A kind of bacterial strain freezing liquid and its application

Abstract

The present application discloses a strain freezing solution and its use. The strain freezing solution contains sclerotium gum and ectoine. By using the strain freezing solution described in the present application, the strains of the genus Sclerotium can be preserved for a long time and can be preserved at -40°C for more than two years. The sclerotia of the genus Sclerotium after preservation still have a 100% germination rate when revived. In addition, the preservation process using the strain freezing solution is simple, the volume of the tube containing the strains is small, and it is convenient to store and find.

Description

Technical Field

[0001] The present application relates to the field of microbial engineering technology, and in particular to a bacterial strain freezing solution and uses thereof. Background Art

[0002] Sclerotium sphericalensis belongs to the subphylum Ascomycota. The number of sclerotia is small, spherical, oval or cylindrical, dark brown, 0.3-2mm in size, with a rough surface and no difference between inner and outer layers.

[0003] Sclerotium uniformis grows radially on PDA medium, with round colonies and white silky hyphae. After 3 days of cultivation at 30℃, it covers a PDA plate with a diameter of 90 mm, and sclerotia begin to form on the surface of the colony around 7 days. The sclerotia are initially white, gradually turn yellow, mature after 10 days, and finally turn brown, spherical, with a smooth and shiny surface and a diameter of 0.4 to 1.7 mm. It does not produce asexual spores, but it is easy to form sclerotia. The sclerotia have strong viability. This characteristic of sclerotia is of great application value in the preservation of strains.

[0004] Ectohydropyrimidine is an osmotic pressure protective substance widely found in halophilic bacteria (Halomonas Elongata) and non-halophilic bacteria such as Streptomyces and Escherichia coli. It can also play a protective role in the dehydration and storage process of Escherichia coli. It is an osmotic pressure compensatory solute and can be used as an osmotic pressure gene inducer. It can balance the osmotic pressure of cells and provide protection for enzymes, DNA, cell membranes and the entire cell under adverse conditions such as high temperature, freezing and drying. At the same time, it can stabilize the enzyme molecular structure of enzyme preparations and ensure that the enzyme does not lose its activity under extreme conditions such as freezing and thawing and high temperature. Therefore, ectohydropyrimidine is a cell all-round protective agent, an effective anti-pressure protective molecule, and a multifunctional active ingredient.

[0005] In the laboratory, the preservation of Sclerotium species is often stored in the form of mycelium grown in a test tube slant medium. Although such a preservation method is simple and effective, it also has some disadvantages, mainly that the preservation time is short. To achieve the purpose of long-term preservation of strains, 3 to 4 transfer activation culture work is required each year, which is labor-intensive and time-consuming; in addition, repeated transfer activation culture will increase the probability of contamination and variation of strains. A method for long-term preservation of rice sheath blight strains is disclosed in Chinese patent CN103396948B. The method uses sclerotia produced by rice sheath blight as the strain preservation form, and uses a clean bench air drying method to dehydrate and dry sclerotia. Although the method is easy to operate, air drying takes too long, there are too many uncontrollable factors, and the degree of dehydration is uncontrollable and cannot be quantified, which easily increases the probability of contamination of strains. At the same time, air drying in a clean bench easily pollutes the environment.

[0006] Chinese patent application CN110878279A discloses a method for preserving Actinomycetes tiecioplanatica, which mainly protects the culture medium and the culture method of the strain. The article mentions the use of glycerol and scleroglucan as protective agents for strain preservation. According to the addition amount of scleroglucan of 2-5%, it can be inferred that scleroglucan is hydrolyzed sclerotinia gum, and the viscosity of 1% aqueous solution is about 2000-3000mpa.s. The effect is not much different from that of glycerol, and it can only ensure that the water does not freeze, and cannot achieve a better protection effect. Summary of the invention

[0007] In view of the above problems, the present application provides a strain freezing solution for Sclerotium, wherein the strain freezing solution contains sclerotium gum and ectoine, wherein the sclerotium gum not only makes water difficult to freeze, but also has a particularly outstanding isolation effect due to its good plasticity, and the ectoine provides protection for enzymes, DNA, cell membranes and the entire cell under adverse conditions such as high temperature, freezing and drying; at the same time, it can stabilize the enzyme molecular structure of the enzyme preparation, ensure that the enzyme does not lose its activity under extreme conditions such as freezing and thawing, high temperature, etc., and can better protect the strain. The strain freezing solution containing sclerotium gum and ectoine is used to preserve the genus Sclerotium, the strain has a long preservation time, and can be preserved at -40°C for more than two years, and still has a germination rate of 100% during recovery.

