## 3 Results and Discussion
### Analysis of the Effect of Incubation Time on the Volatile Composition of the Fermented Beverages  
In general, the results obtained in this study indicate that performing more than two 48-h subcultures led to a decrease in the number of volatile compounds compared to the repeated 24-h fermentation. Additionally, increasing the incubation time from 24 to 48 h provoked a drastic reduction in the total concentrations of the detected volatile compounds (see last two rows of Table 1). The following two hypothesis could explain this phenomenon:  
1. The metabolism of some microorganisms in kefir grains [95; 99; 100] and low pH values [96] could lead to the decrease in the number and/or concentration of these volatile compounds, resulting in the production of other volatile metabolites [95; 99; 100]. For instance, the reduction in the total concentration of hydrocarbons during the spontaneous or inoculated fermentation (with _Lb. curvatus_ BIOTEC) of nham was attributed to the possible microbial oxidation of these compounds for the production of methyl ketones. Additionally, the reduction in the total concentration of aldehydes and ketones was attributed to the microbial conversion of these compounds for the synthesis of alcohols [95]. A decrease in the concentration of most esters, aldehydes, alcohols, and some organic acids was observed with the increase in incubation time during natural and inoculated (with a W-4 LAB culture) fermentation of minced pepper (_Capsicum annuum_ L). cv. \"yanhong\" [99]. The decrease in esters was related to their hydrolysis at low pH values, while the reduction in aldehydes was attributed to their conversion into acids and alcohols by microorganisms. Additionally, the decrease in the concentrations of alcohols was explained by their oxidation to organic acids due to microbial metabolism at low pH values [96], their assimilation by acetic acid bacteria, or microbial transformation or esterification in the production of kombucha [100]. The decline in organic acid levels was assumed to be caused by esterification reactions [100]. 2. A more pronounced acidification of the milk in the 48-h subcultures resulted in final pH values (4.38, 4.02, 4.12, and 4.09) in the fermented samples, which were lower than those (5.17, 4.99, 4.70, and 4.70) in the beverages obtained from fermentation I. This significant decrease in the culture pH values in the 48-h subcultures provoked a greater precipitation and coagulation of proteins (caseins) [101], which could facilitate the release of volatile compounds from the fermented substrate into the air, leading to a partial or total reduction in their concentration in the beverage. In any case, the hypotheses proposed by [PERSON] et al. [95], [PERSON] et al. [99], and [PERSON] et al. [100] could not explain the observed reduction in the total concentrations of volatile compounds in the beverages from repeated 48-h fermentations, compared to those of the unfermented substrate and the beverages from fermentation I. This is mainly because the total concentrations of the seven families of volatile compounds (organic acids, alcohols, aldehydes, ketones, esters, hydrocarbons, and other compounds) decreased when the incubation time of milk with kefir grains increase from 24 to 48 h (Table 1). Thus, the second hypothesis could be a more plausible reason to explain the reduction in the total concentrations of volatile compounds in the beverages from fermentation II. For this reason, the physical structure of the beverages obtained after different incubation times (24, 26, 28, 30, and 48 h) was examined (Figure 3). As can be observed, the kefir beverage obtained at 24 h presents a creamy consistency very similar to that of yogurt with some clumps on the surface (Figure 3A). The gradual formation of gas bubbles becomes apparent from 26 h (Figure 3B), increasing in number and size at 28 h (Figure 3C) and 30 h (Figure 3D).