Correlation Between The Viable Counts Of Bacillus Natto And The Nattokinase Activity And Their Stability In Digestive Fluid

Bacillus natto is the production strain of a traditional fermented food natto,which can produce a variety of physiologically active substances with health care function during the process of fermentation.Nattokinase is one of the most important physiologically active substances.With the strong thrombolytic function,it has been widely used in the prevention of cardiovascular and cerebrovascular diseases.At present,the research of Bacillus natto and nattokinase mainly focuses on how to improve the viable counts and enzyme activity.However,their correlation and the stability to gastrointestinal digestive fluid are rarely reported.Therefore,in this study,the strain with high nattokinase activity was obtained from natto through isolation and screening.Then the correlation between the viable counts and nattokinase activity as well as the stability of this strain and nattokinase to gastrointestinal digestive fluid were studied.The main contents and the results were as follows:?1?Isolation,identification and biological characteristics of the strain producing nattokinase.Based on casein hydrolytic and fibrinolytic activity of nattokinase,37 strains were obtained from commercial natto through isolation and repeated screening using skim milk medium,and one of them was obtained by re-screening using agarose-fibrin plate and named as CN11.The strain CN11 had relatively higher fibrinolytic activity and its activity of crude enzyme was5856.85 U/mL.This strain was identified as Bacillus subtilis through morphological observation of cell and colony as well as identification of physiological and biochemical characteristics.With the result of molecular biology identification and characteristics of nattokinase production,it was further confirmed as Bacillus natto.Its optimal growth temperature was 37?,optimal growth pH was 7.0,and the genetic characteristics keep stable within 20 generations.?2?Correlation between the viable counts of Bacillus natto and nattokinaseactivity.With the nattokinase activity as the index,the optimal liquid and solid fermentation conditions were determined by single factor and orthogonal experiment.Then the correlation between the viable counts of Bacillus natto and nattokinase activity was discussed.The results showed that the optimal medium compositions and conditions of liquid fermentation were as follows: 2.5%sucrose,2% yeast extract,0.05% MgSO₄,0.02% CaCl2,0.1% KH₂PO₄,0.3%K₂HPO₄,initial p H of 7.0,inoculum size of 2%,liquid-loading volume of40 mL/250 mL,fermentation temperature of 37?,fermentation time of 60 h.The optimal solid fermentation conditions were as follows: inoculum size of 6%,fermentation temperature of 37?,fermentation time of 48 h.The viable counts of Bacillus natto were positively related to nattokinase activity.In addition,there was a significant positive correlation in liquid fermentation,and the correlation coefficient was 0.82.?3?Tolerance of Bacillus natto to gastrointestinal digestive fluid.The viable counts were observed when Bacillus natto CN11 was incubated in artificial gastric fluid at different p H values,artificial intestinal fluid and medium of 0.3%?w/v?bile salt,respectively.Then the tolerance of Bacillus natto to gastrointestinal digestive fluid was analyzed.The results showed that the viable counts of Bacillus natto CN11 decreased from 2.8×10⁸ CFU/mL,2.5×10⁸ CFU/mL and 2.6×10⁸ CFU/mL to 8.9×10⁷ CFU/mL,1.7×10⁸ CFU/mL and 2.2×10⁸ CFU/mL after 3 h of incubation in artificial gastric fluid with pH 1.5,pH 2.5 and pH 3.5,respectively.After 4 h of incubation in artificial intestinal fluid,the viable counts increased from 4.2×10⁸ CFU/mL to 5.7×10⁸ CFU/mL.After 4 h of incubation in medium of 0.3%?w/v?bile salt,the viable counts decreased from 4.3×10⁸ CFU/mL to 3.0×10⁸ CFU/mL.In summary,Bacillus natto CN11 has excellent tolerance to artificial gastric fluid,artificial intestinal fluid and bile salt environment.?4?Stability of nattokinase to gastrointestinal digestive fluid.The crude enzyme and solid fermentation product are two forms of nattokinase samples which were obtained by liquid fermentation and solid fermentation of Bacillus subtilis CN11,respectively.The nattokinase activity was observed when nattokinase samples were incubated in artificial gastric fluid at different pH values,artificial intestinal fluid and medium of 0.3%?w/v?bile salt,respectively.Then the stability of nattokinase to gastrointestinal digestive fluid was analyzed.The results showed that when exposed to artificial gastric fluid at pH 1.5,pH 2.5and pH 3.5 for 3 h,the residual activity of crude enzyme and solid fermentation product were 9.6%,50.3%,61.1% and 26.6%,58.3%,78.1%,respectively.After4 h of incubation in artificial intestinal fluid,the residual activity of crude enzyme and solid fermentation product were 65.9% and 83.8%,respectively.After 4 h of incubation in medium of 0.3%?w/v?bile salt,the residual activity were 87.3% and 89.7%,respectively.Therefore,nattokinase has poor tolerance to artificial gastric fluid at pH 1.5.However,it has excellent tolerance to artificial gastric fluid at pH 2.5 and pH 3.5,artificial intestinal fluid and bile salt environment.Nattokinase in the solid fermentation product has higher stability to simulated gastrointestinal digestive fluid.