Microbial protective agent and preservation method

Abstract

The invention relates to the technical field of microorganisms, in particular to a microorganism protective agent and a preservation method. Comprising the following components in concentration: 10-40% of glycerol, 5-10% of DMSO, 2-5% of trehalose, 5-20% of PVP, 0.05-0.2% of vitamin C and a pH buffer system, through cooperation of the components, osmotic pressure can be maintained to be stable, mechanical damage in the cryopreservation process can be reduced, swelling rupture damage caused by the fact that water rapidly enters thalli in the thalli resuscitation process can also be reduced, the resuscitation survival rate is increased, and then the survival rate of genetically engineered bacteria and the plasmid retention rate can be guaranteed at the same time; and repeated freezing and thawing can be realized, so that the thalli are prevented from being damaged and the biological activity of the thalli is ensured.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and in particular to a microbial protective agent and a preservation method. Background Art

[0002] Genetically engineered bacteria, such as E. coli, use plasmids as a host to transfer gene fragments and construct engineered strains that efficiently express desired genes. However, after the plasmid vector containing the foreign gene is transformed into the E. coli recipient cell, the plasmid increases the metabolic burden on the host cell, affecting its own stability. Recombinant genetically engineered bacteria must maintain stability and genetic properties to be meaningful and valuable. Strain stability is the prerequisite and foundation for stable fermentation. Genetically engineered bacteria must be stable during use and must not mutate or exhibit any weakening or loss of certain properties.

[0003] The principle of strain preservation is to slow bacterial metabolism, placing them in a semi-permanent dormant state. Strain preservation is crucial for microbial fermentation. Its purpose is to proactively and effectively prevent aging, mutation, and decline. Different strains require different preservation methods based on their biological characteristics.

[0004] Currently, commonly used methods for preserving bacterial strains include: subculture preservation, which is simple and inexpensive. However, due to the need for regular transfers, this can lead to decreased physiological activity and degeneration of the strain, so the recommended subculture frequency should not exceed three generations. Liquid paraffin preservation, typically stored at 4°C, allows for one to two years and is commonly used to preserve molds and actinomycetes. Freeze-drying involves freezing under vacuum, using sublimation to remove moisture. The sample is then placed in a freeze dryer, ensuring complete dryness of the ampoule and sample. This method can preserve strains for years or even decades, but is the most complex and costly, requiring appropriate strain purity and age. Due to cost and operational considerations, cryopreservation is often used and is suitable for microorganisms with strong freeze tolerance. Glycerol cryopreservation is one of the most commonly used cryopreservation methods. Glycerol serves as a preservative for cryopreservation of bacterial strains, and the amount of glycerol used during cryopreservation affects the survival rate of the strain. Previous studies have shown that increasing glycerol concentrations can lead to plasmid instability, and high glycerol concentrations (>10%) can easily cause plasmid loss. Furthermore, repeated freezing and thawing are generally not recommended for glycerol cryopreservation, as repeated freezing and thawing can damage the cells and reduce their biological activity. Summary of the Invention

[0005] Therefore, the technical problem to be solved by the present invention is to overcome the defects of the prior art freezing preservation method that it is difficult to balance the survival rate and plasmid retention rate, is not suitable for repeated freezing and thawing, and is easy to damage the bacteria and reduce the biological activity of the bacteria, thereby providing a microbial protective agent and preservation method.

[0006] To this end, the present invention provides the following technical solutions:

[0007] The invention provides a microorganism protecting agent, which comprises the following components in the following concentrations: 10-40 v / v% of glycerol, 5-10 v / v% of DMSO, 2-5wt% of trehalose, 5-20wt% of PVP, 0.05-0.2wt% of vitamin C, and a pH buffer system.

[0008] Preferably, the microbial protectant comprises the following components at the following concentrations:

[0009] Glycerol 20-30v / v%, DMSO 5-8v / v%, trehalose 2-3wt%, PVP 10-20wt%, vitamin C 0.15-0.2wt%, and pH buffer system;

[0010] Preferably, the microbial protectant comprises the following components at the following concentrations:

[0011] Glycerol 20v / v%, trehalose 3wt%, DMSO 5v / v%, PVP 10wt%, vitamin C 0.15wt%, and pH buffer system;

[0012] Alternatively, glycerol 30v / v%, trehalose 2wt%, DMSO 8v / v%, PVP 20wt%, vitamin C 0.2wt%, and a pH buffer system.

[0013] Preferably, the pH buffer system contains magnesium ions, and auxiliary ions sodium ions or potassium ions;

[0014] Preferably, the pH buffer system contains magnesium sulfate or magnesium chloride, and potassium chloride or sodium chloride;

[0015] Preferably, the pH buffer system contains MgSO4·7H2O 5-20mM and NaCl 0.85-0.90wt%;

[0016] Preferably, the pH buffer system contains 10 mM MgSO 4 ·7H 2 O and 0.9 wt % NaCl.

