Improvement of Esterifying Power of Isolated Bacillus velezensis from Daqu by Atmospheric Pressure and Room Temperature Plasma Mutagenesis

Strong-flavor Baijiu, a widely popular distilled spirit in China, derives its characteristic aroma and quality largely from ethyl hexanoate, a key flavor compound. The concentration of ethyl hexanoate, influenced by its precursor hexanoic acid, is critical in defining the style and quality of this Baijiu variety. In this study, atmospheric and room temperature plasma (ARTP) mutagenesis technology was applied to strains isolated from Strong-flavor Daqu to enhance their acid and ester production capabilities. A hexanoic acid-producing strain, identified as <i>Bacillus velezensis</i> WY4 through morphological, physiological, biochemical, and molecular analyses, was used as the starting strain. Following 90 s of ARTP exposure, a mutant strain, WY4-3, was successfully developed, achieving a balance between high mutation diversity and moderate lethality. WY4-3 exhibited robust growth across a pH range of 4.2 to 5.0 and demonstrated high ethanol tolerance. After five days of fermentation, WY4-3 produced 0.36 g/L of total acid and 0.528 g/L of total ester, surpassing the wild-type strain. Enzymatic activity assays revealed significant enhancements in amylase (9.13%), saccharifying enzyme (101.72%), and esterification (573.71%) activities in WY4-3. Validation in multiple artificial esterification systems further confirmed the superior ester production capacity of this mutant strain. These findings enrich the microbial germplasm resources for Baijiu brewing and provide a solid foundation for strain selection and genetic improvement in Baijiu production processes. This study highlights the potential of ARTP mutagenesis in optimizing brewing microorganisms and improving the quality and consistency of Strong-flavor Baijiu.