## 3 Results and Discussion
### Static Fermentation of Whole Milk in Consecutive 48-h Batch Subcultures
In previous studies, the fermentation of pasteurized whole cow's milk with Brazilian kefir grains for 24 h [42], and the fermentation of sterilized skimmed cow's milk with milk kefir grains CIDCA AGK1 from the National University of La Plata (Argentina) over seven consecutive 24-h subcultures [43], were conducted under static conditions at 25 \({}^{\circ}\)C.  
The pH values of whole milk kefir (4.42) [42] and the range of pH values of skimmed cow's milk kefir (4.08-4.39) [43] were similar to the pH range (4.02-4.38) of the beverages from the 48-h subcultures but higher than the final pH range (4.70-5.17) observed in the 24-h subcultures (Figures 1A and 2A). The same trend was observed for the final pH values (4.20, 3.69, and 3.61) reported for kefir beverages made from sheep, goat, and cow milk [44]. In contrast, the final pH levels obtained in fermented high heat-treated long-life milk with milk kefir grains at 20 \({}^{\circ}\)C (5.07), 25 \({}^{\circ}\)C (4.56), and ambient temperature (\(\sim\)4.94) [9] were similar to those obtained in the 24-h subcultures (Figure 1A). Additionally, the mean lactic acid concentrations (17.40 and 13.06 g/L) obtained by [PERSON] et al. [42] and [PERSON] et al. [43], respectively, were higher than the LA concentrations obtained in the four 24-h and 48-h subcultures (Figures 1E and 2E). The total LA levels in sheep (6.59 g/L), goat (7.51 g/L), and cow (7.67 g/L) kefir [44], and in millks fermented at different temperatures (\(\sim\)8.5 g/L) [9], were comparable to the LA concentrations obtained in the second and third subcultures of the repeated fermentation I, and lower than those of the repeated fermentation II. However, the mean acetic acid level (1.35 g AA/L) reported by [PERSON] et al. [43] falls within the range of AA concentrations obtained in the 24-h (0.78-1.97 g AA/L) and 48-h (1.08-3.80 g AA/L) subcultures (Figures 1F and 2F). Meanwhile, the AA level (2.70 g/L) reported by [PERSON] et al. [42] falls within the range of AA concentrations obtained in the 48-h subcultures but surpassed the levels observed in the 24-h subcultures. In contrast, only traces of acetic acid were detected in the study by [PERSON] et al. [9]. Additionally, [PERSON] et al. [44] reported no acetic acid production in sheep, goat, and cow kefir beverages. The amounts of lactose consumed (\(\sim\)14.00 and 13.89 g L consumed/L) in the fermentations of whole [42] and skimmed [43] cow's milk fall within the range of lactose consumed in the 24-h (8.47-14.87 g/L) and 48-h (14.59-23.92 g/L) subcultures (Figures 1B and 2B). Considerably higher lactose consumption (28.60, 32.90, and 30.50 g/L) was observed in the kefir beverages obtained at 20 \({}^{\circ}\)C, 25 \({}^{\circ}\)C, and ambient temperature [9], respectively. The levels of lactose reported for cow (19.8 g/L) and goat (24.1 g/L) kefir [44] were comparable to those obtained in the first two subcultures of fermentation II (24.87 and 22.08 g/L, respectively) (Figures 1B and 2B). However, the lactose content in sheep kefir (31.4 g/L) [44] were similar to that of the third subculture of fermentation I (31.13 g/L). Surprisingly, the mean ethanol contents reported by [PERSON] et al. [42] (0.50 g/L) and [PERSON] et al. [43] (0.02 g/L) were considerably lower than those obtained in the 24-h (2.55-3.90 g/L) and 48-h (0.80-5.17 g/L) subcultures (Figures 1G and 2G) and that (9.0 g/L) reported by [PERSON] et al. [9]. Unfortunately, in the study of [PERSON] et al. [44], the concentrations of ethanol in the three kefir beverages (sheep, goat, and cow) were not quantified, making it difficult to compare the concentration of this metabolite. Regarding the microbial composition, the cow's milk fermented with milk kefir grains CIDCA AGK1 provided LAB counts (\(\sim\)10\({}^{8}\)-10\({}^{9}\) CFU/mL) two orders of magnitude higher than those of AAB and yeasts (\(\sim\)10\({}^{6}\)-10\({}^{7}\) CFU/mL) [43]. [PERSON] et al. [42] reported counts of LAB, AAB, and yeasts as high as 12.41, 7.72, and 8.11 log CFU/mL, respectively.