## 3 Results and Discussion
### Acid Sensitivity of Bacteriophage K5  
Pure bacteriophage K5 lysate was used to investigate the survival at pH values between 2.0 and 6.6 (Figure 4). In contrast to thermal inactivation experiments (Section 3.2),sensitivity in acidic environments should investigate the need for a protective measure, such as encapsulation, in order to deliver bacteriophages unscathed through the stomach into the gut. Thereby, the investigated pH range corresponds to the pH of the human stomach. It is lower when empty and increases when food is consumed [65,66]. NaCl solution at a concentration of 2 g L\({}^{-1}\) was used as the dispersion medium, since it corresponds to the salt content in the human stomach and to the salt content of the simulated gastric fluid used for the in vitro digestion experiments (Section 2.4.2) [67]. Bacteriophage K5 incubated at pH 5.0 was stable for 120 min. A reduction by 2.1 log units within 120 min was observed at a pH of 4.0. In contrast, at pH 3.5, the bacteriophage titer was reduced by 3.6 log units after 10 min and then no longer detectable at all. The decrease was even more drastic at a pH value of 3.0, at which no more bacteriophages could be detected after 30 s. At the lowest pH of 2.0, bacteriophage titers were under the quantification limit; therefore, no data is shown in Figure 4. Hereby, it should be considered that the experiment had a limit of quantification of \(2\times 10^{2}\) pfu mL\({}^{-1}\), because the respective acidic solutions had to be neutralized by adding a buffer and were thus diluted accordingly. In contrast to the heat inactivation experiments, all curve progressions were linear (\(n=1\)) and without tailing. This is to be justified with the immediate effect of the acid (no heating-up phase/no adaptation phase necessary). The solution in which the bacteriophages were suspended had no negative influence on the survival behavior, shown by the results of the reference (2 g L\({}^{-1}\) NaCl solution with a native pH). These results demonstrate that bacteriophage K5 is unstable at pH values below 4.0 and should be protected from the acidic pH in the stomach using, e.g., encapsulation. These results correspond to other findings in the literature. In general, bacteriophages are described to be active in solutions with pH values ranging from 5.0 to 8.0 [44]. Several studies found that bacteriophages and other viruses have a general susceptibility to acidic (and basic) solutions [68,69,70,71,64]. They may lose their viability at extreme pH values due to  
Figure 4: Inactivation of bacteriophage K5 in 2.0 g L\({}^{-1}\) NaCl solutions adjusted with HCl to pH values of 3.0 (\(\bigcirc\)), 3.5 (\(\blacktriangle\)), 4.0 (\(\blacksquare\)), 5.0 (\(\blacktriangledown\)), and 6.6 (\(\blacktriangledown\)) (reference). Experimental data are displayed by symbols. \(N^{\star}\) indicates the initial bacteriophage titer before any treatment. Linear regressions are shown by dotted lines. The limit of quantification was \(2\times 10^{2}\) pfu mL\({}^{-1}\). surface protein denaturation or deformation, as well as due to hydrolysis or other changes of the nucleic acid [72]. The T7 bacteriophage was reduced by 4 log units within 10 min when it was exposed to nitrous acid at pH 4.0. At a pH of 5.0, the reduction was below 1 log unit after the same time [68]. [PERSON] et al., investigated the survival of the _Escherichia coli_ bacteriophages MS2 and Q\(\beta\) at pH values between 3 and 11 at different temperatures. Although bacteriophage MS2 had a greater resistance, a significant reduction was seen below pH 5 and above pH 9 [69]. [PERSON] et al., measured up to 6 log unit reductions for _Lactobacillus casei_ bacteriophage J-1 and for _Lactobacillus paracasei_ bacteriophage PL-1 incubated for 30 min in MRS broth with pH values below 4 [70]. Similar observations were made for _Lactococcus lactis_ bacteriophage P680 in a nitric acid cleaning solution with pH values of 2 and 3. After 10 min incubation, P680 was inactivated by 6 log units at pH 2, but by less than 0.5 log units at pH 3 [64].