## 3 Results and Discussion
### Process Design and Economic Analysis  
The preliminary project of the technological process for dried probiotic production was prepared based on laboratory-scale experiments. Its process flow diagram shows the necessary steps and procedures in the variant, including fluid bed drying and coating (Figure 4). Overall, 6 project variants were prepared--for drying with no stress, with pH shock, and with heat shock, and similarly for coating with no stress, with pH shock, and with heat shock. Triggering bacterial stress adaptation was introduced as a way of improving the viability of probiotics during drying and storage [32]. All projects were divided into 3 sections: medium preparation, seed culture and fermentation, and fluid bed drying and coating. Medium preparation is a step where the MRS medium for culturing is mixed from base ingredients, heat sterilized, and distributed to corresponding culturing vessels. The second section contains culturing containers with increasing volumes--from 2 L shake flasks to 250 L bioreactor. In the final stage, the biomass is mixed with a protective substance (5% trehalose) and dried in a fluid bed dryer with crystalline microcellulose as a matrix. The protective effects of using trehalose come from its ability to replace the water in the intracellular macromolecules during drying [33]. In an additional step, the powder can then be coated with 2% HPMC. The final number of cells in the product was set at \(10^{8}\) cfu/g, as recommended by industry standards [34]. Economic calculations have been performed for the process, taking into consideration the cost of raw materials, waste treatment, energy consumption and heat transfer agents, equipment cost, and the direct fixed capital (DFC). DFC consists of total plant direct cost (TPDC), total plant indirect cost  
Figure 3: Subpopulation distribution in fluid bed-dried and coated samples treated with heat shock, pH shock, and untreated. 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. (TPIC), and contractors fee and contingency (CFC). The economic evaluation containing all of the mentioned costs has been summarized in Table 2. The cost of purchasing equipment needed for the process is summarized in Table 3. Additionally, the cost of installation of the equipment was calculated as 1.5\(\times\) the purchase cost. To evaluate the economic profitability of certain technological treatments, such as coating and stresses during culturing, the production costs of obtaining preparations for 6 variants (dried, dried with heat shock, dried with pH shock, coated, coated with heat shock, and coated with pH shock) were compared. As proven by our previous research, the cells that are beneficial for the patient, namely, metabolically active and adherent to the Caco-2 cells, are present in two of the described subpopulations--active and mid-active II. Only those two groups were taken into consideration when assessing the cost of production of probiotic powder, based on the definition of probiotics, which describes them as live cells [35]. Using imaging flow cytometry the mid-active II cell subpopulation was determined to suit the description of VBNC [36] and, therefore, was also included in the project. The results of simulated in vitro digestion experiments were used, where the subpopulation composition of preparation after ingestion was assessed. Using those values, we determined the viable and beneficial (VB) cells, which are the cells that remain useful and confer health benefits to the host after technological processes and digestion. The individual cost of production of 1 kg of VB cells using different technological variants was calculated in Table 4. For the samples measured directly after drying the coated variant treated with heat shock provided the best value in terms of price per 1 kg of VB cells after digestion. As for the samples after 12 months of storage, the coated samples after pH shock showed the best value.