## 4 Discussion
sakazakii_ can survive was 4.1 depending on the strain and the type of acidification medium [51; 52]. The HPLC analysis carried out on the samples showed that the main weak organic acids contributing to the pH were a mixture of formic, lactic, and acetic acid. The latter was the predominant one in the _C. sakazakii_ monoculture, with lactic acid present in lower concentrations, and formic acid detected at significantly lower quantities. With respect to the type of organic acids in the _C. sakazakii_ monoculture, acetic acid dominated over lactic acid. The genus _Cronobacter_ belongs to the broad family of _Enterobacteriaceae_, where the metabolism is of a mixed type [53]; i.e., depending on the substrate and incubation conditions, lactic acid, acetic acid, succinic acid, formic acid, and ethanol are produced. As far as the metabolism of _Bifidobacterium_ spp. is concerned, it is known that they produce acetic and lactic acid in a ratio of 3:2 [54]. These three weak organic acids are known for their inhibitory activity, especially in their non-ionic form, because only in this state are they able to penetrate cell membranes, disrupting the metabolic balance and ultimately leading to cell death [55]. The concentration of these acids in their non-ionic form is determined by (a) their concentration, which is related to pH; (b) their type; and (c) the buffering capacity of the medium in which they are grown. Weak organic acids are characterized by the \(\mathrm{K_{a}}\) constant, which is called the acid ionization constant and is expressed in a logarithmic scale (pKa). pKa expresses the pH value where the acid is 50% in its ionic form. When the pH of the solution tends to be lower than the pKa of the acid, i.e., the pH of the solution becomes more acidic, the equilibrium tends to the opposite side of the non-ionic form, which is the antimicrobial form. In the case of a co-culture, formic, lactic, and acetic were simultaneously present in the RIFs with the pKa in ascending order as follows: formic 3.75 > lactic acid 3.86 > acetic acid 4.76. As the incubation progresses, the concentration of these acids in the RIFs increases, and their equilibria of the ionic/non-ionic state is changed along with their pH. In the cases of a mixture of weak organic acids, the chemical phenomenon of the \"common ion effect\" occurs. Since all three acids release H\({}_{3}\)O\({}^{+}\), the total concentration of H\({}_{3}\)O\({}^{+}\) in the RIFs increases. According to [PERSON]'s principle, the equilibrium of a weak acid shifts to the left as the concentration of H\({}_{3}\)O\({}^{+}\) ions increase, limiting the ionization of the other acids. In the RIFs. as the pH decreases, the differences between the pH and the pKas of the acids change, which subsequently affects the ionic/non-ionic equilibrium of each acid. In particular, as the pH decreases and approaches the pKa of formic and lactic acid, the latter ionizes more readily, releasing H\({}_{3}\)O\({}^{+}\) ions and thus shifting the equilibrium of acetate to the left, in its non-ionic state. For example, in the co-culture at 37 \({}^{\circ}\)C/48 h, the pH was 3.65 and the differences from the pKa of formic (3.75), lactic (3.86), and acetic (1.11) acid were \(-\)0.1, \(-\)0.21, and \(-\)1.11, respectively. The small difference between the pKa\({}_{\mathrm{formic}}\) and pH means that formic acid is close to equilibrium (55.6%), while the relatively larger distance of the pKa\({}_{\mathrm{acetic}}\) from the pH means that its equilibrium is mainly to the left, 76.9% in the non-ionized form. When weak acids that share a common ion are in the same solution, they can actually influence each other's ionization equilibrium through the common ion effect. The weaker an acid is, in a mixture of weak acids, the greater the effect of the common ion it receives from the other acids is.