Optimization of derivatization-gas chromatography/mass spectrometry (Der-GC/MS) for analyzing non-volatile metabolites in soy sauce koji-making process and their evolution patterns.

The composition of metabolites in koji plays a crucial role in determining soy sauce flavor, yet remains insufficiently characterized. This study optimized a derivatization gas chromatography-mass spectrometry (Der-GC/MS) method for analyzing non-volatile metabolites during koji-making. Compared with nuclear magnetic resonance (NMR), which detected 24 compounds, Der-GC/MS showed higher sensitivity and broader coverage, identifying 63 compounds. The final optimized protocol involved extraction with 80 % methanol, ultrasonic treatment for 10 min, shaking for 20 h, followed by derivatization using 100 μL of sample and 120 μL of MSTFA. A total of 63 metabolites were identified, revealing stage-specific metabolic patterns: amino acids and reducing sugars peaked early, sugar alcohols increased mid-process, and organic acids accumulated steadily. These findings provide insights into the metabolic evolution during koji-making and offer a reliable method for studying the development of soy sauce flavor.