A method for continuous fermentation of Clostridium botulinum strains

Abstract

This invention relates to the field of microbial fermentation technology, and more specifically, to a method for continuous fermentation of Clostridium botulinum strains. S1: Place liver blocks into a fermentation tank; S2: Add culture medium to the tank, immersing the fresh liver blocks and agitator, heat to sterilize, and then lower to the inoculation temperature; S3: Inoculate Clostridium botulinum type C strain into the fermentation tank and start the fermentation; S4: After S4, transfer the inoculum solution to a seed bottle for temporary storage; S5: After all materials have been transferred out, start the cleaning procedure; S6: Add liver blocks again as in S1, directly inoculate with the seed solution retained in S4, and replenish with fresh culture medium; S7: Repeat steps S3 to S5. This invention, through an innovative "strain recycling" model, eliminates the cumbersome steps of separate strain activation and expansion for each batch in traditional Clostridium botulinum fermentation processes. After fermentation, only a small amount of fermentation broth needs to be retained as seed for the next batch, eliminating the need to prepare new strains each time, thus simplifying the production process.

Description

Technical Field

[0001] This invention relates to the field of microbial fermentation technology, and more specifically, to a method for continuous fermentation of Clostridium botulinum strains. Background Technology

[0002] Clostridium botulinum poisoning is a serious disease caused by neurotoxins produced by Clostridium botulinum. It is usually caused by carnivores ingesting spoiled meat containing the bacteria and its toxins. The disease has a rapid onset and high mortality rate, posing a significant threat to the livestock economy and wildlife protection. It is a common bacterial infectious disease.

[0003] Currently, the most effective means of preventing botulism is vaccination. The basic principle is to inactivate Clostridium botulinum and add adjuvants such as aluminum hydroxide gel to prepare an inactivated vaccine. This vaccine can stimulate the body to produce a specific immune response, thereby effectively blocking the occurrence and spread of the disease and playing an irreplaceable role in epidemic prevention and control.

[0004] However, in existing vaccine production technologies, the large-scale culture process of Clostridium botulinum still has significant limitations. Traditional culture methods mostly rely on animal-derived raw materials (such as liver blocks, broth, etc.) for static or semi-static fermentation. Although this process can meet the basic requirements for bacterial growth and toxin production, it has many shortcomings in practical operation: Firstly, the operation is cumbersome, involving multiple steps of raw material processing, inoculation, and transfer, with frequent human intervention, which increases the risk of contamination and production volatility. Secondly, the production cycle is lengthy. From raw material processing to final inactivation and harvesting, it often takes several days or even longer, resulting in low production capacity and difficulty in meeting the needs of rapid response to epidemic prevention. Third, the non-standardization of the process leads to significant batch-to-batch variations, posing a challenge to the uniformity and stability control of vaccine quality.

[0005] Therefore, how to simplify the production process of botulinum toxin vaccine, shorten the fermentation cycle, and improve production efficiency and product quality stability has become a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0006] The present invention aims to at least solve one of the technical problems existing in the prior art. To this end, one aspect of the present invention is to provide a method for continuous fermentation of Clostridium botulinum strains, the specific steps of which are as follows: S1. Initial preparation and feeding: Place fresh liver pieces on the circular 316L stainless steel filter screen at the bottom of the fermentation tank; S2. Culture medium preparation and sterilization: Add the prepared culture medium into the tank, immerse the fresh liver pieces and the stirring paddle, turn on the stirring, heat the jacket to sterilize the tank, and after sterilization, cool the culture medium temperature down to the inoculation temperature through the jacket cooling water. S3. Inoculation and fermentation: Under aseptic conditions, the activated Clostridium botulinum type C strain is inoculated into the sterilized fermenter, the fermenter control system is started, and fermentation is carried out according to the process setting parameters until the fermentation endpoint is reached. S4. First feeding and seed retention: After fermentation, let the liver block residue settle. Transfer the supernatant fermentation liquid out through the liquid discharge valve at the bottom of the tank. Accurately measure and transfer the required amount of inoculum liquid, and temporarily store it in a sterile seed bottle for later use. The remaining majority of the fermentation liquid (including products) is transferred to the storage tank through pipelines. S5. Fermentation tank cleaning: After all materials have been transferred out, immediately start the online cleaning program for the fermentation tank. After cleaning, check whether the filter screen inside the tank is clogged. If necessary, backflushing or manual cleaning should be performed. S6. Cyclic Fermentation: Add fresh liver blocks again as required in S1, directly inoculate with the seed liquid retained in the seed bottle in S4, replenish with fresh culture medium, turn on stirring and temperature control, and carry out the next round of fermentation; S7. Continuous cycle: Repeat operations from S3 to S5 to achieve multiple continuous fermentations until strain degeneration, extended fermentation cycle, or contamination occurs. Then, perform comprehensive equipment sterilization and start using new strains.

