Lyoprotectant for Klebsiella pneumoniae phage, preparation method and application thereof

A Klebsiella and phage technology, applied in the field of phage freeze-drying, can solve the problems of relatively few researches on phage freeze-drying preservation methods, no involvement of Klebsiella pneumoniae phage, cumbersome transportation process, etc., and achieve cost-effective preparation Inexpensive, easy to promote, and widely used

Active Publication Date: 2021-03-02
INST OF SOIL SCI CHINESE ACAD OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, the existing patents have never involved the freeze-drying preservation method of Klebsiella pneumoniae phage, and further development of the freeze-drying preservation method of Klebsiella pneumoniae phage is urgently needed
[0006] The main defects in the prior art are: the commonly used phage preparations in the field of food and medicine in my country are mostly stored in liquid form, which has the disadvantages of high preparation cost, low survival efficiency, short storage time, cumbersome transportation process, etc., and the phage preparation or phage application Most of them rely on foreign companies (such as Immunopreparat Research ProductiveAssociation of Russia, Biochimpharm of Georgia and Phage Therapy Center, etc.), and for different types of bacteriophages, it is urgent to develop freeze-dried preservation methods with my country's independent intellectual property rights
In recent years, phage therapy has been gradually applied to fields such as medical treatment, agriculture, and food. Scholars from various countries have studied phages mostly on the isolation and screening of phages and related mechanism research. Relatively small

Method used

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  • Lyoprotectant for Klebsiella pneumoniae phage, preparation method and application thereof
  • Lyoprotectant for Klebsiella pneumoniae phage, preparation method and application thereof
  • Lyoprotectant for Klebsiella pneumoniae phage, preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] 1. Isolation and purification of Klebsiella pneumoniae phage

[0029] The soil samples for the test were collected from the polluted soil around the excrement accumulation pool of the Hengliang Dairy Farm in Nanjing City, Jiangsu Province. Basic physical and chemical properties of soil: 23.8% sand, 45.4% soil, 31.8% clay, pH 7.7, total nitrogen 1.7g kg -1 , water-soluble nitrogen 1.7g·kg -1 , total phosphorus 1.3g·kg -1 , total potassium 17.5g·kg -1 , CEC 19.4cmol·kg -1 .

[0030] Take 10 g of fresh soil sample, add 100 mL of sterile water, culture at 30°C, 250 rpm for 5 h, centrifuge at 8000 rpm for 5 min, filter the supernatant through a 0.22 μm membrane, take 9 mL of the filtrate and 1 mL of Klebsiella pneumoniae grown to the logarithmic phase Add 40mL 3×LB liquid medium to the bacteria suspension, add solid calcium chloride to the final concentration of 1mmol·L -1 , 30°C, 250rpm shaker culture for 12h, the obtained culture solution was 8000rpm, centrifuged for...

Embodiment 2

[0039] Screening of lyoprotectants

[0040] 1) Combining the existing components of the phage freeze-drying protective agent and the accumulation of previous research, the present invention is effective against Klebsiella pneumoniae phage Preliminarily, the selected protective agents for freeze-drying are: skimmed milk powder, peptone, glucose, sucrose, anhydrous magnesium sulfate, gelatin, glycerin. When each protectant is used alone To verify the effect of freeze-drying protection, the above-mentioned freeze-drying protectants were dissolved in 1L of sterile water, sterilized by autoclaving at 115°C for 30 minutes, cooled and mixed with the enriched phage The filtrate was mixed 1:1 and freeze-dried. After storing the lyophilized powder at room temperature for 15 days, its survival rate was verified, and the results are shown in Table 2.

[0041] Table 2 Different protective agents against phage protective effect

[0042]

[0043] It can be seen from Table 2 that ...

Embodiment 3

[0050] Phage liquid Stability verification test.

[0051] Enrichment of phages as described above After enrichment, centrifuge at 8000rpm for 5min, filter through a 0.22μm filter membrane, and take 1 mL of the filtrate was dispensed into 40 2 mL centrifuge tubes under aseptic conditions, and the centrifuge tubes were divided into four groups and stored at room temperature, 4°C, -20°C, and -80°C. And at 30d, 60d, and 90d, the potency was determined by double-plate method. see results image 3 .

[0052] Depend on image 3 It can be seen that the survival rate of phage is the lowest when stored at room temperature and -80°C. After 90 days of storage, the phage The survival rates are 3.4% and 11%, respectively, and it is not recommended for long-term storage; when stored at 4°C and -20°C, the preservation effect of phage is relatively good, and the preservation effect at 4°C is the best. The survival rate reached 52.8%, suggesting that the phage Store at 4°C low tem...

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Abstract

The invention relates to a freeze-drying protective agent for klebsiella pneumoniae bacteriophage and a preparation method and application of the agent. The klebsiella pneumoniae bacteriophage phi YSZKA is involved, the preservation number is CCTCC M 2018513, and the klebsiella pneumoniae bacteriophage is named as Klebsiella phage phi YSZKA through classification. The freeze-drying protective agent is prepared from, by mass, 50 parts of skimmed milk powder, 30 parts of tryptone, 10 parts of glucose, 20 parts of sucrose, 1 part of anhydrous magnesium sulfate, 2 parts of gelatin and 1.5 parts ofglycerinum. The bacteriophage is subjected to vacuum freezing by adding the freeze-drying agent, and freeze-dried powder is placed in a top empty bottle for storage at the normal temperature or 4 DEGC. After the freeze-dried powder is preserved for 90 days, the survival rate of the freeze-dried powder is higher than or equal to 90%, and an optimal selection method for strain storage is providedfor transportation and application of a bacteriophage therapy in the fields such as agriculture, medical treatment, food and environments.

Description

technical field [0001] The invention belongs to the technical field of phage freeze-drying, in particular to a Klebsiella pneumoniae phage (Klebsiella phage ) lyoprotectant and its preparation method and application. Background technique [0002] Klebsiella pneumoniae (Klebsiella.Peneumoniae) is a pathogenic bacterium that widely exists in the natural environment, and it is easy to cause iatrogenic infection to the elderly, infants and other people with low immunity. Klebsiella pneumoniae is a short and thick bacillus, arranged alone, in pairs or in short chains, without spores, without flagella, with thicker capsules, most of which have pili, and mainly exists in the human intestine Tract, respiratory tract, can cause bronchitis, pneumonia, urinary system and trauma infection, and even sepsis, meningitis, peritonitis and so on. The Food and Agriculture Organization of the United Nations / World Health Organization (FAO / WHO) listed it as one of the pathogenic bacteria in ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/00
CPCC12N7/00C12N2795/00011
Inventor 叶茂赵远超黄丹张忠云孙明明蒋新
Owner INST OF SOIL SCI CHINESE ACAD OF SCI
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