Application of a scleroglucan combination protective agent in freeze-drying storage of actinomycetes
By using freeze-dried bacterial strain preservation agent containing hard dextran, the problem of low survival rate of bacterial strains in the prior art is solved, and efficient long-term preservation of bacterial strains is achieved, which significantly improves survival rate and stability.
Patent Information
- Application Number
- CN202011123971.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2020-10-20
- Publication Date
- 2025-05-06
- Estimated Expiration
- 2040-10-20
AI Technical Summary
When existing freeze-drying methods preserve certain bacterial species that are not easy to preserve, their survival rate and stability cannot reach the ideal level, making it difficult to achieve long-term effective and stable storage.
A freeze-dried bacterial seed deposit agent containing hard dextran was used to prepare a water solution of skim milk and hard dextran, and then mixed under sterile conditions after high-pressure steam sterilization to obtain a freeze-dried bacterial seed deposit agent, which was freeze-dried in a vacuum freeze-dryer, and then stored for a long time under a low temperature environment.
It minimizes the loss of water in the bacterial species during freeze-drying process, significantly improves the long-term preservation and survival rate of difficult-to-preservation bacterial species, has stable preservation effect and is easy to operate.
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of strain preservation, and in particular relates to a freeze-dried strain preservative containing hard glucan, a preparation method and its application in difficult-to-preserve strains. Background Art
[0002] Strain preservation is the basis of all microbial work, and a good strain preservation method is the guarantee for obtaining excellent strains. Currently, commonly used strain preservation methods include freeze-drying preservation, ultra-low temperature refrigerator preservation, liquid nitrogen preservation, subculture preservation, carrier preservation, etc. These methods are mainly based on the physiological and biochemical characteristics of different microorganisms, artificially creating hypoxic, low temperature or dry conditions to reduce the metabolic rate of microorganisms, so that they do not mutate while maintaining their vitality, ensuring that the strains still maintain their original vitality, morphological characteristics and excellent traits after preservation. Among them, freeze-drying preservation is recognized as one of the most effective methods for strain preservation.
[0003] The freeze-drying preservation method of bacterial strains is a method of using a bacterial strain preservative to prepare a suspension of the bacterial strain to be preserved in an ampoule tube, pre-cooling and freezing it into a solid, and then sublimating and removing its moisture under vacuum and reduced pressure conditions. This method keeps the bacterial strain in a dry and oxygen-deficient state without severe and direct damage, so as to achieve long-term stable storage. In the process of preserving bacterial strains using freeze-drying, the bacterial strain preservative is a key factor in improving the survival rate of the bacterial strain freeze-dried preservation and extending the storage period of the bacterial strain.
[0004] In the process of scientific research and production, some strains, due to their own characteristics, cannot reach the ideal level of survival rate and stability after preservation using conventional freeze-drying methods. These strains are difficult to preserve and cannot be effectively and stably preserved for a long time, which is a major problem faced by strain preservation work. Summary of the invention
[0005] In order to solve the problem of low survival rate of difficult-to-preserve strains after freeze-drying preservation, the present invention provides a freeze-dried strain preservative containing scleroglucan, a preparation method and application thereof to difficult-to-preserve strains.
[0006] The invention firstly provides a freeze-dried strain preservative containing scleroglucan, which is composed of the following components in mass volume ratio: 10%-20% of skim milk and 0.1%-0.5% of scleroglucan, which is prepared with distilled water.
[0007] The present invention also provides a method for preparing the strain preservative: respectively preparing skim milk and scleroglucan aqueous solutions in different mass percentages, sterilizing them with high-pressure steam at 115°C for 20 minutes, and mixing them evenly in a volume ratio of 1:1 under sterile conditions until the temperature drops to a range of 40-60°C to obtain a freeze-dried strain preservative.
[0008] The present invention also provides a method for applying the freeze-dried bacterial strain preservative containing scleroglucan to difficult-to-preserve bacterial strains, comprising the following steps:
[0009] (1) Under sterile conditions, add 5-30 mL of freeze-dried preservative to the slope of the eggplant bottle where the strain to be preserved has been cultured to maturity, and use a sterile inoculation needle to scrape the spores on the upper layer of the slope to prepare a single spore suspension.
[0010] (2) The single spore suspension to be preserved obtained in step (1) is divided into sterile ampoule tubes in a volume of 0.5-1.0 mL and cooled to -80°C for pre-freezing.
