## 3 Results and Discussion  
### Static Fermentation of Whole Milk in Consecutive 24-h Batch Subcultures  
The chemical composition of different kefir beverages obtained through fermentation of UHT whole milk with kefir grains in consecutive 24-h batch subcultures is depicted in Figure 1. The results showed that the initial pH in each subculture (6.69 \(\pm\) 0.01) decreased to 5.17 \(\pm\) 0.02, 4.99 \(\pm\) 0.09, 4.70 \(\pm\) 0.09, and 4.70 \(\pm\) 0.06 at the end of the first (0 to 24 h), second (24 to 48 h), third (48 to 72 h), and fourth (72 to 96 h) subcultures, respectively (Figure 1A). Consequently, the resulting pH drops (\(\Delta\)pH) were 1.52 \(\pm\) 0.01, 1.70 \(\pm\) 0.08, 1.99 \(\pm\) 0.08, and 1.99 \(\pm\) 0.07 in the first, second, third, and fourth subcultures, respectively. This indicates an increase in the acidification capacity of the microbial population of the kefir grains with the progression of subcultures. The consumption of nutrients such as lactose (8.47 \(\pm\) 1.76, 9.81 \(\pm\) 2.40, 14.87 \(\pm\) 2.88, and 9.02 \(\pm\) 2.10 g/L), total nitrogen (0.96 \(\pm\) 0.09, 1.11 \(\pm\) 0.08, 1.68 \(\pm\) 0.14, and 1.02 \(\pm\) 0.22 g/L), and proteins (5.99 \(\pm\) 1.02, 6.94 \(\pm\) 1.77, 10.51 \(\pm\) 0.98, and 6.38 \(\pm\) 1.66 g/L) exhibited a slight increase from the first to the third subculture, but decreased in the fourth one (Figure 1B-D). In the different subcultures, higher LA concentrations, 5.59 \(\pm\) 0.19, 7.11 \(\pm\) 0.21, 7.38 \(\pm\) 0.19, and 4.49 \(\pm\) 0.11 g/L (Figure 1E) and EtOH, 3.34 \(\pm\) 0.11, 3.90 \(\pm\) 0.15, 3.72 \(\pm\) 0.12, and 2.55 \(\pm\) 0.11 g/L (Figure 1G) were synthesized compared to the productions of AA, 0.78 \(\pm\) 0.10, 0.80 \(\pm\) 0.08, 1.97 \(\pm\) 0.21, and 1.28 \(\pm\) 0.09 g/L (Figure 1F), and GOH, 1.38 \(\pm\) 0.12, 0.35 \(\pm\) 0.10, 0.37 \(\pm\) 0.01, and 0.24 \(\pm\) 0.02 g/L (Figure 1H). Lactic acid is likely synthesized by LAB (2016; 2017; 2018) or yeasts (2012), while ethanol and glycerol are predominantly synthesized by yeasts (2012; 2012). Conversely, acetic acid is mainly produced by AAB (2012). Figure 1: 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) 24-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 TAA titers produced in milk (Figure 11) increased exponentially from the first to the third subculture (5.31 \(\pm\) 0.25, 5.85 \(\pm\) 0.41, and 8.19 \(\pm\) 0.51 AU/mL) and exhibited a slight decrease (6.97 \(\pm\) 0.40 AU/mL) in the fourth passage. TAA is the result of the sum of the antibacterial activity of the metabolic products synthesized by the microorganisms of the milk kefir grains (bacteriocins, lactic acid, acetic acid, ethanol, and glycerol) [10]. Thus, the increase in the TAA values from the first to the third subculture could be related to the exponential increase in acetic acid concentration (Figure 1F) and the logistic increase in the production of lactic acid and ethanol (Figure 1E,G). In the latter case, it could be noted that its concentration in the second and third subculture were not statistically different (\(p\) > 0.05). In contrast, the increase in TAA could not be related to the production of glycerol since the concentration of this metabolite showed an exponential decrease during the whole fermentation period (Figure 1H).