Strain freeze-drying process and effect evaluation method thereof

Abstract

The invention discloses a strain freeze-drying process which comprises the working procedures of strain inoculation amplification, split charging freeze-drying and heat sealing, according to the freeze-drying mode, the one-time library building number of strains can be effectively increased, meanwhile, a gradient temperature control freeze-drying mode is adopted, transfer operation before vacuum heat sealing is optimized, the moisture content of the strains after freeze-drying and heat sealing is reduced to 3% or below, and the yield of the strains is improved. The preservation stability of the strain is improved; the invention synchronously provides an effect evaluation method of the strain freeze-drying process, which comprises a vacuum degree detection mode and a viable bacteria counting mode, provides an evaluation basis for the long-term preservation effect of the freeze-dried strain tube, and realizes the quality control of the whole process of the strain freeze-drying process.

Description

Technical Field

[0001] The present invention relates to the technical field of freeze-drying of microorganisms, in particular to a freeze-drying process of bacterial strains and a method for evaluating the effect thereof. Background Art

[0002] Bacterial strains are an important biological resource. The international community has always attached great importance to the preservation and sharing of microbial strain resources. The current preservation methods used for bacterial strains mainly include regular transplantation, liquid paraffin, sand tube, freeze-drying, -80℃ refrigerator freezing, liquid nitrogen ultra-low temperature freezing, etc. The purpose is to create an environment suitable for long-term dormancy for excellent strains. Usually, dryness, low temperature, lack of oxygen and nutrients are adopted to keep the metabolic activity of microorganisms at the lowest state, but not to the point of death, so as to achieve the purpose of preservation.

[0003] The freeze-drying operation of the above-mentioned strains is usually to redissolve the strains, subculture and amplify them, add excipient solution after collection, pipette and dispense the specified volume into a certain number of strain tubes with a capillary pipette, wrap them and place them in a glass beaker, use a conventional experimental freeze dryer to pre-freeze and vacuum freeze-dry, vacuum seal after freeze-drying, and take samples for inspection.

[0004] The entire preparation process of this strain is relatively simple to control, and the maximum freeze-dried batch size is 70-80 tubes. However, there are the following disadvantages:

[0005] 1. The freeze-drying method of this strain lacks accurate control of the loading amount, which affects the moisture content;

[0006] 2. The freeze-drying method for this strain uses an ordinary experimental freeze dryer, which cannot control the freeze-drying curve and affects the moisture content. The pilot freeze dryer can accurately control the temperature, but the investment requires hundreds of thousands of yuan and has certain requirements for the size of the clean room. It is suitable for vials but not for conventional strain tubes.

[0007] 3. The freeze-drying method of this strain cannot control the vacuum between the completion of freeze-drying and the sealing, which may lead to moisture absorption. The sealing time will affect the moisture content, resulting in the inability to scale up the single batch, affecting the batch size;

[0008] 4. The freeze-drying method of this strain lacks a means of vacuum detection, which affects the evaluation of long-term preservation effectiveness. The existing process has a high moisture content, which affects long-term preservation. There is no method for counting live bacteria, which is a direct evaluation method for the preservation effect of the strain.

[0009] Based on the above research, a new freeze-drying preservation process for bacterial strains needs to be developed, aiming to improve the batch consistency of freeze-dried bacterial strains, reduce moisture, and improve storage stability. At the same time, a method for vacuum leak detection of bacterial strain tubes and evaluation of viable bacteria quantity should be established, so as to achieve quality control of the entire process of bacterial strain freeze-drying process. Summary of the Invention

[0010] The main technical problem solved by the present invention is to provide a new freeze-drying preservation process for bacterial strains. Compared with the original process, the process is more precise in terms of filling quantity control, adopts a gradient temperature increase method in the freeze-drying parameters, adopts vacuum bagging and transfer after the freeze-drying is completed, and uses a vacuum leak detector to detect leaks after sealing. At the same time, a live bacteria counting method is established to achieve quality control of the entire process of the bacterial strain freeze-drying process.

[0011] In order to solve the above technical problems, a technical solution adopted by the present invention is to provide a strain freeze-drying process, including strain inoculation and amplification, packaging and freeze-drying, and sealing steps.

[0012] Preferably, the bacterial strain is inoculated and amplified in a sterile environment by reviving and culturing the first generation of bacterial strains and amplifying them to the ideal generation. After collection, 1.5 ml to 3.0 ml of sterile skim milk is added to each 10% sheep blood ordinary agar medium, and the bacterial strain is scraped into the sterile skim milk with an inoculation stick, and then shaken into a bacterial suspension by a shaker.