[0011] (3) The ampoule tube in step (2) is immediately placed in a vacuum freeze dryer for freeze drying, and then the ampoule tube is sealed with an alcohol burner under vacuum. After passing the inspection, it is stored for a long time in a low temperature environment.
[0012] Furthermore, the pre-freezing and cooling in step (2) is carried out in a step-by-step cooling manner, firstly maintaining the temperature at 4°C for 30 min to 60 min, then rapidly turning to -20°C for 1 h to 2 h, and finally maintaining the temperature at -80°C for 4 to 6 h.
[0013] Furthermore, in step (3), the vacuum drying time is 8 to 10 h, the water content after freeze-drying is controlled at 1 to 3%, and the low-temperature storage temperature is 2 to 8°C.
[0014] The present invention has the positive effects:
[0015] The present invention utilizes existing vacuum freeze-drying equipment, the preservation method is easy to operate, the preservation effect is stable, the water loss damage caused by the strains in the freeze-drying process is reduced to the maximum extent, and the long-term preservation survival rate of the difficult-to-preserve strains is greatly improved. Specific embodiment:
[0017] The following examples are used to illustrate the present invention, but these examples are not intended to limit the scope of the present invention. The norvancomycin-producing bacteria Amycolatopsis orientalis and the rapamycin-producing bacteria Streptomyces hygroscopicus in the examples were provided by North China Pharmaceutical Group New Drug Research and Development Company.
[0018] Example 1
[0019] Preparation of freeze-dried bacterial strain preservative containing scleroglucan: prepare a distilled water solution with a mass volume ratio of 20% skim milk and 1% scleroglucan respectively, sterilize it with high-pressure steam at 115°C for 20 min, wait until the temperature drops to the range of 40-60°C, mix them evenly in a volume ratio of 1:1 under sterile conditions to obtain a freeze-dried bacterial strain preservative.
[0020] The method for applying the prepared freeze-dried bacterial strain preservative containing scleroglucan to the norvancomycin-producing bacteria Amycolatopsis orientalis comprises the following steps:
[0021] (1) Under sterile conditions, add 15 mL of freeze-dried preservative medium to the slope of a mature eggplant bottle cultured with the vancomycin-producing bacterium Amycolatopsis orientalis, and use a sterile inoculation needle to scrape the spores on the upper layer of the slope to obtain a single spore suspension.
[0022] (2) The single spore suspension obtained in step (1) was dispensed into sterile ampoule tubes in a volume of 0.5 mL and cooled step by step, first at 4°C for 60 min, then quickly to -20°C for 1 h, and finally to -80°C for 6 h.
[0023] (3) The ampoule tube in step (2) is immediately placed in a vacuum freeze dryer for freeze drying for 8 hours. The moisture content after freeze drying is controlled at 1-3%. The ampoule tube is then sealed with an alcohol burner under vacuum. After passing the inspection, it is stored for a long time at a low temperature of 2-8°C.
[0024] (4) Determine the survival rate of the preparation. After the prepared cryopreserved tubes were stored at 2-8°C for 3 months, 6 months and 1 year, the survival rate was determined by the live count method. According to the live count determination, the survival rate of the vancomycin-producing bacteria Amycolatopsis orientalis was 63.5% at 3 months, 63.7% at 6 months, and 62.8% at 1 year.
[0025] Example 2
[0026] Preparation of freeze-dried bacterial strain preservative containing scleroglucan: prepare a distilled water solution of 30% skim milk and 0.4% scleroglucan in a mass volume ratio respectively, sterilize it with high-pressure steam at 115°C for 20 min, wait until the temperature drops to the range of 40-60°C, mix them evenly in a volume ratio of 1:1 under sterile conditions to obtain a freeze-dried bacterial strain preservative.
[0027] The method for applying the prepared freeze-dried bacterial strain preservative containing scleroglucan to the norvancomycin-producing bacteria Amycolatopsis orientalis comprises the following steps:
[0028] (1) Under sterile conditions, add 30 mL of freeze-dried preservative medium to the slope of a mature eggplant bottle cultured with the vancomycin-producing bacterium Amycolatopsis orientalis, and use a sterile inoculation needle to scrape the spores on the upper layer of the slope to obtain a single spore suspension.
[0029] (2) The single spore suspension obtained in step (1) was dispensed into sterile ampoule tubes in 1 mL volumes and cooled step by step, first at 4°C for 30 min, then quickly to -20°C for 2 h, and finally to -80°C for 4 h.
