Culture medium for A type clostridium perfringens and C type clostridium perfringens, fermentation culture method and application of fermentation toxin production

Abstract

The invention discloses a culture medium for A type clostridium perfringens and C type clostridium perfringens, a fermentation culture method and application of fermentation toxin production. Wherein the culture medium comprises the following components: tryptone, # imgabs0 # peptone, casein peptone, a yeast extract, lactoalbumin hydrolysate, trihydroxymethyl aminomethane, sodium acetate and glucose. According to the present invention, through the specific formula composition, the A-type clostridium perfringens and the C-type clostridium perfringens can adapt to fermentation in the fermentation tank, and in the fermentation culture process, the toxin production ability of the A-type clostridium perfringens and the C-type clostridium perfringens can be significantly improved by controlling the stirring speed, the pH value of the fermentation liquid and the precise material supplementing manner. And the supernate obtained by fermentation is used as a raw material for preparing the vaccine, so that animals can generate high-level neutralizing antibodies aiming at the clostridium perfringens A and C type toxins after being immunized with the vaccine, the death of the animals caused by infection of the clostridium perfringens A and C type toxins is avoided, and the method has practical popularization and application values.

Description

Technical Field

[0001] The present invention specifically relates to a culture medium for Clostridium perfringens type A and type C, a fermentation culture method, and an application of fermentation to produce toxins. Background Art

[0002] Clostridium perfringens, also known as Clostridium welchii, can decompose sugars in muscles and connective tissues to produce a large amount of gas and cause severe emphysema, thus being named Clostridium perfringens. Clostridium perfringens is widely distributed in soil, sewage, and the intestines of humans and animals. It is one of the pathogens that cause traumatic gas gangrene, necrotic enteritis, and enterotoxemia. Every year, a large number of animals die due to infection with Clostridium perfringens, posing a serious threat to the livestock industry. Clostridium perfringens can produce 12 toxins, among which α, β, ε, and ι are the main lethal toxins. According to toxin neutralization tests, Clostridium perfringens can be divided into five types: A, B, C, D, and E. Type A bacteria (type A) are the main pathogenic bacteria of zoonotic diseases, which can cause necrotic enteritis, enterotoxemia, food poisoning, etc. in humans and animals; Clostridium perfringens type C mainly produces two kinds of exotoxins, α and β, which can cause necrotic enteritis in humans and livestock, enterotoxemia, "sudden death syndrome" in ruminants, especially cattle and sheep, and red dysentery in piglets. In recent years, β2 toxin produced by Clostridium perfringens type A and type C has been confirmed as a new type of exotoxin. A large number of studies abroad have confirmed that intestinal diseases in livestock and poultry are related to β2 toxin. In addition, some studies have shown that β2 toxin can also cause diarrhea in humans.

[0003] The exotoxin secreted by the growth of Clostridium perfringens is a kind of protein. After the exotoxin is detoxified with formaldehyde, a non-toxic substance is obtained, which is called a toxoid. The toxoid retains good immunogenicity. After being prepared into a vaccine and immunizing animals, it can stimulate the body to produce antitoxin (antibody), and the antibody can neutralize the exotoxin, thereby providing a protective effect for the body. In human vaccines, tetanus toxoid vaccine, snake venom antiserum, etc. have been widely used. Therefore, high-density culture of Clostridium perfringens to make it produce a large amount of high-titer exotoxin is the key to preparing toxoid vaccines.

[0004] Traditional antigen culture processes use anaerobic liver broth, liver-gastric enzyme digestion broth, trypsin-digested beef broth, etc. as culture media, and static culture in large glass bottles. The process is complex and requires the collection of fresh beef, animal livers, pancreas, etc. for processing and preparation. The raw material sources and the batch-to-batch stability of the prepared culture media are poor. Static culture in glass bottles also has certain limitations in improving antigen yield and titer, and it is impossible to produce antigens on a large scale at one time. For the Clostridium perfringens type C bacterial solution produced by the traditional process, when using 16 - 20g mice and measuring the toxin titer by intravenous injection, it is 400 - 1000 MLD / ml. For the Clostridium perfringens type A bacterial solution, when using 16 - 20g mice and measuring the toxin titer by intraperitoneal injection, it is 20 - 40 MLD / ml, and the toxin titers are not very high. The latest research, such as patents CN102688484B, CN109943507B, etc., can only culture one of Clostridium perfringens type A or type C, and obtain a bacterial solution with a relatively high toxin titer. It is necessary to provide a culture medium that can simultaneously meet the fermentation culture of Clostridium perfringens type A and type C, and enable this culture medium to be used for fermentation in a fermenter to meet the requirements of industrial production. Summary of the Invention

[0005] The purpose of the present invention is to provide a culture medium for Clostridium perfringens type A and type C, and a method for fermenter culture of Clostridium perfringens type A and type C using this culture medium, so as to mass-produce high-titer toxins of Clostridium perfringens type A and type C for the preparation of high-quality Clostridium perfringens type A and / or type C toxoid vaccines.

