Bacillus amyloliquefaciens ba168 and its fermentation culture method and application
A technology for desolvating amyloliquefaciens and fermenting spores, which is applied to Bacillus amyloliquefaciens Ba168 and its fermentative cultivation and application fields, can solve problems such as crop diseases, industrial losses of traditional Chinese medicinal materials, and impact on crop yields, and achieves good environmental compatibility. Control effect, high biological activity effect
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Embodiment 1
[0037] Example 1:Isolation and purification of Bacillus amyloliquefaciens Ba168:
[0038] (1), soil sample collection:
[0039] Select multiple locations from the original forest soil in the deep mountains of the Qinling Mountains, take 3 to 15cm of soil from each location, mix it through a coarse sieve, and take 25g for separation;
[0040] (2), medium plate preparation:
[0041] Heat and melt NA medium and LB medium and pour it into a petri dish to cool to make NA medium and LB medium plates. The NA medium formula is: glucose 10g, agar 15g, beef extract 3g, yeast extract 1g, peptone 5g, water 1L, pH 6.8~7.0; LB liquid medium formula is: peptone 10g, NaCl 10g, water 1L , yeast extract 5g, pH 7.0;
[0042] (3), the separation of bacteria:
[0043] Plate inoculation was inoculated by smear method and mixed bacteria method.
[0044] Smearing method: Take 0.05ml of the above-mentioned 5 concentrations of soil dilutions, add them to the prepared NA and LB plates respectively...
Embodiment 2
[0048] Example 2: Identification of Bacillus amyloliquefaciens Ba168:
[0049] The identification process to the bacillus amyloliquefaciens Ba168 CGMCC No.6462 of embodiment 1 gained is:
[0050] (1) Morphological characteristics: the colonies on the LB medium are round, with obvious gloss on the surface, not smooth; the edges are irregular, diffuse, and the surface is wrinkled; the colonies are milky white, opaque, turbid; they have certain adhesion; The formula of the LB medium is: 10 g of peptone, 5 g of yeast extract, 10 g of NaCl, 18 g of agar, 1000 ml of water, and the pH is 7.0.
[0051] In the early stage of culture (5~12h), the colonies are colorless and transparent droplets, which are shiny; in the later stage (after 24h), they are white and opaque, relatively dim.
[0052] The thallus is rod-shaped, about 2 μm long, solitary, with peritoneal flagella, and produces spores. Gram stain was positive.
[0053] (2), physiological and biochemical experiment: by the ph...
Embodiment 3
[0061] Example 3: The cultivation method of bacillus amyloliquefaciens (Bacillus amyloliquefaciens) Ba168 of the present invention is:
[0062] (1), strain activation:
[0063] Medium: LB solid medium, the formula of the LB solid medium is: 5g of yeast extract, 10g of peptone, 5g of NaCl, 15-20g of agar, 1000ml of water, pH 7.4-7.6;
[0064] Culture conditions: 30°C, 48h;
[0065] Continuous transfer for 3 generations to ensure the purity of the strain, and then inoculated into the fermentation broth for use.
[0066] (2), preparation of seed solution (shake flask fermentation or eggplant bottle fermentation):
[0067] Culture medium: LB liquid medium, the formula of described LB liquid medium is: yeast extract 5g, peptone 10g, NaCl 10g, water 1000ml, pH 7.0 (flask volume 250ml, filling capacity 50ml; eggplant bottle volume 1000ml, filling Liquid volume 200-300ml);
[0068] Culture conditions: 30°C, 50h-60h;
[0069] Inoculation amount: 6~8%.
[0070] (3), seed tank fe...
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