CN109988753B - Freeze-drying protective agent for Klebsiella pneumoniae bacteriophage and preparation method and application thereof - Google Patents
Freeze-drying protective agent for Klebsiella pneumoniae bacteriophage and preparation method and application thereof Download PDFInfo
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Abstract
The freeze-drying protective agent for the Klebsiella pneumoniae bacteriophage comprises the Klebsiella pneumoniae phi YSZKA with the preservation number as follows: CCTCC M2018513, classified and named Klebsiella phase phi YSZKA. The composition comprises the following components in parts 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 of glycerol, wherein the phage is subjected to vacuum freezing by adding a freeze-drying agent, and the freeze-dried powder is stored in a headspace bottle at normal temperature or 4 ℃. The freeze-dried powder can be preserved for 90 days, the survival rate is not lower than 90%, and a preferable method for storing strains is provided for the transportation and application of the phage therapy in the fields of agriculture, medical treatment, food, environment and the like.
Description
Technical Field
The invention belongs to the technical field of phage freeze-drying, and particularly relates to a Klebsiella pneumoniae phage
(Klebsiella phage
) The freeze-drying protective agent and the preparation method and the application thereof.
Background
Klebsiella pneumoniae (klebsiella. pneumoniae) is a pathogenic bacterium widely existing in natural environment, and is easy to cause iatrogenic infection for people with low immunity such as old people, infants and the like. The Klebsiella pneumoniae is a relatively short and thick bacillus, is arranged singly, doubly or in a short chain, has no spores, flagella and thicker capsules, mostly has pili, is mainly present in intestinal tracts and respiratory tracts of human bodies, and can cause bronchitis, pneumonia, urinary system and wound infection, even septicemia, meningitis, peritonitis and the like. The food and agriculture organization/world health organization (FAO/WHO) of the United nations lists the pathogenic bacteria as one of the pathogenic bacteria in the infant formula milk powder, and the monitoring must be implemented in the finished milk powder and raw materials. The Klebsiella is generally easy to generate drug resistance to cephalosporins, aminosaccharides (streptomycin, gentamicin, kanamycin and the like), chloramphenicol, polymyxin and the like, and is one of hot spots in the field of medical research at present. Therefore, it is necessary to develop a biotechnological method for targeted inactivation of Klebsiella pneumoniae.
Bacteriophages (Phage) are a class of organisms that live by preying on living host bacteria exclusively, and are widely distributed in soil, water, air, and even on the body surface or in the intestinal tract of humans and animals, and their total amount is estimated to be 1031～1032. The applicant obtains a strain of special phage for resistant Klebsiella pneumoniae by sampling the antibiotic-contaminated soil in the earlier stage and separating and screening the antibiotic-contaminated soil in a laboratory by using a double-layer plate method
The bacteriophage has good prevention and treatment effect on antibiotic-resistant Klebsiella pneumoniae, and can be widely applied to the fields of air purifiers, disinfectants, food and feed and the like. However, the long-term preservation method of the Klebsiella pneumoniae phage needs further research and is one of the important difficulties. The common method for preserving the phage is a low-temperature preservation method, namely, the phage liquid is preserved at the low temperature of 4 ℃ to 20 ℃ to 80 ℃, but the survival rate of the phage preserved by the method is low, the preservation time is short, and the method is not beneficial to the practical application of the phage. Therefore, it is highly desired to develop a method capable of preserving phages for a long time.
