A microbial cryopreservation solution and its preservation method

Abstract

The present invention belongs to the technical field of microorganism preservation, and relates to a microorganism cryopreservation solution and a preservation method thereof. The present invention provides a microorganism cryopreservation solution, which is composed of a protective solution and a carrier; the protective solution is composed of skim milk powder, β-cyclodextrin, glycerol and glucose; the carrier is a porous foam metal material modified by acrylic acid. The present invention also provides a preservation method for microorganisms, which can achieve conventional preservation for 30 days at -5 to 0 °C, cryopreservation for 6 months at -20 to -18 °C, and vacuum freeze-drying preservation for 12 months at -90 to -80 °C.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial preservation, and relates to a microbial low-temperature preservation solution and a preservation method thereof. Background Art

[0002] The needs of industry, agriculture, medicine, and scientific research have driven the development of microbial preservation technology. For example, when microorganisms are discovered to be the specific pathogens of certain infectious diseases, the development of vaccines, antibiotics, and chemical drugs requires the isolation, identification, and preservation of microorganisms. The most obvious example in agriculture is the study of nitrogen-fixing bacteria, and there are countless examples of industrial applications of microorganisms. Microbial fermentation also provides a large number of foods, beverages, and medicines. In molecular biology research, advanced DNA recombinant technology can be used to modify microorganisms (US Congress 1981), and microorganisms are the foundation of genetic engineering. Microorganisms are important biological resources, and the purpose of preserving microorganisms is not only to preserve microbial strains in a living state, but also to ensure that their genetic traits remain unchanged from the time of isolation or the beginning of the experiment.

[0003] Currently, most microbial inoculants on the market are stored in solid form. While solid microbial inoculants have a longer shelf life than liquid inoculants, their preparation requires significantly more equipment, processes, labor, and cost. Furthermore, field research activities require the collection and temporary storage of microorganisms. Summary of the Invention

[0004] The present invention aims to provide a microbial cryopreservation solution to extend the shelf life of microbial samples. Another object of the present invention is to provide a microbial preservation method that, when used with the microbial cryopreservation solution, can significantly extend the shelf life of microorganisms while maintaining the effective microbial content.

[0005] Based on the above objectives, the present invention provides a microbial low-temperature preservation solution and a preservation method thereof to address this need in the art.

[0006] On the one hand, the present invention relates to a microorganism low-temperature preservation solution, which is composed of a protective solution and a carrier; the protective solution is composed of skimmed milk powder, β-cyclodextrin, glycerol and glucose; and the carrier is a porous foam metal material modified with acrylic acid.

[0007] Furthermore, in the microorganism cryopreservation solution provided by the present invention, 10 to 15 cm 3 carrier.

[0008] Further, in the microbial cryopreservation solution provided by the present invention, the protective solution is composed of 10 - 30 g / mL skim milk powder, 1 - 5 g / mL β-cyclodextrin, 2 - 10 g / mL glycerol, and 1 - 5 g / mL glucose.

[0009] Preferably, in the microbial cryopreservation solution provided by the present invention, the protective solution is composed of 12.5 g / mL skim milk powder, 1.3 g / mL β-cyclodextrin, 6.5 g / mL glycerol, and 3.4 g / mL glucose.

[0010] Further, in the microbial cryopreservation solution provided by the present invention, the preparation method of the carrier includes: soaking the porous foam metal material in a dilute hydrochloric acid solution to remove the oxide layer, and then immersing it in an aqueous solution of acrylamide for 30 - 60 min to obtain a porous foam metal material with acrylic acid surface modification.

[0011] Further, in the microbial cryopreservation solution provided by the present invention, the mass concentration of the aqueous solution of acrylamide is 20 - 40%.

[0012] On the other hand, the present invention relates to a method for preserving microorganisms, which uses the above-mentioned microbial cryopreservation solution to preserve microorganisms.

[0013] Further, in the method for preserving microorganisms provided by the present invention, it includes:

[0014] By volume, the ratio of the microbial suspension to the protective solution is 1:2 - 5; for every 100 mL of the protective solution, 10 - 15 cm 3 carrier;

[0015] Put the microbial suspension, the protective solution, and the carrier into a container, seal and shake for at least 30 s; then freeze with liquid nitrogen to obtain solid microorganisms, and after vacuum freeze-drying the solid microorganisms, store them under vacuum at -80°C to -20°C.

