Study On Extraction Technology And Properties Of Nattokinase By Liquid Fermentation

Nattokinase is an enzyme which produced by Bacillus subtilis subspecies natto?It is safe,non-toxic,economical,not easy to cause bleeding,no antigen,long half-life.In addition,it can be oral absorption.It is a good thrombolytic health food.In this thesis,we have done the follow work to develop a new generation of nattokinase capsules:?1?In this paper,several protease-producing strains were isolated from Linyi Shandong tempeh and natto powder.Through liquid fermentation and determination of protease activity,a strain of high-yield protease was screened and named as BN.The BN was identified as Bacillus natto by colony characteristics test,cell morphology test,physiological and biochemical tests,along with 16 s rDNA molecular biology identification test.?2?Optimized fermentation enzyme production condition of the above strain BN.The optimal age of BN strain was determined to be 15²0h by measuring OD660 nm in seed liquid.The fermentation carbon source and nitrogen source were determined to be soluble starch and yeast extract respectively by single factor test.The optimum recruitment of inorganic salts were determined to be 0.1% for K2HPO4,0.1% for KH2PO4 and 0.05% for CaCl2 by orthogonal test.With response surface design method,the optimum fermentation conditions were inoculation amount of 3%,temperature 40 ?,pH 7.0,fermentation time84 h by response surface design method,and the activity of nattokinase reached 749.41 U /mL under this optimization condition..?3?Nattokinase was separated and purified by refrigerated centrifugation,fractionated salting out and dialysis desalting.The purified nattokinase showed two band by detection of SDS-PAGE electrophoresis analysis.The darker band with molecular weight of about28 kDa is nattokinase.The recovery and purification multiple of the nattokinase were94.96% and 3.81 times,respectively.?4?The stability of above separated and purified nattokinase was measured.The results of the determination of the properties of nattokinase: optimum reaction temperature is 40?,suitable storage temperature is-22 ?⁴ ?,optimum pH is 6.0,and the activity is relatively stable under the conditions of 6.0⁷.0,Ca²⁺ could activate enzymatic activity,Mg²⁺ has no obvious effect,Zn²⁺?Cu²⁺ and Fe²⁺could inhibit the activity.?5?In the process of making nattokinase capsules,the single factor test confirmed that maltodextrin,soluble starch and trehalose had better protective effect on nattokinase.Through the orthogonal design experiments,which were based on single factor experments,determined?when?the ratio of enzyme solution to starch,maltodextrin and trehalose?v / w?is 1:1:3.5:1,the best protective effect can be achieved.When the dosage of soybean lecithin was 70% of the enzyme solution,the retention rate of the enzyme activity was the highest after treated in simulated gastric juice for 4 h.The capsules were processed insimulated gastric juice for 4 h,and then treated in simulated intestinal juice,the nattokinase residual rate was still up to 39.4%.The nattokinase capsules is not suitable for storage at room temperature.The store effects of 4?and-22?have little difference,but considering the cost,4? is selected as the storage condition for nattokinase capsules.