[0008] The cryoprotectant components used in the prior art generally select glycerol and certain polyol compounds such as mannitol, etc., and some use dimethyl sulfoxide. The main function of glycerol and polyols is to make water difficult to freeze, and the sclerotium gum in the present application can not only achieve such an effect, but also has a particularly outstanding air isolation effect due to its good plasticity. Dimethyl sulfoxide can lower the freezing point of cells, reduce the formation of ice crystals, reduce the damage of free radicals to its cells, and change the permeability of biofilm to electrolytes and metabolites, but dimethyl sulfoxide has a strong toxic effect on cells at room temperature, and tetrahydropyrimidine is an osmotic pressure compensating solute, which can balance the osmotic pressure of cells, and provide protection for enzymes, DNA, cell membranes and the entire cell under adverse conditions such as high temperature, freezing and drying; At the same time, the enzyme molecular structure of the enzyme preparation can be stabilized, and the enzyme is guaranteed not to lose activity under extreme conditions such as freezing and thawing and high temperature, and the strain can be better protected. Therefore, the freezing solution described in the present application can better protect the strain, and the preservation time is long.

[0009] The specific technical solutions of this application are as follows:

[0010] 1. Use of a composition in preparing a bacterial strain freezing solution, wherein the composition comprises sclerotium gum and ectoine.

[0011] 2. Use of a composition for improving the recovery and germination rate of frozen strains, wherein the composition comprises sclerotium gum and ectoine.

[0012] 3. The use according to item 1 or 2, wherein the mass ratio of sclerotium gum to ectoine is 1:1-5.

[0013] 4. A bacterial strain freezing solution, wherein the bacterial strain freezing solution contains sclerotium gum and ectoine.

[0014] 5. The bacterial strain freezing solution according to item 4, wherein the mass ratio of sclerotium gum to ectoine is 1:1-5.

[0015] 6. The bacterial strain freezing solution according to item 4, wherein the viscosity of the bacterial strain freezing solution is greater than or equal to 5000mPa.s, preferably 5000-10000mPa.s.

[0016] 7. The use according to any one of items 1 to 3 or the bacterial strain freezing solution according to any one of items 4 to 6, wherein the bacterial strain is of the genus Sclerotium.

[0017] 8. A method for freezing bacterial strains, wherein the bacterial strain freezing solution described in any one of items 4 to 6 is used.

[0018] 9. The freezing method according to item 8, wherein the freezing method comprises mixing the bacterial strain with the bacterial strain freezing solution and then freezing, wherein the ratio of the bacterial strain to the bacterial strain freezing solution is 8-15 sclerotia / mL.

[0019] 10. A bacterial agent, wherein the bacterial agent comprises bacterial cells, sclerotium gum and ectoine.

[0020] 11. The bacterial agent according to item 10, wherein the bacterial agent is prepared by the freezing method according to item 8 or 9.

[0021] Effects of the Invention

[0022] By using the strain freezing solution described in the present application, the strains of the genus Sclerotium can be preserved for a long time and can be preserved at -40°C for more than two years. The sclerotia of the genus Sclerotium after preservation still have a 100% germination rate when revived. In addition, the preservation process using the strain freezing solution is simple, and the volume of the tube containing the strains is small, which is convenient for storage and retrieval. DETAILED DESCRIPTION

[0023] The present application is described in detail below in conjunction with the described embodiments. Although specific embodiments of the present application are shown, it should be understood that the present application can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided to enable a more thorough understanding of the present application and to enable the scope of the present application to be fully communicated to those skilled in the art.

[0024] It should be noted that certain words are used in the specification and claims to refer to specific components. Those skilled in the art should understand that technicians may use different nouns to refer to the same component. This specification and claims do not use the difference in nouns as a way to distinguish components, but use the functional differences of components as the criterion for distinction. As mentioned throughout the specification and claims, "including" or "comprising" are open-ended terms and should be interpreted as "including but not limited to". The subsequent description of the specification is a preferred embodiment of the present application, but the description is based on the general principles of the specification and is not intended to limit the scope of the present application. The scope of protection of the present application shall be determined by the attached claims.