The appearance of the vacuum freeze-drying method provides a brand new idea for solving the problems, the method is to mix the phage and the freeze-drying protective agent to prepare freeze-dried powder so as to maintain the microbial activity of the phage, but the characteristics of survival rate, acid and alkali resistance, heat resistance and the like of the freeze-dried phage are further researched to the maximum extent. Several phage lyoprotectants have been disclosed by relevant literature review and patent search, but no patents have been found concerning klebsiella pneumoniae phages. The closest to the present invention are three patents, application numbers: CN201710983707.2, CN201610415919.6 and CN201610422048.0 are a Salmonella typhimurium bacteriophage phi Sa-1 freeze-drying protective agent, a phage freeze-drying powder capsule preservation method and a phage freeze-drying preservation method respectively. Application No.: CN201710983707.2 provides a freeze-drying protective agent of Salmonella typhimurium bacteriophage phi Sa-1, which mainly comprises skimmed milk powder, konjac flour, sucrose, sodium glutamate, sodium thiosulfate, microcrystalline cellulose and Tris, wherein the survival rate of the bacteriophage phi Sa-1 is as high as 75 percent; application No.: CN201610415919.6 provides a capsule preservation method, namely phage liquid and phage preservation protective agent are mixed uniformly to prepare freeze-dried powder, the freeze-dried powder is sieved and then is subpackaged in a capsule shell for sealed preservation, and the method can preserve phage at room temperature and is convenient to transport; application No.: CN201610422048.0 provides a preservation method of bacteriophage, lactobacillus bacteriophage, mycobacterium bacteriophage, vibrio parahaemolyticus bacteriophage, which comprises mixing bacteriophage liquid with protective agent (skimmed milk, diatomite, sugar alcohol, DEAE-dextran, arabic gum, bovine serum albumin, maltodextrin, honey, raffinose, aluminum phosphate, sodium chloride, magnesium sulfate, calcium chloride, glycerol, Araexis A and water), preparing into lyophilized powder, and sealing the lyophilized powder in ampoule bottle under aseptic condition for preservation.
These patents all provide methods for freeze-drying preservation of phage, but in practical applications, the optimal preservation methods for different kinds of phage are different. In addition, the existing patents do not relate to the freeze-drying preservation method of the Klebsiella pneumoniae phage, and further development of the freeze-drying preservation method of the Klebsiella pneumoniae phage is urgently needed.
The main defects of the prior art are as follows: most of bacteriophage preparations commonly used in food and medicine fields in China are stored in a liquid form, and have the defects of high preparation cost, low survival efficiency, short storage time, complicated transportation process and the like, and most of the bacteriophage preparations or bacteriophage applications depend on foreign companies (such as Russian immunoproparamat Research Association company, Grougia Biochimpanham company, Phage Therapy Center and the like), aiming at different types of bacteriophages, the development of freeze-drying storage methods with independent intellectual property rights in China is urgently needed.
The main causes of defects are: in 1915, Frederick W.Tport of England scientist discovered a microbe, phage, which can lyse host bacteria and is considered as an effective method for killing pathogenic bacteria. But the development of antibiotics became more and more silent at that time, and the research of bacteriophage became the focus of research again until the 70 th 20 th century. In recent years, phage therapy has been applied to the fields of medical treatment, agriculture, food and the like, and many researchers of various countries research phages through separation and screening of phages and related mechanism research, and practical application of phages, particularly research on methods for freeze-drying and preservation thereof, is relatively little. Therefore, there is a need for technical development work to develop a cryopreservation method optimized for bacteriophages.
Disclosure of Invention
The technical problem to be solved is as follows: aiming at the defects of the prior art, the invention provides a freeze-drying protective agent for a Klebsiella pneumoniae bacteriophage and a preparation method and application thereof. The method screens out a Klebsiella pneumoniae bacteriophage with high sensitivity, high titer and short lysis time as an optimal strain by separating and purifying the specific bacteriophage of the Klebsiella pneumoniae. The phage obtained by screening and a protective agent are mixed by a vacuum freeze-drying method to prepare freeze-dried powder, the freeze-dried powder is stored in a jaw-top hollow bottle at low temperature, and the survival rate, heat resistance, acid and alkali resistance and other related biological characteristics of the freeze-dried powder are periodically measured, so that the optimal freeze-drying storage method of the Klebsiella pneumoniae phage is determined. The method can maintain the activity of the Klebsiella pneumoniae bacteriophage to the maximum extent, and has wide biological control effect and practical application value.
The technical scheme is as follows: the freeze-drying protective agent for the Klebsiella pneumoniae phage comprises the following components in parts by mass: 20-60 parts of skimmed milk powder, 10-50 parts of tryptone, 5-25 parts of glucose, 10-50 parts of sucrose, 1-5 parts of anhydrous magnesium sulfate, 1-5 parts of gelatin and 1-3 parts of glycerol.
