## 3 Results and Discussion
### Static Fermentation of Whole Milk in Consecutive 48-h Batch Subcultures  
To determine the impact of fermentation time on the chemical, microbiological, and volatile composition of milk kefir beverages, a series of consecutive 48 h batch fermentations of UHT whole milk with milk kefir grains was conducted under static conditions(fermentation II). This approach allows for a discussion of the similarities and differences between fermentations I and II. In fermentation II, the final pH values in the first (4.38 \(\pm\) 0.03), second (4.02 \(\pm\) 0.07), and third (4.12 \(\pm\) 0.05) subcultures (Figure 2A) were statistically different (\(p\) < 0.05). However, the final pH value in the fourth subculture (4.09 \(\pm\) 0.03) did not show a statistical difference (\(p\) > 0.05) compared to those of the second and third subcultures. Figure 2: Culture pH (**A**) and chemical composition (as means \(\pm\) standard deviation of triplicate samples and two analytical replications each) of the UHT whole milk (UHT-WM) and milk kefir beverages obtained from the first (Sub-I), second (Sub-II), third (Sub-III), and fourth (Sub-IV) 48-h batch subcultures. L, TN, Pr, LA, AA, EtOH, and GOH are th concentrations of lactose (**B**), total nitrogen (**C**), proteins (**D**), lactic acid (**E**), acetic acid (**F**), ethanol (**G**), and glycerol (**H**), respectively. TAA (**I**) and KGW (**J**) are the total antibacterial activity and drained kefir grain weight, respectively. Similar letters above the bars for each variable indicate that there is no statistical difference between their values (\(p\) > 0.05). The consumptions of lactose, 21.13 \(\pm\) 1.75, 23.92 \(\pm\) 2.165, 17.77 \(\pm\) 2.62, and 14.59 \(\pm\) 1.49 g/L (Figure 2B) and proteins, 13.91 \(\pm\) 1.89, 15.75 \(\pm\) 1.35, 11.69 \(\pm\) 0.83, and 9.60 \(\pm\) 1.90 g/L (Figure 2D) were only not statistically different (\(p\) > 0.05) in the third and fourth 48-h subcultures. In contrast, the consumption of total nitrogen (Figure 2C) in the four subcultures (2.23 \(\pm\) 0.13, 2.52 \(\pm\) 0.08, 1.87 \(\pm\) 0.09, and 1.54 \(\pm\) 0.22 g/L, respectively) showed statistical differences (\(p\) < 0.05). Regarding the synthesis of fermentation products, it can be observed that LA production (Figure 2E) exhibited a significant (\(p\) < 0.05) small linear increase (9.46 \(\pm\) 0.16, 10.58 \(\pm\) 0.18, 10.56 \(\pm\) 0.21, and 11.50 \(\pm\) 0.14 g/L) at an average rate of 0.61 g/L/subculture. Acetic acid production (Figure 2F) increased from the first (1.08 \(\pm\) 0.16 g/L) to the second subculture (3.80 \(\pm\) 0.10 g/L), but then decreased in the third (2.74 \(\pm\) 0.16 g/L) and fourth (2.01 \(\pm\) 0.11 g/L) subcultures. In contrast, the concentrations of EtOH (5.17 \(\pm\) 0.09, 2.84 \(\pm\) 0.11, 1.22 \(\pm\) 0.16, and 0.80 \(\pm\) 0.19 g/L) and GOH (0.44 \(\pm\) 0.08, 0.27 \(\pm\) 0.01, 0.08 \(\pm\) 0.01, and 0.05 \(\pm\) 0.00 g/L) exhibited decreasing profiles (Figure 2G,H). Providing an accurate discussion on the levels of the metabolic products (LA, AA, EtOH, and GOH) synthesized in the different subcultures is very difficult. This is probably due to the existence of a complex microbial population in the milk kefir grains, comprising LAB, AAB, and yeasts [2; 3; 4], which establish complex metabolic relationships among themselves. Thus, it has been reported that certain LAB, such as _Lb. buchneri_, _Lb. parabuchneri_[32], _Lb. plantarum_[33], and _L. lactis_[34], can use lactic acid as a carbon source. Furthermore, some non-lactose-consuming yeasts, such as strains of _S. cerevisiae_[35; 36], _S. exiguous, S. carlsbergensis, and _Debaryomyces hansenii_, can assimilate lactic acid [36], _C. guillermondii_ can metabolize acetic acid [37], and _S. cerevisiae_ can use both lactic and acetic acid [38] as carbon sources. Regarding the EtOH production, it has been recognized that species of AAB such as _Acetobacter_ and _Glucobacter_ can oxidize this metabolite to produce acetic acid [39]. Additionally, assimilation of GOH by some strains of LAB, such as _Lb. collinoides, Lb.