[0013] Preferably, the method for reviving, culturing and subculture amplification of the strain is to scrape an appropriate amount of the first-generation strain with a single-circle inoculation stick in a biological safety cabinet, and evenly spread it on a 10% sheep blood agar medium plate. Each first-generation strain culture can be inoculated with 1 to 2 10% sheep blood agar media. After inoculation, the culture is inverted and placed in an environment of 36.5±0.5°C and 10±1% CO2 for culturing for 16 to 20 hours.

[0014] Preferably, the subpackaging and freeze-drying method is to subpack into sterilized bacterial tubes by a pipette gun for pre-freezing treatment, and then place them in a freeze dryer for gradient temperature control freeze-drying treatment.

[0015] Preferably, the pre-freezing treatment is to wrap the sterilized bacterial tube with sterilized gauze, and then pre-freeze the packed bacterial tube at -35°C for 1 hour or more for 6 hours and 30 minutes.

[0016] Preferably, the gradient temperature-controlled freeze-drying treatment method is to perform gradient vacuum freeze-drying for more than 12 hours and 30 minutes at a vacuum degree of ≤4 Pa, corresponding to a certain vacuum pumping time at -60°C, 20°C, 25°C, 30°C, and 35°C, respectively.

[0017] Preferably, the sealing method is to place the freeze-dried bacteria strain into a disposable sterile sampling bag, connect it to the vacuum in the biosafety cabinet with a sterilized pipe, evacuate the bacteria strain tube in the disposable sterile sampling bag, and use 75% ethanol to disinfect the outer surface of the disposable sterile sampling bag. In the biosafety cabinet, take the bacteria strain tube out of the disposable sterile sampling bag, insert the bacteria strain tube mouth into the vacuum tube, evacuate the vacuum while burning the bacteria strain tube neck with an alcohol burner to seal it.

[0018] In order to solve the above technical problems, another technical solution adopted by the present invention is: a method for evaluating the effect of the freeze-drying process of bacterial strains, including vacuum detection and live bacteria counting methods, wherein the vacuum detection method is to use a vacuum detector to detect the sealed bacterial strain tube, and judge whether the bacterial strain tube is vacuum by whether the vacuum detector generates glow.

[0019] Preferably, the viable bacteria counting method is to take any of the above-mentioned strains obtained by the freeze-drying process, use a capillary pipette or a pipette to draw 0.5 ml of 0.9% sodium chloride solution into the strain tube, repeatedly blow the freeze-dried material until it dissolves into a bacterial suspension, and inoculate the bacterial suspension into 0.9% sodium chloride solution in a gradient dilution manner to obtain several gradient concentration bacterial solutions, use a pipette to take bacterial solutions of different gradient concentrations and add them to 10% sheep blood ordinary nutrient agar medium plates, perform three replicates for each gradient, and culture in an incubator at 36.5±0.5°C and a CO2 concentration of 10±1% for 16-20 hours; after the incubation period, observe the growth of the colonies, select the plates with the same gradient and a colony count of 30-300 for counting, and the number of viable bacteria per milliliter of the original bacterial solution = the average number of colonies in more than three replicate culture plates of the same dilution × the dilution factor × 10.

[0020] Preferably, the gradient dilution method is to add 4.5 ml of 0.9% sodium chloride solution to each sterile centrifuge tube, use a capillary pipette or a pipette to take 0.5 ml of the bacterial solution and add it to the sterile centrifuge tube, for 10 -1 Gradient, from 10 -1 Take 0.5 ml of bacterial solution from the gradient and add it to a sterile centrifuge tube containing 4.5 ml of 0.9% sodium chloride solution. -2 Gradient, 10 -3 , 10 -4 to 10 -n The gradient is operated in this way.

