## 3 Results and Discussion
### Comparison of the Metabolomic Profiles for AAO and ABA  
In addition to the main saccharides, amazake contains over 300 compounds [2]. These include all 20 amino acids derived from rice proteins broken down by _A. oryzae_ proteases, as well as various organic acids formed during carbohydrate metabolism. Both amino acids and organic acids influence the acidity of the culture environment and its habitability for microbes, as well as the final flavor and organoleptic characteristics of the amazake. In this study, we used liquid chromatography-mass spectrometry to evaluate differences in the contents of amazake metabolites. The untargeted and targeted data analyses revealed distinct differences between the metabolite profiles of the two amazakes. Overall, both amazakes showed similar compound intensity rates to each other, and both intensity rates were higher than for the rice control (Supplementary Figure S1). The principal component analysis (PCA) of the untargeted data showed that the two amazakes have clearly different metabolomic profiles from each other (Supplementary Figure S2). PC1 (46.5%) and PC2 (38.2%) accounted for most of the variance, with the main separation occurring between ABA and AAO and the control rice. The replicates for each type of amazake were grouped closely together, which indicates consistent fermentation outcomes. The targeted data analysis (i.e., compounds that were confirmed with a standard) corroborated these findings, with the PCA showing a clear separation between the amazake samples fermented with different microorganisms. PC1 (66.4%) and PC2 (19.65) revealed distinct clustering of the AAO and ABA samples, with AAO exhibiting a richer profile of specific metabolites compared to ABA (Supplementary Figure S3). The free amino acids detected in both the AAO and ABA amazakes were nearly identical (Table 1). One exception was for cysteine, which was only produced in the AAO fermentation. Quantitatively, the AAO samples had higher levels of most amino acids except for alanine, asparagine, and histidine, which were present in higher amounts in the ABA samples. These results line up with our previous observations, where AAO demonstrated higher protease activity (Figure 2D). \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Amino Acids** & **AAO** & **ABA** & \(p\)**-Value** \\ \hline Alanine & \(4\times 10^{9}\) & \(7.27\times 10^{9}\) & \(3.55\times 10^{-2}\) \\ \hline Argin & \(3\times 10^{11}\) & \(1.29\times 10^{11}\) & \(1.56\times 10^{-6}\) \\ \hline Asparagine & \(7\times 10^{8}\) & \(1.57\times 10^{9}\) & \(4.32\times 10^{-2}\) \\ \hline Aspartate & \(1\times 10^{10}\) & \(1.12\times 10^{9}\) & \(6.30\times 10^{-5}\) \\ \hline Cysteine & \(2\times 10^{7}\) & N/F & – \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Free amino acid compositions in _A. oryzae_ and _B. amyloliquefaciens_ amazakes after 16 h of fermentation at 55 \({}^{\circ}\)C.  
The amino acids detected in our study matched those commonly found in amazake fermented with _A. oryzae_, as described in the previous literature [4]. However, we also identified additional amino acids not reported in some studies of koji fermentation with _A. oryzae_ and _B. amyloliquefaciens_[22]. These differences may arise from variations in the type of rice used or the duration of the fermentation. For the organic acids, the AAO samples exhibited significantly higher abundance levels of citric, malic, fumaric, and succinic acids compared to ABA (_p_ < 0.005) (Table 2). Additionally, both amazakes produced coumaric acid, with the highest abundance level found in AAO, while shikimic acid was only detected in ABA samples [20]  
Some reports indicate the presence of citric acid, succinic acid, malic acid, and fumaric acid in traditional amazake [10; 39], which is consistent with our findings. Additionally, pyruvate acid has previously been found, which was not detected in our study. Coumaric acid and shikimic acid were detected for the first time here, with shikimic acid being newly generated in ABA.