[0017] Preferably, the components are included in the following concentrations: glycerol 20v / v%, trehalose 3wt%, DMSO 5v / v%, PVP 10wt%, vitamin C 0.15wt%, MgSO4·7H2O 10mM, and NaCl 0.9wt%.

[0018] Preferably, the components are included in the following concentrations: glycerol 30v / v%, trehalose 2wt%, DMSO 8v / v%, PVP 20wt%, vitamin C 0.2wt%, MgSO4·7H2O 10mM, and NaCl 0.9wt%.

[0019] Preferably, the microorganism protection agent further comprises a solvent for dissolving the components;

[0020] Preferably, the solvent is deionized water.

[0021] The present invention provides use of the microorganism protecting agent in protecting microorganisms.

[0022] Preferably, the microorganism comprises a genetically engineered bacterium;

[0023] Preferably, the microorganisms include Escherichia coli, yeast, Bacillus subtilis or mold

[0024] Preferably, the microorganism is a genetically engineered bacterium.

[0025] The present invention provides a microorganism preservation method, which comprises mixing the microorganism protective agent with the microorganism for preservation.

[0026] Preferably, the storage temperature is -20°C to -80°C.

[0027] Preferably, the storage time is 0 to 15 months.

[0028] Preferably, the microorganism is a bacterium or a fungus;

[0029] Preferably, the microorganism is Escherichia coli, yeast, Bacillus subtilis or mold;

[0030] Preferably, the microorganism is a genetically engineered bacterium.

[0031] The technical solution of the present invention has the following advantages:

[0032] 1. The present invention provides a microbial protective agent comprising the following components at the following concentrations: 10-40% glycerol, 5-10% DMSO, 2-5% trehalose, 5%-20% PVP, 0.05%-0.2% vitamin C, and a pH buffer system. In the microbial protective agent, the combined use of glycerol and DSMO not only reduces the DSMO dosage but also reduces the toxicity of DSMO to genetically engineered bacteria. The combined use of the permeable protective agents glycerol and DSMO, the non-permeable protective agents trehalose and PVP, and the antioxidant vitamin C, and the addition of a pH buffer system, through the coordination of the above components, can maintain osmotic pressure stability, reduce mechanical damage during cryopreservation, and reduce swelling and rupture damage caused by rapid water ingress during bacterial resuscitation, thereby improving the resuscitation survival rate. This can simultaneously ensure the survival rate and plasmid retention rate of the genetically engineered bacteria, and can withstand repeated freezing and thawing, ensuring that the bacteria are not damaged and maintain their biological activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0034] Figure 1 is a map of the recombinant plasmid with the VP1 gene inserted;

[0035] Figure 2 This is the electrophoresis result of the VP1 fragment obtained by PCR amplification of genetically engineered bacteria in the experimental group of Experimental Example 3 of the present invention; in the figure: M: Marker; +: positive control; bands 1 to 10 are plasmids extracted after 1 to 10 freeze-thaw cycles, respectively. DETAILED DESCRIPTION

[0036] The following examples are provided for a better understanding of the present invention and are not intended to limit the best mode of implementation. They do not limit the content and scope of protection of the present invention. Any product identical or similar to the present invention obtained by anyone under the guidance of the present invention or by combining the features of the present invention with other prior arts shall fall within the scope of protection of the present invention.

[0037] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in this field can be used. If the manufacturer of the reagents or instruments is not specified, they are all commercially available conventional reagents.

[0038] Due to cost and operational considerations, cryopreservation is often used, which is suitable for microorganisms with strong antifreeze ability. Glycerol cryopreservation is one of the most commonly used cryopreservation methods. Glycerol is used as a protective agent for the cryopreservation of bacterial strains. During the cryopreservation process, the amount of glycerol content affects the survival rate of the bacterial strain. Studies have shown that as the glycerol content gradually increases, the plasmid will become unstable, and high concentrations of glycerol (>10%) are likely to cause plasmid loss. In addition, the glycerol cryopreservation method is generally not recommended for repeated freezing and thawing. During repeated freezing and thawing, the bacteria are damaged by the freezing process and the biological activity of the bacteria is reduced. In order to solve the defects of the cryopreservation method in the prior art that it is difficult to take into account both the survival rate and the plasmid retention rate, and it is not suitable for repeated freezing and thawing, which can easily damage the bacteria and reduce the biological activity of the bacteria. The present invention provides a microbial protective agent and a preservation method. The microbial protective agent and the preservation method can simultaneously ensure the survival rate and plasmid retention rate of genetically engineered bacteria, and can be repeatedly frozen and thawed to ensure that the bacteria are not damaged and the bacteria are biologically active.

