Fermentation medium and culture method for clostridium tetani

Abstract

The invention discloses a clostridium tetani fermentation medium and a culture method. On one hand, the invention provides a clostridium tetani fermentation culture medium which comprises a clostridium tetani culture medium and a clostridium tetani toxin-producing culture medium according to a specific ratio. On the other hand, the invention also provides a clostridium tetani fermentation culture method using the culture medium, and the method comprises the following steps: S1, carrying out recovery culture on a clostridium tetani cryopreserved strain; s2, inoculating the first generation of clostridium tetani into a clostridium tetani culture medium, and carrying out two-generation culture to obtain a third generation of clostridium tetani; and S3, inoculating the third generation of clostridium tetani into a clostridium tetani toxin-producing culture medium, and carrying out standing culture for 60-72 hours. The non-animal source culture medium is adopted, the safety of vaccine prepared from the prepared toxin is good, and meanwhile the toxin yield is relatively high.

Description

Technical Field

[0001] The present application relates to the technical field of vaccine preparation, and in particular to a Clostridium tetani fermentation medium and a culture method. Background Art

[0002] Vaccination with tetanus toxoid-containing vaccines is currently the most economical and effective way to prevent tetanus, and tetanus toxoid vaccines have been included in the routine immunization programs of children in all countries around the world. Tetanus toxoid is usually produced by the cultivation of toxin-producing Clostridium tetani, which is made after formaldehyde detoxification. It can not only be used to prepare tetanus toxoid vaccines, but also as a carrier protein to prepare polysaccharide protein conjugate vaccines such as Haemophilus influenzae type b conjugate vaccine, meningococcal conjugate vaccine, and pneumococcal conjugate vaccine.

[0003] In the preparation of existing tetanus vaccines, the culture of toxin-producing Clostridium tetani generally uses animal-derived culture medium, and tetanus beef peptic digestion fluid and tetanus cheese trypsin digestion fluid are added to the culture medium to improve the toxin production capacity. However, this traditional culture method uses animal-derived culture medium, and the adverse reaction rate of the vaccine prepared is relatively high, which poses a vaccine safety risk.

[0004] At present, there are a small number of methods for culturing toxin-producing Clostridium tetani in non-animal culture media in the art, which can effectively improve the safety of vaccines. For example, the Chinese invention patent with publication number CN112646747A and invention name Clostridium tetani culture medium discloses a method for culturing non-animal culture media. However, the toxin yield of this type of culturing method using non-animal culture media is significantly lower than that of the existing traditional animal culture media. Summary of the invention

[0005] In order to solve at least one of the above technical problems, a culture method for preparing vaccines using toxins produced using non-animal culture medium is developed, which has better safety and relatively high toxin yield. The present application provides a Clostridium tetani fermentation medium and culture method.

[0006] In one aspect, the present application provides a Clostridium tetani fermentation medium, including a Clostridium tetani culture medium and a Clostridium tetani toxigenic culture medium; The concentration ratio of each component of the Clostridium tetani culture medium includes: 8-12 g / L tryptone, 3-8 g / L yeast extract, 4-6 g / L sodium acetate trihydrate, 0.8-1.2 g / L potassium dihydrogen phosphate, 0.8-1.2 g / L disodium hydrogen phosphate dodecahydrate, 0.015-0.035 g / L ferric chloride hexahydrate, and 2-4 g / L glucose; The concentration ratio of each component of the Clostridium tetani toxin production culture medium includes: 4-6 g / L sodium acetate trihydrate, 0.8-1.2 g / L potassium dihydrogen phosphate, 0.8-1.2 g / L disodium hydrogen phosphate dodecahydrate, 70-80 g / L glycerol, 3-8 g / L yeast extract, 0.02-0.03 g / L ferric chloride hexahydrate, 0.8-1.2 g / L tryptone, 0.8-1.2 g / L peptone, 4-8 ml / L tetanus factor I, 1-4 ml / L tetanus factor II, and 2-4 g / L glucose.