[0016] Further, in the method for preserving microorganisms provided by the present invention, the vacuum freeze-drying includes: storing the solid microorganisms at -50°C to -35°C for 4 - 6 h, then performing primary drying at -35°C to -20°C for 8 - 12 h, and finally performing analytical drying at 10°C to 25°C for 7 - 10 h

[0017] On the other hand, the present invention relates to the application of the above-mentioned microbial cryopreservation solution in the preservation of microorganisms, and the preservation condition is not higher than 25°C.

[0018] Compared with the prior art, the technical solution provided by the present invention has at least the following advantages or beneficial effects:

[0019] (1) The present invention provides a microbial cryopreservation solution, which is directly mixed with a sample containing microorganisms, and can achieve the conventional preservation of microorganisms at -5°C to 0°C for at least 30 days.

[0020] (2) The present invention provides a microbial cryopreservation solution, which is directly mixed with a sample containing microorganisms. Through rapid freezing with liquid nitrogen and vacuum freeze-drying, it can be vacuum cryo-dried and preserved at -90 to -80 °C for 12 months. Detailed implementation manners

[0021] Next, the technical solutions of the present invention will be described in conjunction with the embodiments. However, the present invention is not limited to the following embodiments.

[0022] In the following embodiments, the experimental methods and detection methods are all conventional methods unless otherwise specified; the medicaments and materials can all be purchased on the market unless otherwise specified; the index data are all measured by conventional measurement methods unless otherwise specified.

[0023] Example 1

[0024] This example provides the preparation of a microbial cryopreservation solution.

[0025] Preparation of the protective solution: It consists of 10 g / mL skim milk powder, 1 g / mL β-cyclodextrin, 2 g / mL glycerol, and 1 g / mL glucose, and the solvent is sterile water.

[0026] Preparation of the carrier: The porous nickel foam (purchased from Hebei Zeus Shield Metal Materials Co., Ltd.) is immersed in a dilute hydrochloric acid solution to remove the oxide layer, and then immersed in an aqueous acrylamide solution with a mass concentration of 20% for 30 min, and then taken out and dried with hot air.

[0027] Preparation of the microbial cryopreservation solution: Place 100 mL of the protective solution and 10 cm 3 of the carrier in a glass container.

[0028] Example 2

[0029] This example provides the preparation of a microbial cryopreservation solution.

[0030] Preparation of the protective solution: It consists of 12.5 g / mL skim milk powder, 1.3 g / mL β-cyclodextrin, 6.5 g / mL glycerol, and 3.4 g / mL glucose, and the solvent is sterile water.

[0031] Preparation of the carrier: The porous nickel foam is immersed in a dilute hydrochloric acid solution to remove the oxide layer, and then immersed in an aqueous acrylamide solution with a mass concentration of 20% for 30 min, and then taken out and dried with hot air.

[0032] Preparation of the microbial cryopreservation solution: Place 100 mL of the protective solution and 10 cm 3 of the carrier in a glass container.

[0033] Example 3

[0034] This embodiment provides a method for preparing a microbial cryopreservation solution.

[0035] Preparation of the protective solution: It consists of 30 g / mL skim milk powder, 5 g / mL β-cyclodextrin, 10 g / mL glycerol, and 5 g / mL glucose, and the solvent is sterile water.

[0036] Preparation of the carrier: Soak the porous nickel foam in dilute hydrochloric acid solution to remove the oxide layer, then immerse it in an aqueous acrylamide solution with a mass concentration of 40% for 60 min, and take it out and dry it with hot air.

[0037] Preparation of the microbial cryopreservation solution: Place 100 mL of the protective solution and 15 cm 3 of the carrier in a glass container.

[0038] Example 4

[0039] This embodiment provides a test on the application of the microbial cryopreservation solution for preserving bacteria.

[0040] Tested strains: Commercially available standard Escherichia coli strain (ATCC25922) and standard Staphylococcus aureus strain (ATCC6538).

[0041] The test groups are shown in Table 1.