[0039] The microbial protectant provided by the embodiment of the present invention includes the following components in concentrations: 10-40 v / v% glycerol, 5-10 v / v% DMSO, 2-5wt% trehalose, 5wt%-20wt% PVP, 0.05wt%-0.2wt% vitamin C, and a pH buffer system.

[0040] Among the aforementioned microbial protectants, the present inventors have discovered that while most cryoprotectants produce protective effects, they also produce a number of adverse reactions. To enhance the protective effect of a cryoprotectant, increasing its concentration also increases its adverse reactions. However, when two or more cryoprotectants have the same protective effect but different toxic mechanisms, combining them may yield improved protection while minimizing their toxic effects. Combining a permeable and non-permeable cryoprotectant may produce the most ideal synergistic cryoprotective effect.

[0041] In the microbial protective agent of the present invention, glycerol and DSMO are used in combination, which not only reduces the amount of DSMO used, but also weakens the toxicity of DSMO to genetically engineered bacteria. The permeable protective agent glycerol, DSMO, the non-permeable protective agent trehalose, PVP and the antioxidant vitamin C are used in combination, and a pH buffer system is added. Through the coordination between the above components, the bacteria can be protected both internally and externally, the osmotic pressure can be maintained stable, the mechanical damage of ice crystals during freezing can be reduced, and the swelling and rupture damage caused by the rapid entry of water into the bacteria during the recovery process can be protected, thereby improving the survival rate of the recovery and the plasmid retention rate, thereby ensuring the survival rate and plasmid retention rate of the genetically engineered bacteria at the same time, and repeated freezing and thawing can be carried out to ensure that the bacteria are not damaged and the bacteria are biologically active.

[0042] In some embodiments, in the above-mentioned microbial protective agent, the concentration of glycerol can be any one of 10, 12, 15, 18, 20, 22, 25, 27, 30, 33, 35, 38, 40 v / v% or a range between any two values; the concentration of DMSO can be any one of 5, 6, 7, 8, 9, 10 v / v% or a range between any two values; the concentration of trehalose can be 2, 3, 3.2, 3.5, 3.8, 4 , 4.2, 4.5, 4.8, 5wt% or any range between any two values; the concentration of PVP can be any one of 5, 7, 9, 10, 12, 15, 16, 18, 20wt% or any range between any two values; the concentration of vitamin C can be any one of 0.05, 0.07, 0.09, 0.1, 0.12, 0.15, 0.18, 0.2wt% or any range between any two values.

[0043] In a preferred embodiment, the microbial protective agent comprises the following components at the following concentrations: glycerol 20-30 v / v%, DMSO 5-8 v / v%, trehalose 2-3 wt%, PVP 10-20 wt%, vitamin C 0.15-0.2 wt%, and a pH buffer system.

[0044] In some embodiments, the pH buffer system can be a buffer system containing magnesium ions and auxiliary ions sodium ions or potassium ions; further, the pH buffer system contains magnesium sulfate or magnesium chloride, and potassium chloride or sodium chloride; the applicant has found through experiments that when MgSO4·7H2O and NaCl are used in the pH buffer system, the plasmid retention rate after repeated freezing and thawing is significantly higher than that of traditional Tris-HCl or phosphate buffer systems; when the concentrations of MgSO4·7H2O and NaCl are 5-20mM and 0.85-0.90wt%, respectively, the plasmid retention rate can reach 90% after 6 repeated freezing and thawing at -20°C; further, when the concentrations of MgSO4·7H2O and NaCl are 10mM and 0.9wt%, respectively, the plasmid retention rate can still reach 100% after 6 repeated freezing and thawing at -20°C.

[0045] In a preferred embodiment, the microbial protective agent comprises components with the following concentrations: glycerol 20v / v%, trehalose 3wt%, DMSO 5v / v%, PVP 10wt%, vitamin C 0.15wt%, MgSO4·7H2O 10mM, and NaCl 0.9wt%.

[0046] In a preferred embodiment, the microbial protectant comprises components with the following concentrations: glycerol 30v / v%, trehalose 2wt%, DMSO 8v / v%, PVP 20wt%, vitamin C 0.2wt%, MgSO4·7H2O 10mM, and NaCl 0.9wt%.

[0047] In some embodiments, the microbial protectant further comprises a solvent for dissolving the components;

[0048] In a preferred embodiment, the solvent is deionized water.

[0049] The embodiment of the present invention discloses the use of the microorganism protecting agent in protecting microorganisms.

[0050] In some embodiments, the microorganism is a bacterium or a fungus;

[0051] In some embodiments, the microorganism is Escherichia coli, yeast, Bacillus subtilis or mold.

[0052] In some embodiments, the microorganism comprises a genetically engineered bacterium;

[0053] In a preferred embodiment, the genetically engineered bacteria is Escherichia coli.