[0007] By adopting the above-mentioned technical scheme, the present application adopts a culture medium that does not contain any animal-derived ingredients, which can effectively avoid the problem of adverse reactions to subsequent vaccines caused by the use of animal-derived culture media, thereby greatly improving safety; in addition to the basic nitrogen source and carbon source and other substances required for fermentation, the culture medium of the present application is added with Liuhe water ferric chloride, which can effectively promote the proliferation of Clostridium tetani, and can enable the proliferated Clostridium tetani to have a stronger toxin expression ability, which can effectively improve the toxin yield.

[0008] Optionally, the concentration ratio of each component of the Clostridium tetani culture medium includes: 10-12 g / L tryptone, 4.5-5.5 g / L yeast extract, 4.5-5.5 g / L sodium acetate trihydrate, 0.8-1.2 g / L potassium dihydrogen phosphate, 0.8-1.2 g / L disodium hydrogen phosphate dodecahydrate, 0.02-0.03 g / L ferric chloride hexahydrate, and 2.2-2.8 g / L glucose.

[0009] Optionally, the concentration ratio of each component of the Clostridium tetani toxin-producing culture medium includes: 4.5~5.5g / L sodium acetate trihydrate, 0.8~1.2g / L potassium dihydrogen phosphate, 0.8~1.2g / L disodium hydrogen phosphate dodecahydrate, 74~76g / L glycerol, 4.5~5.5g / L yeast extract, 0.02~0.03g / L ferric chloride hexahydrate, 0.8~1.2g / L tryptone, 0.8~1.2g / L peptone, 4.5~5.5ml / L tetanus factor I, 1.8~2.2ml / L tetanus factor II, and 2.2~2.8g / L glucose.

[0010] Optionally, the pH of the Clostridium tetani culture medium is 7.6-7.8, and the pH of the Clostridium tetani toxigenic culture medium is 6.9-7.1.

[0011] Optionally, the Clostridium tetani toxin-producing culture medium further includes calcium chloride hexahydrate, and the concentration of the calcium chloride hexahydrate is 0.004-0.008 g / L.

[0012] On the other hand, the present application also provides a method for fermenting and culturing Clostridium tetani, using the above-mentioned Clostridium tetani fermentation medium, comprising the following steps: S1. Inoculate the frozen strain of Clostridium tetani into the deep of Clostridium tetani culture medium, and then seal the surface of the culture medium for recovery culture to obtain the first generation of Clostridium tetani; S2, inoculating the first-generation Clostridium tetani obtained in step S1 into a Clostridium tetani culture medium and culturing for two generations to obtain the third-generation Clostridium tetani; S3. The third-generation Clostridium tetani obtained in step S2 is inoculated into a Clostridium tetani toxin-producing culture medium and statically cultured for 60 to 72 hours.

[0013] By adopting the above technical scheme, the present application uses a specific non-animal culture medium, first performs a third-generation fermentation culture on Clostridium tetani, and then performs a toxin production culture based on the third-generation Clostridium tetani. The preparation method is relatively simple and can obtain a higher toxin yield.

[0014] Optionally, in step S1, a semi-solid Clostridium tetani culture medium is used, the culture temperature is 34-36° C., and the culture time is 46-50 hours.

[0015] Optionally, in step S2, the second generation Clostridium tetani is cultured using a semi-solid Clostridium tetani culture medium at a temperature of 34 to 36° C. for a time of 46 to 50 hours.

[0016] Optionally, in step S2, the third generation Clostridium tetani is cultured using a liquid Clostridium tetani culture medium, with a culture tank pressure of 0.003 to 0.030 MPa, a culture temperature of 34 to 36° C., and a culture time of 46 to 50 hours.

[0017] Optionally, in step S3, the culture tank pressure is 0.003-0.030 MPa, and the culture temperature is 34-36°C.