[0021] The beneficial effects of the present invention are:

[0022] The present invention addresses the problems of loading, freeze-drying curve, vacuum sealing, vacuum detection, moisture and live bacteria counting in the existing strain library construction process, and proposes a new strain freeze-drying method. On the one hand, while increasing the number of strains built at one time, a gradient temperature-controlled freeze-drying mode is adopted to optimize the transfer operation before vacuum sealing, so that the moisture content of the strains after freeze-drying and sealing is reduced to below 3%, effectively improving the storage stability of the strains; on the other hand, the present invention simultaneously establishes a vacuum leak detection and live bacteria counting method suitable for strain freeze-drying tubes, which provides an evaluation basis for the long-term storage effect of the strain tubes after freeze-drying. By optimizing the process and technology, the present invention makes the entire strain preparation process controllable, realizes quality control of the entire strain freeze-drying process, and uses simple means to scientifically evaluate the stability of the strains after long-term storage, which has very good technical promotion value and market promotion value. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a photo of the appearance of a bacterial culture tube obtained according to the original freeze-drying process mentioned in the background art;

[0024] Figure 2 This is a photo of the appearance of a bacterial strain tube obtained according to the novel bacterial strain freeze-drying process of the present invention;

[0025] Figure 3 When the live bacteria are counted according to the strain prepared by the freeze-drying process of the new strain of the present invention, 10 -4 Colony growth under gradient;

[0026] Figure 4 When the live bacteria are counted according to the strain prepared by the freeze-drying process of the new strain of the present invention, 10 -5 Colony growth under gradient; DETAILED DESCRIPTION

[0027] The preferred embodiments of the present invention are described in detail below so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby making a clearer and more precise definition of the protection scope of the present invention.

[0028] Example:

[0029] In order to improve the batch consistency of freeze-dried bacteria, reduce moisture, and improve storage stability, the present invention innovatively establishes a new freeze-drying process for bacteria, and at the same time combines a method for evaluating the amount of viable bacteria to achieve quality control of the entire freeze-drying process of bacteria. The freeze-drying process of the present invention includes the steps of inoculation and amplification of bacteria, packaging and freeze-drying, and sealing. The corresponding implementation cases are specifically described below.

[0030] Regarding the inoculation and amplification of bacterial strains, this embodiment takes the working seed meningococcal strain, scrapes an appropriate amount of the first-generation bacterial strain lawn with a single-circle inoculation stick in a biological safety cabinet, and evenly spreads it on a 10% sheep blood ordinary agar medium plate. Each first-generation bacterial strain culture can be inoculated with 2 10% sheep blood ordinary agar media. After the inoculation is completed, it is inverted and placed in a 36.5±0.5℃, 10±1% CO2 environment for 18 hours. In this way, the second-generation bacterial strain is inoculated and amplified to the fifth-generation bacterial strain. This preparation process is a sterile production process.

[0031] After the fifth generation of strains, they are collected. In a biosafety cabinet, 2.0 ml of sterile skim milk is added to each 10% sheep blood ordinary agar medium. Use a single or multiple circles of an inoculation stick to scrape the strain moss into the sterile skim milk, and shake it into a bacterial suspension using a shaker. At this time, the inoculation and amplification process of the strain is completed.

[0032] Use a pipette to draw up the bacterial suspension obtained above and divide it into sterilized culture tubes, with a volume of about 0.3±0.1 ml per tube. Wrap the tube mouth with sterilized gauze, and pre-freeze the divided and wrapped culture at -35°C for about 7 hours. Then, place the pre-frozen culture in a freeze dryer and vacuum it for gradient temperature control freeze-drying. Prevent the pre-frozen culture from melting during the process of moving the culture.

[0033] The gradient temperature-controlled freeze-drying process of the pre-frozen bacteria in the freeze dryer is as follows: first set the temperature and time in the freeze-drying module of the freeze dryer program, call the set time and temperature when the vacuum degree is ≤4Pa, set the vacuuming time to 270 minutes at -60℃, 120 minutes at 20℃, 120 minutes at 25℃, 120 minutes at 30℃, and 605 minutes at 35℃. Put the pre-frozen bacteria into the freeze dryer and vacuum it. When the vacuum degree is ≤4Pa, vacuum freeze-dry it for 20 hours and 30 minutes. At this time, the packaging and freeze-drying process of the bacteria is completed.

[0034] Place the freeze-dried bacteria strain into a disposable sterile sampling bag, connect it to the vacuum in the biosafety cabinet with a sterilized pipe, evacuate the bacteria strain tube in the disposable sterile sampling bag, disinfect the outer surface of the disposable sterile sampling bag with 75% ethanol, and place it into the biosafety cabinet after disinfection. Take the bacteria strain tube out of the disposable sterile sampling bag in the biosafety cabinet, insert the bacteria strain tube mouth into the vacuum tube, evacuate the vacuum while burning the bacteria strain tube neck with an alcohol burner to seal it. After the sealing is completed, check that the appearance is intact and free of cracks. At this time, the bacteria strain sealing process is completed.

