The Characteristics Of Intra-hollow Ca-alginate Capsule And The Study On Immobilization Of Bacillus Subtils For Nattokinase Production

Intra-hollow Ca-alginate capsule comes of the Ca-alginate bead. It not only owns the advantages of broad source, inexpensive cost, gentle immobilization condition, easy operation, good biocompatibility, and less resistance to the substrates and the oxygen, but also can overcome the disadvantages of the difficulty that the substrates and oxygen can hardly transfer into the bead core and the limited space that the cells can only grow on the bead surface and in the gel holes. Therefore, the intra-hollow Ca-alginate capsule is a promising immobilization carrier.In this thesis, we designed the preparation device for intra-hollow Ca-alginate capsule and studied the effects of operation conditions (concentrations of CaCl2 or sodium alginate (SA), dropping velocity and gelation time) on the diameter, membrane thickness and compression intensity of the capsules. The optimal operation conditions for capsule preparation were determined at 100 g L-1 CaCla solution, 10 g L-1 SA solution, 12 g L-1 sodium carboxymethylcellulose solution, dropping velocity of 150 droplets/min and gelation time of 10 min.We investigated the diffusivities of glucose, lactose, lactic acid, glutamic acid, tyrosine, lysine, and phenylalanine from bulk solution into the capsules and calculated the combined diffusion coefficients of the capsule (Dm) and the diffusion coefficients in the capsule membrane (D1) according to the mathematics model. Dm is about 1%~8% smaller than their corresponding diffusion coefficients in pure water while D1 is about 50%~94% smaller than that in pure water. The result indicates that the resistance of intra-hollow Ca-alginate capsule on these low molecular weight substrates is small, and all the tested substrates can diffuse easily into the capsules. In all cases, the diffusion coefficients are hardly affected by SA concentration ranging from 8 g L-1 to12 g L-1 used in preparing the capsules.The molecular weight cut-off is another important parameter that reflects the transport property of the capsule. We measured the diffusivities of global protein bovine albumin bovine (fraction V) (BSA(V)) through intra-hollow Ca-alginate capsule. Though no permeation of BSA(V) from solution into the capsules was observed, BSA(V) can diffuse out from the capsule core. The diffusion of linear macromolecule polyethylene glycol fromsolution into the capsules was also studied. We find that, for the linear molecular, the molecular weight cut-off of infra-hollow Ca-alginate capsule is 4000.Intra-hollow Ca-alginate capsule has a good biocompatibility to the Bacillus subtilis. The optimum culture medium for the encapsulated Bacillus subtilis is obtained at tryptone 2.27%, xylose 2%, YE 1%, CaCl2 0.02%, MgSO4 0.05%, Tris 0.25%, HC1 (0.2M) 9.2mL/100mL substrate, pH 7.0. Moreover, we find that the nattokinase can diffuse out from the capsule core during the cultivation.By using the optimal culture medium, the encapsulated Bacillus subtilis was fermented continuous 4 batches. The activity of nattokinase was nearly 2000IU mL-1 in the first batch, and this value dropped gradually in the following batches. But the lowest activity of nattokinase during the cultivation is still over 1500 IU 'mL-1 This result is better than other reports. Furthermore, the nattokinase can be harvested every batch. Therefore, we can draw a conclusion that intra-hollow Ca-alginate capsule is in favor of nattokinase production. As to the capsule, after cultivated for over 300 hour, the residual capsules are still stable and the cracked capsules are little.