Preferably, 40-60 parts of skimmed milk powder, 20-40 parts of tryptone, 10-20 parts of glucose, 10-30 parts of sucrose, 1-3 parts of anhydrous magnesium sulfate, 1-3 parts of gelatin and 1-2 parts of glycerol.
Further preferably, the milk powder comprises 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 of glycerol.
The phage is Klebsiella pneumoniae phage
The culture is preserved in China center for type culture Collection in 2018, 8 months and 1 days, and the preservation numbers are as follows: CCTCC NO: M2018513, classified and named Klebsiella phase
The address of the depository: china, wuhan university, zip code 430072.
The preparation method of the freeze-drying protective agent for the Klebsiella pneumoniae bacteriophage comprises the steps of mixing and sterilizing tryptone, glucose, sucrose, anhydrous magnesium sulfate, gelatin and glycerol according to a proportion, cooling, and adding skimmed milk powder according to a corresponding proportion.
The lyophilized protectant is used in Klebsiella pneumoniae bacteriophage
Application in freeze-drying preservation.
The working principle of the invention is as follows: 1. the bacteriophage is a micro organism which survives specific 'predation' host bacteria and can be divided into a lytic type and a lysogenic type; 2. the virulent phage can identify host bacteria cell membrane surface receptor protein in the environment migration process, the tail can be specifically adsorbed onto the cell membrane, the nucleic acid injects self DNA into the host bacteria through the hollow tail to execute the invasion process, then the phage DNA can quickly finish self nucleic acid replication and protein synthesis by utilizing the nucleic acid base pair and energy substances in the host, and then a large number of progeny phage are assembled and propagated in the bacteria, and cell wall muramidase is released, so that the host bacteria are cracked and die, the internal structure of the bacteria is damaged, and finally the cracking and releasing process is finished; 3. the vacuum freeze drying technology specifically comprises the steps of firstly freezing a sample at a low temperature, then sublimating redundant moisture of the sample in a vacuum sterile environment, and then analyzing and drying the sample, so as to achieve the final purpose of removing bound water; 4. the freeze-drying protective agent is a class of effective substances for protecting the basic properties and the original functions of the bacteriophage in the freeze-drying process and the storage stage after freeze-drying; 5. the selected phage is from natural environment, and is not subjected to any gene modification and environment-friendly.
Has the advantages that: 1. vacuum freeze drying and adding proper protective agent to ensure bacteriophage
The survival rate of the freeze-dried powder is not less than 90 percent, and the preservation time is more than 3 months; 2. bacteriophage
The freeze-dried powder protective agent has simple condition, low cost and preparation cost, convenient storage and transportation, simple and convenient use and operation, targeted inactivation and easy popularization; 3. freeze-drying protective agent formula for bacteriophage
The pH and heat resistance of the composition have positive promoting effects; 4. the survival rate is improved by 40.1 percent compared with the independent freeze-drying of the bacteriophage; 5. compared with the existing cryopreservation technology, the freeze-drying method can enhance the heat resistance and pH resistance of the bacteriophage. Bacteriophage of the present invention
The freeze-dried powder can be prepared into various forms of products such as feed additives, cleaning agents or disinfectants and the like, and can be widely usedInactivating the resistant Klebsiella pneumoniae in a temperature range and a pH condition. The method has wide application prospect in the repair work of the antibiotics-resistant Klebsiella pneumoniae pollution in the fields of medicine, food and the like in China.
Drawings
FIG. 1 shows a Klebsiella pneumoniae bacteriophage
Double-layer plate plaque-raising pattern;
FIG. 2 shows a Klebsiella pneumoniae bacteriophage
Transmission electron microscopy images of;
FIG. 3 shows a Klebsiella pneumoniae bacteriophage
A graph of the survival rate of the liquid at different temperatures;
FIG. 4 shows a Klebsiella pneumoniae bacteriophage
Survival plots for different preservation methods;
FIG. 5 shows a Klebsiella pneumoniae bacteriophage
Graph of survival rate of lyophilized powder at different temperatures.
Detailed Description
The following detailed description does not limit the technical solutions of the present invention in any way, and all technical solutions obtained by means of equivalent replacement or equivalent transformation fall within the scope of the present invention.