[0007] Preferably, the fresh liver pieces in S1 are minced or bagged fresh liver pieces, and the thickness of the fresh liver pieces stacked is 10cm-20cm.

[0008] Preferably, the circular 316L stainless steel filter screen in S1 is installed 10-15cm away from the bottom of the fermenter.

[0009] Preferably, the culture medium in S2 is Clostridium botulinum culture medium.

[0010] Preferably, the inoculation ratio of the culture medium in S2 to the fermented Clostridium botulinum strain in S3 is 2%.

[0011] Preferably, the S2 jacket heating and sterilization temperature is 116-118℃; the inoculation temperature is 35℃.

[0012] Preferably, the type C botulinum bacteria in S3 is strain C62-4 or C62-6.

[0013] Preferably, the fermentation temperature of the fermenter control system in S3 is 35°C, the fermentation pH is 7.8, and the fermentation time is 144h.

[0014] Preferably, the amount of inoculum solution transferred in step S4 is 1%-2% of the total fermentation volume.

[0015] Preferably, after all the material is transferred out in S5, the online cleaning (CIP) program of the fermenter is started immediately, which is: pre-rinsing with purified water (to remove residue) → cyclic cleaning with alkaline solution heating (to remove protein and impurities) → rinsing with purified water (to neutral) → final cleaning with water for injection (to remove pyrogens).

[0016] The beneficial effects of this invention are as follows: Simplified operation process and reduced labor intensity: This invention, through the innovative "strain recycling" model, eliminates the cumbersome steps of separate strain activation and expansion for each batch in the traditional Clostridium botulinum fermentation process. After fermentation, only a small amount of fermentation liquid (1-2%) needs to be retained as seed for the next batch, eliminating the need to prepare strains anew each time. This design greatly simplifies the production operation process, significantly reduces the workload of frequent inoculation and strain transfer, and effectively reduces manpower input and labor intensity.

[0017] Shortening the production cycle and improving production efficiency: Due to the adoption of a continuous fermentation cultivation method, the time spent on repeated strain preparation between batches (such as time-consuming steps like strain revival and step-by-step expansion) is eliminated, achieving seamless connection of fermentation processes, significantly shortening the overall production cycle, and significantly increasing the number of fermentation batches that can be completed per unit time, thereby effectively improving production capacity and equipment utilization, and meeting the needs of large-scale industrial production.

[0018] Reduce production costs and improve economic efficiency: By reducing the use of consumables such as bacterial culture media and culture vessels, as well as reducing manual operation time, the cost of raw materials and labor is directly reduced. At the same time, the simplification of the process also reduces the risk of contamination caused by multiple aseptic operations, reduces production losses, and brings significant economic benefits to the company's large-scale production.

[0019] To ensure the viability of the strain and maintain fermentation stability, fresh fermentation broth (1-2%) is selected as the seed for the next batch. This ensures that the strain is always in an active physiological state, avoiding the lag period that may exist in traditional slant culture or freeze-dried tube revival. After several generations of adaptive culture, the strain can better adapt to the current fermentation environment, which is conducive to maintaining a stable toxin production level and fermentation performance, and ensuring the consistency of vaccine antigen quality.

[0020] With strong process adaptability and broad application prospects, the method of this invention is specifically designed for the biological characteristics of Clostridium botulinum (type C) and can be perfectly adapted to existing fermentation systems containing animal-derived raw materials such as liver blocks. Its operation is simple, and it is easy to technically modify and promote its application on existing production lines, laying a solid foundation for the continuous and automated production of Clostridium botulinum vaccines.