[0030] (3) The ampoule tube in step (2) is immediately placed in a vacuum freeze dryer for freeze drying for 10 h. The moisture content after freeze drying is controlled at 1-3%. The ampoule tube is then sealed with an alcohol burner under vacuum. After passing the inspection, it is stored for a long time at a low temperature of 2-8°C.
[0031] (4) The survival rate of the prepared cryopreserved tubes was determined, and the prepared cryopreserved tubes were stored at 2-8°C for 3 months, 6 months and 1 year, and then the survival rate was determined by the live count method. The live count determination showed that the survival rate of the vancomycin-producing bacteria Amycolatopsis orientalis was 51.2% at 3 months, 52.3% at 6 months, and 51.8% at 1 year.
[0032] Example 3
[0033] Preparation of freeze-dried bacterial strain preservative containing scleroglucan: prepare a distilled water solution of 40% skim milk and 0.2% scleroglucan in a mass volume ratio respectively, sterilize it with high-pressure steam at 115°C for 20 min, wait for the temperature to drop to the range of 40-60°C, mix them evenly in a volume ratio of 1:1 under sterile conditions to obtain a freeze-dried bacterial strain preservative.
[0034] The method for applying the prepared freeze-dried bacterial strain preservative containing scleroglucan to the norvancomycin-producing bacteria Amycolatopsis orientalis comprises the following steps:
[0035] (1) Under sterile conditions, add 5 mL of freeze-dried preservative medium to the slope of a mature eggplant bottle cultured with the vancomycin-producing bacterium Amycolatopsis orientalis, and use a sterile inoculation needle to scrape the spores on the upper layer of the slope to obtain a single spore suspension.
[0036] (2) The single spore suspension obtained in step (1) was dispensed into sterile ampoule tubes in a volume of 0.5 mL and cooled step by step, first at 4°C for 40 min, then quickly to -20°C for 1.5 h, and finally to -80°C for 6 h.
[0037] (3) The ampoule tube in step (2) is immediately placed in a vacuum freeze dryer for freeze drying for 8 h. The moisture content after freeze drying is controlled at 1-3%. The ampoule tube is then sealed with an alcohol burner under vacuum. After passing the inspection, it is stored for a long time at a low temperature of 2-8°C.
[0038] (4) The survival rate of the prepared cryopreserved tubes was determined, and the prepared cryopreserved tubes were stored at 2-8°C for 3 months, 6 months, and 1 year, and then the survival rate was determined by the viable counting method. The survival rate of the vancomycin-producing bacteria Amycolatopsis orientalis was 38.7% at 3 months, 37.5% at 6 months, and 38.2% at 1 year.
[0039] Example 4
[0040] Preparation of freeze-dried bacterial strain preservative containing scleroglucan: prepare a distilled water solution with a mass volume ratio of 24% skim milk and 0.8% scleroglucan respectively, sterilize it with high-pressure steam at 115°C for 20 min, wait until the temperature drops to the range of 40-60°C, mix them evenly in a volume ratio of 1:1 under sterile conditions to obtain a freeze-dried bacterial strain preservative.
[0041] The method for applying the prepared freeze-dried bacterial strain preservative containing scleroglucan to the rapamycin-producing bacteria Streptomyces hygroscopicus comprises the following steps:
[0042] (1) Under sterile conditions, add 20 mL of freeze-dried preservative of the strain to the slope of a mature eggplant bottle cultured with the rapamycin-producing bacterium Streptomyces hygroscopicus, and use a sterile inoculation needle to scrape the spores on the upper layer of the slope to obtain a single spore suspension;
[0043] (2) The single spore suspension obtained in step (1) was dispensed into sterile ampoule tubes in a volume of 0.5 mL and cooled step by step, first at 4°C for 40 min, then quickly to -20°C for 1.5 h, and finally to -80°C for 5 h.
[0044] (3) The ampoule tube in step (2) is immediately placed in a vacuum freeze dryer for freeze drying for 9 hours. The moisture content after freeze drying is controlled at 1-3%. The ampoule tube is then sealed with an alcohol burner under vacuum. After passing the inspection, it is stored for a long time at a low temperature of 2-8°C.
[0045] (4) The survival rate of the preparation was determined, and the prepared cryopreserved tubes were stored at 2-8°C for 3 months, 6 months and 1 year, and then the survival rate was determined by the live count method. The live count determination showed that the survival rate of rapamycin-producing bacteria Streptomyces hygroscopicus was 59.7% at 3 months, 58.9% at 6 months, and 59.3% at 1 year.
