## 2 Results and Discussion
### Amino Acids Involved in CoA Biosynthesis
#### 2.2.1 Stimulation of Ester Production by \(\beta\)-Alanine. A set of model fermentations were conducted containing one CoA-related amino acid at two concentrations (50 and 100 mg\(\cdot\)L\({}^{-1}\)\(\beta\)-alanine; 10 and 30 mg\(\cdot\)L\({}^{-1}\) l-cysteine; 330 and 650 mg\(\cdot\)L\({}^{-1}\) l-arginine; 30 and 120 mg\(\cdot\)L\({}^{-1}\) l-valine). The cysteine, arginine and valine concentrations fall within, or are slightly higher than, the typical concentrations of these amino acids found in grape juices [38]. The \(\beta\)-alanine concentrations used were slightly higher than those previously reported for _V. vinifera_ grapes [39]. Negative control fermentations did not contain amino acids, and positive control fermentations included all four amino acids at the lowest level added in the individual treatments  
Figure 3: Simplified pathway of CoA biosynthesis in the yeast _S. cerevisiae_ to emphasise the potential role of the four amino acids (boxed) used in this study. described above (_i.e._, 50 mg\(\cdot\)L\({}^{-1}\)\(\beta\)-alanine; 10 mg\(\cdot\)L\({}^{-1}\) l-cysteine; 330 mg\(\cdot\)L\({}^{-1}\) l-arginine and 30 mg\(\cdot\)L\({}^{-1}\) l-valine). The different treatments affected the length of the fermentation lag phase. The model must supplemented with the mixed amino acids had a lag phase of approximately two days, whereas all the other fermentations had a lag phase of four days, despite them having the same total YAN (data not shown). This could be due to the differences in structural complexity of the nitrogen source [40]. It was hypothesised that the four amino acids of interest would influence ester production through their role in CoA biosynthesis (Figure 3). Therefore, the concentrations of these amino acids could theoretically influence the level of CoA produced by yeast during fermentations, and CoA bearing acetyl/acyl groups would impact on the synthesis of ethyl/acetate esters. The \(\beta\)-alanine-supplemented wines had significantly more ethyl hexanoate, ethyl octanoate and ethyl decanoate (115-125 \(\mathrm{\SIUnitSymbolMicro\text{g}}\cdot\)L\({}^{-1}\); 114-122 \(\mathrm{\SIUnitSymbolMicro\text{g}}\cdot\)L\({}^{-1}\); 92-103 \(\mathrm{\SIUnitSymbolMicro\text{g}}\cdot\)L\({}^{-1}\), respectively) than the control wine (16 \(\mathrm{\SIUnitSymbolMicro\text{g}}\cdot\)L\({}^{-1}\); 39 \(\mathrm{\SIUnitSymbolMicro\text{g}}\cdot\)L\({}^{-1}\); 34 \(\mathrm{\SIUnitSymbolMicro\text{g}}\cdot\)L\({}^{-1}\)) or those produced after supplementation of the must with the other three amino acids (Figure 4). However, there were no significant differences between the ethyl ester concentrations in those wines produced from must with either 50 mg\(\cdot\)L\({}^{-1}\) or 100 mg\(\cdot\)L\({}^{-1}\)\(\beta\)-alanine added. This suggests that the effect of the addition of this compound on ethyl ester production may be already saturated at 50 mg\(\cdot\)L\({}^{-1}\). The wines that were produced after the addition of all four amino acids to the must also contained significantly more ethyl esters than the control, but in all three cases the concentrations in the wines were less than those observed in those produced after the addition of 50 mg\(\cdot\)L\({}^{-1}\)\(\beta\)-alanine alone (Figure 4). This may be due to the difference in the time it took for the fermentations to reach dryness, as this happened approximately two days earlier in the musts with all four amino acids added compared to the other treatments. Volatilisation or metabolism of the ethyl esters may have occurred in these wines while the other treatments were still reaching dryness. Individual additions of arginine, cysteine and valine did not have an impact on ester concentrations as they were not significantly different from the negative control in all cases. This suggests that the yeast is not limited in its ability to produce ethyl esters during fermentation by the supply of arginine, cysteine or valine.