Salt-tolerant bacillus and application thereof
A technology of Bacillus and Bacillus, applied in the field of industrial microorganisms, can solve the problems of high application cost, increased dosage of enzyme preparations, inhibition, etc., and achieve the effect of good quality, effective removal and better flavor of soy sauce
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[0049] A preparation method of salt-tolerant protease, said preparation method comprising the steps of:
[0050] Fermentation is carried out with the above-mentioned bacillus as the fermentation strain, and the salt-tolerant protease is extracted from the obtained fermentation product.
[0051] In one example, the step of extracting the salt-tolerant protease comprises:
[0052] 1) centrifuging the fermentation product and collecting the supernatant;
[0053] 2) Add ammonium sulfate to the supernatant, let it stand, and collect the lower layer of turbid liquid,
[0054] 3) Centrifuge the lower turbid liquid to collect the precipitate.
[0055] In one example, in step 1), the centrifugation conditions include: the rotation speed is 11000rpm-13000rpm, the temperature is 2°C-5°C, and the duration is 8min-12min.
[0056] In one example, in step 2), the conditions for standing still include: a temperature of 2° C. to 5° C., and a duration of 50 minutes to 70 minutes.
[0057] I...
Embodiment 1
[0075] Embodiment 1, bacterial strain screening
[0076] 1. Isolation and purification of protease-producing strains:
[0077] Weigh 5g of Aspergillus oryzae solid-state fermented bean koji into a 50mL sterile centrifuge tube, add 20mL of sterile water, seal it, shake it with a vortex oscillator for 2min, then place it in a water bath at 80°C for 20min, then take it out and cool it quickly with tap water to room temperature;
[0078] Take 1mL of the cooled suspension and dilute it step by step with sterile water to 10 -1 、10 -2 、10 -3、 10 -4 、10 -5 Double concentration, draw 100μL 10 -5 The dilution solution of double concentration was evenly spread on the selective plate medium, and placed in a 37°C incubator to cultivate for 18h; wherein, the selective plate medium: skimmed milk powder 2wt%, agarose 2wt% and water 96wt%, 115 Sterilize at ℃ for 20 minutes;
[0079] Select a single colony with a large ratio of transparent circle to colony diameter.
[0080] 2. Enzyme ...
Embodiment 2
[0123] Embodiment 2, the extraction of protease
[0124] 1. Enzyme production fermentation:
[0125]Use an inoculation loop to pick the lawn of Bacillus halotolerant RD19 from the inclined surface or take 500 μL of the frozen bacterial solution from the glycerol cryopreservation tube, inoculate it into 7 mL of the seed medium, and incubate at 37°C and 180 rpm for 12 hours to obtain the seed solution;
[0126] Then inoculate the fermentation medium with an inoculation amount of 2wt%, culture at 37°C and 180rpm for 120h, collect the fermentation liquid, centrifuge at 12000rpm and 4°C for 10min at low temperature and high speed, and collect the supernatant to obtain the crude enzyme liquid.
[0127] Seed medium formula: tryptone 1wt%, yeast powder 0.5wt%, NaCl 1wt%, add water to 100wt%, sterilize at 121°C for 20min.
[0128] Fermentation medium formula: soybean flour 1wt%, bran 0.5wt%, corn flour 1wt%, add water to 100wt%, sterilize at 121°C for 20min.
[0129] 2. Protease extr...
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