Sodium dehydroacetate-based virus preservative as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biology, in particular to a virus preservative based on sodium dehydroacetate and a preparation method and application of the virus preservative. The virus preservative based on sodium dehydroacetate is prepared from the following components in percentage by weight: 1% of sodium dehydroacetate, 1% of potassium sorbate, 0.4% of trehalose, 0.008% of thiomersalate sodium salt and the balance of deionized water. By adopting the liquid preservative, the production standard of the virus preparation is reduced, and the production cost and the technical complexity are reduced; through suitability comparison, it is found that the preservative does not affect the activity of the virus strain, and the virus titer is not reduced after 100 h. Compared with a single preservative, the preservative disclosed by the invention has the advantages that the bacteriostatic ability is stronger, microorganisms in the preparation are obviously inhibited under the room temperature condition, and the detected microbial biomass is the lowest in each group, so that the preservative disclosed by the invention has a good continuous preservative effect on viruses, and the activity of the viruses can be maintained for a longer time.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a virus preservative based on sodium dehydroacetate and its preparation method and application. BACKGROUND

[0002] Virus biological products are widely used in the fields of medicine, agriculture, forestry, and aquaculture, and have played a huge role in medical treatment, scientific research, and animal husbandry. Such biological products are prone to microbial contamination during production, storage, and transportation, which can lead to product failure. Therefore, appropriate virus preservatives are needed to prevent viruses from being contaminated by microorganisms under different storage conditions, while maintaining the virus titer and extending the shelf life of virus products.

[0003] Current preservatives can be divided into several categories according to their mechanisms:

[0004] Acid-type preservatives: such as benzoic acid, sorbic acid, and propionic acid and their salts, which inhibit bacteria by undissociated molecules under acidic conditions, and their preservative effect is related to pH.

[0005] Ester-type preservatives: such as propyl esters, which destroy the cell membrane of microorganisms, denature the proteins inside the cells, and thus disrupt the normal physiological balance of microorganisms.

[0006] Inorganic salt preservatives: such as nitrate, nitrite, and sulfur-containing sulfite, which inhibit the activity of microbial respiratory enzymes and cause microorganisms to die from lack of oxygen.

[0007] Most existing preservatives have some impact on virus activity, leading to the deterioration and failure of virus products due to transportation or storage reasons, resulting in a large amount of waste and financial loss. Therefore, it is of great significance to develop preservatives for virus products. SUMMARY

[0008] To overcome the shortcomings of the prior art, the present application provides a virus preservative based on sodium dehydroacetate and its preparation method and application. The preservative reduces the risk of contamination of virus preparations and improves and prolongs the activity of virus preparations.

[0009] To solve the problems of the prior art, the technical solution adopted by the present application is as follows:

[0010] A virus preservative based on sodium dehydroacetate, consisting of the following components by weight percentage: sodium dehydroacetate 1%, potassium sorbate 1%, trehalose 0.4%, sodium thiomersalate 0.008%, and the balance being deionized water.

[0011] The preparation method of the above virus preservative comprises the following steps:

[0012] a. Sodium dehydroacetate and potassium sorbate were weighed according to the percentage by weight, dissolved in water, sterilized at 121℃ for 15 minutes, and stored at 4℃ for standby, to obtain preservative A liquid;

[0013] b. Sodium thiomersalate and trehalose were weighed according to the percentage by weight, dissolved in water, sterilized by filtration, and stored at 4℃ for standby, to obtain preservative B liquid;

[0014] c. The preservative A liquid and the preservative B liquid were mixed uniformly at a volume ratio of 1:1, to obtain a virus preservative.

[0015] As an improvement, the filter membrane size in the filtering of step b is 0.22 μm.

[0016] A virus preparation with preservative, comprising the virus preservative and a virus, mixed in equal volume under sterile conditions.

[0017] As an improvement, the virus comprises but is not limited to bacteriophage, animal virus, fungal virus, and plant virus.

[0018] Further improvement, the bacteriophage is Vibrio parahaemolyticus bacteriophage.

[0019] Further improvement, the Vibrio parahaemolyticus bacteriophage comprises but is not limited to Vibrio parahaemolyticus bacteriophage VP46, VP48, or VP7.