[0025] The present application provides an application of a composition in preparing a bacterial strain freezing solution, wherein the composition comprises sclerotium gum and ectoine.

[0026] The present application adopts a composition containing sclerotium gum and ectoine as a strain freezing solution, which can prolong the preservation time of the strains, especially for the strains of the genus Sclerotium, which can be preserved at -40°C for more than two years, and the sclerotia of the genus Sclerotium after preservation still have a 100% germination rate when revived

[0027] The present application provides the use of a composition in improving the recovery and germination rate of frozen strains, wherein the composition comprises sclerotium gum and ectoine.

[0028] After the bacterial strains are preserved using the above-mentioned composition, the bacterial strains still have a 100% germination rate when revived.

[0029] In some embodiments, the mass ratio of sclerotium gum to ectoine is 1:1-5.

[0030] For example, the mass ratio of the sclerotium gum to the ectoine (m 小核菌胶 :m 四氢嘧啶 ) can be 1:1, 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, 1:5, etc.

[0031] The present application provides a bacterial strain freezing solution, which contains sclerotium gum and ectoine.

[0032] In the present application, the scleroglucan, also known as scleroglucan, is a non-ionic homogeneous polysaccharide polymer secreted by filamentous fungi of the genus Sclerotium.

[0033] In some embodiments, the cryopreservation solution consists of sclerotium gel, ectoine and water.

[0034] In some embodiments, the mass ratio of sclerotium gum to ectoine is 1:1-5.

[0035] For example, the mass ratio of the sclerotium gum to the ectoine (m 小核菌胶 :m 四氢嘧啶 ) can be 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, 1:5, etc.

[0036] In some embodiments, based on 100 mL of the freezing solution, the freezing solution contains 0.8-1.2 g of sclerotium gel and 1-5 g of ectoine.

[0037] For example, based on 100 mL of the freezing solution, the freezing solution contains 0.8 g, 0.9 g, 1.0 g, 1.1 g, 1.2 g, etc. of sclerotium gum and 1 g, 2 g, 3 g, 4 g, 5 g, etc. of ectoine.

[0038] In some embodiments, the weight average molecular weight of the sclerotium gum is 2000k-3000kDa.

[0039] For example, the weight average molecular weight of the sclerotium gum can be 2000 kDa, 2100 kDa, 2200 kDa, 2300 kDa, 2400 kDa, 2500 kDa, 2600 kDa, 2700 kDa, 2800 kDa, 2900 kDa, 3000 kDa, etc.

[0040] In some embodiments, the viscosity of the cryopreservation solution is greater than or equal to 5000 mPa.s, preferably 5000-10000 mPa.s.

[0041] For example, the viscosity of the freezing solution may be 5000 mPa.s, 6000 mPa.s, 7000 mPa.s, 8000 mPa.s, 9000 mPa.s, 10000 mPa.s, etc.

[0042] In the present application, there is no limitation on the method for measuring the viscosity of the freezing solution, which can be measured according to conventional methods in the art, for example, using a viscometer. In the present application, an NDJ-1 rotary viscometer is used to measure the viscosity of the freezing solution.

[0043] In the present application, the preparation method of the cryopreservation solution is not limited in any way, and it can be prepared according to conventional methods in the art, for example, dissolving sclerotin in water, and then adding ectoine to prepare the cryopreservation solution; preferably, dissolving sclerotin in water to prepare a sclerotin solution, then adding ectoine to mix, and diluting to 100 mL with water. In some embodiments, the method further comprises: sterilizing the solution after dilution, preferably sterilizing at 0.1 MPa and 121°C for 20 min to obtain the cryopreservation solution.

[0044] In some embodiments, the bacterial species is Sclerotium.

[0045] The freezing solution described in the present application has a long freezing time, and the frozen strains have a 100% germination rate when revived, and the tube containing the strains is small in volume, which is convenient for storage and retrieval.

[0046] The present application provides a method for freezing bacterial strains, comprising:

[0047] The above-mentioned bacterial strain freezing solution was used for freezing.

[0048] In some embodiments, the above-mentioned bacterial strain freezing solution is mixed with bacterial strains and then frozen.

