A strain of Bacillus subtilis gzu03 producing β-glucosidase and nattokinase and application method thereof
A technology of Bacillus subtilis and glucosidase, which is applied in the field of microorganisms to achieve the effect of good sensory properties and good application prospects
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Embodiment 1
[0032] 1. Screening of strains, preparation of seed liquid, raw material pretreatment of solid natto fermentation medium
[0033] 1.1 Isolation and purification of strain GZU03
[0034] A single colony with a larger blue circle and a darker color was selected on the geniposide chromogenic plate for purification and culture. After Gram-stained microscope examination was a single bacterial species, the slant was preserved and the glycerol was preserved for future use.
[0035] 1.2 Preparation of seed solution
[0036] Liquid seed medium (g / L): ammonium sulfate 2, magnesium sulfate 0.5, sodium chloride 2, calcium carbonate 2, potassium dihydrogen phosphate 0.5, ginkgo biloba extract 5; accurately weigh the above experimental drugs, fully dissolve and pack After sterilization, it was placed in a high-pressure steam sterilizer for moist heat sterilization for 20 minutes, and then placed in a sterile operating table for cooling;
[0037] 1.3 Raw material pretreatment of solid natt...
Embodiment 2
[0040] Preparation of crude enzyme solution: Use an inoculation loop to select 2 to 3 bacterial lawns on the slant in 1.1, inoculate it on the liquid seed medium in 1.3, and culture at 37°C and 180r / min for 18h. Then, it was inoculated on the fermentation enzyme production medium and cultivated for 72 hours, and it was shaken every 12 hours. Weigh 10 g of fermented natto and dissolve it in 40 mL of sterile physiological saline, leaching at 4°C for 24 hours, then mashed with a beater and centrifuged at 12,000 r / min for 10 minutes in a centrifuge, the supernatant is the solid-state fermentation Crude enzyme solution.
Embodiment 3
[0042] Determination of β-glucosidase and nattokinase activities
[0043] 3.1 Glucose standard curve drawing: draw 1mg / ml anhydrous glucose solution 0, 0.2mL, 0.4mL, 0.6mL, 0.8mL, 1.0mL into a stoppered graduated test tube, add water to 2mL, add 3mL of DNS reagent, mix it in boiling water Boil in medium for 10min, add water to make the volume to 15mL after cooling, and measure the absorbance at the wavelength of 540nm with a spectrophotometer. Take the absorbance as the ordinate and the amount of glucose as the abscissa, draw a standard curve, the linear regression equation is y=0.4657x-0.038, R 2 =0.992( figure 1 ).
[0044] 3.2 Determination of β-glucosidase enzyme activity: take a 15ml stoppered graduated test tube, add 1.8mL of substrate (1% ginkgo flavone glycosides), preheat at 50°C for 3min, add 0.2mL of enzyme solution 2, water bath at 50°C for 30min, add DNS reagent 3mL, boiling water bath for 10min, cooling to 15mL. Taking the inactivated enzyme solution as blank...
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