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Vibrio bacteriophage and bactericidal composition preparation method and application thereof

A bacteriophage and composition technology, applied in the biological field, can solve problems such as antibiotic residues, antibiotic control failures, and lack of scale

Active Publication Date: 2019-01-15
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Research on fish and shrimp bacterial diseases in mariculture shows that the main pathogens are Vibrio, among which Vibrio harveyi, Vibrio alginolyticus and Vibrio parahaemolyticus Vibrio parahaemolyticus is the main pathogenic bacterium, which mainly manifests as ulcer disease in fish, and acute hepatopancreatic necrosis syndrome in shrimp, with a mortality rate as high as 80%. The outbreak of this disease has caused huge economic losses to the aquaculture industry. loss
At present, antibiotics, traditional Chinese medicine preparations, and disinfectants are still used in the prevention and treatment of vibriosis. However, due to the imperfection of the traditional breeding model and the limited use of antibiotics, Vibrio has frequent drug resistance and multi-drug resistance. characteristics, leading to the failure of antibiotic prevention and treatment, serious antibiotic residues in seafood, not only causing economic losses, but also seriously threatening human health

Method used

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  • Vibrio bacteriophage and bactericidal composition preparation method and application thereof
  • Vibrio bacteriophage and bactericidal composition preparation method and application thereof
  • Vibrio bacteriophage and bactericidal composition preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Embodiment 1, phage preparation

[0036] Phage purification

[0037]Take 2 mL of fresh cultures of Vibrio alginolyticus, Vibrio harveyi and Vibrio parahaemolyticus, centrifuge, resuspend in 0.4 mL of 2216E medium, add 0.1 mL of phage in turn (according to the 1:1, 1:10 and 1:100 ratios). Incubate at 30°C for 30min to make the phage particles adsorb to the host bacteria; add 100mL LB medium, and culture at 30°C for 6-8h; take the above culture at 13000×g / min, 4°C, centrifuge for 30min, and take the supernatant; Then filter the supernatant with a 0.22 μm filter membrane to form a phage suspension. Add RNase A and DNase I to 1 μg / mL, incubate at 37°C for 30 minutes; add 9.3g PEG 8000, 5.8g NaCl, shake well until dissolved, keep in ice bath for 1 hour or overnight at 4°C; centrifuge at 10000×g / min for 20 minutes at 4°C, remove Supernatant solution; add 2mL SM solution, fully wash the tube wall and precipitate, and react at room temperature for 1h; add an equal volume of ...

Embodiment 2

[0041] Embodiment 2, in vitro bactericidal effect of bacteriophage

[0042] Take Vibrio alginolyticus, Vibrio harveyi and Vibrio parahaemolyticus cultured to the logarithmic growth phase, and measure their OD 600 value, adjust them to OD 600 About 0.4. Then take 2ml of the correspondingly diluted bacterial solution and 100ul with a titer of about 10 9 Pfu / ml of phage lysate was mixed and allowed to stand at room temperature. Measure OD from 0h 600 Value, measured every 0.5h, measured to 5h.

[0043] The result is as image 3 As shown: the phages can effectively inhibit the corresponding host bacteria, and the concentration of the host bacteria decreased rapidly after 1.5 hours of action, and dropped to about 0.2 at 3 hours, while the concentration of the host bacteria in the control group had risen to above 1.0 at this time; after 5 hours of action, The mixed solution added with phage was obviously clarified, while the control solution was turbid.

Embodiment 3

[0044] Embodiment 3, bacteriophage composite in vitro bactericidal effect

[0045] Take Vibrio alginolyticus, Vibrio harveyi and Vibrio parahaemolyticus cultured to the logarithmic growth phase, and measure their OD 600 value, adjust them to OD 600 about 0.4, and mixed 1:1:1; then take 100 μl of each of the three phages and mix them in 1:1:1 (the titer of each phage is about 10 9 pfu / ml); Take 2ml of the mixed bacterial solution and 300μl of the mixed phage, mix them upside down, and let stand at room temperature. Measure OD from 0h 600 Value, measured every 0.5h, measured to 5h.

[0046] The result is as Figure 4 As shown: the phages can effectively inhibit the corresponding host bacteria. After 1.5 hours of action, the number of host bacteria decreases, and the OD value begins to decline. After 3 hours of action, it has dropped to about 0.3, while the concentration of host bacteria in the control group has risen to above 1.38. ; After 5 hours of action, the mixed solut...

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Abstract

The invention relates to a Vibrio bacteriophage and a bactericidal composition preparation method and application thereof, belonging to the field of biotechnology. The bacteriophage composition comprises Vibrio alginolyticus bacteriophage vB_ValS_PcR-1 (accession number being CCTCC NO: M 2018391), Vibrio harveyi vB_VhaM_PcB-1G (accession number being CCTCC NO: M 2018392) and Vibrio parahaemolyticus phage vB_VhaP_OW (accession number being CCTCC NO: M 2015577). A high-potency culture product can be obtained after host actions, the composition is good in stability at room temperature, has a widehost splitting range, can efficiently inhibit growth of main pathogenic vibrios such as vibrio alginolyticus, vibrio harveyi, vibrio parahaemolyticus, can be applied as a biological antimicrobial agent for vibrio contamination control in food and aquaculture.

Description

technical field [0001] The invention relates to a preparation method of Vibrio phage and its bactericidal composition and its antibacterial application in food and aquaculture as an antibacterial preparation, especially capable of specifically cracking various Vibrio pollution in food and aquaculture. Belongs to the field of biotechnology. Background technique [0002] In recent years, aquaculture, especially marine aquaculture, has become the main production method to increase the global supply of aquatic products. With the continuous increase of the scale and density of aquaculture, diseases frequently appear in the process of cage aquaculture, which has brought great harm to the development of large-scale aquaculture. Great threat. Studies on bacterial diseases of fish and shrimp in mariculture have shown that the main pathogens are Vibrio, among which Vibrio harveyi, Vibrio alginolyticus and Vibrio parahaemolyticus Vibrio parahaemolyticus is the main pathogenic bacteri...

Claims

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Application Information

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IPC IPC(8): C12N7/00A61K35/76A61P31/04A01N63/00A01P1/00A23L3/3571A01K61/00A01K61/13C12R1/92
CPCA01K61/00A01N63/00A23L3/3571A61K35/76A01K61/13A61P31/04C12N7/00C12N2795/10221C12N2795/10231C12N2795/10232C12N2795/10251Y02A40/81
Inventor 张辉王冉庞茂达孙利厂
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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