[0006] The present invention provides a culture medium for Clostridium perfringens type A and type C, which is composed of component A and component B;

[0007] Component A is composed of the following raw materials in parts by weight:

[0008] Tryptone 5 - 15 parts, Peptone 5 - 15 parts, casein peptone 5 - 15 parts, yeast extract 5 - 15 parts, hydrolyzed milk protein 3 - 8 parts, tris(hydroxymethyl)aminomethane 2 - 6 parts, sodium acetate 1.5 - 3.5 parts;

[0009] Component B is glucose 5 - 15 parts;

[0010] The mass ratio of component A to component B is: 26.5 - 77.5∶5 - 15

[0011] Furthermore, component A is composed of the following raw materials in parts by weight:

[0012] Tryptone 10 parts, Peptone 10 parts, casein peptone 10 parts, yeast extract 10 parts, hydrolyzed milk protein 5 parts, tris(hydroxymethyl)aminomethane 4 parts, sodium acetate 2.5 parts;

[0013] Component B is 10 parts of glucose;

[0014] The mass ratio of component A to component B is 51.5:10.

[0015] The present invention also provides a use of the aforementioned culture medium for Clostridium perfringens type A and type C in the fermentative culture of Clostridium perfringens type A and / or type C.

[0016] The present invention also provides a method for fermentative culture of Clostridium perfringens type A and type C, comprising the following steps:

[0017] 1) Take the activated Clostridium perfringens type A or type C, inoculate it into an anaerobic liver broth medium, and statically culture for 16 - 24 hours to obtain a seed solution;

[0018] 2) Take the seed solution and inoculate it into a liquid medium, perform fermentation in a fermenter, and collect the bacterial liquid;

[0019] The liquid medium is prepared by dissolving and sterilizing the aforementioned culture medium for Clostridium perfringens type A and type C with water;

[0020] The conditions for fermentation in the fermenter are as follows: the fermentation temperature is 35 - 38°C, the rotation speed of the liquid medium is 50 - 100 r / min, the inoculation amount of the seed solution is 1 - 5%, and the addition amount of the antifoaming agent is 0.1 - 1‰; when the pH value of the fermentation broth drops to 7.0 - 7.4, add a feeding medium accounting for 5 - 15% of the volume of the fermentation broth, and at the same time maintain the pH value of the fermentation broth at 7 - 7.5 by adding alkali. Continue culturing after feeding until the pH value no longer drops;

[0021] The feeding medium is an aqueous solution containing 4 - 6% glucose, 3 - 5% peptone, 5 - 7% tryptone, and 2 - 4% yeast extract.

[0022] Furthermore, the temperature for static culture in step 1) is 37°C;

[0023] In step 2), the fermentation temperature is 37°C, the rotation speed of the liquid medium is 60 r / min, the inoculation amount of the seed solution is 2%, and the addition amount of the antifoaming agent is 0.5‰; when the pH value of the fermentation broth drops to 7.2, add a feeding medium accounting for 10% of the volume of the fermentation broth, and at the same time maintain the pH value of the fermentation broth at 7 - 7.5 by adding alkali; when the fermenter is a 10 L fermenter, supplement the feeding medium at a flow rate of 3 - 5 ml / min, and when the fermenter is a 500 L fermenter, supplement the feeding medium at a flow rate of 150 - 250 ml / min;

[0024] The feeding medium is an aqueous solution containing 5% glucose, 4% peptone, 6% tryptone, and 3% yeast extract.

[0025] Furthermore, the preparation method of the liquid medium is as follows:

[0026] Take component A, dissolve it in 18 - 19 times the volume of injection water, sterilize it to obtain solution A; take component B, dissolve it in 5 times the volume of injection water, sterilize it to obtain solution B, take solution A and solution B and mix them according to a volume ratio of 19:1, and adjust the pH value to 8.2 - 8.4 to obtain the liquid medium.

