A method for preserving a bacterial strain

Abstract

The application discloses a kind of bacteria preservation methods, suitable for the preservation of the remaining bacteria powder after opening and using in freeze-dried bacteria powder in vacuum freeze-drying tube.The application first mixes the remaining bacteria powder with sterilized salt solution to make bacteria suspension, then incubates under appropriate conditions;The bacteria suspension after incubation is fully mixed with bacteria protectant;The bacteria suspension after mixing is divided into cryogenic tube, and stored at-70 to-85 ℃ under ultralow temperature.The preservation method can avoid the loss and waste of freeze-dried bacteria powder in the opened vacuum freeze-drying tube;The re-preservation of the remaining freeze-dried powder can also prevent the loss of characteristics during bacteria passage, and provides an additional layer of protection for preserving bacterial characteristics.

Description

Technical Field

[0001] This invention relates to a method for preserving microbial strains, applicable to the preservation of residual microbial powder after opening and use of freeze-dried microbial powder in a vacuum freeze-drying tube, belonging to the field of biotechnology. Background Technology

[0002] Traditional methods for preserving microbial strains can be broadly categorized into six types: subculture preservation, liquid paraffin covering preservation, carrier preservation, host preservation, cryopreservation, and freeze-drying preservation. Cryopreservation is the primary method for preserving microbial strains in laboratories, as it is simple to operate and offers a long preservation period.

[0003] Currently, most strains purchased from microbial culture centers by laboratories are preserved using freeze-drying, meaning the microorganisms exist as high-concentration freeze-dried powder. However, when using these purchased freeze-dried strains, laboratories often need to first isolate and purify the freeze-dried powder using plates, then subculture it to prepare liquid working seeds before preserving and using the strain. This process presents two problems: firstly, once opened, a large amount of freeze-dried powder remains in the vacuum freeze-drying tubes, posing a risk of waste due to the inability to preserve it; secondly, some working strains may lose their phenotypic characteristics after subculturing, making it impossible to re-obtain the target strain from the freeze-dried powder. Therefore, effectively preserving microorganisms and avoiding the loss and waste of remaining freeze-dried powder in opened vacuum freeze-drying tubes is an urgent problem to be solved. Summary of the Invention

[0004] To address the aforementioned problems, this invention provides a method for preserving bacterial strains, suitable for storing residual bacterial powder after opening and use of freeze-dried bacterial powder in vacuum freeze-drying tubes. The invention first mixes the residual bacterial powder with a sterilized salt solution to prepare a bacterial suspension, and then incubates it under appropriate conditions. The incubated bacterial suspension is then thoroughly mixed with a bacterial strain protectant. The resulting bacterial suspension is then dispensed into cryovials and stored at -70℃ to -85℃ for ultra-low temperature preservation. This method not only stabilizes the characteristics of the bacterial strain, making it less prone to mutation, but also provides a simple and reproducible method for re-preserving the freeze-dried powder, thus avoiding the loss and waste of freeze-dried bacterial powder in opened vacuum freeze-drying tubes.

[0005] The technical solution proposed in this invention is: a method for preserving bacterial strains, characterized in that it is applicable to the preservation of remaining bacterial powder after opening and use of freeze-dried bacterial powder in a vacuum freeze-drying tube; the preservation method includes the following steps:

[0006] (1) Mix the remaining bacterial powder with the sterilized salt solution to prepare a bacterial suspension;

[0007] (2) Incubate the bacterial suspension from step (1) at 25–37°C for 0.5–1.0 h;

[0008] (3) Thoroughly mix the incubated bacterial suspension with the bacterial strain protectant;

[0009] (4) Dispense the bacterial suspension after mixing in step (3) into cryovials; then store the cryovials at -70℃ to -85℃.

[0010] After opening the cryovial, the first step is to aspirate 0.9 wt% sterile saline solution and mix it thoroughly to obtain the bacterial solution. Then, a portion of the bacterial solution is diluted and spread for use. The remaining bacterial solution is centrifuged and stored. The remaining bacterial powder mentioned in this invention refers to the bacterial powder re-obtained after centrifuging the bacterial solution.