[0042] Table 1: Test groups

[0043]

[0044] (1) Room temperature preservation

[0045] Dilute the activated Escherichia coli in LB liquid medium to prepare a cell suspension of 5×10 4 cfu / mL. After conducting the tests according to the groups shown in Table 1, preserve it at 15 °C at room temperature, and repeat each test group 5 times. Resuscitate it on the 5th, 10th, 30th, and 60th days after preservation, observe the colony morphology and cell morphology of the surviving strains, and conduct biochemical reaction identification when necessary to exclude contamination by miscellaneous bacteria, and calculate the survival rate. The test results are shown in Table 2.

[0046] Table 2: Results of room temperature preservation

[0047]

[0048] As can be seen from Table 2, the survival rate of Escherichia coli preserved in control groups 1 - 3 after 30 days of preservation at 15 °C is 85 - 91%, while the survival rate of Escherichia coli preserved in test groups 1 - 3 after 30 days of preservation at 15 °C is 100%.

[0049] (2) Vacuum freezing preservation

[0050] The activated Staphylococcus aureus was diluted in LB liquid medium to prepare a bacterial suspension of 5×10 4 cfu / mL. Vacuum freeze-drying was carried out according to the groups shown in Table 1, and each test was repeated 5 times.

[0051] Specifically, the microbial suspension, protective solution and carrier were placed in a container and sealed and shaken for at least 30 s; then the solidified microorganism was obtained after freezing with liquid nitrogen. After the solidified microorganism was vacuum freeze-dried (the instrument was FlexiDry vacuum freeze dryer), it was stored in vacuum at -80 °C. Vacuum freeze-drying was as follows: the solidified microorganism was stored at -40 °C for 5 h, then the main drying was carried out at -30 °C for 10 h, and finally the analytical drying was carried out at 15 °C for 8 h.

[0052] Resuscitation was carried out at 6, 12, 15, and 18 months after storage, and the colony morphology and cell morphology of the surviving strains were observed. Biochemical reaction identification was carried out when necessary to exclude contamination by miscellaneous bacteria, and the survival rate was calculated. The test results are shown in Table 3.

[0053] Table 3: Results of vacuum storage at -80 °C

[0054]

[0055] As described in Table 3, the technical solution provided by the present invention achieved a better preservation effect compared with the control group. After vacuum storage at -80 °C for 18 months, the survival rate could reach 50-62.5%. As above, the present invention can be preferably implemented. The above embodiments are only descriptions of the preferred implementation modes of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various changes and improvements made by those of ordinary skill in the art to the technical solution of the present invention should fall within the protection scope determined by the present invention.

Claims

1. A method for preserving microorganisms, characterized in that, Microorganisms are preserved using a low-temperature preservation solution for microorganisms; The low-temperature preservation solution for microorganisms consists of a protective solution and a carrier; the protective solution consists of skim milk powder, β-cyclodextrin, glycerol, and glucose; the carrier is a porous foam metal material modified with acrylic acid; Every 100 mL of the said protective liquid is formulated with 10 - 15 cm 3 of the said carrier; The protective solution consists of 10 - 30 g / mL skim milk powder, 1 - 5 g / mL β-cyclodextrin, 2 - 10 g / mL glycerol, and 1 - 5 g / mL glucose; The preparation method of the carrier includes: soaking the porous foam metal material in a dilute hydrochloric acid solution to remove the oxide layer, and then immersing it in an aqueous solution of acrylamide for 30 - 60 min, which is the porous foam metal material modified with acrylic acid; The mass concentration of the aqueous solution of acrylamide is 20 - 40%; By volume, the ratio of the microorganism suspension to the protective solution is 1:2 - 5; The porous foam metal material is porous foam nickel; 2. The microbial preservation method according to claim 1, wherein The protective solution consists of 12.5 g / mL skim milk powder, 1.3 g / mL β-cyclodextrin, 6.5 g / mL glycerol, and 3.4 g / mL glucose; 3. The microbial preservation method according to claim 1, characterized in that, Including: Placing the microorganism suspension, the protective solution, and the carrier in a container, sealing and shaking for at least 30 s; then freezing with liquid nitrogen to obtain solid microorganisms, and after vacuum freeze-drying the solid microorganisms, storing them under vacuum at -80°C to -20°C.

4. The microbial preservation method according to claim 3, wherein The vacuum freeze-drying includes: storing the solid microorganisms at -50 to -35°C for 4 - 6 h, then performing primary drying at -35 to -20°C for 8 - 12 h, and finally performing analytical drying at 10 to 25°C for 7 - 10 h.

Images