CN113201458A - Composition for improving stability of phage preparation and application thereof - Google Patents
Composition for improving stability of phage preparation and application thereof Download PDFInfo
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Abstract
The invention relates to a composition for improving stability of a phage preparation and application thereof. Specifically discloses a composition for improving the stability of a phage preparation, which comprises the following components: 6g/L of sodium chloride, 20-35g/L of magnesium sulfate, 0.1g/L of dipotassium phosphate, 0.1g/L of sodium dihydrogen phosphate, 3-8mL/L of gelatin, 0.05-0.2g/L of potassium sorbate, 0.1g/L of glycine, 20mL/L of glycerol and the balance of water. Also disclosed is the use of said composition for the preparation of a phage preparation. Compared with other protective agent formulas, the formula of the composition of the invention enables the phage preparation product to keep long-time activity and higher titer, can enable the phage preparation product to maintain longer-time stability under the conditions of thermal acceleration environment and room temperature, and greatly reduces the cost of transportation and storage processes.
Description
Technical Field
The invention belongs to the field of biotechnology and microbial products, and particularly relates to a composition for improving stability of a phage preparation and application thereof.
Background
The problems of drug resistance, ecological environment, drug residue in food and biological health and safety caused by abuse of antibiotics cause extensive attention of the world, and corresponding laws and regulations or measures are also issued by the government of China to promote the nonreactive process. The research and development of novel antibiotic substitutes become an urgent major scientific problem to be solved in animal husbandry. Bacteriophage may play an important role as a naturally occurring bacterial "killer" in the "nonreactive" era. Phages were used for the treatment of human infections as early as 1917, but with the advent of antibiotics in the 40's of the 20 th century, the research on the therapeutic value of phages was essentially shelved. Phages are predators of bacteria, destroying half of the world's bacteria every 48 h. Therefore, in recent years, the research on the use of bacteriophage as an antibiotic substitute has attracted general attention, and particularly, the application of bacteriophage in the fields of medicine, cultivation, food safety, and the like has attracted wide attention of domestic and foreign scholars, and has become one of the current research hotspots. The application of the phage preparation products in food processing and preservation, animal feed additives, animal breeding and human clinical application is sequentially appeared, so that the development prospect of the phage is very wide.
However, in the application process of the bacteriophage, the dosage is large, most of the bacteriophage exists in the form of liquid preparation, and a preservation protective agent is often required to be added to ensure the activity of the bacteriophage. The existing protective agent has poor bacteriophage preservation effect, short bacteriophage preservation time, low survival rate and easy pollution of the bacteriophage, and the bacteriophage must be preserved under low temperature and ultralow temperature conditions, so that the development of the bacteriophage is greatly limited, and therefore, the bacteriophage preservation technology is an important technical problem which restricts the wide application of the bacteriophage.
Disclosure of Invention
The purpose of the invention is as follows: in order to overcome the technical problems, the invention provides a composition for improving the stability of a phage preparation, which increases the storage stability of the phage preparation product in a non-low temperature environment and improves and prolongs the storage period of the phage preparation product.
One aspect of the invention provides a composition for improving the stability of a phage preparation: comprises the following components in percentage by weight:
in the specific technical scheme of the invention, the paint comprises the following components in percentage by weight:
in another aspect of the invention, a composition for improving the stability of a phage preparation is provided: the composition consists of the following components:
in the specific technical scheme of the invention: the anti-aging agent consists of the following components,
in a specific embodiment of the present invention, the gelatin is gelatin with a concentration of 2%.
In a specific embodiment of the present invention, the glycerol is 60% glycerol.
In a specific technical scheme of the invention, the water is deionized water or purified water.
In a particular embodiment of the invention, the pH of the composition is from 6.8 to 7.5.
In another aspect, the present invention provides a phage combination preparation characterized by comprising a phage and the composition of any one of claims 1 to 8;
in a specific technical scheme of the invention, the phage is a phage or a combination of multiple phages;
in a specific embodiment of the present invention, the phage is selected from the group consisting of coliphage, salmonella phage, pseudomonas aeruginosa phage, and vibrio parahaemolyticus phage.
In another aspect, the present invention provides a method for preparing the composition, which comprises the following steps:
1) dissolving sodium chloride, magnesium sulfate, gelatin, dipotassium hydrogen phosphate and sodium dihydrogen phosphate in water, and mixing uniformly;
2) adding glycine and potassium sorbate into the solution obtained in the step 1), adding glycerol, and mixing uniformly;
3) adjusting the pH value of the solution obtained in the step 2) to 6.8-7.5, and then sterilizing to obtain the product.
In another aspect, the invention provides the use of said composition for the preparation of a phage preparation;
in a specific technical scheme of the invention, the composition as defined in any one of claims 1-8 and a phage preparation are uniformly mixed according to a volume ratio of 1:1 to obtain a phage combination preparation;
in a specific technical scheme of the invention, the bacteriophage is one or a combination of multiple bacteriophages; the bacteriophage includes Escherichia coli bacteriophage, Salmonella bacteriophage, Pseudomonas aeruginosa bacteriophage and Vibrio parahaemolyticus bacteriophage.