[0018] In summary, the present invention includes at least one of the following beneficial technical effects: 1. This application uses a culture medium that does not contain any animal-derived ingredients, which can effectively avoid the problem of adverse reactions to subsequent vaccines caused by the use of animal-derived culture media, thereby greatly improving safety.

[0019] 2. In addition to the basic nitrogen source and carbon source and other substances required for fermentation, the culture medium of the present application is added with hexahydrate ferric chloride, which can effectively promote the proliferation of Clostridium tetani, and can make the proliferated Clostridium tetani have a stronger toxin expression ability, which can effectively improve the toxin yield.

[0020] 3. The present application uses a specific non-animal culture medium, first performs a third-generation fermentation culture on Clostridium tetani, and then performs a toxin production culture based on the third-generation Clostridium tetani. The preparation method is relatively simple and can obtain a higher toxin yield. DETAILED DESCRIPTION

[0021] The present application is further described in detail below in conjunction with embodiments.

[0022] The present application designs a Clostridium tetani fermentation medium, including a Clostridium tetani culture medium and a Clostridium tetani toxin production culture medium; The concentration ratio of each component of the Clostridium tetani culture medium includes: 8-12 g / L tryptone, 3-8 g / L yeast extract, 4-6 g / L sodium acetate trihydrate, 0.8-1.2 g / L potassium dihydrogen phosphate, 0.8-1.2 g / L disodium hydrogen phosphate dodecahydrate, 0.015-0.035 g / L ferric chloride hexahydrate, and 2-4 g / L glucose; The concentration ratio of each component of the Clostridium tetani toxin production culture medium includes: 4-6 g / L sodium acetate trihydrate, 0.8-1.2 g / L potassium dihydrogen phosphate, 0.8-1.2 g / L disodium hydrogen phosphate dodecahydrate, 70-80 g / L glycerol, 3-8 g / L yeast extract, 0.02-0.03 g / L ferric chloride hexahydrate, 0.8-1.2 g / L tryptone, 0.8-1.2 g / L peptone, 4-8 ml / L tetanus factor I, 1-4 ml / L tetanus factor II, and 2-4 g / L glucose.

[0023] The present application also provides a method for fermenting and culturing Clostridium tetani, using the above-mentioned Clostridium tetani fermentation medium, comprising the following steps: S1. Inoculate the frozen strain of Clostridium tetani into the deep of Clostridium tetani culture medium, and then seal the surface of the culture medium for recovery culture to obtain the first generation of Clostridium tetani; S2, inoculating the first-generation Clostridium tetani obtained in step S1 into a Clostridium tetani culture medium and culturing for two generations to obtain the third-generation Clostridium tetani; S3. The third-generation Clostridium tetani obtained in step S2 is inoculated into a Clostridium tetani toxin-producing culture medium and statically cultured for 60 to 72 hours.

[0024] The main differences between the present application and the prior art are as follows. First, the present application does not use the traditional animal-derived culture medium. In addition to being able to effectively improve the safety of the vaccine, the quality of the toxin obtained by culture is also relatively good. Secondly, the present application uses a specific culture medium ratio, which, combined with the culture method of the present application, can effectively promote the expression of the toxin and significantly increase the toxin yield.

[0025] The following are preparation examples and embodiments of the present application.

[0026] In the preparation examples and embodiments of the present application, the sources of the main raw materials are as follows: The Clostridium tetani strain was obtained from Wuhan Institute of Biological Products Co., Ltd.; peptone and tryptone were purchased from Wuhan Jiyesheng Chemical Co., Ltd.; tetanus factor I and tetanus factor II were purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0027] The culture medium of the present invention is prepared by the following method.

[0028] The preparation of the semi-solid Clostridium tetani culture medium adopts the following steps: 1) Solution 1: Accurately weigh the components of the Clostridium tetani culture medium, add appropriate amount of water and stir, make up to volume after fully dissolving, and adjust the pH value to 7.6-7.8; 2) Weigh 2 g / L of agar powder and add it to Solution 1 and boil to dissolve. After fully dissolved, dispense into large test tubes or blue-capped bottles for sterilization at 116.5°C for 20 min. After sterilization, store the culture medium in a cold storage at 2-8°C.

