Spray disinfectant containing phage composition in breeding environment and preparation method and application thereof

A bacteriophage and disinfectant technology, applied in the field of biological disinfection preparations, can solve the problem of not carrying out the spray disinfection test of bacteriophage pathogenic bacteria and the like

Inactive Publication Date: 2020-06-05
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the above-mentioned experiments are all simulated disinfection experiments conducted in the laboratory, without using lysing enzymes, and the phages also act alone, and the phages are isolated from hospital sewage, not in the farm environment. Spray Disinfection Test of Environmental Pathogenic Bacteria

Method used

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  • Spray disinfectant containing phage composition in breeding environment and preparation method and application thereof
  • Spray disinfectant containing phage composition in breeding environment and preparation method and application thereof
  • Spray disinfectant containing phage composition in breeding environment and preparation method and application thereof

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Experimental program
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Effect test

Embodiment 1

[0048] The cultivation of embodiment 1 bacteria

[0049] Streptococcus suis 7917, Staphylococcus aureus Pnb25, Escherichia coli Mc1061 and Escherichia coli E20 were streaked and inoculated onto BHI or LB solid culture dishes, incubated at 37°C and 150rpm for 17 hours, and selected single clones were inoculated into 10 mL of BHI or LB liquid In the culture medium, culture at 37°C and 150rpm constant temperature shaking for 17h. On the second day, the host bacteria in the liquid culture medium (the above-mentioned 4 strains of bacteria) were transferred to fresh BHI liquid culture medium (medium for Staphylococcus aureus Pnb25) or LB liquid medium ( Escherichia coli Mc1061, Escherichia coli E20 and Streptococcus suis 7917 medium), 37 ℃, 150rpm culture 2 ~ 4h to logarithmic growth phase (OD 600nm The absorbance value is 0.2, about l×10 8 CFU / mL).

Embodiment 2

[0050] The preparation of embodiment 2 bacteriophage Ecp2, E20-1, SLPW

[0051] The three phage liquids Ecp2, E20-1, and SLPW were serially diluted with BHI or LB medium, and serially diluted 10 1 ~10 8 . Use the double-layer agar plate method to observe the characteristics of the formed phage plaques, select a double-layer plate with uniform growth of phage plaques, select a single phage plaque and inoculate it into 1 mL of BHI or LB medium, incubate at 37°C for 2 to 3 hours, and incubate at 4°C , 12000rpm centrifugation for 10min, the collected supernatant was sterilized by filtration through a 0.22μm filter membrane and stored at 4°C. The liquid is properly diluted, and based on the double-layer agar plate method, the single phage plaque is purified repeatedly as above, and finally the size and shape of the phage plaque on the double-layer plate are consistent, and a single phage is obtained.

[0052] Phage titer (Plaque-forming unit, PFU) refers to the number of plaqu...

Embodiment 3

[0056] Embodiment 3 Preparation of Streptococcus suis lyase LY7917

[0057] Step 1: Induced expression of lyase LY7917. Select 10mL of overnight cultured positive clones and transfer them to 1L LB liquid culture medium bottle containing 50μg / mLKan, culture at 37℃, 180rpm constant temperature shaking for 2-4h to bacterial liquid OD 600 0.6 to 1.0. Add 10mL expression inducer IPTG (100mmol / L) to a final concentration of 1mmol / L, 37°C, 170rpm constant temperature shaking culture for 4h, IPTG-induced expression bacteria 1L washed with 10mM PBS (pH7.2), at 4°C, Centrifuge at 4800rpm for 30min, dissolve the precipitate in 20mL of pre-cooled lysis buffer Binding buffer (pH7.4), sonicate in ice bath, ultrasonic power 200W, work for 5s, interval of 15s, cycle 150 times. After crushing, the bacterial suspension was centrifuged at 4°C and 10,000rpm for 10min, the precipitate was discarded and the supernatant was taken, filtered through a 0.45μm filter to obtain the crude extract of t...

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Abstract

The invention discloses a spray disinfectant containing a phage composition in a breeding environment and a preparation method and application thereof. The spray disinfectant comprises phage or its composition with a total concentration greater than 1010PFU/mL and a streptococcus suis lyase LY7917 with a concentration of 100.0 [mu]g/mL according to a volume ratio of 0.1-100: 1. The phage is derived from a breeding environment and is selected from one or more of escherichia coli phage Ecp2, escherichia coli phage E20-1 and staphylococcus aureus phage. According to the spray disinfectant, a spray sterilization mode is adopted, the synergistic sterilization effect of phage and lyase can be effectively achieved, the total number of bacteria on the ground of an animal house, in air and in a feeding trough is remarkably reduced, the overall disinfection efficiency is superior to that of a chemical disinfectant glutaraldehyde decyl methylammonium bromide solution, and the spray disinfectant is an environment-friendly disinfectant for the livestock and poultry breeding environment.

Description

technical field [0001] The invention relates to a biological disinfection preparation, in particular to a spray disinfectant containing a culture environment bacteriophage composition, a preparation method and application thereof. Background technique [0002] With the intensification and scale of modern breeding, disease outbreaks caused by breeding environment pollution and bacterial infection are becoming more and more serious, endangering livestock production. At present, chemical disinfectants are usually used in livestock and poultry farming to control the spread of pathogenic bacteria through spray disinfection, but chemical disinfectants are highly toxic and may cause harm to animal bodies and pollute the environment, which restricts their use in livestock and poultry to a certain extent. Applications. Moreover, with the emergence of a large number of drug-resistant bacteria, it is becoming more and more difficult to control bacterial pollution in farms. In the face...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01N63/40A01N37/46A01N25/06A01P1/00C12N7/00C12N9/88A61L2/22A61L9/14A61L101/54C12R1/92
CPCA01N25/06A01N37/46A01N63/00A61L2/22A61L9/14A61L2202/25A61L2209/21C12N7/00C12N9/88C12N2795/00051
Inventor 严亚贤谢平林孙建和
Owner SHANGHAI JIAO TONG UNIV
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