Bacillus subtilis mutant strain for high yield of nattokinase and application thereof
A technology of Bacillus subtilis and mutagenic strains, applied in the field of microorganisms, can solve the problems of difficulty in meeting the standards of industrialized functional foods and medicines, low yield and activity of nattokinase, etc. The effect of high and good genetic stability
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Embodiment 1
[0029] Embodiment 1: Mutagenesis of high-yield nattokinase strain
[0030] The bacterial suspension of the laboratory's original starting strain (Bacillus subtilis JNFE0126, CGMCC No.18057) was treated under a 15W ultraviolet lamp at a distance of 20cm for 90s, and the ultraviolet mutagenized strain was obtained through the primary screening of the casein plate and the secondary screening of the fibrin plate, and then Under the irradiation of 400Gy 60Co-γ-rays, the bacterial suspension of the ultraviolet mutagenized strain was first screened on the casein plate and re-screened on the fibrin plate to obtain the mutagenized strain JNFE1126.
[0031]The whole genome of the starting strain and the mutagenized strain JNFE1126 were sequenced respectively by the sequencing method of the second generation + the third generation, that is, Illumina Hiseq+PacBio. Through sequence comparison, it was found that after mutagenesis, compared with the starting strain Bacillus subtilis JNFE0126...
Embodiment 2
[0034] Example 2: Application of Nattokinase Fermented by Mutagenized Strains
[0035] Adopt mutagenic strain JNFE1126 to cultivate and ferment, the specific method is:
[0036] The composition of the seed medium is 0.5% soybean peptone, 1% glucose, 0.5% NaCl, and the pH is 6.5-7.0.
[0037] Fermentation medium consists of 1% soybean peptone, 2% glucose, CaCl 2 0.02%, MgSO 4 0.05%, Na 2 HPO 4 0.2%, NaH 2 PO 4 0.1%, pH at 6.5-7.0.
[0038] (1) Seed liquid preparation:
[0039] Sterilize the spiked Erlenmeyer flask and culture medium, use an inoculation loop or shovel to pick an appropriate amount of bacteria on the slope, insert it into the prepared culture medium, put it in a shaker, and cultivate it at a constant temperature of 37°C for 24 to 36 hours.
[0040] (2) Fermentation culture:
[0041] The fermentation medium was sterilized, and after cooling down to 25°C to 35°C, 2% of the inoculation amount was inserted into the seed culture solution, placed in a shak...
Embodiment 3
[0043] Embodiment 3: Application of mutagenic strain fermented soybean milk
[0044] The mutagenic strain JNFE1126 is adopted for cultivation and fermentation, and the specific method is as follows:
[0045] (1) Seed liquid preparation:
[0046] Sterilize the spiked Erlenmeyer flask and culture medium, use an inoculation loop or shovel to pick an appropriate amount of bacteria on the slope, insert it into the prepared culture medium, put it in a shaker, and cultivate it at a constant temperature of 37°C for 24 to 36 hours.
[0047] (2) fermented milk fermentation:
[0048] Add a single soybean raw material, or a 4:1 blend of soybeans and millet, into 200mL of water, add 20g of soybeans and 5g of millet for beating, and after sterilization, add 2% of the seed bacteria liquid into the slurry, at 37°C, 200r / min fermented for 10 hours. After the fermentation, the enzyme activity and volatile basic nitrogen content in the soybean milk fermented from different raw materials were...
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