[0046] Example 5
[0047] Preparation of freeze-dried bacterial strain preservative containing scleroglucan: prepare a distilled water solution of 30% skim milk and 0.6% scleroglucan in a mass volume ratio respectively, sterilize it with high-pressure steam at 115°C for 20 min, wait until the temperature drops to the range of 40-60°C, mix them evenly in a volume ratio of 1:1 under sterile conditions to obtain a freeze-dried bacterial strain preservative.
[0048] The method for applying the prepared freeze-dried bacterial strain preservative containing scleroglucan to the rapamycin-producing bacteria Streptomyces hygroscopicus comprises the following steps:
[0049] (1) Under sterile conditions, add 5 mL of freeze-dried preservative of the strain to the slope of a mature eggplant bottle cultured with the rapamycin-producing bacterium Streptomyces hygroscopicus, and use a sterile inoculation needle to scrape the spores on the upper layer of the slope to obtain a single spore suspension;
[0050] (2) The single spore suspension obtained in step (1) was divided into sterile ampoule tubes in a volume of 0.5 mL and cooled step by step, first at 4°C for 50 min, then quickly to -20°C for 1 h, and finally to -80°C for 6 h.
[0051] (3) The ampoule tube in step (2) is immediately placed in a vacuum freeze dryer for freeze drying for 8 h. The moisture content after freeze drying is controlled at 1-3%. The ampoule tube is then sealed with an alcohol burner under vacuum. After passing the inspection, it is stored for a long time at a low temperature of 2-8°C.
[0052] (4) The survival rate of the prepared cryopreserved tubes was determined, and the prepared cryopreserved tubes were stored at 2-8°C for 3 months, 6 months and 1 year, and then the survival rate was determined by the live count method. According to the live count determination, the survival rate of rapamycin-producing bacteria Streptomyces hygroscopicus was 48.5% at 3 months, 48.1% at 6 months, and 47.6% at 1 year.
[0053] Example 6
[0054] Preparation of freeze-dried bacterial strain preservative containing scleroglucan: prepare a distilled water solution of 36% skim milk and 0.2% scleroglucan in a mass volume ratio respectively, sterilize it with high-pressure steam at 115°C for 20 min, wait until the temperature drops to the range of 40-60°C, mix them evenly in a volume ratio of 1:1 under sterile conditions to obtain a freeze-dried bacterial strain preservative.
[0055] The method for applying the prepared freeze-dried bacterial strain preservative containing scleroglucan to the rapamycin-producing bacteria Streptomyces hygroscopicus comprises the following steps:
[0056] (1) Under sterile conditions, add 30 mL of freeze-dried preservative medium to the slope of a mature eggplant bottle cultured with the rapamycin-producing bacterium Streptomyces hygroscopicus, and use a sterile inoculation needle to scrape the spores on the upper layer of the slope to obtain a single spore suspension.
[0057] (2) The single spore suspension obtained in step (1) was dispensed into sterile ampoule tubes in a volume of 0.5 mL and cooled step by step, first at 4°C for 60 min, then quickly to -20°C for 1 h, and finally to -80°C for 6 h.
[0058] (3) The ampoule tube in step (2) is immediately placed in a vacuum freeze dryer for freeze drying for 8 hours. The moisture content after freeze drying is controlled at 1-3%. The ampoule tube is then sealed with an alcohol burner under vacuum. After passing the inspection, it is stored for a long time at a low temperature of 2-8°C.
[0059] (4) The survival rate of the preparation was determined, and the prepared cryopreserved tubes were stored at 2-8°C for 3 months, 6 months and 1 year, and then the survival rate was determined by the live count method. The live count determined that the survival rate of rapamycin-producing bacteria Streptomyces hygroscopicus was 34.3% at 3 months, 33.8% at 6 months, and 33.5% at 1 year.
[0060] Comparative Example 1
[0061] In this comparative example, a 10% skim milk distilled water solution without adding scleroglucan was used to freeze-dry and preserve the demethylvancomycin-producing bacteria Amycolatopsis orientalis in Examples 1, 2, and 3. The specific preservation steps were the same as in Example 1.
[0062] (1) Under sterile conditions, add 15 mL of skim milk solution without scleroglucan to the slant of a mature eggplant bottle cultured with the vancomycin-producing bacterium Amycolatopsis orientalis, and use a sterile inoculation needle to scrape the spores on the upper layer of the slant to obtain a single spore suspension;
[0063] (2) The single spore suspension obtained in step (1) was dispensed into sterile ampoule tubes in a volume of 0.5 mL and cooled step by step, first at 4°C for 50 min, then quickly to -20°C for 1 h, and finally to -80°C for 6 h.