[0011] Furthermore, the salt solution composition is as follows (mass ratio): K2HPO4·3H2O 0.8%–1.0%, KH2PO4 0.1%–0.3%, Na2HPO4·12H2O 1.0%–1.2%, MgSO4·7H2O 0.02%–0.03%, with the remainder being water.

[0012] Furthermore, the strain protectant is a mixture of 30%–50% sterile glycerol and 0.3%–0.6% vitamin C aqueous solution (filtered and sterilized); the volume ratio of the two is 5–50:1. The volume ratio of the sterile salt solution to the strain protectant is 1:0.8–1.2, preferably 1:1.

[0013] Preferably, the concentration of the bacterial suspension in step (1) is 0.5*10⁻⁶. 7 ~5*10 7 CFU / mL, preferably 1*10 7 CFU / mL.

[0014] Preferably, the bacterial suspension in step (2) is incubated on a shaker with a shaking speed of 50-100 rpm.

[0015] Preferably, the volume of the cryopreservation tube in step (3) is 1 to 5 mL.

[0016] This invention features a carefully formulated incubation salt solution. The potassium and sodium salts added to the solution maintain cell osmotic pressure, cell permeability, and pH, while the magnesium salt, acting as an activator for many important enzymes, maintains cell activity. This salt solution ensures that the bacteria do not multiply during preservation, maintaining a low metabolic state. Glycerol in the strain protectant has antifreeze and moisturizing effects, protecting the microorganisms. The addition of vitamin C keeps the strain in a low-oxygen concentration environment, preventing free radical damage to the cells.

[0017] The strain preservation method of this invention has the following advantages: First, the lyophilized powder glycerol tubes obtained by this method can be used as the main strain for preservation, or activated and used as working seed. Second, this preservation method avoids the loss and waste of lyophilized bacterial powder in opened vacuum freeze-drying tubes. Third, the re-preservation of remaining lyophilized powder can prevent the loss of traits during cell subculturing, providing an extra layer of protection for the preservation of cell traits. Fourth, the incubation substrate is a salt solution, without the addition of culture medium, avoiding the nutrient conditions necessary for strain growth during preservation and stabilizing the strain traits. Fifth, the addition of vitamin C to the strain protectant maintains the strain in a low-oxygen concentration state. In summary, this method not only stabilizes the strain traits, making them less prone to mutation, but also provides a simple and reproducible re-preservation method for lyophilized powder, which is beneficial for improving production efficiency and the quality of lyophilized powder. Detailed Implementation

[0018] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of skill in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of the present invention.

[0019] Example 1:

[0020] The lyophilized bacterial powder in the vacuum freeze-dried tubes used in this embodiment is Escherichia coli genetically engineered bacteria, with a total bacterial powder content of 0.5 mL in the cryovials. After opening the cryovials, 0.5 mL of 0.9 wt% NaCl sterile physiological saline was aspirated and mixed with a pipette to obtain the bacterial solution; then, 0.25 mL of the bacterial solution was aspirated for dilution and spread for use, and the remaining 0.25 mL of the bacterial solution was centrifuged at 5000 rpm and stored.

[0021] (1) Prepare a sterile salt solution, a 50 wt% sterile glycerol solution, and a 0.6 wt% filtered and sterilized vitamin C aqueous solution; the composition of the sterile salt solution is (mass ratio): K2HPO4·3H2O 0.9%, KH2PO4 0.2%, Na2HPO4·12H2O 1.1%, MgSO4·7H2O 0.025%;

[0022] (2) Take 25 mL of the prepared sterile salt solution and mix it with the remaining bacterial powder in the cryovial (the bacterial powder obtained after centrifuging the above bacterial solution) to make the bacterial concentration approximately 2*10⁻⁶. 7 CFU / mL, to obtain a bacterial suspension;

[0023] (3) Incubate the above bacterial suspension in a shaker at 30°C and 100 rpm for 0.5 h;

[0024] (4) Then add 22.5 mL of 50 wt% sterile glycerol solution and 2.5 mL of 0.6 wt% filtered and sterilized vitamin C aqueous solution to the bacterial suspension after incubation in step (3), and mix thoroughly.

[0025] (5) Then, take 1.2 mL of the bacterial suspension prepared in step (4) into a 1.5 mL cryovial and dispense the cryovial; place the dispensed bacterial strain containing the bacterial suspension at -80℃ for ultra-low temperature storage.