As an embodiment, the present invention provides a method of preparing a composition for phage preparation stability, the method comprising:
1) dissolving the sodium chloride, the magnesium sulfate, the gelatin, the dipotassium phosphate and the sodium dihydrogen phosphate in the water, heating to 20 ℃, and uniformly mixing by magnetic stirring for later use;
2) adding glycine and potassium sorbate with the above component contents into the solution obtained in step 1), adding glycerol with the component contents, and magnetically stirring for 25 min;
3) adjusting the pH of the solution obtained in the step 2) to 7.2, and then sterilizing the solution for 20min at 121 ℃ by high-pressure steam to obtain a composition with stability of the phage preparation;
as an embodiment, the invention also provides a phage preparation comprising a composition and a phage, wherein the volume of the composition and phage is 1: 1.
As an experimental scheme, the invention also provides a method for preparing a phage preparation product, the method comprising: and (3) uniformly mixing the composition and the phage preparation according to the volume ratio of 1:1 under the aseptic condition to obtain a phage preparation product with stability.
The compositions of the present invention may be used in different types of phage preparation products, including, for example, but not limited to, E.coli phage preparation products, Salmonella phage preparation products, Pseudomonas aeruginosa phage preparation products, Vibrio parahaemolyticus phage preparation products.
The invention has the beneficial effects that: screening a combination of a plurality of liquid protective agents; the experiment of accelerating at 37 ℃, the experiment of preserving at 4 ℃ and the experiment of preserving at 25 ℃ prove that the phage preparation has good stable protection effect on the phage preparation product in the composition, compared with other protective agent formulas, the formula of the composition of the invention ensures that the phage preparation product keeps long-time activity and higher titer, the phage preparation product can maintain longer-time stability under the conditions of thermal acceleration and room temperature, and the cost of the transportation and storage process is greatly reduced. The components of the composition of the present invention act synergistically to produce unexpected technical results.
Detailed Description
The invention will be further elucidated by the following specific examples, which are intended to be illustrative only and do not limit the scope of the invention in any way.
EXAMPLE 1 screening of Components of the compositions of the invention
Selecting 12 common protective agent addition components to respectively carry out single-factor experiments: preparing according to the concentration in the table 1, sterilizing, and arranging 13 groups of a control group 1 without adding a protective agent; wherein the percentage of each protective agent in 1-12 groups is g/L, and the balance is deionized water.
Table 113 phage protectant component concentrations and sterilization methods thereof
And (3) uniformly mixing the protective agent components in the table 1 with the coliphage in a volume ratio of 1:1 under an aseptic condition to obtain the coliphage preparation. And placing the mixture at 37 ℃ for a constant-temperature accelerated storage test, detecting the titer of the mixture, and comparing the storage effect of each component.
The results are shown in table 2, at the storage temperature of 37 ℃, the titer of the sucrose, the tween-80 and the dimethyl sulfoxide is reduced by 1 order of magnitude after the storage for 2 days, the titer is reduced by 3 orders of magnitude when the sucrose, the tween-80 and the dimethyl sulfoxide are stored for 10 days, and the phage is inactivated when the sucrose, the tween-80 and the dimethyl sulfoxide are stored for 30 days, wherein 3 components are not selected from the composition; except for the control, the titer of the glycerol, the sodium chloride, the dipotassium phosphate, the sodium dihydrogen phosphate, the gelatin, the potassium sorbate, the glycine and the magnesium sulfate is reduced by 1 order of magnitude when the mixture is stored for 10 days, and the titer is reduced to 6 times when the mixture is stored for 30 days, thereby meeting the screening requirement. Wherein the effects of magnesium sulfate, gelatin and potassium phosphate are better than those of the screened components.
In summary, the components of the screening of the stability composition of the phage preparation were: glycerol, sodium chloride, dipotassium phosphate, sodium dihydrogen phosphate, gelatin, potassium sorbate, glycine and magnesium sulfate.
