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Clostridium perfringens phage, bacteriostatic agent containing phage, preparation method and application

A technology of Clostridium perfringens and bacteriophage, applied in the field of bioengineering, can solve the problems of economic loss, control failure, economic loss and other problems in the livestock and poultry breeding industry, and achieve the effect of preventing and controlling animal intestinal diseases without toxic side effects

Active Publication Date: 2020-09-15
JIANGSU ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the production process of poultry products, Cp is a common bacterium in the intestinal tract of healthy poultry. When the growth environment and diet change, the bacteria will proliferate in large quantities and attach to the intestinal mucosa to produce a large amount of toxins and proteases, causing intestinal mucosal damage. Thus causing diseases such as diarrhea and enteritis, seriously affecting animal production performance and causing huge economic losses
At present, due to the long-term prevention and control of animal necrotic enteritis by adding antibiotics and other growth-promoting agents to the feed, the continuous emergence of drug-resistant strains leads to the failure of control and brings huge economic losses to the livestock and poultry industry.
So far, there are no records and reports on the use of Clostridium perfringens phages to inhibit Clostridium perfringens

Method used

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  • Clostridium perfringens phage, bacteriostatic agent containing phage, preparation method and application
  • Clostridium perfringens phage, bacteriostatic agent containing phage, preparation method and application
  • Clostridium perfringens phage, bacteriostatic agent containing phage, preparation method and application

Examples

Experimental program
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Effect test

Embodiment 1

[0034] Example 1 Isolation, preparation and purification of Clostridium perfringens phage

[0035] 1. Phage isolation

[0036] The sample of the present invention is collected from poultry farm feces, the sample is added to peptone solution, shaken at room temperature for 2 to 4 hours, and the supernatant is taken at 5000×g.min -1 Centrifuge for 30 min, and filter the supernatant with a 0.22 μm filter membrane. Take 1ml of the supernatant and add it to 50ml of TY medium, then add 1ml of ATCC 13124 strain (hereinafter referred to as Cp) overnight culture (BHI overnight culture), and then place it at 37°C and shake it at 150rpm overnight. .min -1 , 4°C, centrifuge for 30 min, take the supernatant; then filter the supernatant with a 0.22 μm filter membrane to form a phage stock solution.

[0037] Take 2ml of the overnight culture and spread it on a 20ml TY agar plate. After absorption, absorb 0.01ml of the phage stock solution and drop it on the surface of the plate; after it ...

Embodiment 2

[0044] Example 2, phage vB_CpeS_JS01 host lineage detection

[0045] Divide the TY plate into several areas, draw the overnight culture of different host bacteria: ATCC 13124, CMCC67422, CMCC 67423 and CMCC 67424 0.1ml drop on the TY plate, spread the bacterial liquid evenly, after it dries, take the phage Stock solution 0.1ml, diluted 10 times, take 10 2 、10 4 and 10 6 0.01ml of the dilution solution was added dropwise to plates coated with different host bacteria, and left to dry naturally. After anaerobic culture at 37°C for 12 hours, the lysis of the phage was observed.

[0046] The results showed that phage vB_CpeS_JS01 had transparent lysis circles for Cp strains CMCC67422, CMCC67423 and CMCC 67424, that is, the phages were all positive for Cp strains.

Embodiment 3

[0047] Embodiment 3, bacteriophage vB_CpeS_JS01 is to the cracking effect of Cp

[0048] Adjust the overnight cultured Cp bacterial solution to OD with TY medium 600 About 0.1 to 0.2, add phage vB_CpeS_JS01 in the bacterial liquid (MOI is 0.1 and 1 respectively), place in 37 ℃ incubator for anaerobic culture, set up negative control at the same time, only add TY medium, respectively at 0h, 2h, 4h , after 6h, 8h, and 10h, take 0.2ml to detect OD 600 value, compare the OD after adding phage 600 Variety.

[0049] The result is as image 3 As shown, after acting at 37°C for 8 hours, compared with the control group, the OD in the phage group 600 The value changed significantly, after 4 hours of action, compared with the control, adding OD in the phage group 600 After 10 hours, the control group grew to 0.54, while the OD value in the MOI0.1 group slightly increased to 0.14, and the OD600 in the MOI1 group was only 0.1. It can be seen that the phage has a significant lytic eff...

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Abstract

The invention provides a clostridium perfringens phage, a bacteriostatic agent containing the phage as well as a preparation method and application of the bacteriostatic agent, and belongs to the technical field of bioengineering. The clostridium perfringens phage is preserved in China Center for Type Culture Collection with a preservation number of CCTCC NO: M 2020047. The phage has a strong cracking effect on clostridium perfringens, and can be used for inhibiting and / or removing clostridium perfringens. The clostridium perfringens phage can effectively prevent and control animal intestinaldiseases by inhibiting excessive growth of the clostridium perfringens. The invention provides a safe bacteriophage antibacterial product source without toxic and side effects for preventing and controlling animal intestinal diseases caused by clostridium perfringens of the livestock and poultry.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a Clostridium perfringens phage, a bacteriostatic agent including the phage, a preparation method and an application. Background technique [0002] Clostridium perfringens (Cp), also known as Clostridium welchii, is a Gram-positive anaerobic bacterium widely distributed in soil, feed, sewage, animal digestive tract and feces, and is the cause of animal necrosis. It is an important foodborne pathogen of necrotic enteritis (NE), enterotoxemia, food poisoning and traumatic gas gangrene in humans. In the production process of poultry products, Cp is a common bacterium in the intestinal tract of healthy poultry. When the growth environment and diet change, the bacteria will proliferate in large quantities and attach to the intestinal mucosa to produce a large amount of toxins and proteases, causing intestinal mucosal damage. Thereby causing diseases such as diarrhea and enteri...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00A61K35/76A61P31/04C12R1/92
CPCC12N7/00A61K35/76A61P31/04C12N2795/10121C12N2795/10151C12N2795/10132
Inventor 张辉包红朵王冉庞茂达吴立婷
Owner JIANGSU ACAD OF AGRI SCI
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