The Strain Mutagenesis And The Optimization Of Chickpea Natto Fermentation

In order to increase the protease activity in natto,chickpea natto submerged fermentation was optimized with Bacillus subtilis natto in this research,protease activity was taken as determing index and three most effective factors were screened out by Plackett-Burman method:liquid volume,rotate speed and chickpea powder.Reaponse surface method was used to optimize the fermentation medium and condition and the quadratic regression model showed the good regression level,which indicated the effect on the improvement of protease activity was significant(p=0.0006).The optimization we got for medium:chickpea powder 5.9%,soya bean meal powder 1.0%,glucose 0.6%,NaCl 0.5%,the best incubation condition:rotate speed 250 r/min,liquid volume 76/500 mL,fermented under the condition of 37?and 48 hours.The protease activity reached 3558.0±1.5 U/mL and had increased 42.8%under this condition compared with initial 2491.4 ±2.8 U/mL,while the dissolved area of nattokinase tested by fibrin plate method had also increased 108.6%.The results indicated that the optimized medium and fermentation condition could significantly improve the protease activity.The suitable dose and intensity of ultrahigh pressure and UV mutagenesis to Bacillus subtilis natto were explored via compound mutagenesis in order to breeding the mutant strain with high yield of protease and strong vitality.The condition of ultrahigh pressure mutagenesis was 250 MPa?5 min and the condition of UV mutagenesis was 15 W?20 cm with 50 s irritation.The lag phase of both single mutagenesis and compound mutagenesis were apparently shortened and the exponential phase was shifted to an earlier date.The protease activity of compound mutant strain P-UV-4 was 2.44±0.12 times of control and 1.32±0.07 times of single ultrahigh pressure mutant strain,which was apparently improved.P-UV-4 showed the lowest biomass while the highest specific activity,which was 14.23±0.71 times of control and about 3 times of ultrahigh pressure mutant strain.Scientific result indicated that compound mutagenesis was more appropriate to this strain as well as the level of positive mutation.This research put the efficiency of compound mutagenesis to the proof.Comparison of control medium,optimized medium control strain and mutant strain indicated that mutant strain P-UV-4 had good adaptability in the optimized medium,that protease activity could reach 4227.3±211.4 U/mL and biomass 4.89×109 CFU/mL.The Origin 9.0 was used to analyze the data and do curve fitting of fermentation kinetic curve,the cell growth model was obtained based on modified Logistic equation:y =-9.06029/1+(x/0.64828)1.42674+9.08638,R2=0.85036;the product generation was obtained based on GaussAmp function:y=-205.73168+4091.30473e-(x-49.50762)2/1028.51533,R2=O.98354;the substrate consumption model was obtained based on ExpDecl function y=49.63199+180.77178e-x/64.61696,R2=0.93639.