[0027] The present invention also provides the use of the bacterial liquid obtained by the aforementioned fermentation culture method in the preparation of Clostridium perfringens type A and / or type C toxoid vaccines.

[0028] Finally, the present invention provides a preparation method of Clostridium perfringens type A and / or type C toxoid vaccine, which comprises the following steps:

[0029] Take the Clostridium perfringens type A and / or type C bacterial liquid obtained by the aforementioned fermentation culture method, centrifuge it, take the supernatant and inactivate it, and then add an adjuvant and mix them to obtain the vaccine.

[0030] Furthermore, the temperature of the centrifugation is 4°C, the rotation speed is 8000 - 10000 r / min, and the time is 15 min; the inactivation is that formaldehyde is added to the supernatant and placed at 37°C until complete detoxification; the concentration of formaldehyde added to the solution is 0.6 - 1.0%; the adjuvant is sterile aluminum hydroxide gel.

[0031] Furthermore, when the vaccine is a bivalent toxoid vaccine of Clostridium perfringens type A and type C, the titer of Clostridium perfringens type A toxin is 50 MLD / ml, the titer of Clostridium perfringens type C toxin is 500 MLD / ml, and the final concentration of aluminum hydroxide gel is 15%.

[0032] The culture medium for Clostridium perfringens type A and type C of the present invention, based on the components of the basic medium such as tryptone, yeast extract, glucose, etc., by adding specific peptone, casein peptone, and hydrolyzed milk protein, enables Clostridium perfringens type A and type C to adapt to fermentation in a fermenter. During the fermentation culture process, by controlling the stirring speed, the pH value of the fermentation broth, and the precise feeding method, the ability of Clostridium perfringens type A and type C to synthesize toxins is significantly improved. Using the supernatant obtained from its fermentation as the raw material for vaccine preparation can enable animals to produce high levels of neutralizing antibodies against Clostridium perfringens type A and type C toxins after immunization with the vaccine, avoiding the death of animals due to infection with Clostridium perfringens type A and type C, and having practical application value for popularization.

[0033] Obviously, based on the above content of the present invention, according to the common general technical knowledge and conventional means in the art, without departing from the above basic technical idea of the present invention, various other forms of modifications, substitutions, or changes can also be made.

[0034] The following is a further detailed description of the above content of the present invention in the form of specific embodiments. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention fall within the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 Antibody levels in neonatal piglets after toxoid vaccine immunization of pregnant sows (The differences in antibody detection of groups A and C between the immunized group and the control group are extremely significant "****")

[0036] Figure 2 Antibody levels in piglets before and after toxoid vaccine immunization (The differences in antibody levels of groups A and C between the immunized group 21 days after immunization and before immunization are extremely significant "****", and the differences between the immunized group and the control group 21 days after immunization are extremely significant "****") DETAILED DESCRIPTION OF THE INVENTION

[0037] Example 1 Preparation of liquid medium for Clostridium perfringens type A and type C of the present invention

[0038] Formulation of 1 L liquid medium:

[0039] Component A: 10 g of tryptone, 10 g of peptone, 10 g of casein peptone, 10 g of yeast extract, 5 g of hydrolyzed milk protein, 4 g of tris(hydroxymethyl)aminomethane, 2.5 g of sodium acetate;

[0040] Component B: 10 g of glucose;

[0041] Preparation method: Take Component A, dissolve and mix it with 950 ml of injection water, sterilize it at 115 °C under high pressure for 30 min to obtain Solution A; take Component B, dissolve and mix it with 50 ml of injection water, sterilize it at 115 °C under high pressure for 30 min to obtain Solution B; mix Solution A and Solution B, and adjust the pH value to 8.2 - 8.4 to obtain the product.

[0042] Example 2 Fermentation culture of Clostridium perfringens type A and type C of the present invention

[0043] 1) Take the activated colony of Clostridium perfringens type A or type C, inoculate it into anaerobic liver broth medium, and culture it statically at 37 °C for 16 - 24 hours to obtain the seed liquid;

[0044] 2) Inoculate the seed liquid obtained in step 1) into a 10 L fermenter. The medium formula in the fermenter is the liquid medium obtained in Example 1. The temperature of the liquid medium is 37 °C, the rotation speed is 60 r / min, the inoculation amount of the seed liquid is 2% of the volume of the liquid medium, and then add a sterile antifoaming agent at 0.5‰ of the volume of the liquid medium. When the fermentation culture reaches a pH value of 7.2, start to supplement the feeding medium at 10% of the volume of the fermentation broth at a flow rate of 3 - 5 ml / min. At the same time, use an alkali solution (sterilized 10% sodium hydroxide solution) to maintain the pH value of the fermentation broth at pH 7.0 - 7.5. After the feeding is completed, continue the culture until the pH value no longer drops, and then harvest the bacterial liquid, and that's it;

