Study On Optimization Of Nattokinase Solid-state Fermentation And Antithrombotic Function

Nattokinase (NK) is a serine protease enzyme which is extracted from a traditional fermented food in Japan. It is reported that NK have fibrinolytic activity and can activate plasminogen in vivo, thereby increasing the amount of endogenous plasmin. So it can prevent and cure thrombosis. Now commonly used in clinical treatment of cardiovascular disease, thrombosis drugs have some flaws:strong toxicity, side effect, short half-life in vivo, shortage of sources, expensive etc. So they are difficult to become popular drugs. NK is expected to compensate for these, so scholars believe that it is likely to become a new generation of antithrombotic drugs.This paper studied NK gene verifying, NK solid-fermentation process optimizing, and its antithrombotic effect in vivo and vitro.The result shows that B.natto 10023 and B.natto 10262 gene are 100% the same with subtilisin NAT gene(aprN), B.subtilis FY99 gene is 96.7% the same with aprN.The medium components of fermentation were studied. The NK activity from high to low is A, B, C, sweet potato waste and distilled grain. The NK activity with B.natto 10262 is much higher than B.natto 10023 in every medium described above. So we use B.natto 10262 as our fermentation strain in the following experiment.To A, B and C for the solid medium of orthogonal test, the factors on the increase in production of nattokinase sequence was C＞A> B. The best combination is A2B2C2 and its result is 5791 IU/g that is better than 4960 IU/g which is fermented only with A.The fermentation conditions for the NK production were studied. The operating factors, including the initial water content, inoculum size, adding amount of A, initial pH, fermentation temperature, fermentation time, seed age, sterilization condition, and drying temperature were determined as follows:80% water content,10% inoculum size,20 g A, natural initial pH, fermentation temperature 28℃, fermentation time 66 h, seed age 19 h, sterilization condition 121℃20 min, and drying temperature 60℃. The optimized condition allowed the NK activity reached to above 10000 IU/g. This is about twice high to initial 4960 IU/g.From the response surface experiment we got its quadratic regression equation. Its multiple correlation coefficient is 0.88, so it can fit our experiment very well. Optimized results for fermentation conditions were got with analysis software SAS. We verified the results and the NK activity can reach to 14338 IU/g which is 107.5% to the highest activity in response surface experiment.The results of mice LD50 of NK and given or injected to mice with NK can guarantee NK safety and will not lead to bleeding tendency.Nattokinase on carrageenan induced mice tail thrombus model and antithrombotic tests in vitro proved nattokinase can significantly inhibit thrombus formation both in vivo and in vitro.