[0049] In some embodiments, the ratio of the bacterial strain to the bacterial strain freezing solution is 8-15 sclerotia / mL). In some embodiments, the freezing is performed at -38 to -42°C.

[0050] For example, the ratio of the bacterial strain to the bacterial strain freezing solution can be 8 sclerotia / mL, 9 sclerotia / mL, 10 sclerotia / mL, 11 sclerotia / mL, 12 sclerotia / mL, 13 sclerotia / mL, 14 sclerotia / mL, 15 sclerotia / mL, etc.

[0051] The cryopreservation can be performed at -38°C, -39°C, -40°C, -41°C, -42°C, etc.

[0052] In some embodiments, the bacterial species is of the genus Sclerotium, preferably Sclerotium truncatum. In some embodiments, the present application does not impose any limitation on the method for culturing the genus Sclerotium, and the genus Sclerotium can be cultured according to conventional methods in the art.

[0053] The present application provides a bacterial agent, which comprises bacterial cells, sclerotin and ectoine. In some embodiments, the bacterial agent is prepared by the above-mentioned freezing method.

[0054] Example

[0055] The present application provides a general and / or specific description of the materials and test methods used in the experiments. In the following examples, unless otherwise specified, % means wt%, i.e., weight percentage. The reagents or instruments used without indicating the manufacturer are all conventional reagent products that can be obtained commercially.

[0056] Example 1

[0057] (1) Preparation of freezing solution:

[0058] Weigh 1g of sclerotium gum powder (M w=2500kDa) was dissolved in 50ml of water and homogenized to prepare a sclerotium gum solution; 3g of tetrahydropyrimidine was weighed and mixed evenly with the sclerotium gum solution, and the mixture was made up to 100ml with water, and sterilized at 0.1MPa and 121°C for 20min to obtain a freezing solution.

[0059] (2) Methods for preserving bacterial strains

[0060] In the clean bench, use an inoculation loop to scrape 5 to 10 neat Sclerotium sclerotia and transfer them to the solid culture medium PDA in the eggplant bottle, culture at 28°C for 6-8 days, and use an inoculation loop to scrape 8 to 15 neat Sclerotium sclerotia in the clean bench into a freezing tube, then use a pipette to draw 1 mL of neat Sclerotium freezing solution and add it to the freezing tube, and then store it at -40°C. The resulting frozen sample is S1.

[0061] Example 2

[0062] The frozen sample was prepared according to the method of Example 1, except that the amount of ectoine added was 1 g, and the obtained frozen sample was S2.

[0063] Example 3

[0064] The frozen sample was prepared according to the method of Example 1, except that the amount of ectoine added was 5 g, and the obtained frozen sample was S3.

[0065] Example 4

[0066] The cryopreserved sample was prepared according to the method of Example 1, except that the amount of sclerotium gel added was 0.8 g, and the obtained cryopreserved sample was S4.

[0067] Example 5

[0068] The cryopreserved sample was prepared according to the method of Example 1, except that the amount of sclerotium gel added was 1.2 g, and the obtained cryopreserved sample was S5.

[0069] Example 6

[0070] The frozen sample was prepared according to the method of Example 1, except that the amount of ectoine added was 0.5 g, and the obtained frozen sample was S6.

[0071] Example 7

[0072] The cryopreserved sample was prepared according to the method of Example 1, except that the amount of ectoine added was 6 g, and the obtained cryopreserved sample was S7.

[0073] Example 8

[0074] The cryopreserved sample was prepared according to the method of Example 1, except that the amount of sclerotium gel added was 0.5 g, and the obtained cryopreserved sample was S8.

[0075] Example 9

[0076] The cryopreserved sample was prepared according to the method of Example 1, except that the amount of sclerotium gel added was 1.5 g, and the obtained cryopreserved sample was S9.

[0077] Comparative Example 1: Preservation of Slant Bacteria in Liquid Paraffin Oil

[0078] Pour paraffin oil into a conical flask, with the liquid amount not exceeding 1 / 3 of the total volume. Sterilize at 121℃ for 30 min twice continuously, and bake in a 105-110℃ incubator for 2 h to remove moisture from the paraffin oil. Culture the microsclerotium on the slant for one week. When plump sclerotia are produced, use a sterile pipette to absorb the paraffin oil and add it to the culture tube. The amount added should be about 1 cm higher than the culture medium at the top of the slant. Insert the rubber stopper and place upright in a 4℃ refrigerator.