[0045] Among them, the preparation method of the feeding medium is as follows:

[0046] Mix the sterilized 25%, g / ml glucose solution, 20%, g / ml peptone solution, 20%, g / ml tryptone solution, 10%, g / ml yeast extract solution in a volume ratio of 2∶2∶3∶3 to obtain it.

[0047] Example 3 Fermentation culture of Clostridium perfringens type A and type C of the present invention

[0048] 1) Take the activated Clostridium perfringens type A or type C colony, inoculate it into an anaerobic liver broth medium, and culture it statically at 37 °C for 16 - 24 hours to obtain a seed liquid;

[0049] 2) Inoculate the seed liquid obtained in step 1) into a 500 L fermenter. The medium formula in the fermenter is the liquid medium obtained in Example 1. The temperature of the liquid medium is 37 °C, the rotation speed is 60 r / min, the inoculation amount of the seed liquid is 2% of the volume of the liquid medium, and then add a sterile antifoaming agent at 0.5‰ of the volume of the liquid medium. When the fermentation culture reaches a pH value of 7.2, start to supplement the feeding medium at 10% of the volume of the fermentation broth at a flow rate of 150 - 250 ml / min. At the same time, use an alkali solution (sterilized 10% sodium hydroxide solution) to maintain the pH value of the fermentation broth at pH 7.0 - 7.5. After the feeding is completed, continue the culture until the pH value no longer drops, and then harvest the bacterial liquid, and that's it;

[0050] Among them, the preparation method of the feeding medium is as follows:

[0051] Mix the sterilized 25%, g / ml glucose solution, 20%, g / ml peptone solution, 20%, g / ml tryptone solution, 10%, g / ml yeast extract solution in a volume ratio of 2∶2∶3∶3 to obtain it.

[0052] Example 4 Clostridium perfringens type A and type C bivalent toxoid vaccine of the present invention

[0053] Take the bacterial liquid harvested in Example 2 or Example 3, centrifuge it at 4°C and 8000 - 10000 r / min for 15 min. After taking the supernatant to measure the toxin titer, add formaldehyde until its concentration in the supernatant is 0.6 - 1.0% (ml / ml), place it at 37°C until complete detoxification, then dilute it with sterile normal saline, and finally mix it with sterile aluminum hydroxide adjuvant to obtain a vaccine with a titer of 50 MLD / ml for Clostridium perfringens type A toxin and 500 MLD / ml for Clostridium perfringens type C toxin, and the final concentration of the aluminum hydroxide adjuvant is 15%.

[0054] The beneficial effects of the present invention are illustrated by the following test examples.

[0055] Test Example 1 Research on the culture method of Clostridium perfringens type A and C of the present invention

[0056] 1 Materials

[0057] 1.1 Strains Clostridium perfringens type A strain C57 - 7 (National Veterinary Microbial Strain (Virus) Preservation Center, preservation number: CVCC38), Clostridium perfringens type C strain C59 - 46 (National Veterinary Microbial Strain (Virus) Preservation Center, preservation number: CVCC2032).

[0058] 1.2 Culture medium reagents Blood agar plate, fresh beef, fresh beef liver, pepsin, hydrochloric acid, tryptone,[[]] Peptone, casein peptone, yeast extract, hydrolyzed milk protein, tris(hydroxymethyl)aminomethane (Tris), glucose, sodium acetate, sodium hydroxide, antifoaming agent, liquid paraffin, anaerobic culture bag.

[0059] 1.3 Instrument and equipment Constant temperature incubator, 10L bacterial fermenter, 500L bacterial fermenter.

[0060] 2 Implementation plan

[0061] 2.1 Preparation of anaerobic meat liver soup medium Prepare it according to the instructions of the commercially available anaerobic meat liver soup medium purchased as the seed liquid medium.

[0062] 2.2 Preparation of meat liver pepsin digestive soup Take materials such as fresh beef, fresh beef liver, pepsin, hydrochloric acid, etc., and prepare them according to the method in the appendix of the "Regulations for Veterinary Biological Products".