[0064] (3) The ampoule tube in step (2) is immediately placed in a vacuum freeze dryer for freeze drying for 10 h. The moisture content after freeze drying is controlled at 1-3%. The ampoule tube is then sealed with an alcohol burner under vacuum. After passing the inspection, it is stored for a long time at a low temperature of 2-8°C.
[0065] (4) The survival rate of the prepared cryopreserved tubes was determined, and the prepared cryopreserved tubes were stored at 2-8°C for 3 months, 6 months and 1 year, and then the survival rate was determined by the live count method. The live count determination showed that the survival rate of the vancomycin-producing bacteria Amycolatopsis orientalis was 2.9% at 3 months, 2.85% at 6 months, and 3% at 1 year.
[0066] Comparative Example 2
[0067] In this comparative example, a 12% skim milk distilled water solution without adding scleroglucan was used to freeze-dry and preserve the rapamycin-producing bacteria Streptomyces hygroscopicus in Examples 4, 5, and 6. The specific preservation steps are the same as those in Example 4.
[0068] (1) Under sterile conditions, add 20 mL of skim milk solution without added scleroglucan to the slant of a mature eggplant bottle cultured with the rapamycin-producing bacterium Streptomyces hygroscopicus, and use a sterile inoculation needle to scrape the spores on the upper layer of the slant to obtain a single spore suspension.
[0069] (2) The single spore suspension obtained in step (1) was dispensed into sterile ampoule tubes in 1.0 mL volumes and cooled step by step, first at 4°C for 60 min, then quickly to -20°C for 1 h, and finally to -80°C for 6 h.
[0070] (3) Immediately place the ampoule tube in step (2) into a vacuum freeze dryer for freeze drying for 8-10 h. After freeze drying, the water content is controlled at 1-3%. Then, the ampoule tube is sealed with an alcohol burner under vacuum. After passing the inspection, it is stored for a long time at a low temperature of 2-8°C.
[0071] (4) The survival rate of the preparation was determined, and the prepared cryopreserved tubes were stored at 2-8°C for 3 months, 6 months and 1 year, and then the survival rate was determined by the live count method. The live count determination showed that the survival rate of rapamycin-producing bacteria Streptomyces hygroscopicus was 0.53% at 3 months, 0.48% at 6 months, and 0.51% at 1 year.
[0072] Although the present invention has been described and illustrated by specific embodiments, it should be understood that various modifications and equivalent substitutions can be made to the present invention by those skilled in the art without departing from the spirit and scope of the present invention. Therefore, the present invention is not limited to the specific embodiments described herein, and more specifically, the protection scope of the present invention is defined by the appended claims.
Claims
1. A method for preserving actinomycete strains, characterized in that The steps include: (1) Under sterile conditions, add the strain freeze-dried preservative to the slope of the eggplant bottle where the strain to be preserved has been cultured to maturity, and use a sterile inoculation needle to scrape the spores on the upper layer of the slope to obtain a single spore suspension; (2) the single spore suspension to be preserved obtained in step (1) is divided into sterile ampoule tubes and cooled to -80°C for pre-freezing; (3) Immediately placing the ampoule tube in step (2) into a vacuum freeze dryer for freeze drying, then sealing the ampoule tube under vacuum, and storing it for a long time at a low temperature environment after passing the inspection; The bacterial strain preservative in step (1) is composed of the following components in a mass volume ratio: 10%-20% skim milk and 0.1%-0.5% scleroglucan, prepared with distilled water; In step (2), the pre-freezing and cooling adopts a step-by-step cooling method, first maintaining at 4°C for 30 min to 60 min, then quickly turning to -20°C for 1 h to 2 h, and finally maintaining at -80°C for 4 to 6 h; In step (3), the freeze-drying time is 8 to 10 hours, the water content after freeze-drying is controlled at 1 to 3%, and the low-temperature storage temperature is controlled at 2 to 8°C; In step (1), the amount of the freeze-dried preservative of the strain is 5 to 30 mL; in step (2), the single spore suspension to be preserved is divided into sterile ampoule tubes in a volume of 0.5 to 1.0 mL.
2. A method for preserving actinomycete strains as claimed in claim 1, characterized in that: Skim milk and scleroglucan solution are prepared separately, sterilized by high-pressure steam, and mixed under aseptic conditions after the temperature drops to a range of 40-60° C. to obtain a freeze-dried strain preservative.