The Optimization Of Fermentation And Purification And Application Of Nattokinase

Natto as an ordinary food was administered by Ministry of Health in2002,Nattokinase as the active ingredient in natto has an excellent efficacy ofprevention and treatment of cardiovascular disease, at present, NK products onthe market are mainly imported from Japan and Taiwan, although the price up toabout2000yuan/kg, it was still favorable by cardiovascular disease patients, sostudies on NK has become a very hot research topic. In this paper, a Bacillusnatto(ID1.1086)which was purchased from the CGMCC as the starting strain,the fermentation, purification and application of nattokinase was selected asresearch object. The main content of the article includes5aspects as below.(1) The preparation and quality identification of natto. The selectedsoybeans were produced natto by washing, soaking, draining, steaming, cooling,draining, inoculation, fermentation, ripening and so on, while its quality weredeterminated. The test showed that natto was yellow, natto-specific odor,viscosity, brushed state, software an hardware suitable for beans, no visibleimpurities; water was57.4g/100g, amino nitrogen(nitrogen) was0.355g/100g,protein was19.56g/100g. The above results met the requirements of the nattoindustry standard SB/T10528-2009Natto.(2) Studies on the solid fermentation of NK. Seven factors which affect theNK activity of the solid fermentation, were investigated, the optimum conditionsfor NK activity were obtained by orthogonal tests as follow: soybean initialmoisture content was50%, soybean cooking time was45min, bacterial inoculumwas10%.(3) Studies on purification of NK by ammonium sulfate salting-out.Ammonium sulfate was used to salting-out, and mapped the salting-out curve,according to the salting-out curve, selected30%ammonium sulfate saturationprecipitate contaminating proteins, and selected70%saturation of ammoniumsulfate precipitate NK, salting-out results as follows: specific activity reached 508.1IU/mg, purification multiples was1.21, the recovery rate was92%.(4) Studies on purification of NK by ultrafiltration and the optimization ofoperating parameters. According to the molecular weigh of NK was27728,select30KD and10KD as ultrafiltration membrane, the NK crude enzymesolution first through the ultrafiltration membrane of30KD, and then permeatethrough the ultrafiltration membrane of10KD, collection of intermediatemolecular weight NK filtrate. The ultrafiltration process parameters effects of onflux and NK content were tested, and the effects of factors like operatingpressure, liquid temperature and liquid pH on flux and NK content are examinedthrough single factor and orthogonal experiments, obtained the optimumconditions as follow: the operating pressure was0.25Mpa, liquid temperaturewas37°C, liquid pH was7.0. By the optimum conditions of ultrafiltrationprocess, obtained the optimum results as follow: specific activity reached1052.4IU/mg, purification multiples was2.50, the recovery rate was81%,compared with ammonium sulfate salting-out, though the recovery rate isrelatively declined, the NK purification multiples greatly improved, and thepurity steps was easy, the purity conditions was mild, reduced the loss of NK.(5) Studies on instant hawthorn and natto powder. Preparation of nattopowder and nattokinase powder by freeze drying method, and its freeze dryingconditions: the freeze drying temperature was-54°C, the vacuum was0.08mbar.Natto powder, hawthorn powder, maltodextrin, xanthan gum, and other rawmaterials through100mesh sieve, mixing, blending and packaging, preparationof instant hawthorn and natto powder. Four factors, which affect the organolepticof the instant hawthorn and natto powder, were investigated, the optimumconditions for organoleptic were obtained by orthogonal tests as follow: the massratio of hawthorn and natto powder was1:0.3, maltodextrin add amount was20%, xanthan gum add amount was0.3%, recombination temperature was60°C,under this optimum condition, hawthorn fruity, instant and no delamination.