Table 237 temperature constant temperature accelerated test of Escherichia coli phage preparation potency (pfu/mL)
| Protectant Components | Initial | 2 days | 5 days | 10 days | 30 days |
| Sucrose | 1.90±0.23×1010 | 7.10±0.18×109 | 5.60±0.13×108 | 7.60±0.13×107 | - |
| Tween-80 | 1.90±0.14×1010 | 5.20±0.12×109 | 2.20±0.12×108 | 1.23±0.13×107 | - |
| Dimethyl sulfoxide | 1.90±0.25×1010 | 4.45±0.09×109 | 5.85±0.32×108 | 3.60±0.13×107 | - |
| Glycerol | 1.90±0.12×1010 | 1.76±0.17×1010 | 1.05±0.21×1010 | 5.30±0.13×109 | 1.50±0.25×106 |
| Sodium chloride | 1.90±0.15×1010 | 1.60±0.21×1010 | 1.02±0.18×1010 | 4.55±0.22×109 | 2.05±0.14×106 |
| Dipotassium hydrogen phosphate | 1.90±0.22×1010 | 1.80±0.28×1010 | 1.06±0.32×1010 | 4.70±0.32×109 | 3.78±0.27×106 |
| Sodium dihydrogen phosphate | 1.90±0.26×1010 | 1.78±0.33×1010 | 1.00±0.09×1010 | 5.05±0.19×109 | 2.20±0.33×106 |
| Gelatin | 1.90±0.16×1010 | 2.00±0.20×1010 | 1.41±0.11×1010 | 8.00±0.31×109 | 5.25±0.12×106 |
| Potassium sorbate | 1.90±0.26×1010 | 1.90±0.17×1010 | 1.40±0.29×1010 | 7.75±0.16×109 | 5.75±0.23×106 |
| Glycine | 1.90±0.10×1010 | 1.75±0.10×1010 | 1.08±0.31×1010 | 3.45±0.09×109 | 1.70±0.20×106 |
| Magnesium sulfate | 1.90±0.09×1010 | 1.88±0.23×1010 | 1.37±0.16×1010 | 7.88±0.22×109 | 5.80±0.14×106 |
| Control | 1.90±0.30×1010 | 8.85±0.07×109 | 3.26±0.22×108 | 7.50±0.11×106 | - |
Example 2 screening of optimum amounts of Components to be added to the compositions of the invention
In the screening of the components of the protective agent, 3 kinds of magnesium sulfate, gelatin and potassium phosphate with the best effect are screened for the working addition amount.
Magnesium sulfate
Magnesium sulfate with different addition amounts is uniformly mixed with coliphage according to the volume ratio of 1:1 under the aseptic condition to obtain the coliphage preparation. And placing the mixture at 37 ℃ for a constant-temperature accelerated preservation test, detecting the titer of the mixture, and comparing the preservation effect of different addition amounts.
As shown in Table 3, at a storage temperature of 37 ℃, the addition amounts of 15g/L and 40g/L decreased by 1 order of magnitude after 5 days of storage, and by 5 orders of magnitude after 30 days of storage; as can be seen from Table 3, the addition amount is between 20 and 35g/L, the titer is reduced by 1 magnitude at the 10 th day of storage, and the titer still has the power of 6 at the 30 th day of storage; the storage effect was best when the amount added was 30g/L, and 30g/L was selected as the amount added of the composition.
TABLE 337 Effect of different magnesium sulfate additions on preservation of E.coli phage preparations (pfu/mL)
Gelatin
And uniformly mixing the gelatin with different addition amounts with the coliphage according to the volume ratio of 1:1 under the aseptic condition to obtain the coliphage preparation. And placing the mixture at 37 ℃ for a constant-temperature accelerated preservation test, detecting the titer of the mixture, and comparing the preservation effect of different addition amounts.
As shown in Table 4, at a storage temperature of 37 ℃, the addition amounts of 1g/L and 11g/L decreased by 1 order of magnitude after 5 days of storage, and by 5 orders of magnitude after 30 days of storage; as can be seen from Table 4, the addition amount is between 3 and 8g/L, the titer is reduced by 1 magnitude at the 10 th day of storage, and the titer still has the power of 6 at the 30 th day of storage; the storage effect was best when the amount added was 5g/L, and 5g/L was selected as the amount added of the composition.
TABLE 437 Effect of different gelatin addition levels on preservation of E.coli phage preparations (pfu/mL)
Potassium sorbate
And (3) uniformly mixing potassium sorbate with different addition amounts with the coliphage according to the volume ratio of 1:1 under the aseptic condition to obtain the coliphage preparation. And placing the mixture at 37 ℃ for a constant-temperature accelerated preservation test, detecting the titer of the mixture, and comparing the preservation effect of different addition amounts.
As shown in Table 5, at a storage temperature of 37 ℃, the addition amounts of 0.01g/L and 0.25g/L decreased by 1 order of magnitude after 5 days of storage, and by 5 orders of magnitude after 30 days of storage; as can be seen from Table 5, the addition amount is between 0.05 and 0.20g/L, the titer is reduced by 1 magnitude order when the mixture is stored for 10 days, and the titer still has the power of 6 when the mixture is stored for 30 days; the storage effect was best when the amount added was 0.10g/L, and 0.10g/L was selected as the amount added of the composition.
TABLE 537 Effect of different potassium sorbate additions on the preservation Effect of coliphage formulations (pfu/mL)
EXAMPLE 3 screening of optimal combinations of the compositions of the invention
And combining the screened optimal protective agent components, and screening an optimal composition.