[0020] Beneficial effects:

[0021] Compared with the prior art, the liquid preservative is used to reduce the production standard of the virus preparation, reduce the production cost and technical complexity; through adaptability comparison, it is found that the preservative does not affect the virus strain activity, and the virus titer is not reduced after 100 hours. Compared with the single preservative, the preservative has stronger bacteriostatic ability, and the microorganisms in the preparation are obviously inhibited under room temperature conditions, and the detected amount of microorganisms is the lowest in each group. Therefore, the preservative has good sustained preservative effect on the virus, and can maintain the virus activity for a longer time. DETAILED DESCRIPTION

[0022] The following examples are used to further illustrate the present application, but do not limit the effective range of the present application in any way.

[0023] Unless otherwise defined, all technical and scientific terms used in the present application have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Generally, the nomenclature used in the present application is well known and commonly used in the art, and if not specifically indicated, the preservative used in the examples of the present application can be commercially available.

[0024] In the following examples, the reagents used are as follows:

[0025] Sodium dehydroacetate is a commercially available product with purity greater than or equal to 99.0%, molecular formula C8H7NaO4, and molecular weight 190.14.

[0026] Potassium sorbate is a commercially available product with purity greater than or equal to 99.0%, molecular formula C6H7KO2, and molecular weight 150.22.

[0027] Merthiolate is a commercially available product with purity greater than or equal to 99.0%, molecular formula C9H9HgNaO2S, and molecular weight 404.81.

[0028] Trehalose is a commercially available product with purity greater than or equal to 99.0%, molecular formula C12H22O11·2H2O, and molecular weight 378.3. 12 H 22 O 11 ·2H2O, molecular weight 378.3.

[0029] The Vibrio parahaemolyticus phage VP46 is preserved in the China Center for Type Culture Collection, Wuhan University, Wuhan, China, with a postal code 430072, on May 26, 2016, and has a preservation number CCTCC NO: M2016290.

[0030] The Vibrio parahaemolyticus phage VP48 is preserved in the China Center for Type Culture Collection, Wuhan University, Wuhan, China, with a postal code 430072, on May 26, 2016, and has a preservation number CCTCC NO: M2016291.

[0031] The Vibrio parahaemolyticus phage VP7 is preserved in the China Center for Type Culture Collection, Wuhan University, Wuhan, China, with a postal code 430072, on May 26, 2016, and has a preservation number CCTCC NO: M2016289.

[0032] Example 1

[0033] Sodium dehydroacetate 20 g and potassium sorbate 20 g were accurately weighed, and then stirred in deionized water until completely dissolved. The solution was diluted with deionized water to 500 mL. The solution was sterilized at 121 ℃ for 15 min to obtain a bacteriophage preservative A solution, which was stored at 4 ℃ for later use.

[0034] Accurately weigh 0.16 of thiomersal sodium salt and 8 g of trehalose, add an appropriate amount of deionized water to stir until fully dissolved, then add deionized water to constant volume to 500 mL; after sterilization through a 0.22 μm filter membrane, prepare B liquid to obtain bacteriophage preservative B liquid, which is stored at 4°C for standby.

[0035] Under sterile conditions, mix the preservative A liquid and B liquid uniformly at a volume ratio of 1:1 to obtain a virus preservative.

[0036] Comparative Example 1

[0037] Accurately weigh 10 g of sodium dehydroacetate, add an appropriate amount of deionized water to stir until fully dissolved, then add deionized water to constant volume to 1 L; sterilize at 121°C for 15 min, cool to room temperature to obtain a virus preservative.

[0038] Comparative Example 2

[0039] Accurately weigh 10 g of potassium sorbate, add an appropriate amount of deionized water to stir until fully dissolved, then add deionized water to constant volume to 1 L; sterilize at 121°C for 15 min, cool to room temperature to obtain a virus preservative.

[0040] Comparative Example 3

[0041] Accurately weigh 0.04 g of thiomersal sodium salt, sterilize through a 0.22 μm filter membrane, and constant volume to 1 L; cool to room temperature to obtain a virus preservative.

[0042] Preparation of Vibrio parahaemolyticus phage preparation in Example 2

[0043] Take Vibrio parahaemolyticus phage VP46 (Vibrio parahaemolyticus phage VP46), accession number CCTCC NO: M2016290; Vibrio parahaemolyticus phage VP48 (Vibrio parahaemolyticus phage VP48), accession number CCTCC NO: M2016291; Vibrio parahaemolyticus phage VP7 (Vibrio parahaemolyticus phage VP7), accession number CCTCC NO: M2016289, and prepare a bacteriophage cocktail combination in a ratio of 1:1:1.

