Integrating gene knockout, GC-O-MS and multivariate statistical analysis: revealing the impact of fadA and bkdB genes on the aroma characteristics of Bacillus subtilis BJ3-2 fermented soybeans

Abstract

Fermented soybean products are an integral part of the Chinese diet, valued for their distinctive aroma. Bacillus subtilis (B. subtilis) BJ3-2 is a highly effective fermentation strain widely used in the industrial production of bacterial-type douchi. However, the specific aroma characteristics of soybeans fermented by this strain remain unclear. This study utilized gene knockout, GC-O-MS, and multivariate statistical analysis to comprehensively investigate the aroma components of soybeans fermented by B. subtilis BJ3-2. For the first time, 12 key aroma skeleton compounds were identified, including guaiacol, 2,5-dimethylpyrazine, and 2-methylbutyric acid, which primarily contribute to the smoky, roasted, and sweaty aroma attributes of fermented soybeans. Additionally, 17 compounds were considered to have important coordinating effects on soybean aroma, such as 3-hydroxy-2-butanone (buttery, creamy). Partial least squares regression (PLSR) analysis demonstrated a good correlation between key aroma compounds and sensory attributes. Gene knockout of fadA and bkdB resulted in a significant increase in 2,5-dimethylpyrazine and 1-octen-3-ol levels, along with a marked reduction in 2-methylbutyric acid levels. These changes enhanced the roasted and beany notes, reduced the sweaty aroma, and ultimately improved the overall aroma profile. These findings provide insights into the genetic regulation of aroma in fermented soybean products and form a foundation for reducing undesirable aromas and improving the overall aroma quality of industrially produced douchi.