[0035] In order to more clearly demonstrate the difference in the effects obtained by the new freeze-drying process of the present invention, the present invention simultaneously provides the preparation results according to the original freeze-drying process involved in the background technology, and compares the preparation effects of the two processes from three aspects: total freeze-dried count, appearance of the culture tube, and water content. The water content is obtained by coulometric detection, as shown in the table below. It can be seen that the total freeze-dried count prepared according to the freeze-drying process of the present invention far exceeds that of the original freeze-drying process. At the same time, the appearance color of the culture tube is uniform, and the moisture content is also significantly reduced.

[0036]

[0037] Regarding the appearance of the above-mentioned bacterial tubes, the appearance photos of the bacterial tubes obtained according to the original freeze-drying process and the new bacterial freeze-drying process of the present invention are as follows: Figure 1 and Figure 2 As shown, it can be clearly seen that the bacterial tubes obtained according to the new freeze-drying process of the present invention have no cracks or damage in appearance, and all of them are flesh-pink loose freeze-dried materials with no shrinkage and very good uniformity. At the same time, the water content of each bacterial tube is less than 3% as tested by the coulometric method, and the total number of freeze-dried batches is 150.

[0038] Based on the bacterial strains obtained by the above-mentioned freeze-drying process, the present invention establishes a vacuum leak detection and live bacteria counting scheme suitable for freeze-dried bacterial strain tubes. The original technology usually uses visual leak detection. The vacuum leak detection provided by the present invention is to detect leaks through vacuum detection. The specific implementation method is to use a vacuum detector to detect the sealed bacterial strain tubes in a biological safety cabinet, and determine whether the bacterial strain tubes are vacuum by whether the vacuum detector produces glow.

[0039] The original public technology does not have a clear counting method. The present invention provides a new method for counting viable bacteria to verify the survival rate of the prepared bacteria. The specific implementation method is to take out five 50ml sterile centrifuge tubes from the biosafety cabinet, mark 10 on the centrifuge tubes, and -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 The gradient was placed on a test tube rack, and 4.5 ml of 0.9% sodium chloride solution was pipetted into each centrifuge tube.

[0040] Take one of the meningococcal cultures prepared above, scratch about 1 / 3 of the upper end of the neck of the culture tube with a sand piece for 1 to 3 circles, wipe the scratched area with ethanol, place the scratched area under a flame, then wrap the culture tube with sterilized gauze and gently break it near the flame; then uncover the gauze, take out the culture tube, use a capillary pipette or pipette to draw 0.5 ml of 0.9% sodium chloride solution into the culture tube and slowly add it, then use the pipette or pipette to repeatedly blow the lyophilized material until it dissolves into a bacterial suspension.

[0041] Use a capillary pipette or a pipette to draw the above bacterial suspension into a sterile centrifuge tube, use a shaker to mix it evenly, for a short time, several times, and visually there is no obvious flocculent matter, which is 10 -1 Gradient, from 10 -1 Take 0.5 ml of bacterial solution from the gradient and add it to a sterile centrifuge tube containing 4.5 ml of 0.9% sodium chloride solution. -2 Gradient, 10 -3 , 10 -4 , 10 -5 The gradient operates in this way;

[0042] Put 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 Take 100ul of each of the five gradients with a pipette and add it to the prepared 10% sheep blood ordinary nutrient agar medium plate. Repeat 3 times for each gradient. After the spreader stick is evenly spread, mark the gradient on the plate and culture it in an incubator at 36.5±0.5℃ and 10±1% CO2 concentration for about 18 hours. After the culture is completed, observe the growth of the colony, such as Figure 3 and Figure 4 According to the viable bacteria counting method of the present invention, -4 , 10 -5 Colony growth under gradient; then, the same gradient was selected, and the plate with the number of colonies between 30 and 300 was counted. The number of viable bacteria per milliliter of the original bacterial solution = the average number of colonies in more than three replicate culture dishes at the same dilution × the dilution factor × 10. A comparative test was also conducted using the original freeze-drying process and the freeze-drying process of the present invention. The data are shown in the table below. It can be seen that the survival rate of the bacteria prepared using the freeze-drying process of the present invention is much higher than that of the bacteria prepared using the original freeze-drying process.

[0043]

[0044] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the contents of the present invention specification, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A bacterial strain freeze-drying process, characterized in that: It includes the processes of bacterial strain inoculation and amplification, packaging, freeze-drying and sealing.