[0054] An embodiment of the present invention provides a method for preserving microorganisms, comprising preserving the microorganisms after mixing the microorganism protective agent with the microorganisms.

[0055] In some embodiments, the storage temperature is -20°C to -80°C. In some embodiments, the storage temperature can be any one of -20°C, -25°C, -30°C, -35°C, -40°C, -45°C, -50°C, -55°C, -60°C, -65°C, -70°C, -75°C, and -80°C, or a range between any two of the values.

[0056] In some embodiments, the storage period is 0 to 15 months. In some embodiments, the storage period is any one of 0 days, 10 days, 20 days, 30 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, and 15 months, or a range between any two of these values.

[0057] In some embodiments, the microorganism comprises bacteria or fungi;

[0058] In some embodiments, the microorganism is Escherichia coli, yeast, Bacillus subtilis or mold.

[0059] In some embodiments, the microorganism comprises a genetically engineered bacterium;

[0060] In a preferred embodiment, the genetically engineered bacteria is Escherichia coli.

[0061] In some embodiments, the microbial protective agent is mixed with the microbial liquid at a volume ratio of 1:1-1:1.5. In some embodiments, the microbial protective agent is mixed with the microbial liquid at a volume ratio of any one of 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, or a range between any two of these volumes.

[0062] The genetically engineered bacteria used in the following examples were based on the amino acid sequence of the VP1 structural protein of foot-and-mouth disease virus (FMDV). This sequence was cloned into the pET-28a vector to obtain a recombinant expression plasmid, which was then transformed into Escherichia coli to obtain the genetically engineered bacteria. The recombinant expression plasmid and the genetically engineered bacteria were prepared using conventional methods as follows:

[0063] The structural protein gene VP1 (see SEQ ID NO. 1) of the A / GDMM / 2013 strain (provided by the National Foot-and-Mouth Disease Reference Laboratory) was cloned into the kanamycin-resistant (Kana) pET-28a vector by molecular cloning technology. After the sequence was confirmed to be correct, the recombinant expression plasmid pET28-VPA was obtained. Figure 1 This process was completed by BGI Biotechnology Co., Ltd. The obtained recombinant plasmid pET28-VPA was transformed into Escherichia coli strain BL21 (DE3) to obtain genetically engineered Escherichia coli bacteria. The genetically engineered Escherichia coli bacteria used in the following experimental examples were all used.

[0064] Example

[0065] This embodiment provides a microbial protective agent as shown in the following table:

[0066] Table 1. Formulation

[0067]

[0068]

[0069] The preparation methods of the above-mentioned Examples 1 to 7 are as follows:

[0070] Weighing the solids (total volume 1 L solution): Accurately weigh trehalose, PVP, vitamin C, MgSO4·7H2O, and NaCl according to the mass ratio. Add the weighed solids to 500 mL of deionized water and stir until completely dissolved.

[0071] Add glycerol: Take glycerol by volume and slowly add it to the above solution of dissolved solids, stirring while adding to ensure uniform mixing.

[0072] Continue to add deionized water to the solution until the volume reaches 900-950 ml (depending on the amount of DMSO added later, the final volume should reach 1 L). Autoclave at 121°C for 20 minutes. After autoclaving, allow to cool at room temperature.

[0073] Add DMSO: After the solution has cooled to room temperature, transfer it to a clean bench. Accurately measure DMSO by volume and, under aseptic conditions, slowly add it to the cooled solution. Stir again to complete the solution.

[0074] Experimental Example 1

[0075] Under the condition that the pH buffer system (pH buffer system (P2): MgSO4·7H2O 10mM, NaCl 0.9%) remains unchanged, five configuration conditions of glycerol, trehalose, DMSO, PVP and vitamin C (the configuration method is the same as that in Example 1) were selected for concentration screening and used for freezing of Escherichia coli genetically engineered bacteria. The Escherichia coli genetically engineered bacteria liquid (bacterial concentration 2.0×10 8 -2.3×10 8 CFU / ml, OD 600 The volume ratio of the preservative was 1:1, and then the preservative was mixed, and then the preservative was frozen and thawed 6 times (the stored Escherichia coli genetically engineered bacteria were first stored at -20 ° C for 24 hours, and then quickly thawed in a 37 ° C water bath; after being completely thawed, they were stored at -20 ° C for 24 hours, and then quickly thawed in a 37 ° C water bath; thus, the preservative was frozen and thawed 6 times). The dissolved bacterial solution was inoculated into LB (Kana resistance) liquid medium at a concentration of 1v / v%, cultured at 37 ° C for 12 hours, and the OD was detected. 600nm The value of L 16 (4 5 ) were used for combination screening using an orthogonal experimental table, with all other culture conditions remaining unchanged. Each treatment was repeated three times, and the average value was taken. The levels of each factor are shown in Table 1.