[0044] The specific phage culture scheme is referred to in CN CN109837251B, and the culture medium and culture conditions used for culture are conventional technical means in the art.

[0045] Under sterile conditions, add an equal volume of the virus preservative prepared in Example 1 to the bacteriophage cocktail combination, mix uniformly to obtain a preparation of Vibrio parahaemolyticus phage cocktail combination with preservative.

[0046] Similarly, the virus preservative prepared in Example 1 was added to an equal volume of Vibrio parahaemolyticus phage VP46; Vibrio parahaemolyticus phage VP48; Vibrio parahaemolyticus phage VP7, respectively, to obtain the corresponding phage preparations.

[0047] Similarly, the comparative preservatives prepared in Comparative Examples 1-3 were prepared into the corresponding preparations of Vibrio parahaemolyticus phage cocktail combination and each phage preparation according to the virus preservative prepared in Example 1.

[0048] Example 3 Virus stability test in comprehensive preservative

[0049] The different kinds of Vibrio parahaemolyticus phage cocktail combination preparations and each phage preparation prepared in Example 3 were subjected to constant temperature acceleration experiment at 37°C, and the original preparation of Vibrio parahaemolyticus phage was used as a control. The titer was determined every 24 h to compare the titer stability of different preservatives.

[0050] Table 1 Titer (PFU / mL) of Vibrio parahaemolyticus phage VP46 in comprehensive preservative at 37°C

[0051] preservative 0h 24h 48h 72h 96h 120h 144h 168h Example 1 2.3 x 10 9 ]] 2.0 x 10 9 ]] 2.0 x 10 9 ]] 1.8 x 10 9 ]] 1.0 x 10 9 ]] 8.3 x 10 8 ]] 5.0 x 10 8 ]]> 2.3 x 10 8 ]]> Comparative Example 1 2.3 x 10 9 ]]> 1.7 x 10 9 ]]> 1.2 x 10 9 ]]> 7.2 x 10 8 ]] 9.0 x 10 7 ]] 6.3 x 10 6 ]] 9.5 x 10 6 ]] 4.0 x 10 5 ]] Comparative Example 2 2.3 x 10 9 ]]> 2.1 x 10 6 ]]> 2.0 x 10 4 ]]> 1.7 x 10 3 ]]> - - - - Comparative Example 3 2.3 x 10 9 ]]> 2.2 x 10 4 ]]> - - - - - - Control 2.3 x 10 9 ]]> 1.5 x 10 9 ]]> 1.0 x 10 9 ]]> 4.9 x 10 8 ]]> 1.0 x 10 8 ]]> 6.2 x 10 7 ]]> 1.8 x 10 7 ]]> 1.0 x 10 7 ]]>

[0052] Table 2 Titer (PFU / mL) of Vibrio parahaemolyticus phage VP48 in comprehensive preservative at 37°C

[0053] preservative 0h 24h 48h 72h 96h 120h 144h 168h Example 1 9.0 x 10 8 ]] 8.9 x 10 8 ]] 8.6 x 10 8 ]] 8.8 x 10 8 ]] 6.0 x 10 8 ]] 5.3 x 10 8 ]]> 2.0 x 10 8 ]]> 9.3 x 10 7 ]]> Comparative Example 1 9.0 x 10 8 ]]> 6.5 x 10 8 ]]> 5.6 x 10 8 ]]> 5.0 x 10 8 ]]> 1.0 x 10 8 ]]> 6.1 x 10 7 ]]> 3.0 x 10 7 ]] 1.8 x 10 7 ]] Comparative Example 2 2.3 x 10 9 ]] 2.0 x 10 6 ]] 1.0 x 10 4 ]] 2.2 x 10 3 ]] - - - - Comparative Example 3 2.3 x 10 9 ]]> 2.0 x 10 4 ]]> - - - - - - Control 9.0 x 10 8 ]]> 5.1 x 10 8 ]]> 4.6 x 10 8 ]] 2.9 x 10 8 ]] 5.9 x 10 7 ]]> 3.4 x 10 7 ]]> 1.2 x 10 7 ]]> 9.1 x 10 6 ]]>

[0054] Table 3 Titer (PFU / mL) of Vibrio parahaemolyticus phage VP7 in comprehensive preservative at 37°C