[0021] Additional aspects and advantages of the invention will become apparent from the description which follows, or may be learned by practice of the invention. Detailed Implementation

[0022] To better understand the above-mentioned objectives, features, and advantages of the present invention, the present invention will be further described in detail below with reference to specific embodiments. It should be noted that, unless otherwise specified, the embodiments and features described in these embodiments can be combined with each other.

[0023] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be implemented in other ways different from those described herein. Therefore, the scope of protection of the invention is not limited to the specific embodiments disclosed below. Example

[0024] S1. Initial preparation and feeding: Place the minced fresh liver pieces on the circular 316L stainless steel filter screen at the bottom of the fermentation tank, with the thickness of the fresh liver pieces being 10cm-20cm; (the circular 316L stainless steel filter screen is installed 10-15cm away from the bottom of the fermentation tank) S2. Culture medium preparation and sterilization: Add the prepared culture medium (the culture medium is type C Clostridium botulinum culture medium) into the tank, immerse the fresh liver block and the stirring paddle, turn on the stirring, heat the jacket to 118°C for tank sterilization, and after sterilization, cool the culture medium temperature down to the inoculation temperature of 35°C by cooling water in the jacket. S3. Inoculation and Fermentation: Under aseptic conditions, the activated Clostridium botulinum type C strain C62-4 was inoculated into the sterilized fermenter. The inoculation ratio of culture medium to activated C62-4 was 2%. The fermenter control system was started, and the fermentation parameters were set as follows: fermentation temperature 35℃, fermentation pH 7.8, and fermentation time 144 hours. Fermentation was carried out until the fermentation endpoint was reached. S4. First discharge and seed retention: After fermentation, let the liver block residue settle. Transfer the supernatant fermentation liquid through the discharge valve at the bottom of the tank. Accurately measure and transfer 2% of the total fermentation liquid as inoculum, and temporarily store it in a sterile seed bottle for later use. The remaining majority of the fermentation liquid (including products) is transferred to the storage tank through pipelines. S5. Fermentation tank cleaning: Immediately after all materials have been transferred out, start the online cleaning (CIP) program for the fermentation tank (pre-rinse with purified water (to remove residue) → heated alkaline solution circulation cleaning (to remove protein and impurities) → rinse with purified water (to neutral) → final cleaning with water for injection (to remove pyrogens)). After cleaning, check whether the filter screen inside the tank is clogged, and backflushing or manual cleaning is necessary if required. S6. Cyclic Fermentation: Add fresh liver blocks again as required in S1, directly inoculate with the seed liquid retained in the seed bottle in S4, replenish with fresh culture medium, turn on stirring and temperature control, and carry out the next round of fermentation; S7. Continuous cycle: Repeat operations from S3 to S5 to achieve multiple continuous fermentations until strain degeneration, extended fermentation cycle, or contamination occurs. Then, perform comprehensive equipment sterilization and start using new strains.

[0025] Testing and Experiment Toxicity testing: Using this continuous fermentation method, a 500L fermenter was inoculated and fermented according to the method of this invention. The supernatant from five consecutive fermentation broths was diluted and intravenously injected into mice weighing 16-20g to determine the minimum lethal dose (MLD). The toxicity test results are shown in Table 1 below: Table 1. Results of toxicity assays

[0026] Inactivation and detoxification test: Inactivation test: Batch samples were taken from thioglycolate fluid medium (TG), casein agar (GA), and tryptic soybean liquid medium (TSB), with 0.2 ml inoculated into each tube for sterility testing. After incubation at 37℃ for 5-10 days, no bacterial growth was observed in any of the samples. Conclusion: Inactivation test passed.

[0027] Detoxification test: During the aseptic test, samples from each batch were centrifuged, and the supernatant was injected into two mice (0.4 ml each). After 5 days of observation, no mice died. Conclusion: Detoxification was successful.

