王丽丰，李 雯，谢 和^*

（贵州大学生命科学学院/农业生物工程研究院，山地植物资源保护与保护种质创新教育部重点实验室，山地生态与农业生物工程协同创新中心，贵州贵阳 550025）

摘要：四甲基吡嗪是白酒重要风味物质的组成成分，赋予白酒一定的健康功效。为提高菌株发酵产四甲基吡嗪的含量，本研究以枯草芽孢杆菌E20为原始菌株，利用同源重组敲除其 2,3-丁二醇脱氢酶（BDH）的编码基因（bdhA），成功构建突变菌株E20-ΔbdhA。将突变菌株和原始菌株接种到黄豆发酵培养基模拟产酱香固态发酵。结果表明，突变菌株发酵物中四甲基吡嗪的含量为100.45 mg/kg，比原始菌株提高了71.97%，酱香风味无明显变化。利用突变菌株E20-ΔbdhA对其固态状态下合成四甲基吡嗪的条件进行优化，确定当种子菌液接种量为5%，发酵培养基为高粱小麦（1∶1），（NH₄）₂HPO₄的添加量为70 g/kg时，发酵物中四甲基吡嗪的含量最高为380.61 mg/kg。

关键词：枯草芽孢杆菌；四甲基吡嗪；基因敲除；发酵条件优化；酱香风味

中图分类号：TS261.13 文献标识码：A 文章编号：1674-506X（2023）01-0008-0007

Study on Improving the Content of Tetramethylpyrazine in Bacillus subtilis by bdhA Gene Knockout 

and Optimization of Fermentation Conditions

WANG Lifeng, LI Wen, XIE He*

(Key Laboratory of Plant Resource Conservation and Germplasm Innovation in Mountainous Region, Collaborative Innovation

Center for Mountain Ecology & Agro-Bioengineering, College of Life Sciences/Institute of Agro-bioengineering，

Guizhou University, Guiyang Guizhou 550025, China)

Abstract: Tetramethylpyrazine is an important component of liquor flavor, which endows liquor with certain health effects. In order to increase the production of tetramethylpyrazine by fermentation, the mutant strain E20-ΔbdhA was successfully constructed by knocking out the encoding gene(bdhA) of 2,3-butanediol dehydrogenase(BDH) of Bacillus subtilis E20 as the original strain by homologous recombination. The mutant strain and the original strain were inoculated into soybean fermentation medium to simulate the solid- state fermentation of soy sauce. The results showed that the content of tetramethylpyrazine in the fermentation of the mutant strain was 100.45 mg/kg, which was 71.97% higher than that of the original strain, and the Maotai-flavor had no obvious change. The conditions for synthesis of TTMP in solid state were optimized by using mutant strain E20-ΔbdhA. It was determined that when the inoculum amount of seed bacteria was 5%, the fermentation medium was sorghum and wheat 1:1 medium, and the addition amount of (NH₄)₂HPO₄ was 70 g/kg, the content of tetramethylpyazine in the fermentation material was the highest 380.61 mg/kg.

Keywords: Bacillus subtilis; tetramethylpyrazine; gene knockout; optimization of fermentation conditions; Maotai-flavor

doi:10.3969/j.issn.1674-506X.2023.01-002

收稿日期：2022-10-24

作者简介：王丽丰（1994-），女，硕士研究生。研究方向：微生物学。

*通信作者：谢和（1963-），男，硕士，副教授。研究方向：资源微生物学和应用微生物学。

引用格式:王丽丰,李雯,谢和.bdhA基因敲除及发酵条件优化提高枯草芽孢杆菌四甲基吡嗪含量研究[J].食品与发酵科技,2023,59(1):8-14.