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This study aimed to investigate the biotransformation of soybean isoflavones to hydroxyisoflavones, and the primary and secondary metabolite change during Aspergillus oryzae KACC40247-mediated fermentation by gas chromatography-time of flight-mass spectrometry and LC-MS with multivariate analysis. The mass spectrometric analysis revealed that acetylglycosides and glycosides decreased during the first 12 h of fermentation, while the aglycones increased up to that time point. This was followed by a decrease in aglycone levels due to the formation of hydroxyisoflavones. The hydroxyflavones, 8-hydroxydaidzein, hydroxygenistein, and hydroxyglycitein, resulting from the biotransformation of the corresponding aglycones, increased up to 24 h, and then subsequently decreased. During fermentation, the levels of monosaccharides, aspartic acid, pyroglutamic acid, gamma-aminobutyric acid, and organic acids gradually decreased, whereas the levels of threonine, serine, and glycine increased. Hydroxyisoflavone was more strongly correlated with antioxidant activity than the other metabolites. Our results suggest that biotransformation has the potential to improve the nutritional properties of soy-based food.

Targeted metabolomics for Aspergillus oryzae-mediated biotransformation of soybean isoflavones, showing variations in primary metabolites