[0026] The strains preserved by the above method, after being frozen at -80℃ for three years, can grow colonies when inoculated into a culture medium and cultured at 37℃, thus achieving the effect of maintaining stable characteristics and stable production levels.

[0027] Cell viability count: Samples of the preserved strains were taken in the first, second and third years of storage, diluted, and spread on agar plates at an appropriate dilution. The plates were then incubated at 37°C for 12 hours, and the cells were observed and counted.

[0028] Fermentation level determination: Strains preserved for 1, 2, and 3 years were fermented in 5L fermenters. The resulting large quantities of wet cells were then subjected to ultra-high pressure disruption in a phosphate buffer solution and centrifuged to obtain crude enzyme solutions. The crude enzyme solutions were subjected to enzymatic reactions, and enzyme activity, i.e., the fermentation level, was calculated by liquid chromatography. The survival rate and activity results of the preserved strains are shown in Table 1.

[0029] The results showed that the cell survival rate of the genetically engineered Escherichia coli under this preservation method reached over 92.3%, and the fermentation level remained basically stable.

[0030] Table 1. Survival rate and activity determination of genetically engineered Escherichia coli strains after preservation.

[0031]

[0032] Example 2:

[0033] The freeze-dried bacterial powder used in this embodiment is *Saccharomyces cerevisiae* genetically engineered bacteria, with a total bacterial powder content of 0.5 mL in the cryovial. After opening the cryovial, 0.5 mL of 0.9 wt% NaCl sterile physiological saline was aspirated and mixed with a pipette to obtain the bacterial solution. Then, 0.25 mL of the bacterial solution was aspirated for dilution and spread for use. The remaining 0.25 mL of the bacterial solution was centrifuged at 5000 rpm and then stored.

[0034] (1) Prepare a sterile salt solution, a 50 wt% sterile glycerol solution, and a 0.5 wt% filtered and sterilized vitamin C aqueous solution; the composition of the sterile salt solution is (mass ratio): K2HPO4·3H2O 0.8%, KH2PO4 0.2%, Na2HPO4·12H2O 1.2%, MgSO4·7H2O 0.02%;

[0035] (2) Take 10 mL of the prepared sterile salt solution and mix it with the remaining bacterial powder in the cryovial (the bacterial powder obtained after centrifuging the above bacterial solution) to make the bacterial concentration approximately 2*10⁻⁶. 7 CFU / mL, to obtain a bacterial suspension;

[0036] (3) Incubate the above bacterial suspension in a shaker at 30°C and 100 rpm for 40 min;

[0037] (4) Then add 9.75 mL of 50 wt% sterile glycerol solution and 0.25 mL of 0.5 wt% filtered sterilized vitamin C aqueous solution to the bacterial suspension after incubation in step (3), and mix thoroughly.

[0038] (5) Then, take 1.2 mL of the bacterial suspension prepared in step (4) into a 1.5 mL cryovial and dispense the cryovial; place the dispensed bacterial suspension into a cryopreservation chamber at -75℃.

[0039] The strains preserved by the above method, after being frozen at -75℃ for three years, can grow colonies after being inoculated into a culture medium and cultured at 30℃, thus achieving the effect of maintaining stable characteristics and stable production levels.

[0040] Cell viability detection: Samples of the preserved strains were taken in the first, second and third years of storage, diluted, and spread on agar plates at an appropriate dilution. The plates were then incubated at 30°C for 24 hours, and the samples were observed and counted.

[0041] Fermentation level determination: Strains preserved for 1, 2, and 3 years were fermented in 5L fermenters. The resulting large quantities of wet cells were then subjected to ultra-high pressure disruption in a phosphate buffer solution and centrifuged to obtain crude enzyme solutions. The crude enzyme solutions were subjected to enzymatic reactions, and enzyme activity, i.e., the fermentation level, was calculated by liquid chromatography. The survival rate and activity results of the preserved strains are shown in Table 2.

[0042] The results showed that the cell survival rate of the genetically engineered Saccharomyces cerevisiae under this preservation method reached over 94.7%, and the fermentation level remained stable.

