## 3 Results
### Acid-Buffering Capacity of Reconstituted Formulae
The lowest pH was observed in the co-culture settings in both RIFs regardless of their acid-buffering capacity. At the end of the 24 h incubation, the pH in the RIF with the lower acid-buffering capacity (LBC) was 5.06, while the pH in the RIF with the higher acid-buffering capacity (HBC) was 5.2. In contrast, the pH in the monocultures in both RIFs was higher in the 5.5 region. [PERSON] et al. [18] studied the growth of 30 different strains of _Cronobacter_ spp. In a laboratory medium at 37 \({}^{\circ}\)C for 24 h and found that they all grew to populations higher than 10\({}^{7}\) cfu/mL at pH > 5.5. In contrast, at pH 5.0, 4 out of the 30 strains failed to grow, while, at pH 4.5, none of the strains grew. Therefore, the inhibition of _C. sakazakii_ growth in co-culture should be attributed to a combination of factors, including the incubation temperature, the very high population of the prebiotic BB-12(r) which is expected to create an antagonistic environment in order to compete with _C. sakazakii_ for growth resources, and the relatively low pH of the RIF. Viability of Microorganisms During Incubation of Reconstituted Infant Formulae at 37 \({}^{\circ}\)C  
Figure 4a shows the changes in _C. sakazakii_, and BB-12\({}^{\text{\textcircled{0}}}\) cell counts in the RIF-1 (LBC) and RIF-2 (HBC) during incubation at 37 \({}^{\circ}\)C for 24 h in monoculture and co-culture. In monocultures, regardless of the type of RIF, the _C. sakazakii_ population remained unchanged during the first 4 h of incubation, and then started to increase until the end of the incubation period (24 h) where different profiles of the _C. sakazakii_ cell counts were observed between RIF-1 and RIF-2. In the latter, which had the higher ABC (HBC), the population of the opportunistic pathogen at the end of the 24 h incubation period was 5.95 (\(\pm\)0.43) log cycles or 4.15 log cycles higher than the inoculation level (1.80 \(\pm\) 0.23 log cycles). In contrast, in RIF-1, with a lower ABC (LBC), the final _C. sakazakii_ population was 4.82 (\(\pm\)0.45) log cycles, about 1 log cycle lower than the population in RIF-2. Figure 3: (**a**) Cell counts of _Cronobacter sakazakii_ (Sak) and _Bifidobacterium animals_ subsp. _lactis_ BB-12\({}^{\text{\textcircled{0}}}\) in two reconstituted infant formulae (RIFs) with different acid-buffering capacities—lower buffering capacity (LBC for RIF-1) and higher buffering capacity (HBC for RIF-2)—during 24 h of incubation at 22 \({}^{\circ}\)C. The _C. sakazakii_ was grown either in monoculture (Mc) or in co-culture (Cc) with the probiotic BB-12\({}^{\text{\textcircled{0}}}\), and (**b**) corresponding pH changes in the respective samples during the incubation period. With regard to the co-cultures, in the RIF-2 (HBC), the initial (1.68 \(\pm\) 0.56 log cycles) population of _C. sakazakii_ after 12 h of incubation increased to 2.34 \(\pm\) 0.55 log cycles and then decreased since its population at the end of the 24 h incubation was 1.48 \(\pm\) 0.33 log cycles, that is, lower than the inoculum. In the RIF with the lower ABC (LBC) at the end of the 24 h incubation at 37 \({}^{\circ}\)C, the population of the _C. sakazakii_ was below the detection limit of the analytical method (10 cells/mL). In Figure 4b, the pH trends of the 24 h RIFs' incubation is illustrated. The pH had a downward trend due to the production of weak acids by the bacteria. The most gradual pH decreasing trend and the highest final pH (4.6 \(\pm\) 0.21) were observed in the _C. sakazakii_'s monocultures in the RIF-2 which had the highest acid-buffering capacity. In contrast, the steepest pH decline gradient was in the co-cultivars and, in particular, in RIF-1 with the lowest acid-buffering capacity (LABC) where the final pH was 3.65 \(\pm\) 0.21. Figure 4: (**a**) Cell counts of _Cronobacter sakazakii_ (Sak) and _Bifidobacterium animalis_ subsp.