## 3 Results and Discussion  
### Production of an Amazake-like Beverage from Bacillus amyloliquefaciens  
A sweet-tasting beverage similar to traditional amazake was successfully created using _B. amyloliquefaciens_. This novel beverage was produced following a process that is equivalent to the traditional method of amazake production, with the exception of using a different microbe, _B. amyloliquefaciens_ (NCIMB 12077), instead of _A. oryzae_ (W-20) (Figure 1). The preliminary tastings showed that the new beverage shares a key characteristic with traditional amazake; it is sweeter than the raw ingredients, suggesting the breakdown of carbohydrates into di- and monosaccharides. It also had a more complex flavor profile than the original substrate (rice), suggesting the generation of diverse volatile and non-volatile flavor compounds during the fermentation reaction. Interestingly, the flavor profile of the novel amazake seemed distinct from that of traditional amazake, highlighting its unique characteristics. The following sections present the characterization of this novel beverage and compares its properties and production process to those of traditional amazake. Although our results show that A. oryzae has a higher saccharification rate and enzymatic activity level, these attributes alone do not ensure identical functionality or yield rates when compared to _B. amyloliquefaciens_. The characteristics of these microorganisms are different, and even if present in the same quantity, their performance and outcomes can vary significantly. Therefore, we measured the ability of _B. amyloliquefaciens_ to produce metabolites and saccharify the medium to understand its viability in the amazake production process. Comparison of Reducing Sugar Contents during Amazake Production with Aspergillus oryzae and Bacillus amyloidiquefaciens  
Saccharification is one of the most distinguishing characteristics of traditional amazake. As the fermentation progresses, enzymes such as amylases and glucosidases that are secreted by _A. oryzae_ during the koji fermentation break down carbohydrates to di- and monosaccharides, which can be detected by our taste buds [10]. Saccharification can be estimated by measuring the reducing sugar content (RS). The sugars produced by _A. oryzae_ are mostly reducing monosaccharides (xylose, fructose, and glucose), whereas _B. amyloliquefaciens_ mainly produces the reducing disaccharide maltose [21; 22]. We initially compared the RS contents for amazakes produced at the traditional fermentation temperature of 55 \({}^{\circ}\)C for both microorganisms, namely an amazake produced with _A. oryzae_ (AAO) and another produced with _B. amyloliquefaciens_ (ABA) (Figure 2A). Both microorganisms produced RS at a higher rate during the first 8 h of the fermentation process; this corresponds to the  
Figure 1: (**A**) Overview of the amazake production process for _A. oryzae_ amazake (AAO) and _B. amyloliquefaciens_ amazake (ABA) at two different temperatures (55 \({}^{\circ}\)C and 70 \({}^{\circ}\)C), each fermented for 0, 8, 16, and 24 h. (**B**) Kojs fermented with _A. oryzae_ (AO) (**top**) and _B. amyloliquefaciens_ (BA) (**bottom**). commonly used fermentation time in traditional amazake recipes [4]. After 8 h, the RS contents for both fermentations began to decrease and eventually approached a plateau. The RS content at each time point was significantly different (\(t\)-test; \(p\) < 0.001). We observed that the RS values were higher for the AAO amazake at every time point of the fermentation process (\(p\) < 0.001), with the highest concentration being found at 24 h (353.0 g/L), as compared to the ABA amazake, which showed the highest concentration at 16 h (127.9 g/L). Ultimately, after 24 h, the saccharification came to an end in both cases, probably due to a substrate or enzyme limitation or as a result of product accumulation and consequent product inhibition [31].