[0043] Table 2. Survival rate and activity determination of preserved Saccharomyces cerevisiae genetically engineered strains

[0044]

[0045] Example 3:

[0046] The freeze-dried bacterial powder used in this embodiment is a type of microfibril, with a total bacterial powder content of 0.5 mL in the cryovial. After opening the cryovial, 0.5 mL of 0.9 wt% NaCl sterile physiological saline was aspirated and mixed with a pipette to obtain the bacterial solution. Then, 0.25 mL of the bacterial solution was aspirated for dilution and spread for use. The remaining 0.25 mL of the bacterial solution was centrifuged at 5000 rpm and then stored.

[0047] (1) Prepare a sterile salt solution, a 50 wt% sterile glycerol solution, and a 0.6 wt% filtered and sterilized vitamin C aqueous solution; the composition of the sterile salt solution is (mass ratio): K2HPO4·3H2O 1.0%, KH2PO4 0.1%, Na2HPO4·12H2O 1.0%, MgSO4·7H2O 0.02%;

[0048] (2) Take 10 mL of the prepared sterile salt solution and mix it with the remaining bacterial powder in the cryovial (the bacterial powder obtained after centrifuging the above bacterial solution) to make the bacterial concentration approximately 2*10⁻⁶. 7 CFU / mL, to obtain a bacterial suspension;

[0049] (3) Incubate the above bacterial suspension in a shaker at 30°C and 100 rpm for 50 min;

[0050] (4) Then add 9.75 mL of 50 wt% sterile glycerol solution and 0.25 mL of 0.6 wt% filtered and sterilized vitamin C aqueous solution to the bacterial suspension after incubation in step (3), and mix thoroughly.

[0051] (5) Then, take 1.2 mL of the bacterial suspension prepared in step (4) into a 1.5 mL cryovial and dispense the cryovial; place the dispensed bacterial suspension into a cryovial at -70℃ for ultra-low temperature storage.

[0052] The strains preserved by the above method, after being frozen at -70℃ for three years, can grow colonies after being inoculated into a culture medium and cultured at 30℃, thus achieving the effect of maintaining stable characteristics.

[0053] Cell viability detection: Samples of the preserved strains were taken at the 1st, 2nd, and 3rd years of preservation, diluted, and spread onto agar plates at appropriate dilutions. The plates were incubated at 30°C for 96 hours, and the cells were observed and counted. The survival rate and activity of the preserved strains are shown in Table 3. The results indicate that the cell viability of the filamentous microbes under this preservation method reached over 92.1%.

[0054] Table 3. Survival rate of strains of *Microbes filamentosa* after preservation.

[0055]

Claims

1. A method for preserving microbial strains, characterized in that, This method is suitable for preserving residual freeze-dried bacterial powder after opening and use in vacuum freeze-drying tubes; the preservation method includes the following steps: (1) Mix the remaining bacterial powder with sterilized salt solution to prepare a bacterial suspension; (2) Incubate the bacterial suspension from step (1) at 25-37 °C for 0.5-1.0 h; (3) Thoroughly mix the incubated bacterial suspension with the bacterial strain protectant; (4) Dispense the bacterial suspension after mixing in step (3) into cryovials; then store the cryovials at -70 ℃ to -85 ℃ for ultra-low temperature storage. The salt solution is composed of, by mass ratio: K₂HPO₄·3H₂O 0.8%~1.0%, KH₂PO₄ 0.1%~0.3%, Na₂HPO₄·12H₂O 1.0%~1.2%, MgSO₄·7H₂O 0.02%~0.03%, with the remainder being water; The strain protectant is a mixture of sterilized glycerol (30%~50% by mass) and a vitamin C aqueous solution (0.3%~0.6% by mass) that has been filtered and sterilized; the volume ratio of the two is 5~50:

1. The volume ratio of the salt solution to the microbial protectant is 1:

1.

2. The method for preserving microbial strains as described in claim 1, characterized in that, The concentration of the bacterial suspension in step (1) is 0.5 × 10⁻⁶. 7 ~5×10 7 CFU / mL.

3. The bacterial strain preservation method according to claim 1, characterized by, In step (2), the bacterial suspension is incubated on a shaker at a speed of 50-100 rpm.

4. The bacterial strain preservation method according to any one of claims 1 to 3, wherein In step (4), the volume of the cryopreservation tube is 1~5 mL.