## 3 Results  
### Acid-Buffering Capacity of Reconstituted Formulae  
The buffering capacity is the ability of a solution or food to resist changes in pH when an acid or base is added [40]. A food with a high buffering capacity (HBC) requires larger amounts of acid or base to have its pH altered. Figure 2 shows the acid-buffering capacity (ABC) of the two RIFs (RIF-1 and RIF-2). It is clear that the RIF-1 has a small but statistically significant (\(p<0.05\)) lower acid-buffering capacity from RIF-2. Both RIFs had approximately the same initial pH, but, gradually, for the same addition of H\({}_{3}\)O\({}^{+}\), the pH of RIF-1 decreased faster and to a lower value than that of RIF-2. Therefore, RIF-1 can be considered to have a lower ABC than RIF-2 since it exhibits a lower \"resistance\" to pH changes. Figure 2: Acid-buffering capacity (ABC) of two reconstituted infant formulae (RIF-1 and RIF-2) as a function of incremental HCl (\(\mu\)M) addition. According to these results, RIF-1 was marked as a low-buffering-capacity infant formula (LBC) and IF-2 as a high-buffering-capacity (HBC) infant formula. Linear, polynomial, and logarithmic regression analyses of the pH (RIF-1, RIF-2) versus HCl (\(\mu\)M) addition were performed. For both RIF-1 and RIF-2, the best fit was obtained from the polynomial regression with R\({}^{2}\) = 0.998 and RMSE 0.023 for RIF-1 and R\({}^{2}\) = 0.999 and RMSE 0.005 for RIF-2. Moreover, a satisfactory fit was obtained by the logarithmic regression with R\({}^{2}\) = 0.985 and RMSE 0.072 for RIF-1 and R\({}^{2}\) = 0.988 and RMSE 0.078 for RIF-2. Viability of Microorganisms During Incubation of Reconstituted Infant Formulae at 22 \({}^{\circ}\)C  
The changes in viable bacterial populations were counted by plating methods during the incubation of the two RIFs, the one with lower acid-buffering capacity (LBC, corresponding to RIF-1) and the other with higher buffering capacity (HBC, corresponding to RIF-2) at 22 \({}^{\circ}\)C, in both a monoculture setting (Mc) with only _C. sakazakii_ and co-culture setting (Cc) with _C. sakazakii_ and BB-12(r) (Figure 3a). The corresponding pH changes were also measured (Figure 3b). It was observed that the population of the strain BB-12(r) during the 24 h at 22 \({}^{\circ}\)C was increased by approximately 1 log cycle in both LBC and HBC formulae (LBC-Cc and HBC-Cc) and the bacterial counts reached approx. 8 log cfu/mL (Figure 3a). This growth could not be characterized as particularly high, although it is not negligible. This should be probably attributed both to the incubation temperature (22 \({}^{\circ}\)C), which is far from the species optimum (37 \({}^{\circ}\)C), and to the rather short incubation period (24 h). It is also possible that the initial physical state of the bacteria, which were added into the PIFs in the form of powder, played a role in this observation, since this may have resulted in a prolonged lag phase. As regards the _C. sakazakii_ cell counts in monoculture, in both RIFs, they were increased. In particular, in the LBC (RIF-1) at the onset of incubation, the inoculum was 1.72 (\(\pm\)0.32), and, 24 h later, it was 3.12 (\(\pm\)0.36), that is, 1.4 log cycles higher. The trend for RIF-2 was similar. In contrast, in the co-culture (LBC/RIF-1) where the inoculum of _C. sakazakii_ was 1.57 (\(\pm\)0.48) log cycles, after 24 h of incubation, it was up to 1.98 (\(\pm\)0.46) log cycles, that is, 0.48 log cycles higher. Overall, in both RIFs, after 24 h of incubation at 22 \({}^{\circ}\)C, the population of _C. sakazakii_ in co-culture with the probiotic BB-12(r) was approximately 0.92 log cycles lower compared to monoculture. Figure 3b illustrates the pH changes in the RIFs inoculated with _C. sakazakii_ and BB-12(r), incubated at 22 \({}^{\circ}\)C in both monoculture (Mc) and co-culture (Cc). Of the two RIFs tested, the RIF-2 had higher ABC--marked as HBC in the graphs--compared to the RIF-1, as mentioned before. Initially, the pH of both RIFs was similar, but, as the incubation progressed, differences in pH trends were observed.