Preparation And Application Research On A Kind Of Layer-by-layer Beads Based On Calcium Alginate Hydrogel

The layer-by-layer encapsulation biomaterial had attracted the attention for theadvantages which the ordinary encapsulation biomaterials do not have. For example,the LbL onion-like encapsulation biomaterial could offer stronger protection for themicroorganism and make the produce of the fermentation products easier to becontrolled than the ordinary encapsulation biomaterials during the process ofimmobilized fermentation. So the LbL structure has been used for drug delivery, cellculture, energy saving, environment protection and so on. In this paper, sodiumalginate (SA) was chosen as main material; Ca2+was chosen as cross-link agency andsuccessfully prepared a kind of layer-by-layer hydrogel bead via dropping method theemulsion technical. Then ten factors during the process of preparation were selectedto find the influence of the preparation conditions to the beads’ size. The morphologyof the beads was observed via scanning electron microscope (SEM). The stability ofthe beads in vitro was examined. The cytotoxicity of the beads was examined by MTTmethod. Finally, the beads were utilized to encapsulate the Bacillus subtilis and doimmobilized fermentation and sustained-release experiment.Based on the preparation method shown above, ten of the factors during theprocess of preparation conditions, specifically, the concentration of CaCl2solutionand SA solution, the ratio of CaCl2solution to SA solution, the ratio of water phase tooil phase, the stirring speed, the length of crosslinking time, the reaction temperature,the pH value of water phase and the viscosity of SA were changed to evaluate theirinfluence of the beads property. The result showed that all of the factors influence thediameter of the beads obviously except gelation time. The beads prepared in thecondition of lower stirring speed, higher concentration of SA solution and CaCl2 solution, lower ratio between water phase and oil phase, higher ratio between SAsolution and CaCl2solution, lower viscosity of SA solution, acidic environment androom temperature were likely to have large size. So the LbL beads with proper sizecould be obtained by changing these factors.From naked eyes, LbL beads were found spherical, smooth-surfaced andnon-aggregated. And the layers could be separated from each other. The result of theSEM observation showed the crinkle, sponge-like and dense shape of the beads’surface and the large gap between layers, which means the beads have the potential tohold a large amount of containers such as microorganism and drugs.In the stability experiment in vitro, the LbL beads were stable in acidenvironment. They dissolved slowly when they were transferred to near neutralenvironment. And they dissolved sharply when they were transferred to weak alkalineenvironment.In the cytotoxicity experiment, the relative survival rate of the MEF cultured inthe extract of the LbL beads for24h,48h and72h was88%,80%and77%respectively. According to the International Standard, it could be realized that thecytotoxicity of this kind of LbL beads is slightly, which means the beads have goodbiocompatibility.In the experiment of immobilized fermentation, five groups were set, specifically,the group of blank fermentation broth, the group of fermentation broth with blankcalcium alginate gel beads (non-LbL structure), the group of fermentation brothwith free Bacillus subtilis, the group of fermentation broth with Bacillus subtilisimmobilized in calcium alginate beads (non-LbL structure) and the group offermentation broth with Bacillus subtilis immobilized in five layers calcium alginatebeads. During the experiment, the group of blank fermentation broth and the group offermentation broth with blank calcium alginate gel beads (non-LbL structure)produced little α-amylase. The group of fermentation broth with free Bacillussubtilis and the group of fermentation broth with Bacillus subtilis immobilized incalcium alginate beads (non-LbL structure) released α-amylase fast in72h, andreleased little α-amylase during72h-240h. The group of fermentation broth with Bacillus subtilis immobilized in five layers calcium alginate beads releasedα-amylase continuously in240h, and amount of α-amylase cumulated in broth wasalmost the same with the group of fermentation broth with free Bacillus subtilis.The result showed that the group of fermentation broth with bacteria coated in LbLbeads could produce α-amylase stability and long-lasting, which means the this kindof system could sustained-release the fermentation protection effectively. So the LbLbeads had the potential to be utilized in fermentation process which needs to avoid theburst release of the production and release the production continuously.