## 3 Results  
### The Influence of Physicochemical Parameters on Free and Encapsulated Lactobacillus casei Bacteria  
The suspensions of the _L. casei_ strain and the microspheres obtained by centrifugation at different speeds (1, 2 and 3) and microdispersion (4) were subjected to various physico-chemical factors, such as a temperature of 40 \({}^{\circ}\)C for 10 min, 60 min and 96 h (Figure 1A); a temperature of 50 \({}^{\circ}\)C after 10 min (Figure 1B); acidic and alkaline pH (Figure 2); UV radiation for exposure times of 24 h and 48 h (Figure 3A); and preservatives--sodium benzoate combined with sodium salts of levulinic and anisic acid (Figure 3B). A 0.2 mol/L sodium citrate solution with pH = 6 was used for the decapsulation process. The viability of free and encapsulated probiotic cells was assessed by counting bacterial colonies grown on MRS agar plates (incubation conditions: 37 \({}^{\circ}\)C, 72 h) after using the pour plate inoculation technique. The obtained results were expressed in log CFU/g. LAB cell reduction was calculated as the difference between the initial number of free LAB cells or LAB cells contained in the microspheres (No) and the number of free LAB cells or LAB cells contained in microspheres exposed to physicochemical agents for a given time (Nt): (No-Nt). In the case of assessing the effect of temperature (Figure 1), the duration of exposure to the free and encapsulated _L. casei_ strain was selected based on trial-and-error analysis, which enabled the observation of visible differences in the survival of the free strain compared with the carriers used. In the case of 50 \({}^{\circ}\)C, it was impossible to observe differences over a longer period of time (60 min), because the samples were biodegradable. Figure 1: The effect of temperature on free and encapsulated _Lactobacillus casei_ bacteria: (**A**) 40 \({}^{\circ}\)C and (**B**) 50 \({}^{\circ}\)C.  
It was shown that exposure of immobilised probiotictic cultures in sodium alginate microspheres (samples 1, 2, 3 and 4) to a temperature of 40 \({}^{\circ}\)C for 10 and 60 min (Figure 1A) had no significant effect on cell viability. In the case of exposure to 40 \({}^{\circ}\)C for 10 min, the reduction was 0-0.05 log, and after 60 min, it was 0.06-0.8 log. Slightly greater decreases in the viability of LAB bacteria were found when the samples were exposed to a temperature of 40 \({}^{\circ}\)C for 96 h. The highest cell reduction was found for free _L. casei_ cells (6.15 log) and microdispersion 4 (3.1 log). For the remaining samples (1, 2 and 3), LAB cell reduction was the lowest in microsphere 2 and amounted to 0.9 CFU log/g. For microsphere samples 1 and 3, the reduction levels were 1.72 CFU log/g and 2 CFU log/g, respectively. A temperature of 50 \({}^{\circ}\)C for an exposure time of 10 min caused a reduction in the number of free _L. casei_ cells of 5.06 log. The greatest decrease in the number of LAB bacteria was observed in the case of microdispersion 4, and it amounted to 1.80 log. In microsphere samples 1, 2 and 3, the decreases in viability were minimal and amounted to 0.37 log, 0.20 log and 0.50 log, respectively. Figure 2: The effect of pH on free and encapsulated _Lactobacillus casei_ bacteria: (**A**) pH = 2.0, (**B**) pH = 5.5 and (**C**) pH = 9.0. The next parameter assessed was pH, with values of 2.0, 5.5 and 9.0 after contact times of 15 min, 30 min and 60 min. In accordance with the literature [42], acidic and alkaline pH values were selected for testing (Figure 2A,C) in order to assess the positive effect of the carriers used on the probiotic strain. In turn, pH 5.5 (Figure 2B) was chosen due to the fact that it corresponds to the physiological pH of the skin, which is the site of the potential application of the product. A pH value of 2 (Figure 2A) had an adverse effect on the viability of both free and sodium alginate-immobilised _L. casei_ cells in samples 2, 3 and 4.