## 2 Results and Discussion
### Diacetyl and Its Metabolic Products  
Diacetyl (2,3-butanedione) is produced by yeast during alcoholic fermentation by a pathway linked to the amino acid metabolism. At the end of alcoholic fermentation, diacetyl is reduced by diacetyl reductase to acetoin (3-hydroxy-2-butanone) and 2,3-butanediol. At this stage of the winemaking process, the concentration of diacetyl thus has no difactive effect. Significantly higher amounts of diacetyl can be produced during malolactic fermentation as an intermediate product in citric acid metabolism (4; 8; 14; 30; 31). In the course of the MLB carbohydrate metabolism pathway, pyruvate is reduced to lactate. However, when the concentration of residual sugars is too low, citric acid begins to be utilized as a carbon source, and additional pyruvate is synthesized. This pyruvate is then a precursor in the process of diacetyl production. The utilization of citric acid starts simultaneously with malic acid degradation, but it is a very slow process. It is particularly observed in states of sugar deficiency (31). Subsequently, because of its low chemical stability, diacetyl can be easily transformed into acetoin and 2,3-butanediol (4; 7; 32). Malolactic fermentation is thus highly recommended to allow diacetyl to enter into the wine aroma profile. During the secondary fermentation, diacetyl, acetoin, and 2,3-butanediol appear in different concentrations, which has a direct effect on wine aroma. Each of these compounds has a different odor detection threshold. Acetoin and 2,3-butanediol have significantly higher threshold values of perceptibility than diacetyl, at an average of 150 and 600 mg/L, respectively (7; 33). The aroma detection threshold value for diacetyl depends on the type and style of wine. In general, for good quality young red wines it ranges from about 0.2 to about 1.84 mg/L, and for aged red wines from 1.25 to 3.39 mg/L (33). The lowest concentration of diacetyl noted in our study occurred in the case of alcoholic fermentation without MLF. The concentration of diacetyl synthesized by the yeasts then ranged from 0.94 to 1.72 mg/L (Table 4), being too low to have an impact on the wine aroma (Figure 1). A significantly higher concentration of diacetyl was noted with malolactic fermentation. In the coinoculation variant this ranged from 2.19 to 4.06 mg/L, and in the sequential inoculation it varied from 3.42 to 5.91 mg/L. For these wines, light, pleasant buttery and nutty aromas were perceptible (Figure 1). The highest concentration of diacetyl was observed for the spontaneous malolactic fermentation (7.44 to 9.22 mg/L); this was characterized as an intensive and unacceptable buttery aroma. According to [PERSON] et al. (2016), the accumulation of diacetyl and acetoin depends on the dynamics of malolactic fermentation. The higher the MLF rate, the lower the concentration of diacetyl and acetoin. In our study, the dynamics of MLF followed the order: COI < SEQI < SPONT (13). This explains the reason for a lesser concentration of diacetyl in the case of coinoculation, and higher concentration for the SEQI and SPONT variants, in which the dynamics of the MLF process were significantly lower. The presence of oxygen during MLF can also affect the diacetyl content in wine. This is directly associated with the oxidation of \(\alpha\)-acetolactate to diacetyl (30; 34). In our study, micro-oxygenation was performed to support the initiation of spontaneous MLF (Figure 2) (13). The additional amount of oxygen present during the vinification process could thus also have led to the significantly higher concentration of diacetyl in this variant (Table 4). The products of diacetyl degradation--acetoin (3-hydroxy-2-butanone) and 2,3-butanediol--were also evaluated. In all the wines, the lowest levels of both compounds were noted when only alcoholic fermentation was performed; this concentration ranged from 0.73 to 1.62 mg/L of acetoin, and from 165 to 288 mg/L of 2,3-butanediol (Table 4). Similarly, as in the case of diacetyl, MLF significantly increased the concentration of both metabolites.