## 3 Results and Discussion
### Miso Fermentation of Cereal Processing By-Products Generates \(\gamma\)-Glutamyl Peptides
koiji, so this confirms the relation between koji substrate and the generation of kokumi peptides [16]. The mechanism of \(\gamma\)-glutamyl dipeptides is led by \(\gamma\)-glutamyl transfer reaction via GCL and GCS. Thus, free amino acids such as glutamic acid are a limiting factor to synthesise \(\gamma\)-EE [39]. Peptide \(\gamma\)-EL was found in all miso samples, with higher abundance observed in the two misos that were made using BSG as the miso substrate (Figure 1A). This could be explained by the higher concentration of free leucine in BSG (6.1 \(w\)/\(w\) %) than in barley (0.3 \(w\)/\(w\) %) [40]. This peptide has also been reported to be present in various fermented foods, including soy sauce, sourdough, parma dry-cured hams, gouda, and parmesan cheese, as well as occurring naturally in various edible beans [33]. By contrast, \(\gamma\)-EF was only detected in wheat bran S1/BSG in the last stage of the miso fermentation (Figure 1D). The abundance of this dipeptide is slightly decreased during the fermentation time in the remaining samples until it reaches zero (Figure 1B-D). It has been found in various fermented products, such as miso and soy sauce [33]. Physicochemical Changes in Fermentation Processes Influence the Generation of \(\gamma\)-Glutamyl Peptides  
It is evident from this study, as well as previous studies, that components in the raw materials used to prepare koji contribute to the formation of precursors such as \(\gamma\)-ECG and the release of different amino acids [16]. Our previous results have also shown that physicochemical changes occurring during the fermentation process, including pH, REDOX, reducing sugar, and colour, are factors that are correlated with differential kokumi peptide formation [16]. Since physicochemical changes can both influence and be influenced by related enzymatic activities during the fermentation process, monitoring the physical and chemical parameters (Supplemental Materials Table S2) not only provides a better understanding of the production of kokumi \(\gamma\)-glutamyl peptides but can also become a useful tool for tracking the progress of fermentation, especially for unconventional substrates like by-products. In this study, we measured the changes in pH levels, protease activity, reducing sugar, water content, and colour, across all samples throughout the 12-week fermentation period, taking samples every 4 weeks. During the fermentation process, the pH levels of three of the four misos (wheat bran S1/barley, wheat bran S2/barley, and barley S1/BSG) followed a similar pattern, whereby the pH level initially decreased from 6 \(\pm\) 0.06 to 4 \(\pm\) 0.06 in the first eight weeks, then increased slightly back to around 6 in the final stage of the fermentation (Figure 2A). This pattern of modulation in pH profile is consistent with what we observed in our previous studies of miso fermentation [16; 40] In contrast, the pH level of the miso made from wheat bran S1/BSG showed an unusual pH profile, where a gradual increase from 6.63 \(\pm\) 0.06 to 9.43 \(\pm\) 0.06 (Figure 2B) was observed over the 12 weeks, without the pH ever reaching below 6, indicating an unusual fermentation process. This unexpected change in pH seems to have an impact on the abundance of the \(\gamma\)-EV peptide in particular (Figure 2B). This pattern and difference suggest that the combination of substrates impacts the pH behaviour and, consequently, kokumi peptide generation. In addition to being a good indicator for monitoring the fermentation process, pH changes may also serve a functional role in the synthesis of \(\gamma\)-dipeptides such as \(\gamma\)-EE, \(\gamma\)-EF, and \(\gamma\)-EL as it influences the ionic form of amino acids, like glutamic acid to glutamate. Glutamate is also an important amino acid for the \(\gamma\)-glutamyl reaction and has been implicated in dampening bitterness from phenylalanine, leucine, and valine [41], a taste attribute that we will further explore in Section 3.4.