Composition 1
Preparing a composition 1 from 6g/L of sodium chloride, 30g/L of magnesium sulfate, 5mL/L of gelatin, 0.1g/L of glycine and water according to the addition amount of the components;
composition 2
Preparing a composition 2 by adding 6g/L of sodium chloride, 30g/L of magnesium sulfate, 5mL/L of gelatin, 0.1g/L of glycine, 0.1g/L of sodium dihydrogen phosphate, 0.1g/L of dipotassium hydrogen phosphate and water according to the above components;
composition 3
Preparing composition 3 from sodium chloride 6g/L, magnesium sulfate 30g/L, gelatin 5mL/L, glycine 0.1g/L, sodium dihydrogen phosphate 0.1g/L, dipotassium hydrogen phosphate 0.1g/L, glycerol 20mL/L, and water according to the above components
Composition 4
Preparing composition 4 from 6g/L sodium chloride, 30g/L magnesium sulfate, 5mL/L gelatin, 0.1g/L glycine, 0.1g/L sodium dihydrogen phosphate, 0.1g/L dipotassium hydrogen phosphate, 20mL/L glycerin, 0.1g/L potassium sorbate and water according to the above-mentioned component adding amounts
And (3) uniformly mixing the 4 compositions with the coliphage in a volume ratio of 1:1 under aseptic conditions to obtain the coliphage preparation. And placing the mixture at 37 ℃ for a constant-temperature accelerated storage test, detecting the titer of the mixture, and comparing the storage effects of different compositions.
The results are shown in table 6, at the storage temperature of 37 ℃, the potency of the composition is reduced by 1 order of magnitude when the composition is stored for 10 days, and the storage results of different compositions are different but have synergistic effect when the composition is stored for 30 days; composition 4 gave the best results, composition 4 being selected as the composition of the invention.
TABLE 637 Effect of different combinations of protectants on preservation of phage preparations of E.coli (pfu/mL)
| Composition comprising a metal oxide and a metal oxide | Initial | 2 days | 5 days | 10 days | 30 days |
| Composition 1 | 1.90±0.23×1010 | 1.80±0.36×1010 | 1.55±0.08×1010 | 6.65±0.10×109 | 6.89±0.33×106 |
| Composition 2 | 1.90±0.31×1010 | 1.90±0.21×1010 | 1.58±0.28×1010 | 7.10±0.11×109 | 7.00±0.24×106 |
| Composition 3 | 1.90±0.22×1010 | 1.85±0.28×1010 | 1.60±0.11×1010 | 7.85±0.27×109 | 7.24±0.18×106 |
| Composition 4 | 1.90±0.15×1010 | 2.00±0.09×1010 | 1.71±0.34×1010 | 9.95±0.31×109 | 8.80±0.28×106 |
Example 4 preparation of Stable phage composition
The composition for improving the stability of the phage preparation comprises the following components in percentage by weight:
a method of making a composition for improving the stability of a phage preparation, the method comprising:
1) dissolving the sodium chloride, the magnesium sulfate, the gelatin, the dipotassium phosphate and the sodium dihydrogen phosphate in the water, heating to 20 ℃, and uniformly mixing by magnetic stirring for later use;
2) adding glycine and potassium sorbate with the above component contents into the solution obtained in step 1), adding glycerol with the component contents, and magnetically stirring for 25 min;
3) adjusting the pH of the solution obtained in the step 2) to 7.2, and then sterilizing the solution for 20min at 121 ℃ by high-pressure steam to obtain a composition with stability of the phage preparation;
preparation of SM liquid as contrast protectant
Preparation of SM solution, sodium chloride 5.89g, magnesium sulfate 2g, 1M Tris-HCl (pH 7.5)50ml, 2% gelatin 5ml, purified water to 1000ml, and filtration sterilized with 0.22 μ M filter membrane were weighed out accurately to obtain SM solution.
Contrast protectant 60% Glycerol protectant
Preparing 60% glycerol, accurately measuring 60 parts of glycerol, accurately measuring 20 parts of deionized water, mixing the glycerol and the deionized water uniformly, and sterilizing at 121 ℃ for 20min to obtain 60% glycerol;
the composition (hereinafter referred to as "composition") for obtaining the stability of the phage preparation of the present invention according to the preparation method of the above 1, the conventional phage protectant SM solution protectant (hereinafter referred to as SM solution) and the conventional phage protectant 60% glycerol protectant (hereinafter referred to as 60% glycerol) according to the preparation methods of the above 2 and 3 were obtained.
EXAMPLE 5 Effect of the compositions of the invention on the accelerated storage test of different bacteriophages at a constant temperature of 37 ℃
Escherichia coli bacteriophage
The composition, SM solution and 60% glycerol from example 4 were mixed with a preparation of E.coli phage.
Mixing Escherichia coli PD07 and Escherichia coli PD185 at a ratio of 1:1 to obtain phage cocktail.
And (3) respectively and uniformly mixing the coliphage cocktail composition with the composition, the SM solution and 60% glycerol according to the volume ratio of 1:1 under the aseptic condition to obtain the coliphage cocktail preparation.
