## 4 Discussion  
In terms of oral vaccination, pathogens should be stably delivered to the intestine since the intestinal mucous membranes are point of stimulus for the immune system [31; 32; 33]. Therefore, a strategy for oral vaccination should satisfy the following conditions: (i) Provide for the selection of an appropriate pathogen, (ii) apply a reservoir of pathogen, (iii) provide for the adequate formulation of the reservoir with fish feed, (iv) offer stable storage of the pathogen in the formulation and in the \begin{table}
\begin{tabular}{c c c c} \hline \hline \multicolumn{1}{c}{**-Loading Parameter**} & **FKC Alone** & **FKC + LDH Mixture** & **FKC + KA Mixture** \\ \hline Encapsulation efficiency (\%) & \(37.75\pm 12.10\) & \(28.06\pm 18.16\) & \(67.22\pm 11.74\) \\ Loading capacity (cells/g) & \(1.89\times 10^{5}\pm 6.05\times 10^{4}\) & \(1.28\times 10^{5}\pm 8.25\times 10^{4}\) & \(3.06\times 10^{5}\pm 5.33\times 10^{4}\) \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Encapsulation efficiency and loading capacity of FKC in alginate beads with and without clays nanoparticles. Figure 6: Linear regression result for FITC- labelled FKC concentration (number/mL) and fluorescence intensity at 520 nm. gastric condition, and (v) provide for a bursting out of the pathogen in an intestinal condition during the residence time. In this regard, _Streptococcus parauberis_, which was found to have pathogenic effect on the starry flounder, was first inactivated by formalin and was encapsulated with alginate hydrogel beads. Furthermore, the alginate was found to form a cross-link with metal cations like Ca2+ [34; 35], and they easily formed hydrogel beads when the alginate solution droplet meets the CaCl\({}_{2}\) solution [36]. We also confirmed that the alginate beads could be formed with a high water content, and that the cross-linked alginate could contain streptococcus-originated FKCs (Figure 3). Furthermore, the streptococci were found to be homogeneously distributed throughout the hydrogel, thus preserving their pair or chain morphologies (Figure 4). Generally speaking, since FKC encapsulation by the alginate beads is a passive process, i.e., FKC suspension in the aqueous system is surrounded by alginate polymers whose cross-linking with Ca2+ happen to encapsulate the FKC moiety. Furthermore, the alginate bead encapsulating the FKC can only be released from the bead when the alginate-Ca2+ cross-link is sufficiently broken. In the meantime, we planned to control the encapsulation and release behaviors of the alginate beads by utilizing the clay nanoparticles that can modify the status of FKC suspension and alginate cross-linking. Various clay moieties have been reported to interact with microbes through surface adsorption, flocculation, agglomeration, etc., [37; 38]. These physicochemical reactions were expected to affect the encapsulation and release of FKC for alginate beads. Furthermore, clay nanoparticles have often been reported to control the swelling property or drug release property of alginate polymers [39; 40]. In this case, we selected two different kinds of clay nanoparticles, KA and LDH, to control the encapsulation and the release of FKC for alginate beads. In this relation, KA and LDH are different in terms of their surface charge although both clays are similar in their plate-like morphology and layer-by-layer stacking structure. Here, KA is known to have an electric point between 4 and 6 [41], implying negative surface charges at a neutral condition. On the other hand, LDHs have a high isoelectric point of \(\sim\)11, being positively charged at a neutral pH [42]. Although the surface charges of streptococci are different from each other depending on the strain, it was generally known that streptococcus bacteria have an isoelectric point \(\sim\)4, indicating negative surface charges in a neutral pH. Therefore, we could expect that the noted dispersion state of _Streptococcus parauberis_ FKC would vary upon the type of clay nanoparticles used.