## 2 Results and Discussion
### VOCs Produced during Fermentation
fermentum_) growing in medium with added glucose, whereas no ethanol was detected in medium with added fructose [24]. Ethanol can also be produced by LAB through the degradation of the amino acid (AA), threonine (Thr). Thr catabolism produces acetaldehyde using either threonine aldolase (TA) or serine hydroxymethyltransferase (SHMT) enzymes, with the acetaldehyde being converted to ethanol in the presence of the enzyme, alcohol dehydrogenase (AlcDH) [46; 47; 48] (Figure 3). While LB672 may or may not have a _glyA_ gene-encoding TA and SHMT enzymes, the Thr present in the defined medium was 50 times lower than the glucose concentration, suggesting that glucose metabolism rather than Thr catabolism was most likely the source of ethanol detected. The concentration of \(m/z\) 45.033 (t.i. acetaldehyde) after 7 days of LB672 fermentation was significantly (\(p<0.05\)) higher in DM at either 25 or 35 \({}^{\circ}\)C than in DMFr at either 25 or 35 \({}^{\circ}\)C (Figure 4a). In DM, the concentration of acetaldehyde was significantly (\(p<0.05\)) higher at 35 \({}^{\circ}\)C than 25 \({}^{\circ}\)C. Acetaldehyde, which is one of the major flavour compounds in yoghurt [49], is produced by LAB either from sugars via the PK pathway [15] or from Thr catabolism using TA or SHMT enzymes [46; 48] (Figure 3). Even though the defined media DM and DMFr used in this study contained Thr at the same proportion, the concentration of acetaldehyde varied significantly (\(p<0.05\)) based on whether glucose or fructose was in the medium. Given the differences in the concentration of sugar (carbon source) and Thr in the defined medium, it is speculated that the acetaldehyde produced was from mainly sugar metabolism. To confirm this, further research using the same experimental design in the defined medium (DM/DMfr) with and without the addition of Thr is required. Acetaldehyde improves desirable yoghurt flavour when present at greater than 8 mg/kg; however, at higher concentrations (200 mg/kg or above), it may negatively influence the overall yoghurt flavour [44; 50]. In the current study, the highest acetaldehyde concentration after LB672 fermentation in the DM was well below the concentration that has been reported to adversely affect flavour. Note that the acetaldehyde concentration is the headspace concentration, not the total concentration in the fermented sample. Further, it is important to note that the concentration of the specific VOC, its perception limit, and presence of other VOCs are crucial factors when considering the desired flavour notes. The concentration of \(m/z\) 89.060 (t.i. ethyl acetate) followed a similar pattern to acetaldehyde (Figure 4c). The ester, ethyl acetate, which is formed by alcohol acetyltrans  
Figure 2: The mean concentration (ppbV) of \(m/z\) 47.049 (t.i ethanol) after 7 days of fermentation by LB672 across defined medium compositions (DM: glucose added, DMfr: fructose added) at either 25 () or 35 (). Values are presented as mean \(\pm\) standard error (\(n=4\)). Different superscript lowercase letters represent significant differences between the carbon sources at either 25 or 35 \({}^{\circ}\)C according to [PERSON]’s test at \(p<0.05\). feras from the reaction between acetyl Co-A and ethanol, can confer fruity notes [41]. It is obvious that the production of ethyl acetate varied significantly (\(p<0.05\)) based on whether glucose (DM) or fructose (DMFr) was present in the defined medium. The signal for \(m/z\) 89.060 was considered to be mainly ethyl acetate, with a minor contribution from ethyl butanoate but not butyric acid, 3-methyl butanol, or acetoin. These contributions were based on the absence of acetoin detected in HS-SPME-GC-MS analysis (Table 2) or in fastGC-PTR-ToF-MS. FastGC-PTR-ToF-MS detected 3-methyl butanol in samples, where the signal was detected at \(m/z\) 71.085 (main fragment) but not at 89.060 (Table 3) and confirmed with authentic standard.