The coliphage cocktail preparation added with the protective agent is placed at 37 ℃ for constant temperature accelerated test, SM liquid and 60% glycerol are used as controls, and the influence of the composition and the conventional protective agent on the titer stability of the phage in the phage cocktail preparation is compared. The results are shown in table 7, at the preservation temperature of 37 ℃, the phage titer of the escherichia coli phage cocktail preparation in the composition starts to decrease by one order of magnitude after being preserved for 10 days, decreases by 4 orders of magnitude after being preserved for 30 days, and the titer still has 4 times when being preserved for 60 days; the titer of the coliphage cocktail preparation in the SM solution begins to decrease by 1 order of magnitude after 5 days, the titer decreases by 2 orders of magnitude after 10 days of storage, the titer decreases to 2 times when 30 days of storage, and the phage is completely inactivated after 60 days of storage; the coliphage cocktail preparation in 60% glycerol starts to decrease by 1 order of magnitude after being stored for 3 days, decreases by 3 orders of magnitude after being stored for 10 days, and completely inactivates when being stored for 30 days. The results show that the composition of the invention has more stable protective effect on the coliphage cocktail preparation than SM liquid and 60% glycerol.
TABLE 737 ℃ potency (pfu/mL) of the coliphage cocktail preparation for isothermal accelerated testing
Salmonella bacteriophage
The composition, SM solution and 60% glycerol from example 4 were mixed with Salmonella phage
Taking salmonella phage SP8, and respectively and uniformly mixing the salmonella phage SP8 with the composition, the SM liquid and 60% glycerol according to the volume ratio of 1:1 under the aseptic condition to obtain the salmonella phage preparation.
The salmonella phage preparation added with the protective agent is placed at 37 ℃ for constant temperature accelerated test, SM liquid and 60% glycerol are used as controls, and the influence of the composition and the conventional protective agent on the titer stability of the phage is compared. The results are shown in table 8, where the salmonella phages in the composition of the invention started to decrease by 1 order of magnitude after 10 days of storage, by 2 orders of magnitude after 30 days of storage, and the titer decreased to the power of 6 at 60 days of storage at 37 ℃; the salmonella phage in the SM liquid starts to reduce by 1 magnitude order after being stored for 5 days, reduces by 2 magnitude orders after being stored for 10 days, and reduces the titer to 4 times after being stored for 60 days; the salmonella phage in 60% glycerol is reduced by 1 magnitude in 5 days of storage, reduced by 2 magnitudes in 10 days of storage, and completely inactivated in 60 days of storage. The results show that the composition of the present invention has more stable protective effect on salmonella phages than SM solution and 60% glycerol.
TABLE 837 titer of Salmonella phage preparation accelerated at constant temperature test (pfu/mL)
| Time | Bacteriophage | Composition comprising a metal oxide and a metal oxide | SM liquid | 60% glycerin |
| 1 day | SP8 | 2.00±0.14×1011 | 2.00±0.15×1011 | 2.10±0.18×1011 |
| 3 days | SP8 | 2.00±0.16×1011 | 2.00±0.17×1011 | 1.90±0.22×1011 |
| 5 days | SP8 | 1.78±0.34×1011 | 8.90±0.14×1010 | 5.00±0.16×1010 |
| 10 days | SP8 | 9.80±0.13×1010 | 8.00±0.24×109 | 2.50±0.12×109 |
| 15 days | SP8 | 3.00±0.24×1010 | 6.80±0.26×108 | 1.20±0.13×107 |
| 30 days | SP8 | 5.20±0.09×108 | 1.10±0.31×106 | 1.90±0.17×105 |
| 60 days | SP8 | 5.50±0.12×106 | 5.60±0.12×104 | - |
Pseudomonas aeruginosa bacteriophage
The composition, SM solution and 60% glycerol obtained in example 4 were mixed with Pseudomonas aeruginosa phage
Taking pseudomonas aeruginosa bacteriophage ASP11, and respectively and uniformly mixing the pseudomonas aeruginosa bacteriophage ASP11 with the composition, the SM liquid and 60% of glycerol according to the volume ratio of 1:1 under the aseptic condition to obtain the pseudomonas aeruginosa bacteriophage preparation.
The pseudomonas aeruginosa bacteriophage preparation added with the protective agent is placed at 37 ℃ for constant-temperature accelerated test, and SM liquid and 60% glycerol are used as controls to compare the influence of the composition and the conventional protective agent on the titer stability of the bacteriophage. The results are shown in table 9, and at the storage temperature of 37 ℃, the titer of the pseudomonas aeruginosa bacteriophage in the composition of the invention begins to decrease by 1 magnitude after the bacillus aeruginosa bacteriophage is stored for 10 days, decreases by 4 magnitudes after the bacillus pyocyaneus is stored for 30 days, and decreases to 4 power after the bacillus pyocyaneus is stored for 60 days; the pseudomonas aeruginosa bacteriophage in the SM liquid starts to reduce by 1 order of magnitude after being stored for 3 days, reduces by 2 orders of magnitude after being stored for 10 days, reduces the titer to 2 times after being stored for 30 days, and completely inactivates after being stored for 60 days; the pseudomonas aeruginosa bacteriophage in 60% glycerol is reduced by 1 order of magnitude after being stored for 3 days, reduced by 4 orders of magnitude after being stored for 10 days, and completely inactivated after being stored for 30 days. The results show that the composition of the present invention has more stable protective effect on pseudomonas aeruginosa bacteriophage than SM solution and 60% glycerol.