[0079] Comparative Example 2: Freeze-dried bacterial strain preservation

[0080] 20g skim milk powder, add 80ml deionized water and mix well, sterilize at 116℃ for 20min, pick 3-5 mature sclerotia from the slope, store the mature sclerotia in ampoules with 20% milk, pre-freeze at -20℃ for 24h, seal the freeze-dried tube, and store in a -20℃ refrigerator.

[0081] Comparative Example 3: Slant Culture Storage

[0082] Pick 3 to 5 mature sclerotia from the slant and transfer them to a small slant. Culture them at 28°C for 7 days. When sclerotia are grown on the small slant, seal the mouth of the test tube with sealing tape and store it at 4°C.

[0083] Comparative Example 4:

[0084] The cryopreserved sample was prepared according to the method of Example 1, except that ectoine was replaced by glycerol with a mass volume ratio of 25%, and the obtained cryopreserved sample was D4.

[0085] Comparative Example 5:

[0086] The frozen sample was prepared according to the method of Example 1, except that the amount of sclerotium gel added was 4 g, and ectoine was not added. The obtained frozen sample was D5.

[0087] Comparative Example 6:

[0088] The frozen sample was prepared according to the method of Example 1, except that the amount of ectoine added was 4 g, and sclerotium gel was not added. The obtained frozen sample was D6.

[0089] Table 1 The dosage of sclerotium gum and tetrahydropyrimidine in the embodiments and comparative examples

[0090] Sclerotium gum (g) Tetrahydropyrimidine(g) Example 1 1 3 Example 2 1 1 Example 3 1 5 Example 4 0.8 3 Example 5 1.2 3 Example 6 1 0.5 Example 7 1 6 Example 8 0.5 3 Example 9 1.5 3 Comparative Example 4 1 Replace ectoine with 25% glycerol Comparative Example 5 4 Comparative Example 6 4

[0091] Test Example 1: Viscosity of cryopreservation solution

[0092] 1. Instrument

[0093] Constant temperature box, NDJ-1 rotary viscometer (rotor No. 3, 12 rpm), thermometer

[0094] 2. Determination

[0095] 200 mL of the cryopreserved solution of Examples 1-9 and Comparative Examples 4-6 was placed in a beaker and kept at a constant temperature of 25° C. for at least 15 min. The solution was measured using a NDJ-1 rotary viscometer (rotor No. 3, rotor No. 4, 12 rpm). The deflection angle (α) was read and the dynamic viscosity was calculated according to the viscometer coefficient (K) under the conditions of the rotor and rotation speed. The results are shown in Table 2.

[0096] η=Kα

[0097] Table 2 Viscosity of cryopreservation solution

[0098] Viscosity(mPa.s) S1 7000 S2 7000 S3 7000 S4 6600 S5 8600 S6 7000 S7 7200 S8 4200 S9 10500 D4 0 D5 20000 D6 0

[0099] As can be seen from Table 2, the viscosity of the freezing solution depends on the sclerotium gel and has nothing to do with the amount of ectoine added. The embedding effect of sclerotia is good within the appropriate viscosity. Too high viscosity is not conducive to the transfer of the freezing solution. For the freezing solution with very low viscosity, the sclerotia are easy to float on the surface of the freezing solution, which is not conducive to the isolation of the freezing solution from the external environment.

[0100] Experimental Example 2 Germination Rate Experiment

[0101] 1. Instrument

[0102] Clean bench, solid culture plate, disposable sterile syringe, 28℃ constant temperature incubator

[0103] 2. Activation

[0104] In a sterile clean bench, take out the cryopreservation tube, transfer the cryopreservation solution and sclerotia to a solid culture plate, use a disposable syringe to absorb the excess cryopreservation solution, evenly distribute the sclerotia on the plate, transfer to a 28°C constant temperature incubator for 3-4 days, observe the germination rate of the sclerotia, and calculate the germination rate according to the following formula. The results are shown in Table 3.