The bacteriophage
Is a prophase preserved bacteriophage which is specially used for 'predation' of Klebsiella pneumoniae, and the preservation number is as follows: CCTCC NO, M2018513, preservation date: 8, 1/8/2018, and named after classificationIs Klebsiella phase
Bacteriophage
An elliptical head and a contracted long tail can be seen, the head length is about 100nm, the transverse diameter is about 72nm, and the tail length is about 110 nm; the plaque is clear and transparent, has neat edges and is a circular spot without a halo, and the diameter of the plaque is about 1-2 mm; incubation period 22min, outbreak period 35 min; the optimal multiplicity of infection (MOI) was 0.001. Phage, according to the ninth report of the International Commission on viral Classification
Belonging to the family of Long-tailed bacteriophages (Siphoviridae bacteriophages).
The bacteriophage
The invention has certain defects in the preservation method, does not invent the preservation method of the Klebsiella pneumoniae bacteriophage, and aims at the bacteriophage according to the methods introduced in CN201710983707.2, CN201610415919.6 and CN201610422048.0 and the early research and accumulation
The biological characteristics of the raw materials and the optimal proportion of the protective agent are determined, and the mass parts of the protective agent are as follows: 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 of glycerol.
Wherein, the defatted milk powder, the tryptone, the glucose and the sucrose in the freeze-drying protective agent surround the liquid drop containing the phage in the suspension through hydrogen bond acting force, so that a layer of stable interface film for protecting the phage is formed, and the influence of temperature on the phage protein can be improved; the anhydrous magnesium sulfate can adjust the pH value in the freeze drying process to the stable state of the phage active substance; the gelatin and the glycerol can enhance the protection of the bioactive material in the aspects of physics and chemistry, greatly increase the viscosity of the freeze-dried sample and improve the stability of the sample.
And (3) after the freeze-dried powder is prepared, filling the freeze-dried powder into a 10mL jaw headspace bottle under an aseptic condition, sealing an aluminum cover, and storing at low temperature.
Example 1:
1. separation and purification of Klebsiella pneumoniae phage
The soil sample to be tested is collected from polluted soil around a beam dairy farm excrement accumulation pool in Nanjing City of Jiangsu province. Basic physicochemical properties of soil: 23.8 percent of sand grains, 45.4 percent of soil grains, 31.8 percent of clay grains, 7.7 percent of pH and 1.7 g/kg of total nitrogen-11.7 g.kg of water-soluble nitrogen-11.3 g.kg of total phosphorus-117.5 g/kg of total potassium-1，CEC 19.4cmol·kg-1。
Adding 10g of fresh soil sample into 100mL of sterile water, performing shaking culture at 30 ℃ and 250rpm for 5h, centrifuging at 8000rpm for 5min, sterilizing the supernatant with a 0.22 mu m filter membrane, adding 9mL of filtrate and 1mL of Klebsiella pneumoniae suspension growing to logarithmic phase into 40mL of 3 XBL liquid culture medium, and adding calcium chloride solid until the final concentration of the solution is 1 mmol.L-1Shaking-culturing at 30 deg.C and 250rpm for 12h to obtain culture solution at 8000rpm, centrifuging for 5min, and sterilizing with 0.22 μm filter membrane to obtain bacteriophage stock solution; screening phages by adopting a double-layer plate method, purifying, uniformly mixing 100 mu L of filtrate with 100 mu L of Klebsiella pneumoniae suspension, standing for 20min at room temperature, adding 5mL of 0.75% semisolid LB agar culture medium, uniformly mixing, flatly spreading and pouring onto an LB solid plate, culturing at 30 ℃ for 10-12 h, observing plaques, picking a single clear and transparent plaque with a clear edge into LB liquid containing host bacteria when the plaque (the diameter of the plaque in figure 1 is about 1-2mm), and culturing at 30 ℃ and 250rpm for 8 h; centrifuging at 8000rpm for 5min, sterilizing with 0.22 μm filter membrane, and naming the obtained phage as a strain
Stored in SM buffer at 4 ℃ and stored at low temperature.
2. Microbiological characterization of Klebsiella pneumoniae phages
For bacteriophage
And (5) observing by using an electron microscope. Dropping the phage stock on a copper net, negatively dyeing with 2% phosphotungstic acid with pH 7.0 for 90s, absorbing the excessive dye solution with filter paper, drying, and observing the form under Hitachi H-7650 type transmission electron microscope.