TABLE titre of constant temperature accelerated test of Pseudomonas aeruginosa phage preparation at 937 ℃ (pfu/mL)
| Time | Bacteriophage | Composition comprising a metal oxide and a metal oxide | SM liquid | 60% glycerin |
| 1 day | ASP11 | 2.00±0.14×1010 | 2.10±0.18×1010 | 1.90±0.11×1010 |
| 3 days | ASP11 | 1.90±0.22×1010 | 9.80±0.11×109 | 4.50±0.17×109 |
| 5 days | ASP11 | 1.55±0.12×1010 | 1.60±0.08×109 | 7.80±0.22×108 |
| 10 days | ASP11 | 9.75±0.10×109 | 1.00±0.14×108 | 5.50±0.14×106 |
| 15 days | ASP11 | 5.60±0.08×108 | 9.80±0.25×106 | 7.20±0.19×104 |
| 30 days | ASP11 | 8.45±0.13×106 | 6.20±0.12×102 | - |
| 60 days | ASP11 | 3.50±0.30×104 | - | - |
Vibrio parahaemolyticus phage
The composition obtained in example 4, SM solution and 60% glycerol were mixed with the Vibrio parahaemolyticus phage
Taking the vibrio parahaemolyticus phage PG31, and respectively and uniformly mixing the vibrio parahaemolyticus phage PG31 with the composition, the SM liquid and 60% glycerol according to the volume ratio of 1:1 under the aseptic condition to obtain the vibrio parahaemolyticus phage preparation.
The vibrio parahaemolyticus phage preparation added with the protective agent is placed at 37 ℃ for constant temperature acceleration test, SM liquid and 60% glycerol are used as controls, and the influence of the composition and the conventional protective agent on the titer stability of the phage is compared. The results are shown in Table 10, and at the storage temperature of 37 ℃, the titer of the phage of Vibrio parahaemolyticus in the composition of the invention starts to decrease by 1 magnitude after 10 days of storage, decreases by 4 magnitudes after 30 days of storage, and decreases to 4 power after 60 days of storage; the vibrio parahaemolyticus phage in the SM liquid is reduced by 1 order of magnitude after being stored for 3 days, reduced by 3 orders of magnitude after being stored for 10 days, and completely inactivated after being stored for 60 days, wherein the titer is reduced to 2 power after being stored for 30 days; the vibrio parahaemolyticus phage in 60% glycerol is reduced by 1 order of magnitude after being stored for 3 days, reduced by 4 orders of magnitude after being stored for 10 days, and completely inactivated after being stored for 30 days. The results show that the composition of the present invention has more stable protective effect on the bacteriophage Vibrio parahaemolyticus than SM solution and 60% glycerol.
TABLE 1037 ℃ potency of constant temperature accelerated test of Vibrio parahaemolyticus phage preparation (pfu/mL)
| Time | Bacteriophage | Composition comprising a metal oxide and a metal oxide | SM liquid | 60% glycerin |
| 1 day | PG31 | 2.50±0.15×1010 | 2.20±0.14×1010 | 2.30±0.15×1010 |
| 3 days | PG31 | 2.35±0.26×1010 | 8.10±0.16×109 | 5.50±0.09×109 |
| 5 days | PG31 | 1.80±0.13×1010 | 6.20±0.13×108 | 1.80±0.12×108 |
| 10 days | PG31 | 9.88±0.31×109 | 2.00±0.25×107 | 2.50±0.33×106 |
| 15 days | PG31 | 1.20±0.11×108 | 1.80±0.12×106 | 5.10±0.19×104 |
| 30 days | PG31 | 9.80±0.18×106 | 3.00±0.21×102 | - |
| 60 days | PG31 | 1.50±0.15×104 | - | - |
EXAMPLE 6 Effect of the compositions of the invention on Long-term stability storage tests at 4 ℃ and 25 ℃ without using phages
Escherichia coli bacteriophage
Taking the coliphage, and respectively and uniformly mixing the coliphage with the composition or the SM liquid obtained in the example 4 under the aseptic condition according to the volume ratio of 1:1 to obtain the coliphage preparation.
The coliphage preparation added with the protective agent is placed at 4 ℃ and 25 ℃ for long-term stability storage test, meanwhile, SM liquid is used as a control, the titer is measured every 30 days in the first half of the year of storage, and the titer is measured every half of the year after half of the year, so that the influence of the composition and the conventional protective agent on the titer stability of the phage is compared.
