## 3 Results and Discussion
### Static Fermentation of Whole Milk in Consecutive 48-h Batch Subcultures
diolivorans, Lb. reuteri_[40], or _Lb. buchneri_[41], has also been reported. Therefore, it seems logical to assume that the final concentration of the different metabolic products in the different subcultures is the result of the difference between their production and consumption by the corresponding producing and consuming strains present in the milk kefir grains, respectively. TAA production increased logistically (\(p\) < 0.05) during the fermentation (Figure 2I), contrasting with the exponentially decreasing profiles exhibited by ethanol and glycerol production (from the first to the fourth subculture), and acetic acid synthesis (from the second to the fourth subculture). Thus, although lactic acid production showed a slightly linear increase, the rise in TAA levels appeared to be associated with the increase in bacteriocin production by the LAB strains present in the grains [10; 18; 23; 24]. The drained KGW again exhibited no significant increase (\(p\) > 0.05) throughout the fermentation process (Figure 2I), consistent with the observations from the 24-h subcultures (Figure 1I). The viability in microbial populations increased to 7.40 \(\pm\) 0.09, 9.03 \(\pm\) 0.03, 8.48 \(\pm\) 0.05, and 9.12 \(\pm\) 0.06 log CFU/mL (in the case of LAB), 8.48 \(\pm\) 0.06, 9.32 \(\pm\) 0.09, 8.57 \(\pm\) 0.06, and 8.97 \(\pm\) 0.07 log CFU/mL (in the case of AAB), and 7.42 \(\pm\) 0.05, 6.69 \(\pm\) 0.06, 6.18 \(\pm\) 0.07, and 5.60 \(\pm\) 0.04 log CFU/mL (in the case of yeasts) in the first, second, third, and fourth subcultures, respectively. As observed, only the yeast count corresponding to the fourth subculture did not surpass the threshold level of 6.0 log CFUs of probiotic cells per mL needed to observed beneficial effects on the host [26; 27]. As occurred in the previous 24-h subcultures, the counts of Enterobacteriaceaeae and _Pseudomonas_ were considerably lower than the minimum detection limit of 30 colonies, indicating once again that these beverages are in good hygienic condition. Comparing the fermentations I and II (Figures 1 and 2), it is evident that the 48-h batch subcultures provided higher (\(p\) < 0.05) \(\Delta\)pH values (2.31 \(\pm\) 0.04, 2.67 \(\pm\) 0.08, 2.57 \(\pm\) 0.06, and 2.60 \(\pm\) 0.04) and showed greater (\(p\) < 0.05) consumption of nutrients (L: 21.13 \(\pm\) 1.75,23.92 \(\pm\) 2.16, 17.77 \(\pm\) 2.62, and 14.59 \(\pm\) 1.49 g/L, TN: 2.23 \(\pm\) 0.13, 2.52 \(\pm\) 0.08, 1.87 \(\pm\) 0.09, and 1.54 \(\pm\) 0.22 g/L, and Pr: 13.91 \(\pm\) 1.89, 15.75 \(\pm\) 1.35, 11.69 \(\pm\) 0.83, and 9.60 \(\pm\) 1.90 g/L). However, the increase in nutrient consumption did not lead to an increase in product synthesis (LA, AA, EtOH, and GOH) as observed in Figure 2E-H. These results suggest that extending the duration of each subculture from 24- to 48 h, the microbiota of the kefir grains consumed higher amounts of nutrients to support the growth and maintenance of cells rather than for product synthesis. In terms of microbial content, the four beverages obtained from fermentation I had counts of LAB (except in the first subculture) and AAB (except in the second subculture) that were almost similar to those from fermentation II. Meanwhile, yeast counts at the end of the subcultures from fermentation II decreased as the number of subcultures increased, contrasting with their behavior in the 24-h subcultures. Moreover, the 48-h kefir beverages exhibited a slightly higher acidic, lower alcohol content (except in the first subculture), and increased antibacterial activity (\(p<0.05\)) compared to the 24-h kefir beverages (Figures 1 and 2). However, the 24-h kefir beverages, particularly those obtained from the first two subcultures, showed statistically significant higher final lactose concentrations (\(p<0.05\)) compared to those from fermentation II.