[0055] preservative 0h 24h 48h 72h 96h 120h 144h 168h Example 1 2.8 x 10 9 ]] 2.6 x 10 9 ]]> 2.0 x 10 9 ]] 2.1 x 10 9 ]]> 1.9 x 10 9 ]] 1.0 x 10 9 ]]> 8.6 x 10 8 ]]> 5.3 x 10 8 ]]> Comparative Example 1 2.8 x 10 9 ]] 2.2 x 10 9 ]]> 1.8 x 10 9 ]]> 1.6 x 10 9 ]]> 9.2 x 10 8 ]] 7.1 x 10 8 ]]> 4.3 x 10 8 ]] 1.0 x 10 8 ]]> Comparative Example 2 2.3 x 10 9 ]]> 2.0 x 10 7 ]]> 1.0 x 10 4 ]]> 2.2 x 10 3 ]]> - - - - Comparative Example 3 2.3 x 10 9 ]]> 2.0 x 10 4 ]] 1.0 x 10 4 ]]> 2.2 x 10 3 ]]> - - - - Control 2.8 x 10 9 ]]> 2.6 x 10 9 ]]> 8.5 x 10 8 ]]> 6.3 x 10 8 ]]> 1.2 x 10 8 ]]> 5.8 x 10 7 ]]> 4.0 x 10 6 ]]> 3.0 x 10 6 ]]>

[0056] Table 4 Titer (PFU / mL) of cocktail combination in comprehensive preservative at 37°C

[0057]

[0058]

[0059] The results are shown in Tables 1-4: The titer of the virus preparation with the preservative prepared in Example 1 did not change in magnitude for 96 h, while the control titer decreased by 11-20 times compared with the initial titer; 72 h after the preparation, the virus strain titer of the sodium dehydroacetate group decreased by about 90%, and the titer of the other groups was almost completely lost; while the titer of Example 1 was almost not lost. From 96 h to 168 h, the effect of Example 1 and Comparative Example 1 differed, and the titer of the new preservative was 500 times that of Comparative Example 1 after 168 h. Further, the preservative ratio of Example 1 did not affect the phage titer and could protect the phage.

[0060] Example 4: Determination of the effect of different preservatives on the contamination of microorganisms in the virus preparation

[0061] The different types of Vibrio parahaemolyticus phage cocktail preparations prepared in Example 2 were placed in an indoor environment, and the amount of microorganisms in each preparation was determined every day. The determination method was a conventional technique in the art. The original Vibrio parahaemolyticus phage preparation was used as a control.

[0062] Table 5: Count of contamination microorganisms (PFU / mL) in Vibrio parahaemolyticus phage VP46 under different preservatives

[0063] preservative 0h 24h 48h 72h 96h 120h 144h 168h Example 1 - - - - - 8.0 x 10 0 ]]> 5.0 x 10 1 ]]> 2.3 x 10 2 ]]> Comparative Example 1 - - - - 9.0 x 10 2 ]]> 6.3 x 10 3 ]]> 9.5 x 10 4 ]]> 4.0 x 10 5 ]]> Comparative Example 2 - - 9.0 x 10 2 ]]> 6.3 x 10 3 ]]> 9.0 x 10 4 ]]> 4.1 x 10 5 ]]> 9.0 x 10 6 ]]> 9.0 x 10 6 ]]> Comparative Example 3 - - - - 3.0 x 10 2 ]]> 6.3 x 10 4 ]]> 9.5 x 10 5 ]]> 4.0 x 10 6 ]] Control - 1.5 x 10 7 ]]> 1.0 x 10 9 ]]> 4.9 x 10 9 ]] 1.0 x 10 9 ]]> 6.2 x 10 9 ]]> 1.8 x 10 9 ]]> 1.0 x 10 8 ]]>

[0064] Table 6: Count of contamination microorganisms (PFU / mL) in Vibrio parahaemolyticus phage VP48 under different preservatives

[0065] preservative 0h 24h 48h 72h 96h 120h 144h 168h Example 1 - - - - - 1.0 x 10 1 ]]> 3.0 x 10 1 ]]> 1.0 x 10 2 ]]> Comparative Example 1 - - - - 3.0 x 10 2 ]]> 3.3 x 10 3 ]]> 9.7 x 10 4 ]] 1.0 x 10 5 ]]> Comparative Example 2 - - 2.0 x 10 2 ]]> 4.3 x 10 3 ]]> 2.0 x 10 4 ]]> 6.1 x 10 5 ]]> 7.0 x 10 6 ]]> 8.0 x 10 6 ]] Comparative Example 3 - - - 1.0 x 10 1 ]]> 3.0 x 10 2 ]]> 5.3 x 10 4 ]] 2.5 x 10 5 ]]> 9.0 x 10 6 <!-- 4 -->]]> Control - 1.5 x 10 7 ]]> 1.0 x 10 9 ]]> 4.9 x 10 9 ]]> 1.0 x 10 9 ]]> 1.2 x 10 9 ]]> 2.8 x 10 9 ]]> 4.0 x 10 8 ]]>

