## 3 Results  
The pathogenicity experiments confirmed that _Streptococcus parauberis_ could be utilized as an antigen to stimulate the immune system of starry flounder. Thus, we tried to estimate the potential of _Streptococcus parauberis_ as an oral vaccine after appropriate formalin inactivation and encapsulation. Upon review, the colloidal property of FKC with or without clay nanoparticles in an aqueous system was also tested and monitored. As shown in Figure 1a, the hydrodynamic radius of the FKC + LDH mixture was much larger than that of FKC or LDH itself before mixing, suggesting the formation of agglomeration between FKC and LDH nanoparticles. On the other hand, the reviewed FKC + KA mixture showed a similar hydrodynamic radius as compared to the FKC or KA itself (Figure 1b). As we have seen, the quantitative colloidal parameters are summarized in Table 1, showing the different effects of LDH and KA on the colloidal stability of FKC. The Z-average of FKC + LDH was much larger than those of the FKC and LDH. Furthermore, the PdI values, which indicate the degrees of homogeneity with lower values, dramatically increased upon mixing. On the other hand, the FKC + KA mixture showed still a comparable Z-average with FKC or KA, and the PdI value of the mixture even decreased upon mixing. These results strongly suggest that LDH and KA respectively acted as an agglomerating agent and dispersant for FKC under the reviewed aqueous system. The formation of agglomerates or dispersion was also visualized using SEM measurements (Figure 2). As shown in Figure 2a, the FKC of _Streptococcus parauberis_ exhibited round shaped cells of more or less 1 \(\upmu\)m in size. Both clays showed particle dimension of hundreds of nanometers with a characteristic plate-like morphology (Figure 2b, d). The SEM images of the mixture were fairly different from each other depending on the type of clay nanoparticles that were used. We could not observe any FKC cells on the FKC + LDH mixture (Figure 2c). It seems that smaller LDH particles cover all FKC cells to hide them. On the other hand, the SEM image (Figure 2e) of the FKC + KA showed plate-like KA particles, as well as round FKCs (white dotted circles in Figure 2e). The SEM images also showed an agglomeration and dispersion of the FKCs under the existence of LDH and KA, respectively. To confirm the possibility of FKC encapsulation by alginate bead, we carried out a SEM measurement on the dried alginate beads containing FKC. As shown in Figure 3a, a half-crushed bead showed hollow spaces that could be attributed to the dehydration process. In the area of the wall inside the hollow (Figure 3b), we could find homogeneously patterned texture at the background, where several assemblies of spheres (white dashed line in Figure 3b,c) were observed. The patterned texture was considered as the alginate-Ca\({}^{2+}\) cross-linked network and the assemblies were linked FKCs. This result showed that FKC could be encapsulated within the alginate bead while several cells were linked together. \begin{table}
\begin{tabular}{c c c c c c} \hline \hline \multirow{2}{*}{**Parameters**} & \multirow{2}{*}{**FKC**} & \multicolumn{2}{c}{**Clay**} & \multicolumn{2}{c}{**FKC + Clay Mixture**} \\  & & **LDH** & **KA** & **LDH** & **KA** \\ \hline Z-average (nm) & 1571 & 244.3 & 1146 & 2847 & 1176 \\ PdI [FOOTNOTE:]Footnote : footnotemark: [ENDFOOTNOTE] & 0.495 & 0.069 & 0.403 & 0.899 & 0.396 \\ \hline \hline \end{tabular} a) PDI: polydispersity index equals to (standard deviation)\({}^{2}\)/average.  
\end{table} 
Table 1: Dynamic light scattering results of FKC, clays, and FKC + clay mixture. Figure 1: Hydrodynamic radius of formalin- killed cells (FKC) (solid line), clays (dashed line), and FKC + clay mixture (circle and line) with respect to (**a**) layered double hydroxide (LDH) and (**b**) kaolinite (KA). The location of the FKC inside the alginate beads in the hydrogel state was visualized by the use of fluorescence and confocal microscopy, respectively.