[0076] Table 1. Factor and level settings

[0077]

[0078] This experiment uses L 16 (4 5 The orthogonal experiment table of the cryopreservation effect is as follows. From the range analysis in the table, we can see that the five influencing factors are trehalose, DMSO, glycerol, PVP, and vitamin C in descending order of significance; that is, trehalose has the greatest impact on the cryopreservation effect.

[0079] Table 2. Orthogonal table of glycerol, trehalose, DMSO, PVP and vitamin C

[0080]

[0081]

[0082] Therefore, according to the sizes of K1, K2, K3, and K4, the optimal levels are 20 v / v% glycerol, 3 wt% trehalose, 5 v / v% DMSO, 10 wt% PVP, and 0.15 wt% vitamin C.

[0083] Experimental Example 2 Verification of the Optimal Formula

[0084] In this experiment, the synergistic protective effects of different permeable protective agents, different non-permeable protective agents, and different antioxidants on bacteria were investigated.

[0085] Experimental Group: 100 mL of cryopreservation solution was prepared according to the optimal conditions selected in Experimental Example 1. The specific formula was: 20% glycerol (v / v), 3% trehalose (v / v), 5% DMSO (v / v), 10% PVP (v / v), 0.15% vitamin C (v / v), 10 mM MgSO₄·7H₂O, 0.9% NaCl (v / v), with the balance being deionized water. Comparative Groups: Group AE: While maintaining the same non-permeable and antioxidant compositions, the permeable and antioxidant compositions were varied to verify the optimal permeable and antioxidant compositions; Group FJ: While maintaining the same permeable and antioxidant compositions, the permeable and antioxidant compositions were varied to verify the optimal permeable and antioxidant compositions; Group KL: While maintaining the same permeable and non-permeable compositions, the antioxidant compositions were varied to verify the optimal antioxidant compositions. The protective agent formulas for Groups A through L are shown in Table 2.

[0086] After the protective agent was prepared according to the formula, it was sterilized by high pressure at 121°C for 20 min. Under aseptic operation conditions, 500 μL of bacterial solution (Escherichia coli genetically engineered bacteria concentration 2.0×10 8 -2.3×10 8 CFU / ml, OD 600 1.2-1.4) and 500 μL of protective agent solution, mix gently and make sure to mix thoroughly; the mixed product should be stored at -20℃.

[0087] The genetically engineered bacteria frozen with the above-mentioned different cryoprotectants (see Table 2 for details) were frozen and thawed repeatedly 6 times. The dissolved bacterial solution was inoculated into LB (Kana resistance) liquid medium at a concentration of 1 v / v%, cultured at 37°C for 12 hours (refer to Experimental Example 1), and the following test items were tested:

[0088] Test 1: Colony growth

[0089] Take the bacterial solution cultured for 12 hours and use blank LB (Kana resistance) medium as a control to measure OD 600nm To preliminarily identify whether the growth condition has changed, each treatment was tested 3 times, and the OD 600nm The bacterial solution was gram-stained and the morphology and coloration of the bacteria were observed under a microscope.

[0090] For each group, 30 frozen bacterial suspensions (obtained by repeated freezing and thawing six times for the genetically engineered bacteria frozen with different cryoprotectants (see Table 2 for details)) were randomly selected and numbered. After complete thawing, the revived bacterial suspension was picked with an inoculating loop and evenly spread on LB solid (Kana resistance) medium. The suspension was incubated at 37°C for 12-14 hours, and the formation of colonies was observed. The survival rate was calculated. The test was repeated three times for each treatment, and the average value was taken.

[0091] Survival rate (%) = number of frozen tubes with colony formation / total number of coated tubes × 100%.

[0092] Test 2: Measurement of viable bacteria count

[0093] Take the bacterial solution cultured for 12 hours and dilute it accurately to 10 with sterile LB (without Kana resistance) liquid medium in a clean bench. -6 -10 -7 Dilution solution, label it. Use a pipette to accurately draw 0.20 mL of dilution solution onto a solid LB plate. After incubation at 37°C for 16-24 hours, count the number of colonies on each plate. Make at least three replicates for each concentration. Calculate the average number of colonies on the plate, and then multiply the average by the dilution factor to obtain the total number of viable bacteria.

[0094] Test 3: Determination of plasmid retention rate of bacterial strains

[0095] Take 1 mL of the bacterial suspension that has been frozen and thawed 6 times, and dilute the bacterial suspension to a dilution factor of 10. -5 , 10 -6 , 10 -7 Spread the bacterial suspension onto LB solid medium (without Kana resistance) and incubate at 37°C for 15-17 hours. Select a dish with 30-300 colonies and pick 100 well-grown colonies. Inoculate each dish onto LB solid medium containing antibiotics (with Kana resistance) and onto LB solid medium without antibiotics (without Kana resistance). Incubate at 37°C for 12 hours. Count the number of colonies on each medium and calculate the plasmid retention rate.

