## 2 Results and Discussion
### VOCs Produced during Fermentation
Further, based on the retention time (RT) of main and fragment ions detected in fastGC-PTR-ToF-MS (Table 4), the \(m/z\) 89.060 detected by PTR-ToF-MS was not butyric acid, though a small signal was observed for ethyl butanoate. The concentration of \(m/z\) 49.011 (t.i. methanethiol) was significantly (\(p<0.05\)) higher in DM at 35 \({}^{\circ}\)C than in all other treatments after 7 days of fermentation. In addition, methanethiol in DMFr at 35\({}^{\circ}\)C was significantly higher (\(p<0.05\)) than in DM and DMFr at 25 \({}^{\circ}\)C (Figure 4b). Methanethiol, which is an odour-active flavour compounds in meat [51] and cheese [52], can be produced by LAB from methionine (Met), a sulphur-containing AA, through either a transamination reaction, demethylation, or through methanethiol using various enzymes [53; 46; 54; 55] (Figure 3). Even though Met was present in the two defined media (DM and DMFr) used at the same proportion, the concentration of methanethiol was higher in the glucose-containing medium compared to the fructose-containing medium. This difference could be because the extra NADH generated during the metabolism of glucose by LB672 via the PK pathway is used in the methanethiol production pathway (Figure 3). Furthermore, the concentration of \(m/z\) 91.027 (t.i. methyl thiolacetate/ mercaptoacetone) followed a similar pattern to methanethiol (Figure 4d). The concentration of \(m/z\) 91.072 (t.i. 2,3-butanediol) was significantly (\(p<0.05\)) higher in DM at either 25 or 35 \({}^{\circ}\)C compared to DMFr at either 25 or 35 \({}^{\circ}\)C (Figure 4e). 2,3-butanediol can be produced from pyruvate which is produced either from sugar metabolism or aspartic acid (Asp) catabolism through an intermediate acetoin using the diacetyl acetoin reductase enzyme (DAR) (Figure 3) [57; 43; 56]. Even though the defined media DM and DMFr contained Asp at the same proportion, the concentration of \(m/z\) 91.072 varied significantly (\(p<0.05\)) based on whether glucose or fructose was present in the medium. As a result, it is speculated that the \(m/z\) 91.072 produced in the current study mainly resulted from pyruvate, which is produced from glucose metabolism. To confirm this, further research using the same experimental design in the defined medium (DM/DMFr) with and without the addition of Asp is required. Once 2,3 butanediol is produced, it can subsequently be converted to acetoin by the 2,3-butanediol dehydrogenase enzyme (BDH) (Figure 3). As acetoin was not detected in this study, it is speculated that DAR but not BDH is present in LB672. The concentration of \(m/z\) 121.057 (t.i. 2,3 benzeneacetaldehyde) was significantly (\(p<0.05\)) higher in DM at 35 \({}^{\circ}\)C compared to all other treatments (Figure 4f). Benzeneacetaldehyde, which is one of the key flavour compounds in cheese [52], can be produced by LAB from phenylalanine through a transamination reaction, followed by decarboxylation reaction (Figure 3) [46]. \begin{table}
\begin{tabular}{c c c c c c} \hline \hline \multirow{2}{*}{**Compound Name**} & \multirow{2}{*}{**Molecular Formula**} & \multicolumn{4}{c}{**Main/fragment Ions Checked**} \\ \cline{3-6}  & & \(m/z\) & \(m/z\) & \(m/z\) & \(m/z\) \\ \hline \multirow{2}{*}{Ethyl acetate} & \multirow{2}{*}{C\({}_{4}\)H\({}_{8}\)O\({}_{2}\)} & 89.060 & 61.028 & 43.018 & \\  & & (C\({}_{4}\)H\({}_{8}\)O\({}_{2}\)H\({}^{+}\) & (C\({}_{2}\)H\({}_{4}\)O\({}_{2}\)H\({}^{+}\) & (C\({}_{2}\)H\({}_{3}\)O)H\({}^{+}\) & \\  & & 89.060 & 71.049 & 43.054 & 29.039 \\ \multirow{2}{*}{Butyric acid} & \multirow{2}{*}{C\({}_{4}\)H\({}_{8}\)O\({}_{2}\)} & (C\({}_{4}\)H\({}_{8}\)O\({}_{2}\))H\({}^{+}\) & (C\({}_{4}\)H\({}_{6}\)O)H\({}^{+}\) & (C\({}_{3}\)H\({}_{7}\))H\({}^{+}\) & (C\({}_{2}\)H\({}_{5}\))H\({}^{+}\) \\ \hline \hline \end{tabular}
\end{table} 
Table 4: Main and fragment ions checked for \(m/z\) 89.060 in fastGC-PTR-ToF-MS.