Bacteriophage
An elliptical head and a contracted long tail (FIG. 2) were seen, with a head length of about 100nm, a transverse diameter of about 72nm, and a tail length of about 110 nm; the plaque is clear and transparent, has neat edges and is a circular plaque without halo, the diameter of the plaque is about 1-2mm (figure 1), and the bacteriophage is reported according to the ninth report of the international committee for virus classification
Belonging to the family of Long-tailed bacteriophages (Siphoviridae bacteriophages).
Multiplicity of Infection (MOI), also known as phage titer, refers to the ratio of phage to host bacteria count before Infection occurs. Two phage strains are obtained based on the above operation, 100 μ L phage filtrate is taken, and 100 μ L log-phase host bacterial suspension is added according to the infection complex numbers of 100: 1, 10: 1, 1: 100, 1: 1000 and 1: 10000 respectively. Shaking and culturing for 5h at 30 ℃. Phage titers were determined for each group by the double-plate method (table 1). Bacteriophage
The optimal multiplicity of infection is 0.001.
TABLE 1 determination of optimal multiplicity of infection
The phage one-step growth curve was determined. According to the optimal multiplicity of infection, 500. mu.L of phage and 500. mu.L of host bacterial suspension are added into 9ml of LB liquid medium, cultured with shaking at 37 ℃ and 150rpm, sampled every 10min, centrifuged, filtered and the phage titer is determined by the double-layer plate method. MacrophageThallus
The incubation period of (1) is 22min, and the outbreak period is 35 min.
Example 2:
screening of lyoprotectants
1) The invention combines the existing components of the phage freeze-drying protective agent and the early research accumulation, and the invention can be used for the phage of the Klebsiella pneumoniae
The protective agent selected for freeze drying is preliminarily planned as follows: skimmed milk powder, peptone, glucose, sucrose, anhydrous magnesium sulfate, gelatin, and glycerol. When each protective agent is used independently, the pair
The freeze-drying protective effect verification research comprises the steps of respectively dissolving the freeze-drying protective agent in 1L of sterile water, sterilizing at 115 ℃ for 30min under high pressure, cooling, and mixing with the enriched phage
Filtrate 1: 1, mixing uniformly, and freeze-drying. The survival rate of the lyophilized powder was verified after being stored at room temperature for 15 days, and the results are shown in table 2.
TABLE 2 different protective agent pairs phage
Protective effect of
As can be seen from Table 2, the increase of phage was observed after 15 days of preservation with the addition of 8 lyoprotectants alone
The survival rate of (1). The freeze-drying protection effect is anhydrous magnesium sulfate>Peptone>Glycerol>Defatted milk powder>Sucrose>Gelatin>Glucose, among which anhydrous magnesium sulfate has relatively best protective effect, bacteriophage
The survival rate reaches 69.35 percent.
2) Further screening the protective effect of the combination of different protective agents, selecting anhydrous magnesium sulfate as a substrate according to the determined optimal concentration, namely 50g/L of skimmed milk powder, 30g/L of peptone, 10g/L of glucose, 20g/L of sucrose, 1g/L of anhydrous magnesium sulfate, 2g/L of gelatin and 1.5g/L of glycerol, sequentially adding the rest protective agents according to the sequence of freeze-drying protective effect, and verifying the protective effect of the combination of different protective agents on the bacteriophage
The protective effect of (1). The specific experimental operation is the same as 1).
TABLE 3 different combinations of protective agents against phages
Protective effect of
Note: "√" indicates the addition of this component; "-" indicates that the component was not added.
As shown in Table 3, the best results were obtained by mixing 8 types of protective agents at the optimum concentrations, and using phage in combination with the protective agent components gradually increased
The survival rate of the cells reaches 90.58 percent.
Example 3:
phage liquid
And (5) stability verification test.
Enrichment of phages according to the above method
After enrichment, 8000rpm, centrifuging for 5min, filtering with 0.22 μm filter membrane, and collecting
1mL of filtrate is subpackaged into 40 2mL centrifuge tubes under sterile conditions, and the centrifuge tubes are divided into four groups and are respectively preserved at room temperature, 4 ℃, 20 ℃ and 80 ℃. And titers were determined at 30d, 60d, 90d using the double plate method. The results are shown in FIG. 3.
