## 3 Results and Discussion
### Miso Fermentation of Cereal Processing By-Products Generates \(\gamma\)-Glutamyl Peptides
We observe in this study that pH levels are strongly correlated with the changes in enzymatic activities during fermentation (\(p<0.01\)), as shown in Supplemental Materials Figure S1. The results show the lowest activity for barley S1/BSG (0.80 \(\pm\) 0.01 U/g), followed by wheat bran S2/barley (1.09 \(\pm\) 0.02 U/g), wheat bran S1/BSG (1.23 \(\pm\) 0.01 U/g), and the highest for wheat bran S1/barley (1.70 \(\pm\) 0.02 U/g) (Supplementary Table S1) for the final time point. It was also found that the koji and miso substrates are not the only determining factors for the fermentation process. Based on results from the two misos that used the same koji and miso substrates (wheat bran S1/barley and wheat bran S2/barley), we observed very different enzymatic activities (Figure 2C). This suggests that the _A. oryzae_ strain also has an influence on the enzymatic activity during fermentation. According to a study by [PERSON] et al. in [42], protease activity reaches its highest point at pH levels ranging from 6 to 7, depending on the specific _A._ strain. Meanwhile, the synthesis of \(\gamma\)-glutamyl peptides involves three distinct enzymes, including GGT (\(\gamma\)-glutamyl transferase), glutamatergic, and either GCS (\(\gamma\)-glutamylcysteine synthetase) or GCL (glutamate-cysteine ligase). In vitro, these enzymes catalyse the creation of \(\gamma\)-glutamyl dipeptides from glutamate and amino acids with an optimal pH above 7.5 [43]. These observations suggest that the strain of _A. oryzae_ used could also have an influence on \(\gamma\)-glutamyl peptide formation and remains to be further explored. Another physicochemical property we investigated were the parameters in relation to colour (a*, b*, and L*). Our previous study showed that a* and b* were also correlated  
Figure 2: Physicochemical characteristics during the miso fermentation. (**A**) pH and \(\gamma\)-EV for the first cluster, (**B**) pH and \(\gamma\)-EV for the second cluster, (**C**) proteases activity (U/g), (**D**) L* luminosity vs. pH. One-way ANOVA and post hoc analysis (Tukey test) with 95% confidence levels are shown in Supplemental Materials Table S1. All parameters were measured in triplicate, excluding colour, where \(n=10\). Legend: pH: dotted line, peptides: continuous line. with kokumi peptides, particularly for \(\gamma\)-EL and \(\gamma\)-EV [16]. According to a study by [PERSON] et al. in [44], the impact of colour on consumer preferences in product appearance was examined, where on average, high a* (reddish) was associated with higher miso quality, whereas b* (blueish) and L* (luminosity) showed the opposite effect. Luminosity (L*) affects the darkness/reddish characteristic of miso and is closely related to its palatability and acceptance. The study described in this paper found that L* was negatively correlated with \(\gamma\)-EE, \(\gamma\)-EF (\(p<0.05\)), and \(\gamma\)-EL (\(p<0.01\)) (Supplemental Materials Figure S1). It was also observed that L* is negatively correlated with pH (Supplemental Materials Figure S1), and there is an especially pronounced decrease in L* accompanying the increase in pH for the wheat bran S1/BSG miso (Figure 2D). Based on our earlier conclusion of pH as a potential indicator for fermentation progress (Figure 2D), as well as subsequent characterisations of volatile aroma profile in Section 3.3, this suggests that luminosity could serve as a potential indicator for predicting the desirable characteristics for miso-based fermentation. Volatile Aroma Compound Profiles of Upcycled Miso Reveal Correlation with \(\gamma\)-Glutamyl Peptides  
Aroma plays an important role in food acceptance. Some interactions of plant-based protein and aromas have been studied, suggesting that compounds such as hexanal, (E)-2-nonenal, and (E,E)-2,4-decadenal can lead to consumer rejection. However, fermentation is able to reduce these compounds, making plant-based ingredients more palatable [9].