## 3 Results and Discussion
### Microbial Community of Amazake Cultures Based on AAO and ABA Production  
Fermented products rely on the specific microbial communities present in them, which can influence the flavor, nutritional value, and safety of the final product [43]. Our study investigated the initial and final microbial communities to uncover any shifts in species dominance and diversity, as well as potential implications for food quality and safety. This has not previously been done for amazakes produced with either of the two microbes. First, we compared the microbial community structure of each amazake at time 0 and found that the initial number of microbial species (richness) in ABA was nearly double that of AAO (Figure 4A; F\({}_{1,10}\) = 53.15, \(p\)-value \(<\) 0.001). Simultaneously, the microbial community compositions differed significantly between AAO and ABA (Figure 4B; R\({}^{2}\) = 0.876, \(p\)-value = 0.001). These results show that the primary organism in an amazake culture greatly determines the whole microbial community, thereby determining the physicochemical, gastrointestinal, and nutritional properties of the final product. Secondly, we investigated the changes in microbial community structure between the initial and final time points of each amazake. After 16 h of fermentation, the microbial richness was greater than at time 0 for both AAO (Figure 4A; X\({}^{2}\) (1, N = 6) = 428.41, \(p\)-value \(<\) 0.001) and ABA (Figure 4A; X\({}^{2}\) (1, N = 6) = 17.72, \(p\)-value \(<\) 0.001). Likewise, the microbial community composition was influenced, although to a lesser extent, by the time point (Figure 4B; R\({}^{2}\) = 0.01516, \(p\)-value = 0.290). Interestingly, we noted that some of the 16 h amazakes were still very similar in community composition to the initial amazakes (0 h) for both microorganisms (Figure 4A), potentially signaling some variability in the rates of compositional change. Specifically, the _B. amyloliquefaciens_ amazake demonstrated a shift towards predominance by the inoculated bacteria from 0 to 16 h, with _B. amyloliquefaciens_ increasing in abundance over time to become the dominant species. This is interesting because it shows that _B. amyloliquefaciens_ did not establish dominance during the koji fermentation but only  
Figure 4: Microbial communities of _A. oryzae_ and _B. amyloliquefaciens_ amazakes: (**A**) numbers of different species in the microbial communities of ABA and AAO amazakes at 0 and 16 h; (**B**) Bray–Curtis dissimilarity rates between compositions of microbial communities in ABA and AAO amazakes at 0 and 16 h; (**C**) taxonomic characterization of the dominant microorganisms in the microbial communities of ABA amazake and (**D**) AAO amazake at 0 and 16 h. All amazakes were fermented at 55 \({}^{\circ}\)C.  
managed to do this during the amazake process. The top species present at time 0 h were _Bacillus cereus_ and _Streptococcus pneumoniae_, which are commonly associated with food poisoning and human diseases [44; 45]. By the end of the fermentation (16 h), these were replaced by microorganisms such as _Bacillus licheniformis_, a common probiotic that can be found in a variety of foods [46], as well as _Bacillus velezensis_ (Figure 4C). The latter shares a close phylogenomic resemblance with _B. amyloliquefaciens_ and is commonly found in fermented food products; it is characterized by its strong proteolytic activity during fermentation processes [47]. Certain strains of _B. amyloliquefaciens_ have been reported to inhibit the synthesis of toxic substances, including toxic compounds produced by _Bacillus cereus_ in the food industry [12], thereby highlighting the potential for producing fermented products inoculated with these beneficial bacteria. In contrast, fungi maintain dominance throughout the fermentation period of AAO amazakes, indicating stability in the microbial community. In addition to the dominant species _Aspergillus oryzae_, many other _Aspergillus species_ were detected, including _Aspergillus minisclerotigenes_ and _Aspergillus arachidica_ (Figure 4D).