Application of soybean meal as strain protective agent and strain preservation method

Abstract

The invention provides application of soybean meal as a strain protective agent and a strain preservation method, and belongs to the technical field of microorganism preservation. The strain preservation method comprises the following steps: preparing a soybean meal solution with the volume concentration of 10-40%, mixing the soybean meal solution with a strain culture solution in the logarithmic phase according to the volume ratio of 4: 6-6: 4 to prepare a suspension, and performing freeze-drying treatment for strain preservation. According to the present invention, the plant-derived soybean powder is adopted as the strain protection agent, such that the virus or bacterial pollution risk possibly caused by the use of the animal-derived protection agent is avoided, the strain storage safety is improved, and the good stability and the good reliability are provided. The soybean meal is convenient to obtain and store, so that the production and storage cost is reduced; and meanwhile, the use of the soybean meal has small influence on the environment, and meets the green and environment-friendly requirements.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial preservation, and in particular relates to an application of soybean powder as a strain protective agent and a strain preservation method. Background Art

[0002] In the field of microbiology, strain preservation is a vital task, which is of key significance for ensuring the quality, activity and genetic stability of strains. The traditional method of strain preservation is mainly through vacuum freeze-drying, which can preserve strains for a long time in a low temperature and low pressure environment to maintain their activity. However, vacuum freeze-drying requires the use of protective agents to prevent the strains from being damaged during the freeze-drying process. Strain protective agents play an indispensable role in strain preservation. However, the currently commonly used strain protective agents have certain limitations and potential problems.

[0003] Traditional strain protective agents mainly include animal-derived protective agents and plant-derived protective agents. Animal-derived protective agents, such as serum and peptone, can protect strains to a certain extent, but because they come from animals, there is a risk of carrying viruses or bacteria, which may cause strains to be contaminated, thereby affecting the safety and reliability of strain preservation. In addition, the acquisition of animal-derived protective agents is often subject to many restrictions, the cost is high, and the production and storage process is relatively complicated. In terms of environmental protection, the production and use of animal-derived protective agents may put a certain amount of pressure on the environment. Summary of the invention

[0004] In view of this, the purpose of the present invention is to provide an application of soybean powder as a strain protective agent and a strain preservation method, which has good stability and reliability for strain preservation and improves the safety of strain preservation compared to animal-derived strain protective agents.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] Application of soybean powder as strain protectant in strain preservation.

[0007] Preferably, the bacterial species is Amycolatopsis orientalis.

[0008] The present invention also provides a strain preservation method, which comprises preparing a soybean powder solution with a volume concentration of 10%-40%, mixing it with a logarithmic growth phase strain culture solution at a volume ratio of 4:6-6:4 to prepare a suspension, and freeze-drying the suspension to preserve the strain.

[0009] Preferably, the solvent of the soybean powder solution is deionized water.

[0010] Preferably, the soybean powder solution and the culture solution of the bacteria in the logarithmic growth phase are mixed in a volume ratio of 1:1 to prepare the suspension.

[0011] More preferably, the bacterial species is Amycolatopsis orientalis.

[0012] Preferably, the suspension is divided into thin glass tubes for freeze-drying and sealed for storage.

[0013] More preferably, the freeze-drying treatment is performed at a temperature of -25°C, a vacuum degree of ≤0.067 mbar, and a time of ≥24 h.

[0014] Preferably, the strain is stored at a temperature of -75°C to -85°C.

[0015] The present invention also provides application of the bacterial strain preservation method in microbial culture or reproduction.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The present invention uses soybean powder of plant origin as a strain protective agent, thereby avoiding the risk of virus or bacteria contamination that may be caused by using a protective agent of animal origin, and improving the safety of strain preservation.

[0018] The soybean powder used in the present invention is a common plant raw material, which is easy to obtain and store, and reduces the production and storage costs. At the same time, the use of soybean powder has little impact on the environment, and meets the requirements of green environmental protection.

[0019] The present invention verifies the effect of soybean powder as a protective agent through experiments. The results show that soybean powder can effectively protect the activity of bacterial strains, and there is no significant difference in the protective effect of soybean powder of different brands on bacterial strains, indicating that soybean powder has good stability and reliability as a protective agent.