The test results are shown in table 11, and at the storage temperature of 4 ℃, the titer of the coliphage in the composition of the invention is reduced by 1 magnitude after 90 days of storage, reduced by 2 magnitudes after 1 and half years of storage, and reduced by 3 magnitudes after 2 years of storage; the coliphage in the SM liquid is reduced by 1 magnitude order when stored for 60 days, reduced by 2 magnitude orders when stored for 1 year, reduced by 3 magnitude orders when stored for 1 year and half, and reduced by 4 magnitude orders when stored for 2 years; at the preservation temperature of 25 ℃, the coliphage in the composition starts to decrease by 1 order of magnitude after being preserved for 30 days, and the titer is 2 times when being preserved for 2 years; after being stored for 30 days, the coliphage in the SM solution begins to decline by 1 order of magnitude, and the titer is 1 power when the coliphage is stored for 2 years. The experiment shows that compared with SM liquid, the composition has more stable protection effect on the coliphage, can ensure that the coliphage has longer storage time at room temperature, and is beneficial to the production and application of phage preparations.
TABLE titer in the E.coli phage and Long-term stability storage assay at 114 ℃ and 25 ℃ (pfu/mL)
Salmonella bacteriophage
Taking salmonella phage, and mixing the salmonella phage with the composition or SM liquid obtained in example 4 respectively under aseptic condition according to the volume ratio of 1:1 to obtain the salmonella phage preparation.
The salmonella phage preparation added with the protective agent is placed at 4 ℃ and 25 ℃ for long-term stability storage test, meanwhile, SM liquid is used as a control, the titer of the salmonella phage preparation is measured every 30 days in the first half year of storage, and the titer of the salmonella phage preparation is measured every half year after the first half year, so that the influence of the composition and the conventional protective agent on the titer stability of the phage is compared.
The test results are shown in table 12, and at the storage temperature of 4 ℃, the titer of the salmonella phage in the composition of the invention is reduced by 1 order of magnitude when the composition is stored for 1 year, and is reduced by 2 orders of magnitude when the composition is stored for 2 years; the salmonella phage in the SM liquid is reduced by 1 order of magnitude when being stored for 1 year, and is reduced by 2 orders of magnitude when being stored for 2 years, and the titer is about 50 percent of that of the composition. At the storage temperature of 25 ℃, the salmonella phage in the composition starts to decrease by 1 order of magnitude after being stored for 90 days, decreases by 2 orders of magnitude when being stored for 1 year, and decreases by 2 orders of magnitude when being stored for 2 years; salmonella phage in SM fluid began to decline by 1 order of magnitude after 90 days of storage and by 4 orders of magnitude when stored for 2 years. The experiment shows that compared with SM liquid, the composition has more stable protection effect on the salmonella bacteriophage, and can ensure that the salmonella bacteriophage has longer storage time at room temperature, thereby being beneficial to the production and application of bacteriophage preparations.
TABLE titer in Salmonella phage and Long term stability preservation test at 124 ℃ and 25 ℃ (pfu/mL)
Pseudomonas aeruginosa bacteriophage
And (3) taking the pseudomonas aeruginosa bacteriophage, and respectively and uniformly mixing the pseudomonas aeruginosa bacteriophage with the composition or the SM liquid obtained in the example 4 under the aseptic condition according to the volume ratio of 1:1 to obtain the pseudomonas aeruginosa bacteriophage preparation.
The pseudomonas aeruginosa bacteriophage preparation added with the protective agent is placed at 4 ℃ and 25 ℃ for long-term stability storage test, meanwhile, SM liquid is used as a control, the titer of the pseudomonas aeruginosa bacteriophage preparation is measured every 30 days in the first half year of storage, the titer of the pseudomonas aeruginosa bacteriophage preparation is measured every half year after the first half year, and the influence of the composition and the conventional protective agent on the titer stability of the bacteriophage is compared.
The test results are shown in table 13, at the preservation temperature of 4 ℃, the titer of the pseudomonas aeruginosa bacteriophage in the composition is reduced by 1 order of magnitude after being preserved for 30 days, the titer is reduced by 2 orders of magnitude when being preserved for 120 days, the titer is reduced by 3 orders of magnitude when being preserved for 1 year and half, and the titer is reduced to 5 times after being preserved for 2 years; the pseudomonas aeruginosa bacteriophage in the SM liquid is reduced by 1 magnitude order when being stored for 30 days, reduced by 2 magnitude orders when being stored for 90 days, reduced by 3 magnitude orders when being stored for 1 year, and the titer is reduced to 4 times when being stored for 2 years. At the preservation temperature of 25 ℃, the pseudomonas aeruginosa bacteriophage in the composition starts to be reduced by 1 order of magnitude when being preserved for 30 days, and the price is reduced to 2 times when being preserved for 2 years; the Pseudomonas aeruginosa bacteriophage in SM liquid is reduced by 1 order of magnitude when stored for 90 days, and is completely inactivated when stored for 2 years. The experiment shows that compared with SM liquid, the composition has more stable protection effect on the pseudomonas aeruginosa bacteriophage, and meanwhile, the pseudomonas aeruginosa bacteriophage can be stored for a longer time at room temperature, and the production and application of a bacteriophage preparation are facilitated.
