## 3 Results and Discussion  
### Miso Fermentation of Cereal Processing By-Products Generates \(\gamma\)-Glutamyl Peptides  
Kokumi peptides have been shown to enhance the palatability and perceived mouthfulness of foods [33]. In a previous study, we demonstrated that the miso fermentation of regional cereal grains results in the generation of \(\gamma\)-glutamyl peptides [16]. We were, therefore, interested in whether the miso fermentation of cereal byproducts is able to produce kokumi peptides as well. We selected two main cereal production and processing by-products, wheat bran and brewer's spent grain (BSG), and introduced them at different stages of the miso fermentation. Wheat bran was used as the key substrate for the preparation of koji in this study based on evidence of wheat bran being utilised in traditional and industrial koji-making practices [34; 35; 36; 37], and BSG was used as the miso substrate based on its high residual protein and nutrient contents that are comparable to other common miso substrates [6; 20]. We also included barley, a substrate that was demonstrated to produce \(\gamma\)-glutamyl peptides through miso fermentation [16], and tested different combinations of miso fermentation as detailed in Table 1. To investigate the potential effect of the genetic influence of the koji fungus, _A. oryzae_, on the fermentation process, we also tested different strains of _A. oryzae_ (S1 and S2) for specific combinations (Table 1). A previous study demonstrated that all but one of the \(\gamma\)-glutamyl peptides that were identified in grain-based miso fermentation [16] were also present in the miso samples prepared using cereal-processing by-products, including \(\gamma\)-ECG (oxidized), \(\gamma\)-EE, \(\gamma\)-EL, \(\gamma\)-EF, \(\gamma\)-EV, and \(\gamma\)-EVG. Likewise, the reduced form of \(\gamma\)-ECG was not detected, most likely due to natural oxidation during the fermentation process (Figure 1) [16]. Cluster analysis of the different kokumi peptides and miso shows that the miso made with wheat bran koji is clustered into one group, and the one made with barley koji is clustered into another (Figure 1A). This observation demonstrates that the koji substrate is an important parameter for the abundance and identity of kokumi peptides in subsequent miso fermentations, as suggested previously [16]. Looking more closely at the individual \(\gamma\)-glutamyl peptides identified, \(\gamma\)-ECG oxidized was present in all of the samples after 12 weeks of fermentation (Figure 1A). Notably, the abundance of \(\gamma\)-ECG is inverse to the abundance of \(\gamma\)-EVG and \(\gamma\)-EV in the different samples (Figure 1A). This trend might be explained by the mechanism suggested by [PERSON] et al. [38], where \(\gamma\)-ECG is the precursor of \(\gamma\)-EV and \(\gamma\)-EVG. The synthesis of \(\gamma\)-EV and \(\gamma\)-EVG is mediated through the transfer of the \(\gamma\)-glutamyl residue from \(\gamma\)-ECG to valine via the GGT (glutamyl transferase) pathway or the dipeptide Val-Gly (VG), respectively. The abundance of \(\gamma\)-EVG is promising because it is the most potent kokumi peptide found and studied in a wide range of fermented products [33]. Interestingly, \(\gamma\)-EE was identified in all samples at the end of the fermentation process (Figure 1A). In our previous study on alternative miso, this peptide was identified in barley  
Figure 1: (**A**) \(\gamma\)-Glutamyl peptides detected in barley, BSG, and wheat bran miso after 12 weeks of fermentation, including cluster analysis for samples. The abundance of \(\gamma\)-glutamyl peptides is based on chromatography areas, which are unitless. Relative abundance can be compared for the same peptide but not across different peptides. (**B**–**E**) Evolution of \(\gamma\)-glutamyl peptides over time: (**B**) barley S1/BSG, (**C**) wheat bran S2/barley, (**D**) wheat bran S1/barley, (**E**) wheat bran S1/BSG. Supplemental Materials Table S1 shows MS information.