[0029] The preparation of liquid Clostridium tetani culture medium adopts the following steps: 1) Solution 1: Accurately weigh the components of the Clostridium tetani culture medium, add appropriate amount of water and stir, make up to volume after fully dissolving, and adjust the pH value to 7.6-7.8; 2) Divide into large test tubes or blue-capped bottles for sterilization at 116.5°C for 20 min. After sterilization, store the culture medium in a cold storage at 2-8°C.

[0030] The preparation of Clostridium tetani toxin-producing culture medium adopts the following steps: 1) Solution 1: Accurately weigh the formula amount of sodium acetate trihydrate, potassium dihydrogen phosphate, disodium hydrogen phosphate dodecahydrate, glycerol, yeast extract, ferric chloride hexahydrate, tryptone and neopeptone, add appropriate amount of water and stir, make up to volume after fully dissolving, and adjust the pH value to 7.3-7.5; 2) Sterilize at 116.5°C for 20 minutes; 3) Add the sterilized and filtered tetanus factor I, tetanus factor II and tetanus glucose solution into the sterilized culture medium, adjust the pH value to 6.9-7.1, and finally prepare the tetanus toxigenic culture medium.

[0031] The following are examples of the present application: The clostridium tetani fermentation culture method used in the embodiment of the present application comprises the following steps: S1. Pipette 0.9 mL of liquid Clostridium tetani culture medium and add it into the culture tube, repeatedly blow and beat the lyophilized material to dissolve the lyophilized bacteria in the culture tube into a bacterial suspension; prick the bacterial suspension and inoculate it into the depth of the semi-solid Clostridium tetani culture medium, the inoculation amount is about 0.3 mL / tube, and then add 8 mL of liquid paraffin to cover the surface of the culture medium; put the inoculated semi-solid Clostridium tetani culture into an incubator, maintain the temperature at 34-36° C., and culture for 48 hours to obtain the first generation Clostridium tetani; S2, the first generation Clostridium tetani obtained in step S1 is inoculated into the depth of the semi-solid Clostridium tetani culture medium, the inoculation amount is about 3%, and finally 100mL tetanus liquid paraffin is added to cover the surface of the culture medium, and the sealing bandage is applied; the inoculated semi-solid Clostridium tetani culture is placed in an incubator, maintained at a temperature of 34-36°C, and cultured for 48 hours to obtain the second generation Clostridium tetani; a 60L fermentation tank is taken, 40L liquid Clostridium tetani culture medium is loaded, the second generation Clostridium tetani is inoculated into the 60L fermentation tank, the tank pressure is maintained at 0.003-0.030Mpa, the temperature is maintained at 34-36°C, and the culture is allowed to stand for 50 hours to obtain the second generation Clostridium tetani; S3, take a 1400L large fermentation tank, load it with 1000L Clostridium tetani toxin-producing culture medium; start the transplanting program to transfer the liquid in the 60L small tank of step S2 into the 1400L large fermentation tank, and control the transplanting operation time within 20min; maintain the tank pressure at 0.003-0.030Mpa and the temperature at 34-36°C, and culture it statically for 60-72 hours. During the culture process, record the pH, temperature and tank pressure every 6 hours. After 55 hours, record and take samples every 1 hour for floc unit detection. When the continuous detection of floc value tends to be stable, terminate the culture.

[0032] Embodiments 1 to 8 The difference between Examples 1 to 8 of the present application is that the ratio of the culture medium is different. The specific ratio of the culture medium of Examples 1 to 8 of the present application is shown in Tables 1 and 2 below.