[0020] The technical solution of the invention is simple and feasible, and can be used not only for the preservation of Amycolatopsis orientalis strains, but also for the preservation of strains of other bacteria, and has broad application prospects. DETAILED DESCRIPTION

[0021] The present invention provides an application of soybean powder as a strain protective agent in strain preservation. Soybean powder is a common plant raw material that is easy to obtain and store. The present invention does not specifically limit the source of soybean powder, and it can be directly obtained through market channels. As an implementable embodiment, the soybean powder of the present invention is purchased from Imuraya Food Co., Ltd., and the soybean powder product specifications are: protein: 23%, crude fat: 4.7%, drying loss: 4.7%, fineness (60 mesh): 100%; purchased from Shanghai Yicun Food Co., Ltd., the soybean powder product specifications are: protein: 43%, crude fat: 5.1%, drying loss: 4.5%, fineness (60 mesh): 97%. The present invention preferably performs physical and chemical properties and hygienic index tests after purchasing the soybean powder to meet the strain preservation requirements.

[0022] In the present invention, the preferred strain is Amycolatopsis orientalis.

[0023] The present invention also provides a strain preservation method, which comprises preparing a soybean powder solution with a volume concentration of 10% to 40%, preferably 20%, and mixing it with a culture solution of the strain in the logarithmic growth phase at a volume ratio of 4:6 to 6:4 to prepare a suspension, and freeze-drying the suspension to preserve the strain. The strain activity is highest during the logarithmic growth phase, which is conducive to preserving the survival rate.

[0024] In the present invention, the solvent of the soybean powder solution is preferably deionized water to avoid other components affecting the preservation of the strains.

[0025] In the present invention, preferably, the soybean powder solution and the culture solution of the bacterial strain in the logarithmic growth phase are mixed in an equal volume ratio to prepare the suspension, and more preferably, the bacterial strain is Amycolatopsis orientalis.

[0026] In the present invention, the suspension is preferably divided into thin glass tubes for freeze-drying and sealed for storage to ensure the stability of the strain.

[0027] In the present invention, the freeze-drying treatment is preferably performed at a temperature of -25°C, a vacuum degree of ≤0.067 mbar, and a time of ≥24 h.

[0028] In the present invention, the preferred temperature for storing the strains is -75°C to -85°C, more preferably -80°C.

[0029] The present invention also provides application of the bacterial strain preservation method in microbial culture or reproduction.

[0030] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0031] Example 1

[0032] A strain protective agent is prepared from soybean powder as a raw material and deionized water as a solvent to form a soybean powder solution with a volume concentration of 20%.

[0033] Example 2

[0034] A strain protective agent is prepared by using soybean powder as raw material and deionized water as solvent to form a soybean powder solution with a volume concentration of 10%.

[0035] Example 3

[0036] A strain protective agent is prepared by using soybean powder as raw material and deionized water as solvent to form a soybean powder solution with a volume concentration of 40%.

[0037] Example 4

[0038] A method for preserving bacterial strains, comprising the following steps:

[0039] The culture solution of Amycolatopsis orientalis cultured to the logarithmic growth phase is mixed with the strain protective agent in Example 1 in equal volumes to prepare a suspension; the suspension is divided into thin glass tubes and placed in a freeze dryer for freeze drying. The freeze drying conditions are: temperature -25°C, vacuum degree below 0.067 mbar, time more than 24 hours, and sealing after freeze drying to ensure the stability of the strain; the freeze-dried vacuum freeze-dried tube is stored in a -80°C freezer for strain preservation.

[0040] Example 5

[0041] A method for preserving bacterial strains, comprising the following steps:

[0042] The culture solution of Amycolatopsis orientalis cultured to the logarithmic growth phase was mixed with the strain protective agent in Example 2 at a volume ratio of 4:6 to prepare a suspension; the suspension was divided into thin glass tubes and placed in a freeze dryer for freeze drying. The freeze drying conditions were: temperature -25°C, vacuum degree below 0.067 mbar, time more than 24 hours, and sealing after freeze drying to ensure the stability of the strain; the freeze-dried vacuum freeze-dried tube was stored in a -75°C freezer for strain preservation.

[0043] Example 6

[0044] A method for preserving bacterial strains, comprising the following steps:

[0045] The culture solution of Amycolatopsis orientalis cultured to the logarithmic growth phase was mixed with the strain protective agent in Example 3 at a volume ratio of 6:4 to prepare a suspension; the suspension was divided into thin glass tubes and placed in a freeze dryer for freeze drying. The freeze drying conditions were: temperature -25°C, vacuum degree below 0.067 mbar, time more than 24 hours, and sealing after freeze drying to ensure the stability of the strain; the freeze-dried vacuum freeze-dried tube was stored in a -85°C freezer for strain preservation.

