## 3 Results and Discussion
### Volatile Composition of Beverages Obtained through Static Discontinuous Fermentations I and II
#### 3.3.2 Alcohols  
The formation of alcohols in milk fermented with kefir grains is mainly due to the metabolism of yeasts [19; 20; 21; 89]. Like ketones, alcohols that are generated by the reduction in the corresponding aldehydes [90] are important contributors to the flavor of dairy products [85]. In this study, the fermentation of milk in repeated 24-h subcultures increased both the number and total concentration of alcohols compared to the unfermented milk (Table 1). Two alcohols were detected in the unfermented milk, whereas 9, 10, 10, and 6 alcohols were found in the fermented samples from the first, second, third, and fourth 24-h subcultures, respectively. The total concentration of alcohols increased in the first two subcultures (82.71 \(\pm\) 5.84 \(\upmu\)g/L and 108.89 \(\pm\) 16.56 \(\upmu\)g/L) but decreased in the last two (89.47 \(\pm\) 13.96 \(\upmu\)g/L and 31.63 \(\pm\) 8.45 \(\upmu\)g/L), although it did not reach the lower concentration (15.13 \(\pm\) 0.00 \(\upmu\)g/L) found in unfermented milk (Table 1). These results are consistent with the statement of [PERSON] et al. [91], who described kefir as a fermented dairy product with a wide variety of volatile alcoholic compounds. The increase in the number and concentrations of these compounds during fermentation is the result of increasing biochemical reactions [92]. However, the concentration of some alcohols can also decrease during fermentation due to their reaction with organic acids to form esters [48]. The only two alcohols detected in unfermented milk were 2-phenylethanol and 2-ethyl hexanol, indicating that the other alcohols found in the kefir beverages originated during repeated 24- or 48-h subcultures. In the 24-h subcultures, the concentration of 2-phenylethanol increased from 8.86 \(\pm\) 0.00 \(\upmu\)g/L (unfermented milk) to 12.36 \(\pm\) 5.54, 23.09 \(\pm\) 9.51, 22.90 \(\pm\) 3.73, and 11.41 \(\pm\) 1.74 \(\upmu\)g/L in the first, second, third, and fourth subcultures, respectively. The detection of relatively high concentrations of 2-phenylethanol was also described by [PERSON] et al. [53] in whey samples mixed with beet juice and fermented _K. lactis_. The presence of this alcohol in the milk kefir samples is related to the metabolism of amino acids [54]. In the repeated 48-h fermentation, the concentration of 2-phenylethanol increased to 10.70 \(\pm\) 4.84 \(\upmu\)g/L in the first subculture, but decreased gradually in the second (9.06 \(\pm\) 0.39 \(\upmu\)g/L), third (5.34 \(\pm\) 1.00 \(\upmu\)g/L), and fourth (3.20 \(\pm\) 0.87 \(\upmu\)g/L) passages (Table 1). As observed, the concentrations of this compound in the second, third, and fourth subcultures of fermentation II were significantly lower (\(p\)! 0.05) than those detected in the corresponding subcultures of fermentation I. However, the concentrations of 2-phenylethanol in the first subcultures of fermentations I and II were not statistically different (\(p\)! 0.05). 2-Ethyl-1-hexanol exhibited the same behavior as 2-phenylethanol in the 24-h subcultures, as it was detected in both unfermented milk (6.27 \(\pm\) 0.00 ug/L) and in the four fermented beverages (12.00 \(\pm\) 0.59, 19.23 \(\pm\) 3.27, 17.59 \(\pm\) 0.83, and 8.03 \(\pm\) 7.34 ug/L). In contrast, the concentration of this compound decreased in the beverages from fermentation II, reaching only mean concentrations between 2.43 and 3.37 ug/L (Table 1). The alcohol detected at the highest concentration during fermentations I and II was 1-pentanol, although in both cases, its concentration decreased gradually from the first to the fourth subculture. However, as observed for 2-phenylethanol and 2-ethyl-1-hexanol, the concentrations of 1-pentanol in the 24-h subcultures were higher than those found in the 48-h subcultures (Table 1). This volatile compound was also identified in dairy beverages fermented with kefir grains [54], and in yogurts produced with cocultures of traditional LAB strains, although at relatively low concentrations [55].