Bdellovibrio bacteriovorus freeze-dried powder preparation and application thereof

A technology for phagocytosing leech vibrio and freeze-dried powder is applied in the application, bacteria, biocide and other directions, and can solve problems such as unfavorable large-scale use of leech vibrio preparations, lack of leech vibrio function, and decreased infectivity of pathogenic bacteria, etc. To solve the problem of phage monogeneity and environmental pollution, ensure phage diversity, and facilitate growth and reproduction.

Active Publication Date: 2019-08-30
XIAMEN HUIYING ANIMAL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Through the investigation of Bdellovibrio related patent documents, it was found that most freeze-dried Bdellovibrio preparations are generally cultured with live host bacteria Escherichia coli, which has several defects: on the one hand, it is necessary to separate unlysed Escherichia coli after the cultivation, which is cumbersome to operate; on the other hand On the one hand, if the li

Method used

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  • Bdellovibrio bacteriovorus freeze-dried powder preparation and application thereof
  • Bdellovibrio bacteriovorus freeze-dried powder preparation and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 Bdellovibrio Bdellovibrio Isolation Test in Seawater

[0040] This example is used to illustrate the source of Bdellovibrio and its acquisition method of the present invention.

[0041] Take seawater samples and sediment from the coastal waters of Jimei District, Xiamen, mix them well and centrifuge at 3000r / min for 5min, take the supernatant, put 100mL in 250mL triangular flasks, and add 10 8 CFU / mL of Vibrio parahaemolyticus and Vibrio alginolyticus enrichment culture, 30 ℃, 100r / min aerobic culture for 48h. After the enrichment culture, seawater agar double-layer plates were used for separation and detection, and Vibrio parahaemolyticus and Vibrio alginolyticus were used as host bacteria for testing. The plates were cultured at 30°C for 2 days, and multiple transparent phage plaques appeared. Pick multiple upper layer of phage plaque culture medium and add a small amount of sterilized seawater, vortex and oscillate, so that the solid small pieces of cultur...

Embodiment 2

[0042] Example 2 Bdellovibrio phage spectrum test

[0043] This example is used to illustrate the phage spectrum and test method of the bacteriophage Bdellovibrio of the present invention.

[0044] Escherichia coli, Salmonella, Staphylococcus aureus, Vibrio alginolyticus, Vibrio vulnificus, Vibrio parahaemolyticus, Vibrio harveylia, Aeromonas hydrophila, Pseudomonas aeruginosa, Elizabeth mir, respectively, prepared The resulting bacterial suspension is the host bacterium, and the lysing experiment is performed after diluting the suspension of Example 1. Using seawater double-layer agar plate method, do 3 parallels for each gradient, culture at 30°C for 2-3 days, and observe whether there are phage plaques formed on the plate.

[0045] The cracking effect of table 1 Bdellovibrio to each host bacterium

[0046]

[0047] Note: "+" indicates that Bdellovibrio has the ability to lyse the host bacteria, and "-" indicates that Bdellovibrio has no ability to lyse the host bacteri...

Embodiment 3

[0048] Example 3 Preparation of Bdellovibrio Bacteria Liquid

[0049] This example is used to illustrate the bacteriophage Bdellovibrio liquid provided by the present invention and its preparation method.

[0050] The Bdellovibrio phage described in this example is provided by Example 1.

[0051] 1. Preparation of inactivated host bacteria concentrate: inoculate Vibrio alginolyticus, Vibrio parahaemolyticus, Aeromonas hydrophila, and Pseudomonas aeruginosa respectively in 10g / L peptone, 3g / L beef extract, 10g / L Sodium chloride, pH 7.0-7.5 fermentation broth, after aerobic culture at 30°C for 24 hours, the sludge was collected by centrifugation, mixed and placed in a water bath at 60°C for 30 minutes, cooled for later use.

[0052] 2. Preparation of bacteriophage Bdellovibrio bacteria liquid: take the uninactivated mixed host bacteria in the above step 1 and dilute them with sterilized seawater, so that the concentration of bacteria is 10 9 CFU / mL was inoculated with Bdellovi...

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Abstract

The invention discloses a bdellovibrio bacteriovorus freeze-dried powder preparation. The preparation is prepared by the following steps: culturing bdellovibrio bacteriovorus by adopting a plurality of mixed live host bacteria; performing filtration to remove host pathogenic bacteria so as to obtain a bdellovibrio bacteriovorus seed liquid; culturing the seed liquid by using a plurality of mixed inactivated host bacteria; adding a freeze-drying protective agent into the obtained bdellovibrio culture solution, and performing uniform mixing; adding a freeze-drying carrier, and performing mixingto obtain a preparation to be freeze-dried; and finally performing freeze-drying to obtain the preparation. The invention also discloses a preparation method and application of the preparation. The preparation disclosed by the invention has a lysis effect on various aquatic pathogenic bacteria, is efficient and safe, solves the problems of phage singleness and environmental pollution of the bacteriophage bdellovibrio generated by taking single active pathogenic bacteria as host bacteria in the prior art, ensures the phage diversity of the bacteriophage bdellovibrio, and improves the lysis activity of the bacteriophage bdellovibrio.

Description

technical field [0001] The invention relates to the technical field of mariculture, in particular to a bacteriophage Bdellovibrio freeze-dried powder preparation and its application. Background technique [0002] The quality of aquaculture water often determines whether aquatic animals can grow healthily, and the infestation of various pathogenic bacteria in the water body will reduce the disease resistance of aquatic animals and even lead to death. Commonly used water disinfection generally uses chemical agents or antibiotics. Long-term use will cause environmental water pollution and continuously enhance the drug resistance of pathogenic bacteria. [0003] Bdellovibrio phage is a type of bacteria that parasitizes other bacteria and can cause their lysis, mainly Gram-negative bacteria, such as Escherichia coli, Salmonella, Pseudomonas aeruginosa, Aeromonas hydrophila and Vibrio, etc. Negative pathogenic bacteria, and harmless to humans and animals. In recent years, researc...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12N1/04A01N63/00A01P1/00C02F3/34C12R1/01C02F103/20
CPCA01N63/00C02F3/34C02F2103/20C12N1/04C12N1/20
Inventor 林松泉黄榕林章秀李惠静庄建军
Owner XIAMEN HUIYING ANIMAL TECH
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