[0033] Table 1 Example 1 to 8 Clostridium tetani culture medium ratio table Tryptone Yeast Extract Sodium acetate Potassium dihydrogen phosphate Sodium dihydrogen phosphate Ferric chloride glucose Example 1 8g / L 3g / L 3g / L 0.8g / L 0.8g / L 0.015g / L 2g / L Example 2 9g / L 4g / L 4g / L 0.9g / L 0.9g / L 0.025g / L 3g / L Example 3 10g / L 5.5g / L 5.5g / L 1.0g / L 1.0g / L 0.035g / L 4g / L Example 4 11g / L 7g / L 7g / L 1.1g / L 1.1g / L 0.015g / L 2g / L Example 5 12g / L 8g / L 8g / L 1.2g / L 1.2g / L 0.025g / L 3g / L Example 6 12g / L 4.5g / L 4.5g / L 1.2g / L 1.2g / L 0.030g / L 2.2g / L Example 7 11g / L 5.5g / L 5.5g / L 0.8g / L 0.8g / L 0.020g / L 2.8g / L Example 8 10g / L 5g / L 5g / L 1g / L 1g / L 0.025g / L 2.5g / L Table 2 Example 1 to 8 Clostridium tetani toxin production culture medium ratio table Tryptone Neopeptone yeast Sodium acetate Potassium dihydrogen phosphate Sodium dihydrogen phosphate Ferric chloride glucose Example 1 0.8g / L 0.8g / L 3g / L 3g / L 0.8g / L 0.8g / L 0.015g / L 2g / L Example 2 0.9g / L 0.9g / L 4g / L 4g / L 0.9g / L 0.9g / L 0.025g / L 3g / L Example 3 1.0g / L 1.0g / L 5.5g / L 5.5g / L 1.0g / L 1.0g / L 0.035g / L 4g / L Example 4 1.1g / L 1.1g / L 7g / L 7g / L 1.1g / L 1.1g / L 0.015g / L 2g / L Example 5 1.2g / L 1.2g / L 8g / L 8g / L 1.2g / L 1.2g / L 0.025g / L 3g / L Example 6 1.2g / L 1.2g / L 4.5g / L 4.5g / L 1.2g / L 1.2g / L 0.030g / L 2.2g / L Example 7 0.8g / L 0.8g / L 5.5g / L 5.5g / L 0.8g / L 0.8g / L 0.020g / L 2.8g / L Example 8 1g / L 1g / L 5g / L 5g / L 1g / L 1g / L 0.025g / L 2.5g / L In the Clostridium tetani toxin-producing culture medium of Examples 1 to 8 of the present application, the concentration of glycerol was 75 g / L, the concentration of tetanus factor I was 5 ml / L, and the concentration of tetanus factor II was 2 ml / L.

[0034] Comparative Example 1 Example 5 of the Chinese invention patent with publication number CN112646747A and invention name Clostridium tetani culture medium was adopted as comparative example 1 of the present application.

[0035] Comparative Example 2 The animal-derived fermentation medium and fermentation method of Wuhan Institute of Biological Products Co., Ltd. were used as Comparative Example 2 of this application.

[0036] Detection 1) Toxin yield The flocculent unit detection method recorded in the Chinese Pharmacopoeia was used to detect the toxin yields of Examples 1 to 8 of the present application and Comparative Examples 1 to 2.

[0037] 2) Virulence identification The fermentation products of Examples 1 to 8 of the present application and Comparative Example 2 were purified by the existing ammonium sulfate salting-out method, and the purified toxin culture was collected.

[0038] 9 groups of healthy experimental guinea pigs were prepared, with 5 in each group; the 9 groups of experimental guinea pigs were respectively injected with the toxin culture of Examples 1 to 8 and Comparative Example 2, with each guinea pig being injected with 0.12 mL. The death time of the guinea pigs after injection was recorded, and the average death time of each group of guinea pigs was calculated.

[0039] The results are shown in Table 3 below.

