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Enterobacter hormaechei bacteriophage YZU.P.A-5 and application thereof

A technology of YZU.P.A-5, Enterobacter hallii, applied in the field of Enterobacter hallii bacteriophage, bacteriostatic application, can solve the problem that there is no report on the application, and achieve the effect of efficient lysis activity, reducing pollution and controlling growth

Active Publication Date: 2020-12-18
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although Enterobacter hallii is an important harmful biological factor in animal breeding and food production, due to the lack of research on its biological characteristics at home and abroad, so far there has been no report on the phage inhibitor of Enterobacter hallii and its application

Method used

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  • Enterobacter hormaechei bacteriophage YZU.P.A-5 and application thereof
  • Enterobacter hormaechei bacteriophage YZU.P.A-5 and application thereof
  • Enterobacter hormaechei bacteriophage YZU.P.A-5 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Phage isolation and purification preparation

[0035] Phage isolation

[0036] Sewage samples were collected from farmers' market in Yangzhou, Jiangsu. Take 30mL sewage sample and put it into a 50mL centrifuge tube, centrifuge at 5000×g for 10min, take 5mL supernatant and add it to 5mL LB liquid medium, add 100μL Enterobacter hallii strain SYZU.2-5 in logarithmic growth phase, 37℃ , 100rpm shaker culture overnight; transfer the culture in the test tube to a sterile centrifuge tube, centrifuge at 5000×g for 10min at 4°C, and pass the supernatant through a 0.22μm filter membrane to obtain the phage stock solution.

[0037] With SM buffer (1L: NaCl 5.8g, MgSO 4 .7H 2 O 2.0g, 1M Tris-HCl (pH7.4) 50mL) carry out 10-fold gradient dilution to the phage stock solution by volume ratio, take 100 μ L of diluted phage suspension and 100 μ L of logarithmic growth phase Enterobacter hallii and mix at room temperature After 10 minutes, add 5 mL of LB semi-solid medium, mix well, a...

Embodiment 2

[0042] Characteristic Analysis of Phage YZU.P.A-5

[0043] Phage Morphological Characteristics

[0044] The microscopic morphological characteristics of phage were observed by transmission electron microscope. Using phosphotungstic acid negative staining method, with the membrane side of the copper mesh facing up, take 10 μL of the YZU.P.A-5 purified solution obtained in Example 1 (10 12 PFU / mL) was dropped on the copper grid, absorbed for 15 minutes, then blotted dry, took out the copper grid, and dried naturally for 2-3 minutes. Then add 2% phosphotungstic acid (PTA) aqueous solution to the copper mesh for staining, take it off after 2 minutes, absorb the water with absorbent paper, dry it in the air for 5 minutes, observe with a transmission electron microscope, and select a clear phage image for photo analysis. The microscopic morphology of bacteriophage YZU.P.A-5 is as follows: figure 2 As shown, the phage YZU.P.A-5 presents a symmetrical head, a diameter of about 77....

Embodiment 3

[0049] Inhibitory Effect of Bacteriophage YZU.P.A-5 on Biofilm of Enterobacter hallii

[0050] Take the culture of Enterobacter hallii in the logarithmic growth phase (mixed strain of Enterobacter hallii SYZU.2-5 and ATCC 700323, equal volume of bacteria liquid), dilute with LB medium, and adjust the final concentration to about 1×10 7 CFU / mL. The test was divided into P.A-5 treatment group, negative control group and blank group. YZU.P.A-5 treatment group: Add 150 μL concentration of 10 to each well of 96-well plate 7 CFU / mL Enterobacter hallii diluted bacteria liquid (strain SYZU.2-5 and ATCC 700323 mixed in equal volume) and 150 μL phage YZU.P.A-5 suspension (mixed with 10 12 PFU / mL YZU.P.A-5 purified liquid obtained by 10-fold dilution 5 PFU / mL, B treatment is 10 6 PFU / mL, C treatment is 10 7 PFU / mL, D treatment is 10 8 PFU / mL, E treatment is 10 9PFU / mL); Negative control group: Add bacterial solution (10 7 150 μL each of CF U / mL) and SM buffer; blank group: add 30...

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Abstract

The invention discloses enterobacter hormaechei bacteriophage YZU.P.A-5 and application thereof. The bacteriophage is identified to have unique genome structure characteristics, and belongs to a novelkind of bacteriophage; and the bacteriophage has been preserved in China Center for Type Culture Collection since June 15, 2020 under the preservation number of CCTCC NO: M 2020206. The bacteriophagedisclosed by the invention can efficiently crack enterobacter hormaechei so as to effectively inhibit enterobacter hormaechei in various matrices; and thus, the bacteriophage can be applied to preparation of a bacterial depressant for controlling enterobacter hormaechei pollution. The bacterial depressant prepared by the bacteriophage can effectively control growth of enterobacter hormaechei in culture media and food matrices, as well as effectively inhibit formation of enterobacter hormaechei mycoderm on surfaces of equipment, utensils and raw material solids; and moreover, the bacterial depressant is simple to prepare and convenient to use, as well as capable of reducing risks of enterobacter hormaechei propagation and food-borne diseases.

Description

technical field [0001] The invention belongs to the field of bioengineering, and in particular relates to an enterobacterium hallii bacteriophage YZU.P.A-5 and its antibacterial application as a biological antibacterial agent in food production and storage environments. Background technique [0002] Enterobacter hormaechei (Enterobacter hormaechei) belongs to Enterobacteriaceae (Enterobacteriaceae) intestinal flora 75 (unknown Enterobacter hybrid group, this group includes 23 strains, 75 is the flora code), is an important zoonosis The opportunistic pathogenic bacteria are widely distributed in human and animal intestinal tract and natural environment, and can cause animal and human infection under certain conditions. Studies have shown that the bacteria caused respiratory diseases in animals and sepsis cases caused by neonatal infections have broken out in many countries and regions, and the bacteria is gradually showing a trend of drug resistance. It is of great significa...

Claims

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Application Information

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IPC IPC(8): C12N7/00A01N63/40A01P1/00C11D3/38C11D3/48C12R1/92
CPCC12N7/00A01N63/40C11D3/381C11D3/48C12N2795/10121C12N2795/10131C11D2111/20C11D2111/14C11D2111/12
Inventor 杨振泉陈曹伟周文渊高璐郑香峰袁磊饶胜其
Owner YANGZHOU UNIV
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