[0096] Plasmid retention rate (%) = number of single colonies containing kanamycin / number of single colonies not containing kanamycin × 100%.

[0097] Table 3. Configuration of protective agents with different components and proportions

[0098]

[0099] Experimental results:

[0100] Table 4. Comparison results of protective agents with different components and proportions

[0101]

[0102]

[0103] The above results indicate that both colony growth (survival rate) and plasmid retention rates in the experimental group were 100%, and the bacteria were in good condition under microscopic examination. In Group AE, after changing the permeability protectant formula, both survival and plasmid retention rates fell below 90%, particularly in Group C, where both rates fell below 80%. This may be due to the toxicity of both DMSO and ethylene glycol, which may have harmed the bacteria. In Group FJ, the combination of non-permeability protectants was changed. Only Group H had a survival rate and plasmid retention rate above 90%, while all other groups were below 90%. In Group KL, the antioxidant composition was changed. Both survival and plasmid retention rates fell below 80%. Overall, compared with other cryoprotectant combinations, the experimental cryoprotectant formula achieved the best cryopreservation results for genetically engineered bacteria without damaging the cells, thus preserving their biological activity.

[0104] Experimental Example 3: Determination of survival after repeated freezing and thawing

[0105] Repeated freeze-thaw survival: Three tubes of genetically engineered Escherichia coli preserved with two groups of protective agents were randomly selected for repeated freeze-thaw tests. The dissolved bacterial liquid was inoculated into LB (Kana resistance) liquid culture medium at a concentration of 1% and cultured at 37°C for 12 hours. One group was the experimental group and the other group was the glycerol frozen control group.

[0106] Experimental group: 100 ml of cryopreservation solution was prepared according to the optimal configuration conditions. The specific formula is: 20 v / v% glycerol, 3 wt% trehalose, 5 v / v% DMSO, 10 wt% PVP, 0.15 wt% vitamin C, 10 mM MgSO4·7H2O, 0.9 wt% NaCl, and the balance is deionized water. After preparing according to the above formula, autoclave sterilization was carried out at 121°C for 20 min. Under aseptic operation conditions, 500 μL of 12 h cultured bacterial solution (E. coli genetically engineered bacteria concentration 2.0×10 8-2.3×10 8 CFU / ml, OD 600 1.2-1.4) and 500 μL of protective agent solution, mix gently and make sure to mix thoroughly; the mixed product should be stored at -20℃.

[0107] Control group: Prepare 100 ml of glycerol 40% (V / V), autoclave at 121°C for 20 min, and add 500 μL of bacterial solution (E. coli genetically engineered bacteria concentration 2.0×10 8 -2.3×10 8 CFU / ml, OD 600 1.2-1.4) and 500 μL 40% glycerol solution, mix gently and make sure to mix thoroughly; the mixed product should be stored at -20°C.

[0108] The stored genetically engineered Escherichia coli bacteria were first frozen at -20°C for 7 days and then quickly thawed in a 37°C water bath; after complete thawing, they were frozen at -20°C for 7 days and then quickly thawed in a 37°C water bath; the bacteria that had been repeatedly frozen and thawed 12 times were inoculated into LB liquid culture medium containing Kana and cultured to measure the colony growth (the method was referred to Experimental Example 2, repeated 3 times, and the average value was taken), the number of viable bacteria was measured (the method was referred to Experimental Example 2, repeated 3 times, and the average value was taken), and the plasmid retention rate of the bacteria was determined (the method was referred to Experimental Example 2).

[0109] Plasmid identification: Spread each freeze-thawed bacterial suspension onto LB (Kana resistance) solid medium. Pick a single colony and culture it in LB (Kana resistance) liquid medium for 12-14 hours overnight. Collect the bacterial suspension and extract the plasmid from the bacterial suspension according to the operating instructions of the high-purity plasmid extraction kit of Tiangen Biochemical (Beijing) Co., Ltd. Use primers designed for VP1 to perform PCR identification and sequencing of the corresponding extracted recombinant expression plasmids.