[0066] Table 7: Count of contamination microorganisms (PFU / mL) in Vibrio parahaemolyticus phage VP7 under different preservatives

[0067]

[0068]

[0069] Table 8: Count of contamination microorganisms (PFU / mL) in the cocktail under different preservatives

[0070] preservative 0h 24h 48h 72h 96h 120h 144h 168h Example 1 - - - - - 9.0 x 10 0 ]]> 1.0 x 10 1 ]]> 1.3 x 10 2 ]]> Comparative Example 1 - - - - 1.0 x 10 2 ]]> 3.3 x 10 3 ]]> 1.5 x 10 4 ]]> 1.0 x 10 5 ]]> Comparative Example 2 - - 2.0 x 10 2 ]] 4.3 x 10 3 ]] 2.0 x 10 4 ]] 9.1 x 10 5 ]] 6.0 x 10 6 ]] 6.0 x 10 6 ]]> Comparative Example 3 - 1.5 x 10 2 ]]> 1.1 x 10 2 ]]> 1.0 x 10 2 ]]> 5.0 x 10 2 ]]> 7.3 x 10 4 ]] 3.5 x 10 5 ]]> 1.0 x 10 6 ]]> Control preservative Example 1 Comparative Example 1 Comparative Example 2 Comparative Example 3 Control - 1.2 x 10 7 ]]> 1.0 x 10 9 ]]> 4.9 x 10 9 ]] 1.4 x 10 9 ]]> 6.4 x 10 9 ]] 7.8 x 10 9 ]] 6.0 x 10 8 ]]>

[0071] The results are shown in Tables 5-8: After 24 h of culture, the number of contaminating bacteria in the control group reached 10 7The comparative examples 1-3 all detected a large amount of contaminant bacteria between 48h and 96h. The virus preparation prepared in the example 1 has the least amount of contaminant bacteria, and the low content of contaminant bacteria is detected all the time, and the effect is better than that of other preservative groups. It is illustrated that the preservative of the application can well maintain the survival stability of Vibrio parahaemolyticus bacteriophage and control the contaminant bacteria at a low level, which is beneficial to the production and application of the bacteriophage preparation.

[0072] The above description of the application and its embodiments is illustrative, and is not limiting, and the shown is only one of the embodiments of the application, and the actual structure is not limited thereto. Therefore, if a person skilled in the art is inspired thereby, without departing from the purpose of the application, similar structure modes and embodiments are not creatively designed, which should all belong to the protection scope of the application.

Claims

1. A viral preservative based on sodium dehydroacetate, characterized in that, It consists of the following components by weight percentage: 1% sodium dehydroacetate, 1% potassium sorbate, 0.4% trehalose, 0.008% sodium thimerosal, and the balance being deionized water.

2. The method for preparing the viral preservative according to claim 1, characterized in that, Includes the following steps: a. Weigh out sodium dehydroacetate and potassium sorbate according to their weight percentage, dissolve them in water, sterilize at 121℃ for 15 min, and store at 4℃ for later use to obtain preservative solution A; b. Weigh out sodium thimerosal and trehalose according to their weight percentages, dissolve them in water, filter to remove bacteria, and store at 4°C for later use to obtain preservative solution B. c. Mix preservative A solution and preservative B solution at a volume ratio of 1:1 until homogeneous to obtain a virus preservative.

3. The preparation method according to claim 2, characterized in that, The filter membrane used in step b has a size of 0.22 μm.

4. A virus preparation with antiseptic properties, characterized in that, It comprises the viral preservative as described in claim 1 and the virus, mixed in equal volumes under sterile conditions.

5. The antiseptic viral preparation according to claim 4, characterized in that, The viruses mentioned include, but are not limited to, bacteriophages, animal viruses, fungal viruses, and plant viruses.

6. The antiseptic viral preparation according to claim 5, characterized in that, The bacteriophage is Vibrio parahaemolyticus bacteriophage.

7. The antiseptic viral preparation according to claim 6, characterized in that, The Vibrio parahaemolyticus phages include, but are not limited to, Vibrio parahaemolyticus phages VP46, VP48, or VP7.

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