FCT).It is significant to improve the quality of FCT to alleviate the adverse effects caused by the high protein content with screening a microorganism with protease-producing. In our study, a protease-producing strain were screened from FCT. The strain was identified by molecular biology methods, and the fermentation conditions were optimized to improve the enzyme-producing activity of the strain. FCT was fermented by the strain with protease-producing, and using near-infrared spectrometer and headspace solid-phase microextraction-gas chromatography-time-of-flight-mass spectrometry, six conventional chemical indexes and volatile components in tobacco leaves before and after fermentation were detected. The strain was identified as Bacillus kochii. The optimized medium composition was: 1% (w/v ) carbon source (wheat flour:glucose＝2:1), 2.5% (w/v) corn steep liquor, 30% (v/v) tobacco extract, and the optimal fermentation temperature was 40 ℃. Fermentation for 24 h, the protease activity increased to 51.05 u/g. After fermentation, the total nitrogen and total alkaloids of FCT decreased, the ratio of two sugars increased. The content of β-ionone, β-citral, geranyl isovalerate and other carotenoid degradation products in FCT were significantly increased, sensory evaluation of FCT was improved. Bacillus kochii with protein degradation ability screened from tobacco leaves had good functions in reducing the irritation and improving the quality of FCT through fermentation, showing good application potential.

Keywords：Bacillus kochii; protease; fermentation optimization;  flue-cured tobacco; quality improvement and aroma enhancement

doi：10.3969/j.issn.1674-506X.2022.03-012

收稿日期：2022-03-23

基金项目：烟叶醇化过程中微生物群落变化规律与关键风味物质形成关系及菌剂制备项目（No.KJSB201808020001）

作者简介：蔡文（1994-），男，硕士，助理工程师。研究方向：生物发酵。

*通信作者：朱鹏程（1986-），男，硕士，助理工程师。研究方向：生物发酵，烟草产品开发。

引用格式：蔡文,吴鑫颖,张倩颖,等.高斯芽孢杆菌产中性蛋白酶条件优化及其对烟叶发酵的影响[J].食品与发酵科技,2022,58(3):92-98,118.