[0040] Table 3 Test results of Examples 1 to 8 and Comparative Examples Toxin yield (Lf / ml) Virulence identification results (h) Example 1 102 15.4 Example 2 108 15.2 Example 3 106 15.5 Example 4 105 15.1 Example 5 104 14.9 Example 6 106 15.3 Example 7 111 15.4 Example 8 113 15.1 Comparative Example 1 90 / Comparative Example 2 112 15.2 From the data in Table 3, it can be seen that the results of toxicity identification of the toxin cultures prepared in Examples 1 to 8 of the present application and the toxin cultures prepared in the traditional culture medium and method of Comparative Example 2 have no significant difference, and the toxicity of the toxin cultures is very similar. It can be seen that the culture medium of the present application, in combination with the method of the present application, can stably express the target toxin by fermenting and culturing Clostridium tetani, and the toxicity of the obtained toxin fully meets the requirements for vaccine preparation.

[0041] From the data in Table 3, it can be seen from the data of Examples 1 to 8 and Comparative Examples 1 to 2 that the culture medium of the present application, after adding hydrated ferric chloride and optimizing the ratio, can effectively improve the toxin production ability of Clostridium tetani, and the toxin yield is close to or even exceeds the existing traditional culture method; compared with the existing culture method using non-animal source culture medium, the toxin yield can be increased by more than 10%.

[0042] From the data comparison of Examples 1 to 8 in Table 3, it can be seen that after the ratio of the culture medium of the present application is further optimized, the toxin yield can be further improved.

[0043] The applicant has conducted relevant experimental studies on the parameters of the culture method of the present application. Within the parameter range of the present application, adjusting the method parameters has little effect on the toxin yield of the present application. In addition, the applicant has also conducted relevant experimental studies on the addition amounts of glycerol, tetanus factor I and tetanus factor II in the toxin-producing culture medium. Within the parameter range of the present application, the addition amount of glycerol has little effect on the toxin yield of the present application; the most preferred addition amounts of tetanus factor I and tetanus factor II are the addition amounts used in Examples 1 to 8 of the present application, and changing the addition amounts of tetanus factor I and tetanus factor II has a slight effect on the toxin yield.

[0044] Embodiments 9 to 13 Examples 9 to 13 of the present application are based on Example 8 of the present application, and the only difference is that calcium chloride hexahydrate is added to the Clostridium tetani toxin production culture medium. In the Clostridium tetani toxin production culture medium of Examples 9 to 13, the concentrations of calcium chloride hexahydrate are 0.004 g / L, 0.005 g / L, 0.006 g / L, 0.007 g / L and 0.008 g / L, respectively.

[0045] The same test was performed on Examples 9 to 13, and the results are shown in Table 4 below.

[0046] Table 4 Test results of Examples 9 to 13 Toxin yield (Lf / ml) Example 9 118 Example 10 124 Embodiment 11 122 Example 12 119 Embodiment 13 117 It can be seen from the data in Table 4 that in Examples 9 to 13 of the present application, adding a trace amount of hydrated calcium chloride to the toxin-producing culture medium can further promote the expression of the toxin and further increase the toxin yield.

[0047] The above are all preferred embodiments of the present application, and the protection scope of the present application is not limited thereto. Therefore, any equivalent changes made according to the structure, shape, and principle of the present application should be included in the protection scope of the present application.

Claims

1. A Clostridium tetani fermentation medium, characterized in that: Includes Clostridium tetani culture medium and Clostridium tetani toxigenic culture medium; The concentration ratio of each component of the Clostridium tetani culture medium includes: 8-12 g / L tryptone, 3-8 g / L yeast extract, 4-6 g / L sodium acetate trihydrate, 0.8-1.2 g / L potassium dihydrogen phosphate, 0.8-1.2 g / L disodium hydrogen phosphate dodecahydrate, 0.015-0.035 g / L ferric chloride hexahydrate, and 2-4 g / L glucose; The concentration ratio of each component of the Clostridium tetani toxin production culture medium includes: 4-6 g / L sodium acetate trihydrate, 0.8-1.2 g / L potassium dihydrogen phosphate, 0.8-1.2 g / L disodium hydrogen phosphate dodecahydrate, 70-80 g / L glycerol, 3-8 g / L yeast extract, 0.02-0.03 g / L ferric chloride hexahydrate, 0.8-1.2 g / L tryptone, 0.8-1.2 g / L peptone, 4-8 ml / L tetanus factor I, 1-4 ml / L tetanus factor II, and 2-4 g / L glucose.