[0110] The experimental results are shown in the following table:

[0111] Table 5 Verification results of optimal configuration conditions

[0112]

[0113]

[0114] The primary task of cryoprotectants is to effectively protect genetically engineered bacterial strains from damage during freezing and long-term storage, and to maintain the viability of the strains. From the results in the table above, it can be seen that through the comparison of repeated freezing and thawing between the experimental group and the control group (7 days as a freeze-thaw cycle), after 6 repeated freeze-thaws, the colony growth (survival rate) of the glycerol-frozen control group was lower than 50%, and staining microscopy showed that the state of the bacteria had deteriorated, the edges were rough, and the edges were unclear; the plasmid retention rate and the number of viable bacteria also decreased significantly, indicating that repeated freezing and thawing had caused great damage to the glycerol-frozen bacteria, and subsequent experiments were no longer possible. After 10 freeze-thaws, the colony growth (survival rate) and plasmid retention rate of the experimental group were both above 90%, and the bacteria were identified to have good growth and uniform morphology by staining, and the bacterial concentration was measured at OD 600nm The data of the number of viable bacteria in the repeated freezing and thawing process are not much different. It is confirmed that the freezing formula of the present invention can be used for repeated freezing and thawing of genetically engineered bacteria without damaging the bacteria and ensuring the biological activity of the bacteria.

[0115] The recombinant expression plasmid was extracted from the bacterial solution of the experimental group that was repeatedly frozen and thawed 10 times. The target band of VP1 of 636 bp was amplified by PCR. Figure 2 ,After sequencing, the DNA sequence contained in the plasmid was 100% homologous to the original sequence, and no site mutation occurred.

[0116] Experimental Example 4: Freezing Temperature Range and Survival Rate Detection

[0117] Two groups of Escherichia coli genetically engineered bacteria were preserved using two groups of protective agents, one for the experimental group and the other for the glycerol-frozen control group:

[0118] Experimental group: 100 ml of cryopreservation solution was prepared according to the optimal configuration conditions. The specific formula is: 20 v / v% glycerol, 3 wt% trehalose, 5 v / v% DMSO, 10 wt% PVP, 0.15 wt% vitamin C, 10 mM MgSO4·7H2O, 0.9 wt% NaCl, and the balance is deionized water. After preparing according to the above formula, autoclave sterilization was carried out at 121°C for 20 min. Under aseptic operation conditions, 500 μL of bacterial solution (E. coli genetically engineered bacteria concentration 2.0×10 8 -2.3×10 8 CFU / ml, OD 600 1.2-1.4) and 500 μL of protective agent solution, mix gently and make sure to mix thoroughly; the mixed product should be stored at -20℃ or -80℃.

[0119] Control group: Prepare 100 ml of glycerol 40% (V / V), autoclave at 121°C for 20 min, and add 500 μL of bacterial solution (E. coli genetically engineered bacteria concentration 2.0×10 8 -2.3×10 8 CFU / ml, OD 600 1.2-1.4) and 500 μL 40% glycerol solution, mix gently and make sure to mix thoroughly; the mixed product should be stored at -20℃ or -80℃.

[0120] The mixed products of the experimental group and the control group were stored at -20 ° C and frozen for 0 months, 1 month, 2 months, 3 months, 6 months, 9 months, 12 months, 15 months and 18 months, respectively. 30 tubes were randomly selected (30 tubes were randomly selected for each freezing temperature corresponding to each group) for resuscitation treatment (the dissolved bacterial solution was inoculated into LB (Kana resistance) liquid culture medium at a concentration of 1 v / v% and cultured at 37 ° C for 12 h), and the colony growth (repeated 3 times and the average value was taken) and the plasmid retention rate of the strain were detected (the method was referred to Experimental Example 2).

[0121] Experimental results:

[0122] Table 6 Colony growth (survival rate) results of different freezing ranges and storage periods

[0123]

[0124]

[0125] Table 7 Plasmid retention rates at different freezing ranges and storage periods

[0126]

[0127] Generally speaking, the survival rate and plasmid retention rate of genetically engineered bacteria after resuscitation are both above 90%, and experiments have confirmed that this does not affect subsequent fermentation culture. As can be seen from the above results, the experimental and control groups were placed at -20°C for 18 months. The survival rate and plasmid retention rate of the control group's bacteria at -20°C were less than 50% within 3 months, and only 20% after 6 months, almost completely ineffective. However, after 12 months, the survival rate and plasmid retention rate of the experimental group were still above 95%. After 15 months, the survival rate and plasmid retention rate of the experimental group at -20°C were still above 90%, and at 18 months, the survival rate and plasmid retention rate had fallen below 90%. This shows that this freezing formula can extend the freezing time of the bacteria at -20°C to 15 months, while the control group can only be preserved for 1 month.

