Vibrio mimicus phage QY-1 with variety-crossing cracking characteristic, preparation method and application thereof

A technology of mimetic Vibrio, OY-1, applied in the field of bioengineering, can solve the problem of limited mixed contamination effect of various Vibrio, and achieve the effect of reducing food pollution, reducing pollution and reducing food-borne diseases

Active Publication Date: 2019-03-26
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Usually, phages can only produce inhibitory effects against specific species of bacteria, and have limited effect on mixed contamination of ...

Method used

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  • Vibrio mimicus phage QY-1 with variety-crossing cracking characteristic, preparation method and application thereof
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  • Vibrio mimicus phage QY-1 with variety-crossing cracking characteristic, preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1, phage isolation and purification preparation

[0037] Phage isolation

[0038] The sample of the present invention is collected from the sewage of Shitasi farmer's market in Yangzhou, Jiangsu. Take 30 mL of sewage sample and put it into a 50 mL centrifuge tube, centrifuge at 8000×g for 10 min, take 4 mL of supernatant and add it to 5 mL of LBS liquid medium, and add 100 μL of Vibrio mimicus CICC 21613 in the logarithmic growth phase, placed on a 37°C shaker for overnight culture, the next day, transfer the culture in the test tube to a sterile centrifuge tube, centrifuge at 8000×g for 10 min at 4°C, and take the supernatant Pass through a 0.22 μm filter membrane to obtain the phage stock solution, and store in a refrigerator at 4°C.

[0039] Doubling dilution of the phage stock solution with sterile SM buffer, take 100 μL of the appropriate dilution of the phage solution and 100 μL of Vibrio mimicus CICC 21613 in the logarithmic growth phase, mix at room ...

Embodiment 2

[0052] Embodiment 2, the inhibitory effect of bacteriophage OY-1 in culture medium

[0053] Take 100 μL each of Vibrio mimicus CICC 21613, Vibrio parahaemolyticus CICC21617, and Vibrio alginolyticus isolate J1-4 in the logarithmic growth phase, add 8 mL of LBS liquid medium, and add phage OY-1 with an MOI value of 100 100 μL each (original bacteria + OY-1); with the same volume of LBS liquid medium as the control (original bacteria), cultured at a constant temperature at 25°C, measured the absorbance at 600 nm after 6 hours, and set 3 parallels for each group , taking the average value to make a histogram, such as Figure 5 As shown, the bacteriophage OY-1 was effective against Vibrio mimicus CICC21613 (OD value decreased from 0.39 to 0.05), Vibrio parahaemolyticus CICC21617 (OD value decreased from 0.53 to 0.09) and Vibrio alginolyticus J1-4 (OD value decreased from 0.47 down to 0.1) have a certain inhibitory effect on the growth in the culture medium.

Embodiment 3

[0054] Embodiment 3, the inhibitory effect of phage minimal vibrio bacteriophage OY-1 to minimal vibrio biofilm

[0055] Take Vibrio mimicus CICC 21613 in the logarithmic growth phase and dilute it with LBS medium, and the final concentration is 1×10 7 CFU / mL, experimental groups (groups A, B, C, D, E, F): add diluted bacterial solution (10 7 CFU / mL) and phage OY-1 suspension 10 9 PFU / mL, 10 8 PFU / mL, 10 7 PFU / mL, 10 6 PFU / mL, 10 5 PFU / mL, 10 4 150 μL for each PFU / mL; control group: add diluted bacterial solution (10 7CFU / mL) and 150 μL of LBS medium each; blank group: add 300 μL of LBS medium to each well; incubate at 37°C for 24 h; remove the 96-well plate, suck out the suspended bacteria, and wash the 96-well plate 3 times with sterile PBS , blow dry, and fully remove planktonic cells; add 200 μL of 0.2% crystal violet staining solution to each well, and stain for 30 min; suck out the staining solution, and thoroughly wash the 96-well plate with sterile PBS u...

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Abstract

The invention relates to a vibrio mimicus phage QY-1 with a variety-crossing cracking characteristic, a preparation method and an application thereof. The separated vibrio mimicus phage is named as vibrio mimicus phage QY-1 (the preservation number is CCTCC NO: M2017588). The vibrio mimicus phage QY-1 has efficient variety-crossing cracking activity to vibrio mimicus, vibrio parahemolyticus and vibrio alginolyticus. A phage culture and a preparation thereof can serve as a biological bacteriostatic agent to control breeding of pathogenic vibrio and formation of a biofilm, so that the risk of pathogenic vibrio pollution of aquatic food and in a breeding processing environment is reduced.

Description

technical field [0001] The invention relates to a vibrio mimic phage with cross-species lysis characteristics, and its antibacterial application as a biological antibacterial agent, water body purifier and feed additive in aquatic product breeding, storage, transportation and processing environments, reducing pathogenicity Pollution caused by Vibrio, to ensure food safety and hygiene, belongs to the field of bioengineering. Background technique [0002] Vibrio is a kind of Gram-negative bacilli widely distributed in the world, and it is one of the most common, widespread and harmful bacterial pathogens in aquaculture. With the development of aquaculture, vibriosis The outbreaks of the disease are becoming more frequent and spreading faster and faster, which has brought huge economic losses to the aquaculture industry all over the world. Among the currently discovered Vibrio species, Vibrio parahaemolyticus ( Vibrio parahemolyticus ), Vibrio alginolyticus ( Vibrio alginol...

Claims

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Application Information

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IPC IPC(8): C12N7/00A01N63/02A01P1/00A23K10/18A23L5/20C12R1/93
CPCA23K10/18A23L5/28C12N7/00C12N2795/00021A01N63/10
Inventor 高璐杨振泉欧阳敏李熠胡钦饶胜其
Owner YANGZHOU UNIV
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