2. The Clostridium tetani fermentation medium according to claim 1, characterized in that The concentration ratio of each component of the Clostridium tetani culture medium includes: 10-12 g / L tryptone, 4.5-5.5 g / L yeast extract, 4.5-5.5 g / L sodium acetate trihydrate, 0.8-1.2 g / L potassium dihydrogen phosphate, 0.8-1.2 g / L disodium hydrogen phosphate dodecahydrate, 0.02-0.03 g / L ferric chloride hexahydrate, and 2.2-2.8 g / L glucose.

3. The Clostridium tetani fermentation medium according to claim 1, characterized in that The concentration ratio of each component of the Clostridium tetani toxin-producing culture medium includes: 4.5-5.5 g / L sodium acetate trihydrate, 0.8-1.2 g / L potassium dihydrogen phosphate, 0.8-1.2 g / L disodium hydrogen phosphate dodecahydrate, 74-76 g / L glycerol, 4.5-5.5 g / L yeast extract, 0.02-0.03 g / L ferric chloride hexahydrate, 0.8-1.2 g / L tryptone, 0.8-1.2 g / L peptone, 4.5-5.5 ml / L tetanus factor I, 1.8-2.2 ml / L tetanus factor II, and 2.2-2.8 g / L glucose.

4. The Clostridium tetani fermentation medium according to claim 1, characterized in that The pH of the Clostridium tetani culture medium is 7.6-7.8, and the pH of the Clostridium tetani toxin-producing culture medium is 6.9-7.

1.

5. The Clostridium tetani fermentation medium according to claim 1, characterized in that The Clostridium tetani toxin-producing culture medium also includes calcium chloride hexahydrate, and the concentration of the calcium chloride hexahydrate is 0.004-0.008 g / L.

6. A method for fermenting and culturing Clostridium tetani, characterized in that: Using the Clostridium tetani fermentation medium according to any one of claims 1 to 5, comprising the following steps: S1. Inoculate the frozen strain of Clostridium tetani into the deep of Clostridium tetani culture medium, and then seal the surface of the culture medium for recovery culture to obtain the first generation of Clostridium tetani; S2, inoculating the first-generation Clostridium tetani obtained in step S1 into a Clostridium tetani culture medium and culturing for two generations to obtain the third-generation Clostridium tetani; S3. The third-generation Clostridium tetani obtained in step S2 is inoculated into a Clostridium tetani toxin-producing culture medium and statically cultured for 60 to 72 hours.

7. The method for culturing Clostridium tetani by fermentation according to claim 6, characterized in that: In the step S1, a semi-solid Clostridium tetani culture medium is used, the culture temperature is 34-36° C., and the culture time is 46-50 hours.

8. The method for culturing Clostridium tetani by fermentation according to claim 6, characterized in that: In the step S2, the second generation Clostridium tetani is cultured in a semi-solid Clostridium tetani culture medium at a temperature of 34 to 36° C. for a time of 46 to 50 hours.

9. The method for culturing Clostridium tetani by fermentation according to claim 6, characterized in that: In the step S2, the third generation of Clostridium tetani is cultured using a liquid Clostridium tetani culture medium, the culture tank pressure is 0.003-0.030 MPa, the culture temperature is 34-36° C., and the culture time is 46-50 hours.

10. The method for culturing Clostridium tetani by fermentation according to claim 6, characterized in that: In step S3, the pressure of the culture tank is 0.003-0.030 MPa, and the culture temperature is 34-36°C.