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Nattokinase was a neutral serine protease which extracted from the traditional fermented food , had remarkable effect of thrombolytic. Taking Nattokinase can prevent and inhibit thromboxane’s production and amplification. However, liquid enzyme had poor stability and inactivated easily. The main purpose of this study was to determine the technical parameters and establish technology condition to improve the stability of micro-capsules of nattokinase. In this experiment, took nattokinase as raw materials which obtained by submerged fermentation. Concentrated by ultra-rate, took a single factor test, orthogonal experiment, response surface data analysis to research the conditions by the coacervation method, immobilization method and CD DEA, and analysis the stability of the micro-capsules of Nattokinase.1. Research on technology conditions of purification of nattokinase used ultrafiltration. The effect on the yield of ultrafiltration pressure, ultrafiltration temperature, liquid pH were researched. The results were :ultrafiltration pressure 0.25 Mp, ultrafiltration temperature 37℃, liquid pH7. It was obtained for the 5122.21 IU/mg, purification factor of 1.26, the recovery 95.7%. After agarose gel electrophoresis it had a same single band with Nattokinase , for about 28 KD. It was feasible to use ultrafiltration technology to separate and purify nattokinase.2. Research on technology conditions and stability of micro-capsules nattokinase by coacervation. Studied the main influence on the embedding rate of wall material concentration, gelatin concentration in inner phase, gelatin concentration in outer phase, water-oil phase ratio. The results were : CMC concentration 2%, gelatin concentration in outer phase 0.7%, gelatin concentration in outer phase 5% gelatin, water-oil ratio 0.6, embedding rate 85.03%. Through electron microscopy, micro-capsules had spherical compact structure and good shape.It had high tolerance on pH and temperature as compared with the original Nattokinase, but thrombolysis rate about 6.0% lower.3. Research on technology conditions and stability of micro-capsules nattokinase by immobilized. Used single factor ctor experiment, orthogonal experiment to optimize the parameters. The results were : sodium alginate concentration 3.0%, glutaraldehyde concentration 0.30%, CaCl2 concentration 0.14%, embedding rate 86.83%. Through electron microscopy, micro-capsules had compact structure, spherical, smooth and good shape.It had high tolerance on pH and temperature as compared with the original Nattokinase, but lower than coacervation, and thrombolysis rate about 1.9% lower.4. Research on technology conditions and stability of micro-capsules nattokinase by CD DEA. Used single factor ctor experiment and quadratic rotary combinational design to study core-wall ratio,β-cyclodextrin concentration, solids content and ultrasonic time on the embedding rate. The results were: core-wall ratio 0.667 ,β-cyclodextrin concentration 11.85% solids content 17.14%, ultrasonic time 26.64 min, embedding rate 69.52%. Through electron microscopy, micro-capsules had compact structure, smooth and good shape by starch adsorpted.5. In the normal acidic environment of the gastrointestinal, micro-capsules of nattokinase still had activity 2451 IU/mg by CD DEA, but original nattokinase almost lost by in vitro simulated gastric environment. Conclusion: through a comparative analysis of three kinds of embedding methods, storage stability, economy and safety determine the CD DEA method was the best, embedding rate 69.52%, activity rate 85.17%, thrombolysis rate 43.9%.
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