[0046] Example 7

[0047] Effects of different volume concentrations of strain protectants on the preservation of Amycolatopsis orientalis strains

[0048] The oriental pseudomycotic acid bacteria cultured to the logarithmic growth phase were mixed with the strain protective agents in Examples 1, 2, and 3 in equal volumes to prepare a suspension. The suspension was divided into thin glass tubes and placed in a freeze dryer for freeze drying. The freeze drying conditions were: temperature -25°C, vacuum degree below 0.067 mbar, time more than 24 hours, and sealing after freeze drying to ensure the stability of the strain. The freeze-dried vacuum freeze-dried tubes were stored in a -80°C freezer for strain preservation.

[0049] After 30 days, samples were taken for viable bacterial count detection. The method for detecting the viable bacterial count was as follows: the freeze-dried tube was cut open, dissolved in 9 mL of sterile water, and then 800 μL was added from another 9 mL of sterile water using a graduated pipette, and this was used as 10 -1 Diluent, add 1 mL of diluent to 9 mL of sterile water using a graduated pipette and mix thoroughly to make 10 -2 Dilution, repeatedly mix and dilute to 10 -7 . 10 -3 -10 -7 Apply 0.1 mL of the dilution solution to the plate culture medium according to N=3, and culture at 28.0±1.5℃ for 5-7 days. Select the test area where the number of colonies can be accurately counted from the plate after culture, and use it to calculate the number of viable bacteria (optimal number: 30-100). Calculation formula: average number of colonies of n=3 / dilution rate / 0.1 mL (CFU / tube).

[0050] Table 1 Effects of different volume concentrations of strain protectants on the preservation of Amycolatopsis orientalis strains

[0051]

[0052] As shown in Table 1, using soybean powder as a raw material as a strain protectant for strain preservation can ensure a higher number of viable bacteria. The number of viable bacteria is different at different final concentrations of the protectant. When the final concentration of the protectant is 10%, the number of viable bacteria is the highest, and the preservation effect is the best.

[0053] Example 8

[0054] On the basis of the treatment with the final concentration of 10% of the protective agent in Example 7, a comparative experiment was conducted using skimmed milk powder, an animal-derived protective agent, as a strain protective agent. The results are shown in Table 2:

[0055] Table 2 Effects of strain protectants from different sources on the preservation of Amycolatopsis orientalis strains

[0056]

[0057] As shown in Table 2, the strain preservation effect of using soybean powder as a strain protectant is better than that of skimmed milk powder at the same final concentration of the protectant. Compared with animal-derived strain protectants, plant-derived soybean powder as a strain protectant has application prospects.

[0058] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Application of soybean powder as strain protectant in strain preservation.

2. The use according to claim 1, characterized in that: The strain is Amycolatopsis orientalis.

3. A method for preserving bacterial strains, characterized in that: A soybean powder solution with a volume concentration of 10% to 40% is prepared, and the solution is mixed with a culture solution of bacteria in the logarithmic growth period at a volume ratio of 4:6 to 6:4 to prepare a suspension, and the suspension is freeze-dried to preserve the bacteria.

4. The method for preserving bacterial strains according to claim 3, characterized in that: The solvent of the soybean powder solution is deionized water.

5. The method for preserving bacterial strains according to claim 3, characterized in that: The soybean powder solution and the logarithmic growth phase bacterial culture solution are mixed in a volume ratio of 1:1 to prepare a suspension.

6. The method for preserving bacterial strains according to claim 3 or 5, characterized in that: The strain is Amycolatopsis orientalis.

7. The method for preserving bacterial strains according to claim 3, characterized in that: The suspension is divided into thin glass tubes for freeze-drying and sealed for storage.

8. The method for preserving bacterial strains according to claim 3 or 7, characterized in that: The freeze-drying treatment is performed at a temperature of -20°C to -30°C, a vacuum degree of ≤0.067 mbar, and a time of ≥24 hours.

9. The method for preserving bacterial strains according to claim 3, characterized in that: The temperature for storing the strain is -75°C to -85°C.

10. Use of the strain preservation method according to any one of claims 3 to 9 in microbial culture or propagation.

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