## 3 Results and Discussion  
### In Vitro Simulated Digestion  
In this part of the research, different samples were treated with two-step gastrointestinal digestion and subjected to adhesion assay afterward. The following samples were used: free bacteria cells, cultured in optimal conditions, and treated with heat shock and pH stress (Figure 1); samples after fluid bed drying and coating were analyzed both fresh and after 12 months of storage after no treatment, heat shock, and pH shock (Figures 2 and 3). Detailed results are available as supplementary materials (Table S1). Heat shock and pH shock were chosen as two types of the most common stress factors for lactic acid bacteria. These stresses are also present during drying (heat shock) and digestion (pH shock). Additionally, cross-protection systems can help the cells adapt to different types of stresses sharing similar resistance mechanisms [29]. The obtained results were used in the project of the technological process to represent the subpopulation spread for different variants. Four subpopulations of cells were determined using IFC, based on their metabolic activity (measured by RedoxSensor green) and cellular membrane damage (measured by propidium iodide). The active subpopulation includes the cells with high levels of metabolic activity and no cellular membrane damage, the mid-active I cells show low levels of both metabolic activity and cellular membrane damage, while the mid-active II cells show high levels of both; the dead cell subpopulation contains cells with no metabolic activity and a high degree of cellular membrane damage. Free cells presented in Figure 1 show high levels of activity before treatment and after pH shock. The cells after heat shock already show much lower levels of active cells than two other variants. After in vitro digestion, all free cell samples show a significant decrease in active cell subpopulation, especially after the second stage, representing the small intestine. For both dried and coated samples (also after storage) the results show that the first stage of digestion (S1), representing the gastric conditions, led to a decrease in active cells in all samples. For free cells cultured in optimal conditions and after pH shock, the mid-active II subpopulation has increased after S1, which shows that the damage may not be permanent, as these cells have shown the ability to regenerate in optimal conditions [24]. A similar increase in mid-active II cells was also observed in dried cells treated with pH and heat shock after 12 months of storage. The second stage of digestion (S2), representing the small intestine, was more lethal for cells than S1, as also observed by [PERSON] et al. [30]. In all samples reduction of active cells was observed with a simultaneous decrease in overall observed cells, caused by cell breakdown. An increase in cellular debris that could not be assigned to any of the four subpopulations was observed in IFC as a result of the cell decomposition. Cells after coating were overall less affected by the GI conditions than uncoated cells. Similar results were observed for Lactobacillus salivarius  
Figure 1: Subpopulation distribution in samples from bacterial cultures grown in optimal conditions, with heat shock and pH shock. Shown in four stages—pre-adhesion, after the first stage of in vitro digestion, after the second stage of in vitro digestion, and after adhesion. NRRL B-30514 coated with rice protein-shellac composite [31]. Cells after S2 were subjected to adhesion assay, which showed an increase in active subpopulation, further proving that the mid-active cells were able to resume their activity after being placed in optimal conditions during adhesion assay. Mid-active cells would not be enumerated using plate cell counts, and with such methods, a reduction in the number of micro-organisms would be noticed. However, the preparations evaluated using our method show activity even after a year, thanks to the detection of VBNC cells.