Comprehensive insights into the mechanism of flavor formation in Cheonggukjang: Integration of metagenomics, volatomics, and metabolomics

Microbial metabolism shapes the unique flavor profile of <i>Cheonggukjang</i>; however, the formation pathways of characteristic flavor compounds mediated by microbiota remain unclear, hindering precise quality control. To fill this gap, this study innovatively integrated metagenoics, volatilomics, and metabolomics to systematically decode the flavor formation mechanism during <i>Cheonggukjang</i> fermentation. Volatile compound analysis defined three fermentation stages for <i>Cheonggukjang</i> (0-18, 18-60, and 60-72 h), identifying the 60-72 h period as the most critical for flavor formation. A total of 15 key flavor compounds were identified, with 10 designated stage-specific flavor markers. LefSe analysis revealed that <i>Bacillus subtilis</i>, <i>Bacillus velezensis</i>, <i>Caldibacillus thermoamylovorans</i>, and <i>Bacillus licheniformis</i> were the key biomarkers across different fermentation stages, while redundancy analysis (RDA) indicated that total sugar as the key driver of microbial succession. Additionally, this study reconstructed the metabolic network responsible for characteristic flavor formation and identified <i>C. thermoamylovorans</i>, <i>B. licheniformis</i>, <i>B. velezensis</i>, <i>B. subtilis</i>, <i>Bacillus paralicheniformis</i>, and <i>Caldibacillus hisashii</i> as core functional microbiota modulating amino acid metabolic to drive flavor development<i>.</i> This study lays a theoretical framework for standardizing <i>Cheonggukjang</i> production and targeted regulating its flavor quality.