Salmonella broad-spectrum virulent bacteriophage as well as preparation method and application thereof

A Salmonella broad-spectrum potent, Salmonella technology, applied in the field of microorganisms, can solve the problems of inability to achieve broad-spectrum treatment, weak acid resistance of bacteriophages, loss of efficacy, etc., and achieve the effects of reducing animal mortality, improving flora, and low production costs.

Active Publication Date: 2021-04-16
山东仙普爱瑞科技股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, there are also obvious limitations in the practical application of phages.
(1) Due to their strong host selectivity, most phages have a narrow host spectrum and cannot achieve broad-spectrum therapeutic effects like antibiotics
(2) At present, phage preparations are mostly used orally to control Salmonella, but phages are weak in acid resistance, and will be inactivated due to high acidity when passing through gastric acid, thus losing their curative effect

Method used

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  • Salmonella broad-spectrum virulent bacteriophage as well as preparation method and application thereof
  • Salmonella broad-spectrum virulent bacteriophage as well as preparation method and application thereof
  • Salmonella broad-spectrum virulent bacteriophage as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1 Isolation of Salmonella broad-spectrum virulent phage XPAR_CPS02

[0046] Sample treatment: collect the feces and litter of sick chickens with salmonellosis from chicken farms, soak them in physiological saline overnight in a 37 °C incubator, filter out excess impurities with gauze, and filter them with a 0.22 μm microporous membrane before use to obtain Processed samples.

[0047] Preparation of host bacteria: Pick a single colony of Salmonella pullorum virulent strain C79-13 and inoculate it in 100 mL of LB broth. Shake culture at 180 r / min at 37°C for 16-18 h to obtain a suspension of host bacteria for later use.

[0048] Proliferation of phage: Take 10 mL of the above-mentioned treated sample and add it to the above-mentioned Erlenmeyer flask containing the bacterial suspension of the host bacteria. After mixing, shake and culture in an air bath shaker at 37°C at 160 rpm overnight to obtain a phage proliferation solution.

[0049] Phage isolation: After t...

Embodiment 2

[0053] Example 2 Morphological observation of bacteriophage XPAR_CPS02

[0054] Using phosphotungstic acid negative staining method, immerse the copper mesh in 20 μL ultracentrifugation and resuspended phage suspension (purified phage filtrate in the preservation step) for 10 minutes, and absorb excess liquid with filter paper. The copper mesh was then stained with phosphotungstic acid dye for 10 min, and allowed to dry naturally. The morphology of the phage was observed under a transmission electron microscope, and its size was measured with the software DigitalMicrograph Demo 3.9.1.

[0055] Phage XPAR_CPS02 presents typical characteristics of a lytic phage, with translucent plaques, neat and clear edges, no halos, and a plaque diameter of 2.85 mm, see figure 1 . The resulting phage was scanned by a transmission electron microscope, and the results showed that under the electron microscope, the phage XPAR_CPS02 had a regular polyhedral head (about 60nm in length and about ...

Embodiment 3

[0056] Example 3 Determination of the host spectrum of bacteriophage XPAR_CPS02

[0057] The test selects Salmonella standard strains, multidrug-resistant strains, different serotype strains and other species strains to determine the host spectrum of phage XPAR_CPS02. The multidrug-resistant strains are preserved by our company's microbiology experiment and are mainly isolated from sick chickens in various chicken farms. Specific steps are as follows:

[0058] The host spectrum was determined by spot method. Take 100 µL of the above-mentioned test strains cultured to the logarithmic phase, add them to 5 mL of LB broth medium containing 0.7% agar after cooling to 50 °C, mix well and pour into the pre-prepared NA medium plate as the bottom layer, After it was solidified, 5 µL of phage was dropped on the center of the plate, dried and cultured upside down at 37°C overnight to observe whether there were lysis spots. The experimental results are shown in Table 1.

[0059] The exp...

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Abstract

The invention provides a salmonella broad-spectrum virulent bacteriophage. The preservation number of the bacteriophage is CCTCC NO: M 2020839. The invention also provides a preparation process and application of the salmonella broad-spectrum virulent bacteriophage, and the preparation process comprises fermentation and extraction; in the step of fermentation, a salmonella gallinarum attenuated vaccine strain is used as host bacteria for bacteriophage fermentation. The bacteriophage XPARCPS02 disclosed by the invention can be used for cracking salmonella of various serotypes and also has different degrees of cracking effects on drug-resistant bacteria; the salmonella typhimurium lysate lysate has a cracking capability on salmonella typhimurium, salmonella typhimurium drug-resistant bacteria, salmonella gallinarum, salmonella enteritidis, salmonella enteritidis drug-resistant bacteria, salmonella typhimurium, salmonella pullorum, salmonella paratyphi A, salmonella paratyphi B and salmonella dubicularis.

Description

technical field [0001] The invention relates to the field of microorganisms, in particular to the preparation of Salmonella broad-spectrum virulent phage and the application of the mixed preparation with Bacillus licheniformis. Background technique [0002] salmonella( Salmonella ) belonging to the family Enterobacteriaceae, is a common Gram-negative bacterium and an important foodborne pathogen. There are many serotypes of Salmonella, about 2600 are currently known, and all of these serotypes can cause salmonellosis. In my country, the positive rate of Salmonella in chickens reaches 30%-50%, which has caused great economic losses to the breeding industry of our country. Antibiotics are widely used in the prevention and control of animal diseases due to their quick effect and low price. However, with the abuse of antibiotics, multidrug-resistant pathogens and even superbugs have emerged, and the problem of bacterial resistance has also been listed by the World Health Organ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00A61K35/76A61K35/742A61P31/04A61P1/12C12R1/92
CPCY02A50/30
Inventor 李琦王兴业韩国英刘刚王晓冉王茂超郭海岩
Owner 山东仙普爱瑞科技股份有限公司
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