2. A bacterial strain freeze-drying process according to claim 1, characterized in that: The bacterial strain inoculation and amplification method is as follows: the first generation bacterial strain is revived and cultured in a sterile environment and subcultured to an ideal generation; after collection, 1.5 ml to 3.0 ml of sterile skim milk is added to each 10% sheep blood ordinary agar medium; the bacterial strain is scraped into the sterile skim milk with an inoculation stick, and then shaken into a bacterial suspension by a shaker.

3. A bacterial strain freeze-drying process according to claim 2, characterized in that: The method for resuscitating, culturing and subculture amplification of the bacterial strain is as follows: scraping an appropriate amount of first-generation bacterial strain with a single-circle inoculation stick in a biological safety cabinet, evenly smearing it on a 10% sheep blood ordinary agar medium plate, each first-generation bacterial strain culture can be inoculated with 1 to 2 10% sheep blood ordinary agar medium plates, and after the inoculation is completed, the plates are inverted and placed in an environment of 36.5±0.5°C and 10±1% CO2 for incubation for 16 to 20 hours.

4. The freeze-drying process of bacterial strains according to claim 1, characterized in that: The method of subpackaging and freeze-drying is to subpack into sterilized bacterial tubes through a pipette gun for pre-freezing treatment, and then place them in a freeze dryer for gradient temperature control freeze-drying treatment.

5. A bacterial strain freeze-drying process according to claim 4, characterized in that: The pre-freezing treatment method is to wrap the sterilized bacterial tube with sterilized gauze, and pre-freeze the packed bacterial tube at -35°C for 1 hour or more for 6 hours and 30 minutes.

6. A bacterial strain freeze-drying process according to claim 4, characterized in that: The gradient temperature-controlled freeze-drying treatment method is to perform gradient vacuum freeze-drying for more than 12 hours and 30 minutes at a vacuum degree of ≤4 Pa, at -60°C, 20°C, 25°C, 30°C, and 35°C for a certain vacuum time.

7. The freeze-drying process for bacterial strains according to claim 1, characterized in that: The sealing method is as follows: placing the freeze-dried bacterial strain into a disposable sterile sampling bag, connecting the bag to the vacuum system in a biosafety cabinet with a sterilized pipe, evacuating the bacterial strain tube in the disposable sterile sampling bag, disinfecting the outer surface of the disposable sterile sampling bag with 75% ethanol, taking the bacterial strain tube out of the disposable sterile sampling bag in the biosafety cabinet, inserting the bacterial strain tube mouth into the vacuum tube, evacuating the bag while burning the bacterial strain tube neck with an alcohol burner to seal the bag.

8. A method for evaluating the effect of a freeze-drying process of a bacterial strain according to any one of claims 1 to 7, characterized in that: It includes vacuum detection and live bacteria counting methods, wherein the vacuum detection method is to use a vacuum detector to detect the sealed culture tube, and judge whether the culture tube has vacuum by whether the vacuum detector produces glow.

9. The method for evaluating the effect of a freeze-drying process of bacterial strains according to claim 8, wherein: The viable bacteria counting method comprises taking any of the above-mentioned strains obtained by the freeze-drying process, using a capillary pipette or a pipette to draw 0.5 ml of a 0.9% sodium chloride solution into a strain tube, repeatedly blowing and stirring the freeze-dried material until it dissolves into a bacterial suspension, inoculating the bacterial suspension into a 0.9% sodium chloride solution according to a gradient dilution method to obtain bacterial solutions of several gradient concentrations, using a pipette to take bacterial solutions of different gradient concentrations and add them to 10% sheep blood normal nutrient agar culture plates, performing three replicates for each gradient, and culturing the plates in an incubator at 36.5±0.5°C and a CO2 concentration of 10±1% for 16-20 hours. After the incubation period, observing the growth of the colonies, selecting plates with the same gradient and a colony count of 30-300 for counting, the number of viable bacteria per milliliter of the original bacterial solution = the average number of colonies in three or more replicate culture plates of the same dilution × the dilution factor × 10.

10. The method for evaluating the effect of a freeze-drying process of bacterial strains according to claim 9, wherein: The gradient dilution method is to add 4.5 ml of 0.9% sodium chloride solution to each sterile centrifuge tube, use a capillary pipette or a pipette to take 0.5 ml of the bacterial solution and add it to the sterile centrifuge tube, for 10 -1 Gradient, from 10 -1 Take 0.5 ml of bacterial solution from the gradient and add it into a sterile centrifuge tube containing 4.5 ml of 0.9% sodium chloride solution. -2 Gradient, 10 -3 , 10 -4 to 10 -n The gradient is operated in this way.

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