[0028] According to the "Regulations for Veterinary Biological Products of the People's Republic of China" (2000 edition) and the "Veterinary Pharmacopoeia of the People's Republic of China" (2025 edition, Part III), the virulence MLD (minimum lethal dose) and inactivation and detoxification of Clostridium botulinum fermentation liquid (C61-4) meet the regulations and standards, fully meet the production requirements, and can be used to prepare qualified Clostridium botulinum poisoning (type C) antigen.

[0029] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the invention by those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the invention should be included within the scope of protection of the invention.

Claims

1. A method for continuous fermentation cultivation of Clostridium botulinum strain, characterized in that: The specific steps of the cultivation method are as follows: S1. Initial preparation and feeding: Place fresh liver pieces on the circular 316L stainless steel filter screen at the bottom of the fermentation tank; S2. Culture medium preparation and sterilization: Add the prepared culture medium into the tank, immerse the fresh liver pieces and the stirring paddle, turn on the stirring, heat the jacket to sterilize the tank, and after sterilization, cool the culture medium temperature down to the inoculation temperature through the jacket cooling water. S3. Inoculation and fermentation: Under aseptic conditions, the activated Clostridium botulinum type C strain is inoculated into the sterilized fermenter, the fermenter control system is started, and fermentation is carried out according to the process setting parameters until the fermentation endpoint is reached. S4. First feeding and seed retention: After fermentation, let the liver block residue settle. Transfer the supernatant fermentation liquid out through the liquid discharge valve at the bottom of the tank. Accurately measure and transfer the required amount of inoculum liquid, and temporarily store it in a sterile seed bottle for later use. The remaining majority of the fermentation liquid is transferred to the storage tank through pipeline. S5. Fermentation tank cleaning: After all materials have been transferred out, immediately start the online cleaning program for the fermentation tank. After cleaning, check whether the filter screen inside the tank is clogged. If necessary, backflushing or manual cleaning should be performed. S6. Cyclic Fermentation: Add fresh liver blocks again as required in S1, directly inoculate with the seed liquid retained in the seed bottle in S4, replenish with fresh culture medium, turn on stirring and temperature control, and carry out the next round of fermentation; S7. Continuous cycle: Repeat operations from S3 to S5 to achieve multiple continuous fermentations until strain degeneration, extended fermentation cycle, or contamination occurs. Then, perform comprehensive equipment sterilization and start using new strains.

2. The method for continuous fermentation of Clostridium botulinum strain according to claim 1, characterized in that: The fresh liver pieces in S1 are minced or bagged fresh liver pieces, and the thickness of the fresh liver pieces stacked is 10cm-20cm.

3. The method for continuous fermentation of Clostridium botulinum strain according to claim 1, characterized in that: The circular 316L stainless steel filter screen in S1 is installed 10-15cm away from the bottom of the fermenter.

4. The method for continuous fermentation of Clostridium botulinum strain according to claim 1, characterized in that: The culture medium in S2 is Clostridium botulinum type C culture medium.

5. The method for continuous fermentation of Clostridium botulinum strain according to claim 1, characterized in that: The inoculation ratio of the culture medium in S2 to the fermented Clostridium botulinum strain in S3 is 2%.

6. The method for continuous fermentation of Clostridium botulinum strain according to claim 1, characterized in that: The S2 jacket is heated to a sterilization temperature of 116-118℃; the inoculation temperature is 35℃.

7. The method for continuous fermentation of Clostridium botulinum strain according to claim 1, characterized in that: The type C botulinum bacteria in S3 are strains C62-4 or C62-6.

8. The method for continuous fermentation of Clostridium botulinum strain according to claim 1, characterized in that: The fermentation temperature of the fermenter control system in S3 is 35℃, the fermentation pH is 7.8, and the fermentation time is 144h.

9. The method for continuous fermentation of Clostridium botulinum strain according to claim 1, characterized in that: The amount of inoculum required for transfer in S4 is 1%-2% of the total fermentation volume.

10. The method for continuous fermentation of Clostridium botulinum strain according to claim 1, characterized in that: In S5: after all the material has been transferred out, the online cleaning program of the fermenter is started immediately, which is: pre-rinse with purified water → alkali heating and circulation cleaning → rinsing with purified water → final cleaning with water for injection.

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