## 3 Results & Discussion
### Total acidity and Production of Secondary Metabolites  
After the end of alcoholic fermentations, an average reduction of 20.1% in tartaric acid was observed (Table 3). No significant differences were observed between fermentations containing different amounts of YAN. These results are in agreement with preview studies for _S. cerevisiae_ and could be due to biochemical utilization or physical losses through decreased solubility or tartaric acid's adsorption to the surface of yeast cells [38]. Based on the absence of statistically important differences between different yeast strains, it is surmised that physiochemical phenomena have a greater impact on the alterations of tartaric acid concentrations. There were no differences in malic acid concentrations, nor was any lactic acid production noted (data not shown) even when, in some cases, an up to 22% consumption of malic acid could be observed, being mainly strain-dependent [38; 39]. The ability to metabolize extracellular malic acid depends on an efficient system for malic acid transport (i.e., active import via a malate transporter) and a malic acid converting enzyme [40] that seemed to be absent in the investigated strains. Reduction in citric acid concentration was also observed (Table 3). Fermentations carried out with the commercial, Sa and Sb strains presented a 74.5%, 74.8% and 47.6% reduction in citric acid content, respectively. Overexpression of CIT1 and CIT3 genes encoding mitochondrial citrate synthase, which catalyzes the first step in the tricarboxylic acid (TCA) pathway, and/or of the KGD1, KGD2 and LPD1 genes encoding the oxoglutate dehydrogenase (OGDH) complex, which helps the formation of succinic acid and fumaric acid in commercial and Sa strains, could be a possible explanation for these results [29]. Absence of significant differences between fermentations of the same strain with different YAN contents, constitutes indication of suppressed expression of GLN3 and URE2 genes associated with alternative nitrogen assimilatory pathways connected with the TCA cycle [35]. The more limited alterations in citric acid concentration in fermentations carried out with Sb strain led to higher total acidity (TA) and lower pH, compared to wines fermented with the commercial and Sa strains. The observed relatively high TA (and low pH), were within the range commonly measured in wines produced from Assyrtiko grapes [41]. Apart from the higher TA values, a more moderated increase in volatile acidity (VA) was also achieved in Sb wines compared to the commercial and Sa wines (Table 3). These results are in line with previous works that demonstrated a positive correlation between acetic acid and glycerol production. As reported by [PERSON] et al., 1994, the production of acetaldehyde and consequently, the production of acetic acid and other compounds such as acetoin and higher alcohols, is mainly strain-dependent [42]. The different strains examined could be categorized according to phenotypes as low, medium and high acetaldehyde producers, respectively. Moreover, accumulation of acetic acid in fermentations carried  
\begin{table}
\begin{tabular}{l c c c c c c} \hline \hline  & **Must** & \multicolumn{2}{c}{**Commercial**} & \multicolumn{2}{c}{**Sa**} & \multicolumn{2}{c}{**Sb**} \\ \hline
**YAN (mg/L)** & **80** & **150** & **250** & **150** & **250** & **150** & **250** \\ \hline EtOH \% (\(\mathrm{e}/\mathrm{\gamma}\)) & 0.00 \(\pm\) 0.00 & 12.76 \(\pm\) 0.12 \({}^{\mathrm{a}}\) & 12.91 \(\pm\) 0.01 \({}^{\mathrm{a  
out with the commercial strain, with the possibly suppressed expression of HAA1 gene (responsible for the adaptation of yeast cells upon exposure to toxic concentrations of acetic acid), could have led to decreased cell viability and retarded fermentations [43; 44]. In addition, a slightly higher (however, not significantly important) increase in VA was observed in fermentations with 250 mg N/L compared to those with 150 mg N/L YAN, when carried out with the commercial and Sb strains, unlike with the Sa strain.