[0128] Experimental Example 5

[0129] The microbial protective agents prepared in Examples 1-7 were implemented according to Experimental Example 3, and the results were as follows:

[0130] Table 8 Verification results of Example 1

[0131]

[0132]

[0133] Table 9 Verification results of Example 2

[0134]

[0135]

[0136] Table 10 Verification results of Example 3

[0137]

[0138] Table 11 Verification results of Example 4

[0139]

[0140]

[0141] Table 12 Verification results of Example 5

[0142]

[0143] Table 13 Verification results of Example 6

[0144]

[0145] Table 14 Verification results of Example 7

[0146]

[0147]

[0148] Experimental Example 6

[0149] The microbial protective agents prepared in Examples 1-7 were implemented according to Experimental Example 4, and the results were as follows:

[0150] Table 15 Colony growth (survival rate) results of different freezing ranges and storage periods in Example 1

[0151]

[0152] Table 16 Colony growth (survival rate) results of different freezing ranges and storage periods in Example 2

[0153]

[0154] Table 17 Colony growth (survival rate) results of different freezing ranges and storage periods in Example 3

[0155]

[0156]

[0157] Table 18 Colony growth (survival rate) results of different freezing ranges and storage periods in Example 4

[0158]

[0159] Table 19 Colony growth (survival rate) results of different freezing ranges and storage periods in Example 5

[0160]

[0161] Table 20 Colony growth (survival rate) results of different freezing ranges and storage periods in Example 6

[0162]

[0163] Table 21 Colony growth (survival rate) results of different freezing ranges and storage periods in Example 7

[0164]

[0165]

[0166] In summary, the present invention adopts L 16 (4 5 ) was used for combined screening using an orthogonal experimental table. The optimal freezing formula was 20% glycerol (v / v), 3% trehalose (v / v), 5% DMSO (v / v), 10% PVP (v / v), 0.15% vitamin C (v / v), 10mM MgSO4·7H2O (v / v), and 0.9% NaCl (v / v). Sterilization was performed at 121°C for 20 minutes. Under aseptic conditions, the preservative was mixed with the bacterial solution in a 1:1 volume ratio and stored at -20°C. A freeze-thaw cycle of 7 days was used. The experimental group, which underwent 10 freeze-thaw cycles, was used within 2.5 months, demonstrating no damage to the bacteria while maintaining their biological activity. Sequencing confirmed that the DNA sequence contained in the plasmid was 100% homologous to the original sequence, with no site mutations. Compared with the conventional formula (glycerol freezing), this freezing formula extends the storage time at -20°C from 1 month of the conventional formula to 15 months; in summary, the freezing formula of the present invention can stably preserve genetically engineered bacterial strains for a long time, and does not damage the bacteria during the freezing process, ensuring the smooth progress of subsequent experiments.

[0167] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will readily appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A microbial protective agent, characterized in that The invention comprises components with the following concentrations: 10-40 v / v% of glycerol, 5-10 v / v% of DMSO, 2-5 wt% of trehalose, 5-20 wt% of PVP, 0.05-0.2 wt% of vitamin C, and a pH buffer system.

2. The microbial protective agent according to claim 1, characterized in that The following concentrations of components are included: Glycerol 20-30v / v%, DMSO 5-8v / v%, trehalose 2-3wt%, PVP 10-20wt%, vitamin C 0.15-0.2wt%, and pH buffer system; Preferably, the microbial protectant comprises the following components at the following concentrations: Glycerol 20v / v%, trehalose 3wt%, DMSO 5v / v%, PVP 10wt%, vitamin C 0.15wt%, and pH buffer system; Alternatively, glycerol 30v / v%, trehalose 2wt%, DMSO 8v / v%, PVP 20wt%, vitamin C 0.2wt%, and a pH buffer system.

3. The microbial protective agent according to claim 1 or 2, characterized in that The pH buffer system contains magnesium ions, and sodium ions or potassium ions; Preferably, the pH buffer system contains magnesium sulfate or magnesium chloride, and potassium chloride or sodium chloride; Preferably, the pH buffer system contains MgSO4·7H2O 5-20mM and NaCl 0.85-0.90wt%; Preferably, the pH buffer system contains 10 mM MgSO 4 ·7H 2 O and 0.9 wt % NaCl.

4. The microbial protective agent according to any one of claims 1 to 3, characterized in that The microorganism protecting agent further comprises a solvent for dissolving the components; Preferably, the solvent is deionized water.

5. Use of the microorganism protecting agent according to any one of claims 1 to 4 in protecting microorganisms.

6. The use according to claim 5, characterized in that The microorganism is a bacterium or a fungus; Preferably, the microorganism is Escherichia coli, yeast, Bacillus subtilis or mold; Preferably, the microorganism is a genetically engineered bacterium.

7. A method for preserving microorganisms, characterized in that: The method comprises mixing the microorganism protecting agent according to any one of claims 1 to 4 with microorganisms for preservation.

8. The microorganism preservation method according to claim 7, characterized in that: The storage temperature is -20°C to -80°C.

9. The microorganism preservation method according to claim 7 or 8, characterized in that: The storage time is 0 to 15 months.

10. The microorganism preservation method according to any one of claims 7 to 9, characterized in that: The microorganism is a bacterium or a fungus; Preferably, the microorganism is Escherichia coli, yeast, Bacillus subtilis or mold; Preferably, the microorganism is a genetically engineered bacterium.

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