## 2 Results and Discussion
### Leu/Ile-Derived VOCs
This finding suggests that LB672 in the presence of added Leu, Ile, or Phe at the lower acetate concentrations is using a pathway other than an ethanol-producing pathway. Acetic acid was detected in all media, with its concentration being significantly (\(p<0.05\)) higher at 1.2% acetate-containing DM compared to 0.1% acetate-containing DM (Figure 6B). Though _Lev. Brevis_ strains have been shown to produce acetic acid in addition to lactic acid [66], owing to the fact that acetate was present in the DM, it is not possible to confirm that LB672 produced additional acetate. Acetic acid, however, is the building block for fatty acids consisting of an even number of carbons [67; 32]. In the current study, butanoic, hexanoic, octanoic, and decanoic acids were present in a higher abundance in media containing 1.2% acetate compared to 0.1%. This result demonstrates in a concentration-dependent manner that acetate could be converted into fatty acids with even carbon chain-length numbers. Figure 5: Normalized peak area of 2-methyl butanol (**5A**) and 3-methyl butanol (**5B**) across different medium compositions (see Table 1) at 0.1% and 1.2% acetate. Values are presented as mean \(\pm\) standard error (\(n=6\)). Different letters represent significant difference between different medium compositions according to [PERSON]’s test at \(p<0.05\). Figure 6: Normalized peak area of ethanol (**6A**) and acetic acid (**6B**) across different medium compositions (see Table 1) at 0.1% and 1.2% acetate. Values are presented as mean \(\pm\) standard error (\(n=6\)). Different letters represent significant difference between the different medium compositions according to [PERSON]’s test at \(p<0.05\). In the current study, the addition of some individual amino acids (Phe, Met, Leu, and Ile) or the addition of the amino acid mixture (Aamix) had an impact on the relative proportions of VOCs formed by LB672 fermentation. In contrast, no particular VOCs were detected in the medium supplemented with Thr. Thr catabolism by LAB is initiated by threonine aldolase (TA) or serine hydroxymethyltransferase (SHMT), which converts Thr to acetaldehyde and glycine [35; 36]. Acetaldehyde was not detected after LB672 fermentation in the current study. As acetaldehyde is an intermediate compound in the ethanol production pathway [64], it was considered that it was most likely not detected rather than not being present. The inability to detect acetaldehyde was due to the fact that the HS-SPME method used was not optimised for its detection--rather, it was designed to detect a broad range of compounds. The addition of different concentrations of acetate (0.1, or 1.2%) in the DM significantly influenced the relative abundance of VOCs generated after LB672 fermentation. The mechanism(s) underpinning this observation were, however, unclear, as acetate's role in LAB growth and VOC generation is complicated. In addition to serving as a buffering agent, acetate has also been reported to demonstrate growth-stimulating activities for several LAB [47], and omitting acetate from the media can result in diminished growth [48]. Acetate can also serve as a precursor to a number of lipids/fatty acids [32; 68] and ethanol [65]. The presence of aminotransferases such as BCAT, ArAT, and AspAT and other enzymes involved in amino acid catabolism, including KdcA, AlcDH, AldDH, and CBL/CGL, in LB672 is likely in the current study because of the production of VOCs derived from branched-chain, aromatic, or sulphur (Met) amino acids. This could be confirmed by applying a genomic analysis of LB672. In the current study, the VOCs produced after 16 days of fermentation were analysed using GC-MS. Although GC-MS is a commonly used reference method for VOC analysis, it cannot be used to track changes in VOCs in real time. However, over time, the concentration of VOC produced can fluctuate dramatically. It is therefore possible that compounds were produced but were not detected in the analysis performed at the end of the fermentation (16 days).