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Enterobacter sakazakii phage EspYZU05 and purpose thereof

A technology of Enterobacter sakazakii and bacteriophage, applied in the field of Enterobacter sakazakii bacteriophage isolates, can solve the problems of dairy products production and processing industry hazards, enhanced resistance, difficult to remove, etc., to reduce food-borne diseases, inhibit bacteria Membrane formation, no toxic side effects

Active Publication Date: 2018-03-23
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, manufacturers often use chemical disinfectants to disinfect processing equipment, pipelines, and production environments to ensure product safety. However, when Enterobacter sakazakii grows in a natural environment, the capsule-like structure produced by Enterobacter sakazakii makes it very easy to Adsorbed on the surface of food, processing utensils, pipelines, etc. to form a biofilm, the resistance of the bacteria in the film to chemical disinfectants and other adverse environments is significantly enhanced, and it is difficult to be removed, resulting in continuous pollution and bringing serious consequences to the dairy production and processing industry. to seriously endanger

Method used

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  • Enterobacter sakazakii phage EspYZU05 and purpose thereof
  • Enterobacter sakazakii phage EspYZU05 and purpose thereof
  • Enterobacter sakazakii phage EspYZU05 and purpose thereof

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Effect test

Embodiment 1

[0032] Embodiment 1, phage isolation and purification preparation

[0033] Phage isolation

[0034]The sample of the present invention is collected from the sewage of Yangda Kangyuan Dairy Co., Ltd., Jiangsu Province. Take 30mL sample into a 50mL centrifuge tube, centrifuge at 5000×g for 10min, take 5mL supernatant and add it to 5mL nutrient broth liquid medium, add 100μL Enterobacter sakazakii CICC 21569 in the logarithmic growth phase at the same time, place at 37℃ Cultivate overnight on a shaker, and the next day, transfer the culture in each test tube to a sterile centrifuge tube, centrifuge at 5000×g for 10 minutes at 4°C, take the supernatant and pass it through a 0.22 μm microporous membrane to sterilize, and obtain the phage stock solution. It can be stored in a refrigerator at 4°C.

[0035] Dilute the phage stock solution with sterile SM buffer, take 100 μL of phage liquid with appropriate dilution and 100 μL of Enterobacter sakazakii CICC 21569 in the logarithmic g...

Embodiment 2

[0046] Embodiment 2, the inhibitory effect of phage EspYZU05 on Enterobacter sakazakii bacterial biofilm

[0047] Enterobacter sakazakii in the logarithmic growth phase was diluted with nutrient broth medium, and the final concentration was 1×10 7 CFU / mL, experimental group: add diluted bacterial solution (10 7 CFU / mL) and phage EspYZU05 suspension (10 5 PFU / mL, 10 8 PFU / mL) each 150μL; control group: add diluted bacterial solution (10 7 CFU / mL) and 150 μL of nutrient broth medium; blank group: add 300 μL nutrient broth medium to each well; incubate at 37°C for 24 hours; take out the 96-well plate, suck out the suspended bacteria, and wash the 96-well plate with sterile PBS 3 Once, blow dry to fully remove planktonic cells; add 200 μL of crystal violet staining solution with a concentration of 0.2% to each well, and stain for 30 minutes; suck out the staining solution, and thoroughly wash the 96-well plate with sterile PBS until the eluate is colorless, blow Dry; add 200 μ...

Embodiment 3

[0049] Embodiment 3, the bacteriostasis of bacteriophage EspYZU05 in culture medium

[0050] The prepared nutrient broth medium was divided into 20mL stoppered test tubes and sterilized at 121°C for 15min. Enterobacter sakazakii (10 5 CFU / mL) to inoculate 100 μL into 10mL medium, add 100 μL phage EspYZU05 suspension (10 7 PFU / mL), 100 μL of SM buffer was added to the control group, and the inoculum was placed in a 37°C incubator and a 25°C incubator for constant temperature cultivation. The culture was sampled 1mL every 6h, and the wavelength at 600nm was measured with a spectrophotometer. For the absorbance value, 3 parallels were set for each group, and the average value was used for analysis.

[0051] The result is as Figure 6 As shown, as the culture time prolongs, the OD of the control group 600nm Rapid growth, reaching 0.5, 0.25 and 0.1 at 24h (37°C) and 24h (25°C) respectively, while the OD of the experimental group added with EspYZU05 suspension 600nm Always kept...

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Abstract

The invention belongs to the field of bioengineering, and relates to an enterobacter sakazakii phage EspYZU05 and a purpose thereof. A collection serial number of the enterobacter sakazakii phage EspYZU05 is CCTCC (China Center for Type Culture Collection) NO: M 2016716. The invention also discloses bacteriostatic application of the phage as a food additive component or a biological bacteriostaticagent in food production and storage; the pollution caused by the amplification of enterobacter sakazakii in the production and processing processes of a dairy product particularly can be inhibited,and the safety and the sanitation of a product are guaranteed.

Description

technical field [0001] The present invention relates to a bacteriophage isolate of Enterobacter sakazakii and its antibacterial application in food production and storage as a food additive or biological antibacterial agent, especially capable of inhibiting the production and processing of dairy products by Enterobacter sakazakii The pollution caused by proliferation ensures the safety and sanitation of products. Belongs to the field of bioengineering. Background technique [0002] Enterobacter sakazakii is a foodborne pathogen that can cause neonatal meningitis, enterocolitis, and bacteremia. At present, many cases of Enterobacter sakazakii infection have been reported worldwide, and the mortality rate is as high as 50% to 80%. Infant formula milk powder is the main source of neonatal infection, and the bacteria is often isolated from other dairy products and their production environments. According to the contamination characteristics of Enterobacter sakazakii in dairy ...

Claims

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Application Information

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IPC IPC(8): C12N7/00A01N63/00A01P1/00A23C9/12C12R1/92
Inventor 杨振泉杨晓珺高璐饶胜其尹永祺
Owner YANGZHOU UNIV
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