## 3 Results and Discussion
### Miso Fermentation of Cereal Processing By-Products Generates \(\gamma\)-Glutamyl Peptides
To explore this further, we conducted an HS-SPME-GC/MS analysis and found a total of 125 volatile organic compounds across the different miso samples (Supplemental Materials Table S3). We first performed a correlation analysis between the kokumi peptides and volatile aroma compounds and identified 67 compounds that showed correlative association with the \(\gamma\)-glutamyl peptides (Supplemental Materials Figure S2) and 63 in particular that were present in the last stage of the miso fermentation (Figure 3). These compounds included three acids, four alcohols, six aldehydes, thirty-one esters, ten ketones, five phenols, and four pyrazines. These results corresponded with our previous study on alternative grain-based miso, where the most represented volatile aroma group present was esters [16]. Cluster analysis of the volatile aroma compound profiles revealed that three of the misos (wheat bran S1/barley, wheat bran S2/barley, and barley S1/BSG) belong to the same cluster, whereas the miso made of wheat bran S1/BSG belongs to a separate second cluster (Figure 3). This observation may be related to the unexpected differences in physicochemical changes that were exhibited during the fermentation process, especially the non-acidic pH levels for the wheat bran S1/BSG miso (Figure 2B), which could serve as a potential explanation for the unusual aromatic attributes of this sample, a point that we will return to later in this section. First, focusing our analysis on the volatile aroma profile of the main clusters, it was observed that the esters were amongst the group of volatile compounds most strongly associated with kokumi peptides, especially high-fatty acid ethyl esters like hexadecanoic acid methyl ester, hexadecanoic acid ethyl ester, 9,12-octadecanenoic acid (Z,Z)-methyl ester, elaidic acid methyl ester, linoleic acid ethyl ester, and ethyl oleate (Figure 3). This observation is in line with our previous study on alternative grain-based miso, where we also observed high concentrations of ester aroma compounds due to the degradation of fatty acids [16]. These molecules are known to have waxy, fatty, oily, and fruity aromas. Most of them are produced during long-term fermentation processes lasting 3-6 months [45; 46]. They might be involved in the relationship between the koku sensation and fat, contributing to a mouthfeel of coating and lingering as well as an aftertaste, as has also been suggested in a previous study that showed that kokumi peptides can enhance the fatty taste [26]. Compounds that cause fruity aromas are also present prominently in the main cluster; these include compounds from the ester group, such as isobutyl acetate, 2-methyl-butanoic acid- ethyl ester, 3-methyl-butanoic acid- ethyl ester and isoamyl lactate, as well as phenylethyl alcohol (Figure 3); the latter has been associated with a rose-honey-like aroma [44]. Phenylethyl alcohol is particularly noteworthy, as it is found to be present in all three misos in the cluster (Figure 3) and has been reported to be produced by degrading the amino acid phenylalanine [46], which could be a possible explanation for why these misos do not contain \(\gamma\)-EF (Figure 1A). Figure 3: Volatile aroma compounds were identified in the miso after 12 weeks of fermentation. All the volatile compounds shown have a significant correlation with the kokumi peptides (Pearson’s coefficient, 95% confidence level). Kokumi peptides correlation plot and statistical analysis are available in Supplemental Materials Figure S2 and Table S3, respectively. Compounds in bold are present in the highest concentrations and are mentioned in the manuscript. Within the main cluster, the three misos can be further sub-clustered into two different groups based on their volatile aroma profiles. Interestingly, wheat bran S1/barley and wheat bran S2/barley did not cluster into the same group, suggesting that the strain of _A.