[0105] Sclerotium germination rate = (germinated sclerotia number / total number of sclerotia) × 100%

[0106] Table 3 Germination rate

[0107] Germination rate (month) Example 1 Comparative Example 1 Comparative Example 2 Comparative Example 3 1 100％ 22.22％ 0 100％ 2 100％ 14.28％ 0 100％ 3 100％ 14.28％ 0 100％ 4 100％ 0 0 77.8％ 5 100％ 0 0 41.18％ 6 100％ 0 0 22.22％ 12 100％ 0 0 14.28％ 18 100％ 0 0 0

[0108] From Table 3, it can be found that the methods of slant refrigeration, freeze drying and paraffin oil sealing for preserving strains are obviously not suitable for the preservation of Sclerotium micrococcus strains.

[0109] Table 4 Sample germination rate table (-40℃)

[0110] sample Germination rate (12 months) Germination rate (24 months) S1 100％ 100％ S2 100％ 100％ S3 100％ 100％ S4 100％ 100％ S5 100％ 100％ S6 91.7％ 83.3％ S7 100％ 100％ S8 87.5％ 75.0％ S9 100％ 100％ D4 100％ 62.5％ D5 37.2％ 0 D6 54.2％ 0

[0111] From Table 4, it can be found that the germination rate of samples S1-S5 at 24 months can reach 100%. The amount of ectoine added in S6 of Example 6 is 0.5%. Ectoine is also used as an antifreeze agent. The amount of ectoine added is too little, which cannot effectively prevent the formation of ice crystals to damage hyphae and sclerotia during freezing, which may be one of the reasons for the low germination rate of sclerotia. The amount of ectoine added in S7 is 6%, and too high a concentration may also affect the germination rate of sclerotia. The amount of sclerotium gum added in S8 was 0.5%, and the sclerotium and mycelium floated on the surface of the cryopreservation solution, which could not achieve an effect of embedding and isolating from the air. The amount of sclerotium gum added in S9 was 1.5%, and the cryopreservation was also relatively viscous, making it difficult to separate the sclerotium from the cryopreservation solution, which was not convenient for operation. The germination rate of D4 using glycerol as the cryopreservation solution at 12 months could also reach 100%, but the germination rate at 24 months was far lower than that of S1; D5 and D6 could also germinate at 12 months using sclerotium gum and tetrahydropyrimidine alone, but the germination rate was less than 60%, and the germination rate at 24 months was zero. From the above experimental results, it can be seen that the simultaneous use of sclerotium gum and tetrahydropyrimidine can synergistically enhance the germination rate of sclerotium to the greatest efficiency.

[0112] The above is only the preferred embodiment of the present application, and does not limit the present application in other forms. Any technician familiar with the profession may use the above disclosed technical content to change or modify it into an equivalent embodiment with equivalent changes. However, any simple modification, equivalent change and modification made to the above embodiment based on the technical essence of the present application without departing from the technical solution of the present application still falls within the protection scope of the technical solution of the present application.

Claims

1. Use of the composition in preparing a bacterial strain freezing solution, characterized in that: The composition comprises sclerotium gum and ectoine; The mass ratio of sclerotium gum to ectoine is 1:1-5; The bacterial species is of the genus Sclerotium.

2. The use of the composition in improving the recovery and germination rate of frozen strains, characterized in that: The composition comprises sclerotium gum and ectoine; The mass ratio of sclerotium gum to ectoine is 1:1-5; The strain is of the genus Sclerotium.

3. A bacterial strain freezing solution, characterized in that: The bacterial strain freezing solution contains sclerotium gum and ectoine; The mass ratio of sclerotium gum to ectoine is 1:1-5; The bacterial species is Sclerotium.

4. The bacterial strain freezing solution according to claim 3, characterized in that: The viscosity of the bacterial strain freezing solution is greater than or equal to 5000 mPa.s.

5. The bacterial strain freezing solution according to claim 4, characterized in that: The viscosity of the bacterial strain freezing solution is 5000-10000 mPa.s.

6. A method for freezing bacterial strains, characterized in that: Use the bacterial strain freezing solution described in any one of claims 3 to 5.

7. The cryopreservation method according to claim 6, characterized in that: The freezing method comprises mixing the bacterial strain with the bacterial strain freezing solution and then freezing, wherein the ratio of the bacterial strain to the bacterial strain freezing solution is 8-15 sclerotia / mL.

8. A bacterial agent, characterized in that The bacterial agent is prepared by the freezing method according to claim 6 or 7.