As can be seen from FIG. 3, the survival rate of the phage was the lowest when the phage was stored at room temperature and-80 ℃ and after 90 days of storage, the phage was stored
The survival rates are 3.4% and 11%, respectively, and long-term storage is not recommended; the phage has relatively good preservation effect at 4 ℃ and-20 ℃, wherein the preservation effect at 4 ℃ is the best, and the phage is preserved at 90 days
The survival rate reaches 52.8 percent, and the phage is recommended to be used
Storing at 4 deg.C.
Example 4:
bacteriophage
Freeze-drying preservation test study
The existing phage storage method is used for research, and the combination of the Klebsiella pneumoniae phage
The biological characteristics of the invention, the independently researched and developed freeze-dried powder protective agent, mainly comprises the following components: 50g/L of skimmed milk powder, 30g/L of peptone, 10g/L of glucose, 20g/L of sucrose, 1g/L of anhydrous magnesium sulfate, 2g/L of gelatin, 1.5g/L of glycerol and 1L of deionized water. And verifying the comparison of the freeze-drying method and the liquid low-temperature method (4 ℃) on the phage
The effect is preserved.
Three sets of processing are set according to the above situation: treatment of one, bacteriophage
Storing the liquid at low temperature, and measuring 1mL of the enriched liquid by using a pipette
Filtrate (10)7pfu/mL) into 2mL centrifuge tubes, and preserving at 4 ℃; treating II, the bacteriophage
The liquid and the protective agent are mixed according to the proportion of 1: 1, uniformly mixing, and measuring 1mL of uniformly mixed phage by using a pipette gun
The liquid is stored in a 2mL centrifuge tube at 4 ℃; treating step three, mixing phage
The liquid and the protective agent are mixed according to the proportion of 1: 1, mixing uniformly, packaging into centrifuge tubes, pre-cooling at-20 deg.C, pre-heating vacuum freeze dryer (ALPHA 1-4/2-4LD plus, width x height x depth, 390x415x540 mm; freezing efficiency, 4kg/24h, Germany) for 30min, placing into freeze-dried sample, freeze-drying, and collecting bacteriophage
The lyophilized powder was dispensed into 10mL jar (diameter. times.height, 22.5X 46mm) and stored at 4 ℃. The above treatments were sampled every 15 days to determine titer and to calculate survival rate. Under the aseptic condition, 1mL of sterile water is injected into the jaw headspace bottle by a disposable syringe, the sterilized water is lightly shaken, the dissolved phage liquid is diluted, and the survival rate of the phage liquid is verified by a double-layer plate method. From the results of FIG. 4, it was found that the phages after addition of the protective agent (treatment two, treatment three) were more preserved at day 90 than when they were preserved at low temperature alone
Survival rate is obviously increased, and phage is treated in the third step
The highest survival rate of the filtrate after being prepared into dry powder is 93.3 percent, and the phage is verified to be protected by the protective agent provided by the invention
The activity of the compound has obvious retention effect, and provides a technical support for the application of the bacteriophage in the fields of agriculture, medical treatment, food and the like.
Example 5:
bacteriophage
Stability test of storage of powder
Phage were tested according to the test methods described above
Subpackaging the lyophilized powder into jaw headspace bottles in equal parts, respectively storing at room temperature, 4 deg.C and-20 deg.C, taking one bottle of lyophilized powder every 15 days, adding 1mL sterile water, slightly shaking for dissolving, and verifying bacteriophage by double-layer flat-copy method
Judging the most suitable condition for storing the freeze-dried powder. As can be seen from the results in FIG. 5, the activity of the lyophilized powder was relatively stable under the storage conditions of room temperature, 4 ℃ and-20 ℃, and the phage was
The survival rate is above 85%, wherein the effect is best when the product is stored at 4 deg.C, and the bacteriophage is
The survival rate was 93.4%. Illustrative phage
Is more suitable for being stored by freeze-dried powder at 4 ℃.
