The Optimization Of Nattokinase Fermentation Process And The Activity Research In Digestive Juice

Cardiovascular diseases (CVDs) has a high mortality rate in the world. The people die from the disease reaches to15million each year, ranking the first cause of death of a variety of diseases. Compared to traditional thrombolytic agents, nattokinase is deemed to thrombolytic therapy medicine because of its good fibrinolytic activity, long half-life, oral administration and small side effect. Nattokinase production mainly use soybean to finish the fermentation, but the soybean meal as the main raw material for nattokinase production has not been reported. Nowadays, nattokinase activity measured methods are either expensive or time-consuming, not suitable for online detection of nattokinase production. This experiment treated reducing fermentation costs while increasing the activity as the entry point, study the nattokinase economy, rapid detection method, optimal fermentation process and its activity in the digestive juices. The main results are as follows:Three activity measured methods such as Agarose-fibrin plate method, Folin-phenol method, and Tetrapeptide substrate method were compared for research. An economic and rapid new method to determine the nattokinase activity was found: Established the relationship between activity and A680nm, shortened the enzymatic activity and reduceed the cost of the determination.We did the univariate analysis in nattokinase solid fermentation’s seven aspects of the selection of the carbon source, carbon and nitrogen ratio, water content, fermentation time, inoculum, fermentation temperature and the addition of metal ions.Then the optimal fermentation conditions were obtained by orthogonal experiments of four factors and three levels:40℃fermentation temperature,140%water content of soybean meal,10%Inoculum and36h fermentation time; The activity of nattokinase production was3203.260IU/g. increased12.5%compared to the optimized before. The optimum drying conditions of70℃,8h were get by the single factor experiments of drying temperature and time, the enzyme recovery after dry experiment was61.4%. The basic optimization of liquid fermentation conditions was the nitrogen source, carbon source and whether to add a soybean oil, the enzyme activity was about200IU/mL, and increased19.5IU/mL. In addition,60Co γ-ray mutagenesis breeding screened out an activity increased by4.5%natto strain.Three different solid forms of nattokinase(no crushed, mechanical crushing, and ultra-fine powder samples) were treated by artificial gastric juice for4h, the activity maintained about80%of its initial activity, while the activity of crude enzyme solution rapidly decreased by45%after treated for0.5h, then decreased slowly. The activity became48.6%of the initial activity when treated for4h. Treated by artificial gastric juice for4h and artificial intestinal for4h, the activity of mechanical crushing and ultra-fine powder were still47.0%and51.0%of their initial activity. When treated by the human digestive juice, the activity of mechanical crushing and ultra-fine powder gradually increased to the initial activity, while the crude enzyme solution began to decline when treated for0.5h, and then reduced to79.8%of the initial activity when at4h.