High Cell Density Cultivation Of Soybean Endophyte Bacillus And Preparation And Evaluation Of Freeze-dried Bacterial Powder

During the Tang Dynasty in China,tempeh was introduced to Japan.After that,the fermentation process was continued to be improved,and a soybean fermented product called"Natto" appeared.Natto is of various physiological functions.For example it can dissolve blood clots,prevent osteoporosis,and regulate the intestinal environment.At the beginning of the nineteenth century,Japanese scholars isolated a bacterial strain from soil,which was suitable for soy fermentation to produce natto.The bacterial strain was called "Bacillus natto".Later,"Bacillus natto" was identified as Bacillus subtilis.So far,all strains appeared on the markets in both Japan and in China are Bacillus subtilis.Previously,our laboratory isolated a series of nattokinase-producing bacillus from soybean endophytes.The steamed soybean was fermented with the isolated soybean endophytes.We found that the nattokinase content,antioxidant capacity and anti-immune suppression capacity in mice are significantly higher compared to that of the commercially available natto.At present,there are many commercially sold brands of natto fermentation powder on the market.Natto fermentation with bacterial powder is simple and quick,therefore,it is suitable for market demand.The vacuum freeze-drying method for preparing natto bacterial powder has various advantages,such as single equipment requirements,simple operation,and high survival rate of the bacterial powder.The fermentation capacity of freeze-dried bacterial powder is determined by the number of viable bacterial powder.High cell density culture technology can significantly increase the concentration of bacterial cells by optimizing the medium composition and cultural conditions.In this study,the high-yield nattokinase soybean endophyte isolated previously by our laboratory was used as the test material,the bacterium of which was identified as Bacillus licheniformis GN.The endophytic bacterium GN was used to study the high cell density cultivation,the preparation and preservation methods for freeze-dried bacterial powder.The activity of nattokinase was determined by fermenting natto with self-made GN freeze-dried powder.In order to explore the anti-aging activity,the self-made GN freeze-dried powder was given by gavage to both healthy mice and D-galactose aging mice.The main conclusions are as follows:1.After optimization,the composition of the medium for strain GN was determined as follows:glucose 35.034 g/L,yeast extract 27.993 g/L,MgSO4 1.285 g/L.Under the cultivation of 37℃,3%inoculum,and initial pH 6.5,the number of viable endophytic Bacillus GN was increased from 2.01×108 CFU/mL to 2.36×109 CFU/mL,OD600 increased from 1.35 to 12.66,and the dry cell weight from 2.45 g/L to 6.03 g/L.2.After the screening test,the protective agent for soybean endophytic Bacillus GN is:sucrose 7.5(w/v)%,skimmed milk powder 10.0(w/v)%,maltodextrin 12.5(w/v)%.Vacuum freeze-dried bacterial powder was prepared by the method,and the bacterial number reached 1.095×1010 CFU/g.After 7 months of storage,the survival rate reaches 84.4%.3.The freeze-dried bacterial powder was used to ferment steamed soybean.The average nattokinase activity of the fermented soybeans was 57.67 U/mL,and that which was made by the commercially available bacterial powder was in the range from 19.22 U/mL to 40.47 U/mL.4.The healthy mice was given the self-made GN freeze-dried powder for 30 days by gavage method.The mice were observed in good eating and mental state,and no abnormal lesions in internal organs were observed.5.Gavage D-galactose aging mice with self-made strain GN freeze-dried powder were studied the antioxidant activity.The results showed that the GN freeze-dried powder improved the spleen index of mice,significantly increased the total antioxidant capacity of mice serum(p<0.05)and significantly reduced the lipid peroxidation product MDA concentration(p<0.05).