Example 6:
freeze-drying preservation method for bacteriophage
Heat resistance of
Verification of phage after lyophilization
Influence of heat resistance, two sets of treatments were set: treatment A freshly enriched liquid (10) was taken at a ratio of 1 mL/tube9pfu/mL) in 6 centrifuge tubes, standing the centrifuge tubes in water bath at 30 deg.C, 40 deg.C, 50 deg.C, 60 deg.C, 70 deg.C and 80 deg.C for 30min, diluting to appropriate concentration, and verifying phage by double-layer plate method
The potency of (a); taking 6 bottles of 4 deg.C phage lyophilized powder, dissolving in 1mL sterile water, standing in 30 deg.C, 40 deg.C, 50 deg.C, 60 deg.C, 70 deg.C, 80 deg.C water bath for 30min, diluting to appropriate concentration, and verifying phage by double-layer plate method
The potency of (A). Results Table 2 shows the phages after a water bath at 70 ℃ and 80 ℃ for 30min
The heat resistance of the freeze-dried powder (treatment II) is obviously enhanced, the titer is respectively reduced to 5.75 multiplied by 106 pfu/mL and 8.65 multiplied by 104pfu/mL, and the heat resistance is improved by 1-2 orders of magnitude compared with that of the filtrate (treatment I). The freeze-drying preservation method is proved to be helpful for improving the heat resistance of the bacteriophage phi YSZKA.
TABLE 4 phages
Titer (pfu/mL) after 30min water bath at different temperatures
Example 7:
freeze-drying preservation method for bacteriophage
Resistance to the influence of pH
Verification of phage after lyophilization
Resistance to the influence of pH, two sets of treatments were set: treatment A freshly enriched liquid (10) was taken at a ratio of 1 mL/tube9pfu/mL) in 6 centrifuge tubes, adjusting pH to 2, 4, 6, 8, 10, standing for 30min, diluting to appropriate concentration, and verifying phage by double-layer plate method
The potency of (a); processing two 6 bottles of 4 deg.C phage lyophilized powder, dissolving with 1mL buffer solution with adjusted pH, standing for 30min, diluting to appropriate concentration, and verifying phage by double-layer plate method
The potency of (A). The results are shown in Table 3, after standing for 30min in different pH systems, when the pH is 2-6, the tolerance capacity to acid and alkali of the second treatment phase is obviously enhanced compared with that of the first treatment phase, the titer of the phage of the second treatment phase is improved by 1-2 orders of magnitude, and when the pH is 8-10, the titer of the second treatment phase is slightly improved compared with that of the first treatment phase, namely, the titer is respectively 8.15 multiplied by 108 pfu/mL、4.75×107pfu/mL. The freeze-drying preservation method is proved to be helpful for improving the pH tolerance of the bacteriophage phi YSZKA.
TABLE 5 phages
Titers at different pH (pfu/mL)
The invention provides a Klebsiella pneumoniae bacteriophage
The freeze-drying preservation method proves that the preservation of the freeze-drying powder can effectively maintain and prolong the bacteriophage
The preservation time, the heat resistance, the acid and alkali resistance and the survival rate of the fertilizer are improved. The freeze-dried powder provided by the invention has good preservation effect at normal temperature and 4 ℃, and can be used as bacteriophage
Provides a technical support for transportation and application in the fields of agriculture, medical treatment, food, environment and the like.
Claims (1)
1. The freeze-dried powder of the Klebsiella pneumoniae bacteriophage is characterized by comprising the following components in parts 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, 1.5 parts of glycerol and phage; the phage is Klebsiella pneumoniae phage phi YSZKA, is preserved in China center for type culture Collection in 2018, 8 months and 1 days, and has the preservation number: CCTCC M2018513, classified and named asKlebsiellaphase φ YSZKA, deposit Unit Address: china, wuhan university, zip code 430072.
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| CN110438091B (en) * | 2019-07-12 | 2020-12-22 | 广西大学 | Novel Klebsiella pneumoniae phage and application thereof |
| CN113201458B (en) * | 2020-12-21 | 2022-05-27 | 青岛诺安百特生物技术有限公司 | Composition for improving stability of phage preparation and application thereof |
| CN114836337B (en) * | 2022-03-04 | 2024-04-02 | 广东省实验动物监测所 | Freeze-drying protective agent and freeze-drying powder for pasteurella pneumophila and preparation method thereof |
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