## 4 Discussion
Accordingly, we speculate that the modification of amino acid profile in spine grape must by supplementing alanine, phenylalanine, and isoleucine has the potential to increase fruity ester production. Studies have shown that amino acid addition affects wine ester concentration [7; 12; 34]. Generally, nitrogen management during winemaking is based on the catabolism of the amino acids combined with de novo synthesis of proteinogenic amino acids [16; 17]. However, there are limited studies on amino acid supplementation based on the amino acid profile of grape must and on the correlation between amino acids and aroma compounds. We found that goal-directed supplementation of amino acids led to significant increase in the production of AHAs and MCFAEEs. In contrast, IN had no significant effect on fruity ester content. Earlier, [PERSON] et al. reported no effect of DAP supplementation on the ester content of synthetic juice [35]. Meanwhile, [PERSON] et al. [4] showed an increase in ethyl ester content with DAP supplementation. These differences in findings may be due to the differences in nutrient availability and yeast strains [2]. Amino acid utilization via Ehrlich pathway had pronounced effects on the balance between higher alcohols and corresponding acetate esters. In the present study, AA-Phe and AA-Ile caused a higher production of \(\beta\)-phenylethylal acetate and isoamyl acetate due to increase in 2-phenylethylal and isoamyl alcohol precursors. However, AA-Ile had no marked effect on total MCFAEE content and, similar to AA-Phe, had reduced ethyl acetate production. In contrast to AA-Phe and AA-Ile, AA-Ala significantly enhanced ethyl acetate production. This may be because alanine is converted to pyruvate [36], a main precursor of acetyl-CoA, and the accumulation of acetyl-CoA could have led to increase in ethyl acetate and fatty acid biosynthesis [15; 37]. To conclude, AA-Ala enhanced fruity ester production; however, AHA and MCFAEE levels in AA-Ala were less than that in IN+MAA. Despite similar YAN content, differences in amino acid composition significantly influenced the ester content in wine. The production of fruity esters was considerably higher under both MAA and IN+MAA treatments; however, a higher (2.1-fold) increase in ethyl acetate content with MAA treatment may lead to off-flavor and finally decreased wine quality [38]. Meanwhile, IN did not contribute to fruity ester production. Single amino acid supplements led to an imbalance in the fruity ester profile. Therefore, IN+MAA, a balanced and goal-directed amino acid supplement, can be used to enhance fermentative aroma quality of spine grape wine. In addition to the aroma compounds produced during fermentation, changes in metabolic intermediates also greatly affect wine ester profile. Amino acids act as precursors of fermentation-derived aroma compounds principally via the Ehrlich pathway [14]. Research has indicated that transamination of amino acids is essential for the subsequent redistribution of nitrogen for the de novo synthesis of proteinogenic amino acids [16], which consequently releases \(\alpha\)-keto acid intermediates [39]. [PERSON] et al. [16] showed that the carbon skeletons of consumed amino acids contribute less to the production of volatile compounds. This is in contrast to the findings of [PERSON] et al. who reported that even minor changes in the amino acid profile of synthetic grape must significantly influence volatile production [7]. These contradicting results indicated an interaction between carbon and nitrogen metabolism for the production of aroma compounds. Accordingly, we analyzed fruity ester precursors and corresponding extracellular metabolites to illustrate the mechanism involved in fruity ester production. CK-SP, CK-CS, IN, and AA-Ile showed lower production of MCFAs, which are markers for limited acetyl-CoA levels [40]. [PERSON] et al. [41] showed that the level of fatty acids is the most limiting factor in ethyl ester production. An increase in MCFA production enhanced MCFAEE production [25].