## 3 Results
### Analysis of Extracellular Metabolites and Precursors Involved in Fruity Ester Synthesis  
Fruity ester precursors and corresponding extracellular metabolites, including higher alcohols (isobutanol, isopentanol, 1-hexanol, and 2-phenylethanol), volatile linear fatty acids (hexanoic acid, octanoic acid, and decanoic acid), and carbon-metabolism-derived extracellular metabolites (L-malic acid, acetic acid, citric acid, succinic acid, and glycerol), under different treatments were analyzed (Tables 3 and 4). Nitrogen supplementation significantly increased the levels of higher alcohols and fatty acids. AA-Phe and AA-Ile dramatically increased the higher alcohol level; AA-Phe increased 2-phenylethanol content (5-fold compared with CK-SP); and AA-Ile increased the isoamyl alcohol level (1.8-fold compared with CK-SP), whereas AA-Ala and IN+MAA did not contribute to higher alcohol production. In contrast, AA-Ala, AA-Phe, and IN+MAA treatments significantly enhanced the production of medium-chain fatty acids (MCFAs). Analysis of carbon-metabolism-derived extracellular metabolites indicated that CK-CS had the highest organic acid content while AA-Ile produced the highest amount of glycerol. All nitrogen treatments reduced citric acid content in spine grape wine. Compared with CK-SP, the treatments AA-Ala, AA-Phe, AA-Ile, and MAA led to a significant increase in glycerol content in spine grape wine while IN+MAA led to a significant decrease in glycerol, malic acid, and citric acid levels. Other nitrogen treatments, except AA-Phe, increased acetic acid content. In addition, IN+MAA produced the highest level of extracellular succinic acid, while other treatments had no significant effect on succinic acid production. Furthermore, the differences in yeast extracellular metabolites involved in MCFA and acetate synthesis pathways are illustrated in Figure 3. Only MAA and IN+MAA treatments, which resulted in significant modification of fruity ester profile, were selected in Figure 3 while CK-SP and IN were used as controls. Compared with CK-SP and IN, IN+MAA treatment decreased glycerol and acetic acid levels and increased citric acid, succinic acid, ethyl octanoate, and ethyl decanoate levels. In contrast, IN had no significant influence compared with CK-SP. \begin{table}
\begin{tabular}{c c c c c c c c c} \hline
**Compounds** & **CK-CS** & **CK-SP** & **IN** & **AA-Ala** & **AA-Phe** & **AA-Ile** & **MAA** & **IN+MAA** \\ \hline Glycerol & 7.47 \(\pm\) 0.12 & 7.26 \(\pm\) 0.22b & 7.36 \(\pm\) 0.28 ab & 7.65 \(\pm\) 0.24 & 7.63 \(\pm\) 0.19 & 7.76 \(\pm\) 0.26 & 7.73 \(\pm\) 0.30a & 6.98 \(\pm\) 0.15c \\ Malic acid & 2.51 \(\pm\) 0.14 & 0.78 \(\pm\) 0.04a & 0.72 \(\pm\) 0.01a & 0.65 \(\pm\) 0.01 & 0.71 \(\pm\) 0.01 & 0.67 \(\pm\) 0.01 & 0.73 \(\pm\) 0.02a & 0.64 \(\pm\) 0.01b \\ Acetic acid & 1.41 \(\pm\) 0.01 & 0.71 \(\pm\) 0.04b & 0.67 \(\pm\) 0.03b & 0.77 \(\pm\) 0.07 & 0.66 \(\pm\) 0.03 & 0.74 \(\pm\) 0.01 & 0.85 \(\pm\) 0.03a & 0.81 \(\pm\) 0.01a \\ Citric acid & 0.70 \(\pm\) 0.06 & 0.33 \(\pm\) 0.08a & 0.33 \(\pm\) 0.04a & 0.25 \(\pm\) 0.01 & 0.22 \(\pm\) 0.01 & 0.25 \(\pm\) 0.01 & 0.22 \(\pm\) 0.01 ab & 0.19 \(\pm\) 0.01b \\ Succinic acid & 4.61 \(\pm\) 0.10 & 1.01 \(\pm\) 0.02b & 1.06 \(\pm\) 0.02b & 0.92 \(\pm\) 0.01 & 1.11 \(\pm\) 0.01 & 0.92 \(\pm\) 0.04 & 1.03 \(\pm\) 0.01b & 1.22 \(\pm\) 0.04a \\ \hline \end{tabular}
\end{table} 
Table 4: Concentrations of extracellular metabolites in wines obtained from different nitrogen nutrient treatments (g/L). Data are mean \(\pm\) standard deviation. Values displaying different letters within each row are significantly different according to the Duncan test at 95% confidence level. The Duncan test was carried out in columns CK-SP, IN, MAA, and IN+MAA.