TABLE titer in Pseudomonas aeruginosa phage and Long term stability storage experiments at 134 ℃ and 25 ℃ (pfu/mL)
Vibrio parahaemolyticus phage
And (3) taking the vibrio parahaemolyticus phage, and respectively and uniformly mixing with the composition or SM liquid obtained in the example 4 under an aseptic condition according to the volume ratio of 1:1 to obtain the vibrio parahaemolyticus phage preparation.
The vibrio parahaemolyticus phage preparation added with the protective agent is placed at 4 ℃ and 25 ℃ for long-term stability storage test, meanwhile, SM liquid is used as a control, the titer is measured every 30 days in the first half of a year of storage, the titer is measured every half of a year after the first half, and the influence of the composition and the conventional protective agent on the titer stability of the phage is compared.
The test results are shown in Table 14, and at the storage temperature of 4 ℃, the titer of the phage of Vibrio parahaemolyticus in the composition of the invention is reduced by 1 order of magnitude when stored for 60 days, reduced by 2 orders of magnitude when stored for 120 days, and reduced to 7 times when stored for 2 years; the bacteriophage of vibrio parahaemolyticus in SM liquid is reduced by 1 order of magnitude when stored for 30 days, reduced by 2 orders of magnitude when stored for 90 days, reduced by 3 orders of magnitude when stored for 1 year, and reduced to 6 times when stored for 2 years. At the preservation temperature of 25 ℃, the vibrio parahaemolyticus phage in the composition is reduced by 1 order of magnitude when being preserved for 30 days, reduced by 2 order of magnitude when being preserved for 90 days, and the titer is reduced to 3 times when being preserved for 2 years; the bacteriophage of vibrio parahaemolyticus in SM liquid is reduced by 1 order of magnitude when stored for 30 days, and the price is reduced to 2 power when stored for 2 years. The experiment shows that compared with SM liquid, the composition of the present invention has stable protective effect on the vibrio parahaemolyticus phage, and can make the vibrio parahaemolyticus phage have long storage time at room temperature, which is favorable for the production and application of phage preparation.
TABLE titer in Vibrio parahaemolyticus phage and Long-term stability preservation test at 144 ℃ and 25 ℃ (pfu/mL)
From the above experimental results, it can be seen that the scheme of the present invention achieves several orders of magnitude improvement in phage stability for conventional glycerol protectors as well as SM protectors, resulting in unexpected technical effects. The compositions of the invention include both a portion of the salt in the SM protectant and glycerol in the glycerol reagent, but produce effects far exceeding those of the combination of the two products, and demonstrate that the cooperation between the components of the compositions of the invention can produce a synergistic effect rather than a simple superposition of the components.
Claims (10)
1. A composition for improving stability of a phage preparation, comprising: comprises the following components in percentage by weight:
2. the composition of claim 1, wherein: comprises the following components in percentage by weight:
3. a composition for improving stability of a phage preparation, comprising: the composition consists of the following components:
4. the composition according to any one of claims 1 to 3, characterized in that: the gelatin is gelatin with a concentration of 2%.
5. The composition according to any one of claims 1 to 3, characterized in that: the glycerol is 60% glycerol.
6. The composition according to any one of claims 1 to 3, characterized in that: the water is deionized water or purified water.
7. The composition according to any one of claims 1 to 3, characterized in that: the pH value of the composition is 6.8-7.5.
8. A phage combination preparation, characterized by comprising a phage and the composition of any one of claims 1-8;
preferably, the bacteriophage is one or a combination of multiple bacteriophages;
more preferably, the bacteriophage is selected from the group consisting of a coliphage, a salmonella phage, a pseudomonas aeruginosa phage, a vibrio parahaemolyticus phage.
9. A process for the preparation of a composition according to any one of claims 1 to 8, characterized in that it comprises the following steps:
1) dissolving sodium chloride, magnesium sulfate, gelatin, dipotassium hydrogen phosphate and sodium dihydrogen phosphate in water, and mixing uniformly;
2) adding glycine and potassium sorbate into the solution obtained in the step 1), adding glycerol, and uniformly mixing;
3) adjusting the pH value of the solution obtained in the step 2) to 6.8-7.5, and then sterilizing to obtain the product.
10. Use of a composition according to any one of claims 1 to 8 for the preparation of a phage preparation;
preferably, the composition of any one of claims 1 to 8 is mixed with a phage preparation uniformly according to a volume ratio of 1:1 to obtain a phage combination preparation;
more preferably, the bacteriophage is one or a combination of bacteriophages; the bacteriophage includes Escherichia coli bacteriophage, Salmonella bacteriophage, Pseudomonas aeruginosa bacteriophage and Vibrio parahaemolyticus bacteriophage.
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