## 3 Results
For this process, FKC was previously labelled with green fluorophore, FITC (Figure 4). Upon review, the fluorescence microscopy image showed a green fluorescence that spread throughout the alginate bead, suggesting that the FKCs could be homogeneously encapsulated in the beads. Furthermore, the green fluorescence in the microscopic images was seen to be slightly blurred at some points. Therefore, to confirm the existence of FITC-labelled FKC in detail, confocal microscopy was carried out. When we focused on a certain plane along the z-axis of the alginate bead, we could clearly observe green dots that indicate the location of the FITC-labelled FKC. Notably, the green dots were found to be linked to each other forming assemblies of \(\sim\)10 FKCs, which corresponded to the findings of the SEM observation (Figure 3). Figure 3: Scanning electron microscopy images of the inside of dried alginate bead encapsulating FKC. The dashed circles indicated the location of the FKCs on the alginate network. (**a**) half-crushed dried alginate bead; (**b**) assemblies of FKC; (**c**) magnified images of FKC. Figure 2: Scanning electron microscope images of FKC, clays, and FKC + clay mixtures. (**a**) FKC; (**b**) LDH; (**c**) FKC + LDH; (**d**) KA; and (**e**) FKC + KA. We further investigated the inside of the dried alginate beads containing either FKC + LDH or FKC + KA mixture (Figure 5). When compared to alginate beads with FKC only, the bead wall was shown to be less homogeneously patterned. However, we could clearly observe a difference between the FKC + LDH containing alginate bead and the one containing FKC + KA. The former showed several points that were characteristic of intensive small dots (white dotted circle in Figure 5a), which were attributed to the FKC + LDH agglomerates. On the other hand, the latter showed a more homogenous aspect, which indicated that the FKC cells and KA were uniformly distributed throughout the alginate beads. Figure 4: Fluorescence and confocal microscopy of wet alginate beads containing FITC-labelled FKC. The specimens were prepared by simply compressing wet alginate beads between slide glasses. EX: 492 nm, EM: 520 nm. Figure 5: Scanning electron microscopy images for the inside of the dried alginate bead encapsulating (**a**) FKC + LDH and (**b**) FKC + KA mixture. The dashed circles indicated the location of the FKC + LDH agglomerates. To trace the FKC inside the alginate bead, the cells were labelled with FITC. In order to check that this FITC-FKC could be utilized in the quantification of FKC, the relationship between the FKC concentration and fluorescence intensity was fitted into a linear equation. As shown in Figure 6, the regression factor, R\({}^{2}\) was larger than 0.999, suggesting the possibility of FKC quantification with FITC measurement. In the calibration, we prepared six standard solutions with concentrations of \(5\times 10^{4}\), \(1\times 10^{5}\), \(2\times 10^{5}\), \(4\times 10^{5}\), \(6\times 10^{5}\), \(8\times 10^{5}\) cells/mL. This quantification method showed the presence of a determined limit of detection (LOD) and limit of quantification (LOQ) of \(3.2\times 10^{4}\) cells/mL and \(9.6\times 10^{4}\) cells/mL, respectively. Incidentally, these values corresponded to OD = 0.0032 and OD = 0.0096 for conventional spectrophotometric quantification, and thus the current fluorescence quantification was considered to be more sensitive than the conventional method. Based on this quantification method, the encapsulation efficiency and loading capacity of the FKC in the alginate beads with or without clay nanoparticles were analyzed. As summarized in Table 2, the encapsulation efficiency and loading capacity were noted to be highly dependent on the type of clay nanoparticles used as ingredients. Here, the existence of LDH was shown to reduce the encapsulation efficiency while KA dramatically increased the encapsulation efficiency.