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Bacillus velezensis DH82 and preparation method and application of antibacterial protein thereof

A technology of Bacillus and DH82, applied in the field of preparation of Bacillus velesi DH82 and its antibacterial protein, can solve the problems of no Bacillus velesi and few prevention and control patents, etc., to improve immunity and protect the body. The effect of healthy growth and good application prospects

Inactive Publication Date: 2019-05-17
THIRD INST OF OCEANOGRAPHY STATE OCEANIC ADMINISTATION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there are very few patents on the application of Bacillus velaisi to the prevention and control of bacterial diseases in aquaculture
At present, there is no patent report on Bacillus Velez in this regard

Method used

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  • Bacillus velezensis DH82 and preparation method and application of antibacterial protein thereof
  • Bacillus velezensis DH82 and preparation method and application of antibacterial protein thereof
  • Bacillus velezensis DH82 and preparation method and application of antibacterial protein thereof

Examples

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Effect test

preparation example Construction

[0037] (2) Preparation of fermentation supernatant: Inoculate the activated bacterial liquid in step (1) into the improved LB medium at 1-5% (V / V), 25-40°C, 150-220rpm, 10-35h, All fermentation broths were centrifuged at 5000-12000rpm, 4°C for 10-30min, and the supernatant was filtered through a 0.22μm microporous membrane to obtain the fermentation supernatant;

[0038] (3) Preparation of antibacterial protein: in step (2) fermentation supernatant 50 ~ 2000mL, add solid ammonium sulfate to make solution ammonium sulfate saturation reach 40 ~ 100%, 4 ℃ overnight, centrifuge 20 ~ 30min at 6000 ~ 10000rpm to get The precipitate was dissolved in ultrapure water, dialyzed with a 3000Da dialysis bag for 24-48 hours, and freeze-dried to obtain the antibacterial protein.

[0039] The LB medium component used in the embodiment of the present invention is: 1% peptone, 0.5% yeast powder, 1% sodium chloride, pH7.3; the improved LB medium component is: 1% tryptone, 0.5% yeast powder, 0.5...

Embodiment 1

[0041] Embodiment 1: the preparation of antibacterial protein

[0042] (1) Fermentation supernatant: take Bacillus Velez DH82 and inoculate it on the improved LB medium, 25°C, 150rpm, 16h, then inoculate the improved LB medium according to the inoculation amount of 5% (V / V), After 35 hours at 25°C and 220rpm, all the fermentation broth was centrifuged at 5000rpm and 4°C for 30min, and the supernatant was filtered through a 0.22μm microporous membrane to obtain the fermentation supernatant;

[0043] (2) Antibacterial protein: Add solid ammonium sulfate to the fermentation supernatant to make the ammonium sulfate saturation of the solution reach 40%, overnight at 4°C, centrifuge at 6000rpm for 30min to obtain a precipitate, dissolve it in ultrapure water, dialyze with a 3000Da dialysis bag for 24h, freeze-dry get antimicrobial protein.

Embodiment 2

[0044] Embodiment 2: antibacterial protein preparation

[0045] (1) Fermentation supernatant: take Bacillus Velez DH82 and inoculate it on the improved LB medium, 40°C, 220rpm, 8h, inoculate the improved LB medium according to 1% (V / V) inoculum, 40 ℃, 150rpm, after 10h, all the fermentation broth was centrifuged at 12000rpm, 4℃ for 10min, and the supernatant was filtered through a 0.22μm microporous membrane to obtain the fermentation supernatant;

[0046] (2) Antibacterial protein: Add solid ammonium sulfate to the fermentation supernatant to make the ammonium sulfate saturation of the solution reach 100%, overnight at 4°C, centrifuge at 10000rpm for 20min to obtain a precipitate, dissolve it in ultrapure water, dialyze with a 3000Da dialysis bag for 48h, freeze-dry get antimicrobial protein.

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Abstract

The invention discloses a bacillus velezensis DH82 which is deposited on December 4, 2018 in China General Microbiological Culture Collection Center with the preservation number of CGMCC No. 16865. The method further discloses a preparation method of an antibacterial protein thereof by using the bacillus velezensis DH82 and the application of the antibacterial protein in an aquaculture feed additive. The antibacterial protein can effectively prevent and control diseases of aquaculture animals caused by aquatic pathogenic bacteria, thereby protecting the healthy growth of aquatic animals. The protein can be used as a feed additive for fish farming to significantly improve the immunity of the aquatic animals and has a good application prospect.

Description

technical field [0001] The invention relates to a microbial strain and application thereof, in particular to a preparation method and application of a Bacilus velezensis DH82 and an antibacterial protein thereof. Background technique [0002] In recent years, with the rapid development of marine fishery and aquaculture, the purification ability of aquaculture water in my country has weakened, the water environment has been polluted, and pathogenic bacteria have proliferated, which has greatly increased the chance of cross-infection of pathogens among aquatic animals. Edwardsiela tarda, Aeromonas hydrophila, Vibrio campbelii and other aquatic bacterial pathogens often infect fish in the breeding process of sea and freshwater fish skin and gills, causing sepsis, posing a greater threat to the aquaculture industry and causing economic losses. Therefore, it is imminent to seek safe, efficient and ecological control methods. [0003] For the prevention and control of bacterial ...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12P21/00A23K20/147A23K50/80C12R1/07
CPCY02A40/70
Inventor 徐长安王青华黄仕新唐旭罗曼
Owner THIRD INST OF OCEANOGRAPHY STATE OCEANIC ADMINISTATION
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