## 3 Results and Discussion
### Stability in Simulated Gastric Fluid and Release in Simulated Intestinal Fluid  
The encapsulation of bacteriophages should ensure their transmission through the digestive tract. One the one hand, the bacteriophages should be protected from acidic conditions in the stomach, which usually lie in a pH range from 1 to 2.5 [65]. On the other hand, they should be released from their matrix, as it is essential for their effect in the gut of individuals. After the K5 bacteriophage capsules were produced, they were subjected to a static in vitro digestion to study their mode of action, as visualized in Figure 6. The compositions of SGF and SIF were based on conditions in the human stomach and intestinal tract. Incubation conditions of 37 \({}^{\circ}\)C, 120 min, and moderate rotating (100 rpm) should simulate the physical conditions in the stomach and intestine [33]. Figure 6a shows that K5 bacteriophage capsules incubated in SGF for 120 min withstand the low pH value of 2.5 and bacteriophages stay intact. The bacteriophage titers show a non-significant reduction of 0.1-0.2 log units after incubation. The same is true for SGF having a pH value of 2.0 (data not shown). Any enzymatic reactions of pepsin did not have a measurable effect on the bacteriophage titer. In general, no differences were observed for the two variants of capsule matrices. As proven, bacteriophage K5 is quickly inactivated by acidic conditions, especially at a pH value of 3.0 or below (Figure 4). From this, the effect of encapsulation becomes clear, because inadequately encapsulated or free bacteriophages could not have survived the incubation of 120 min. Figure 6b depicts the release of the bacteriophage K5 capsules from their matrix. Within the first 30 min, the bacteriophage titers were slightly lower (by 0.6-1.0 log units) than the values between 30 and 120 min. This indicates that there was a very quick dissolution of the capsules and release of the K5 bacteriophages into the liquid phase. It should be noted that the given values (log \(N_{\text{C},t}\)# in pfu mL\({}^{-1}\)) show the released bacteriophages in the liquid phase of the experimental approach (after adding 1% capsules to SIF) and are, therefore, lower than the values from Figure 6a (log \(N_{\text{C},t}\) in pfu g\({}^{-1}\)), which represent the surviving bacteriophages in the capsules. Static in vitro digestion is a method that is frequently used in the literature to investigate the efficacy of different encapsulation methods. In contrast to dynamic in vitro digestion, static systems are less complex, require small quantities of samples and enzymes, and are independent of any specific laboratory devices. However, all average parameters (e.g., pH value) are set at the beginning of the experiment, the dynamic release of enzymes is not feasible, the accumulation of metabolites is not considered, and the complex physiology of the digestive tract is not imitated. Therefore, there is a need for further research using dynamic in vitro digestion protocols in order to come as close as possible to what occurs in vivo [87]. Taking into account the advantages and disadvantages mentioned here, only static systems will be used for discussion in the following section. Figure 6: Static in vitro digestion of bacteriophage K5 capsules composed of alginate and micellar casein () or alginate and whey protein isolate (). (a) Incubation in simulated gastric fluid (SGF). Results are given as bacteriophage titer of solid capsules (\(N_{\text{C}}\) in pfu g\({}^{-1}\)). (b) Incubation in simulated intestinal fluid (SIF). Results are given as bacteriophage titer of dissolved capsules (1% in SIF) (\(N_{\text{C}}\)# in pfu mL\({}^{-1}\)). \(N_{\text{C}}\)* and \(N_{\text{C}}\)*# indicate the initial bacteriophage titers before any treatment, respectively. The limits of quantification were \(10^{3}\) pfu g\({}^{-1}\) and \(10^{1}\) pfu mL\({}^{-1}\) for bacteriophage titers in SGF and SIF, respectively.