Extraction And Properties Analysis Of Nattokinase From Vegetable Soybean Fermented By Bacillus Natto

Natto is a traditional fermented food with multiple health effects.Nattokinase(Nattokinase,NK)is a serine protease produced by Bacillus natto,which can significantly dissolve thrombus in vivo and in vitro,and is considered to be a very researchable thrombolytic agent.In this experiment,the surplus products such as vegetable soybeans remaining after the enterprise's export were used to ferment with natto bacteria,and the activity of nattokinase was used as an indicator to optimize the fermentation process of natto,extract and preliminary purify nattokinase,and investigate some properties of it.It will bring a new direction for the development of vegetable soybean deep-processed products and the treatment of the surplus products after export.The optimal fermentation scheme determined by single factor and orthogonal experiments is as follows.Fermentation temperature is 37 ?.Inoculation amount is 0.02 g/100 g.Fermentation time is 36 hours,and post-ripening time is 18 hours.Under this condition,the activity of nattokinase in fermented Natto is 2236.60 IU/g.The appearance of the matured natto is yellow-green,with a uniform white film on the surface,without obvious ammonia odor,and it will be difficult to break after drawing.20% and 60% saturated ammonium sulfate was separately used to remove salt protein and precipitate nattokinase,and then DEAE anion exchange chromatography was used to separate and purify nattokinase.By the SDS-PAGE electrophoresis,the molecular weight of the extracted nattokinase was between 25 and 35 k Da,the purification factor reached 16.3 times,and the recovery rate was 22.5%.The investigation of some properties of nattokinase showed that the optimum temperature of nattokinase is 37 ?,and its stability is better below 40 ?.The optimal p H value is 8.0,and it is stable in the range of p H 6.0?8.0.The results of in vitro thrombolysis experiment of nattokinase showed that after adding the crude enzyme solution(20 mg/m L)to the test tube,the dissolution rate of the blood clot reached 77.55±0.45% after 5 hours,which is 41.93% higher than the control group.The dissolution rate reached 90.25±1.90% after 10 hours,which is 59.11% higher than the control group.It indicates that the nattokinase produced by the surplus products such as vegetable soybeans ferment with natto bacteria has a certain in vitro thrombolytic effect.