[0063] 2.3 Preparation of self - made medium for Clostridium perfringens

[0064] Culture medium formula 1: Tryptone 30 g / L, yeast extract 10 g / L, tris(hydroxymethyl)aminomethane (Tris) 4 g / L, sodium acetate 2.5 g / L, glucose 10 g / L, pH 8.2 - 8.4.

[0065] Culture medium formula 2: Tryptone 20 g / L, Peptone 10 g / L, Yeast extract 10 g / L, Tris (Tris(hydroxymethyl)aminomethane) 4 g / L, Sodium acetate 2.5 g / L, Glucose 10 g / L, pH 8.2 - 8.4.

[0066] Culture medium formula 3: Tryptone 10 g / L, Peptone 10 g / L, Peptone from casein 10 g / L, Yeast extract 10 g / L, Tris (Tris(hydroxymethyl)aminomethane) 4 g / L, Sodium acetate 2.5 g / L, Glucose 10 g / L, pH 8.2 - 8.4.

[0067] Culture medium formula 4: Tryptone 10 g / L, Peptone 10 g / L, Peptone from casein 10 g / L, Yeast extract 10 g / L, Lactalbumin hydrolyzate 5 g / L, Tris (Tris(hydroxymethyl)aminomethane) 4 g / L, Sodium acetate 2.5 g / L, Glucose 10 g / L, pH 8.2 - 8.4.

[0068] Preparation method: Taking 1 L of culture medium preparation as an example: According to their respective formulas, weigh all components except glucose and dissolve them successively in 950 ml of injection water, and sterilize at 115 °C under high pressure for 30 min; weigh 10 g of glucose and dissolve it in 50 ml of injection water, and sterilize at 115 °C under high pressure for 30 min. Mix the two before use, and adjust the pH value to 8.2 - 8.4 with 2 mol / L hydrochloric acid or / and 5 mol / L sodium hydroxide solution.

[0069] 2.4 Clostridium perfringens culture

[0070] 2.4.1 Bacterial resuscitation Take the freeze-dried Clostridium perfringens type A strain C57 - 7 (National Veterinary Microbial Strain (Virus) Preservation Center, preservation number: CVCC38), Clostridium perfringens type C strain C59 - 46 (National Veterinary Microbial Strain (Virus) Preservation Center, preservation number: CVCC2032). After adding 1 ml of normal saline to dissolve, inoculate them respectively on blood agar plates and culture anaerobically at 37 °C for 48 h. Pick 3 - 5 single colonies and inoculate them into 100 ml of anaerobic liver broth medium. Culture statically at 37 °C for 16 - 24 hours and harvest as the seed liquid.

[0071] 2.4.2 Subculture Take the harvested seed liquids of Clostridium perfringens type A strain C57 - 7 and Clostridium perfringens type C strain C59 - 46, and inoculate them into liver, stomach and enzyme digestion broth and the self-made culture medium for Clostridium perfringens respectively according to the inoculation ratio of 1%, and culture statically at 37 °C for 16 hours, and harvest for inspection.

[0072] 2.4.3 Inspection After appropriately diluting the harvested bacterial liquid, use a spectrophotometer to measure OD 600Value. Another 4℃ harvested bacterial liquid was centrifuged at 8000 - 10000 r / min for 15 min, and then filtered through a 0.22μm syringe filter to obtain exotoxin for toxin titer determination. The exotoxin was serially diluted with a diluent (2 g of gelatin, 9.25 g of Na2HPO4·12H2O, 8.34 g of NaH2PO4·2H2O, dissolved by heating with 1000 ml of purified water, filtered through a 0.45μm filter and autoclaved at 115℃ for 30 min). The dilution factors of Clostridium perfringens type A exotoxin were 2, 4, 8, 12, 16, 20 times; the dilution factors of Clostridium perfringens type C exotoxin were 40, 100, 200, 400, 600, 800 times. Sixteen to twenty grams of SPF mice were taken, and two mice were injected for each dilution. Clostridium perfringens type A exotoxin was intraperitoneally injected into mice at 0.1 ml / mouse, and Clostridium perfringens type C exotoxin was injected into mice via the tail vein at 0.1 ml / mouse. After injection, the mice were observed for 3 days, and the death situations of mice in each group were counted. The minimum lethal dose (MLD) titer of the exotoxin for mice was calculated based on the highest dilution factor at which all 2 mice died (for example, if all 2 mice died at the highest dilution of 10 times, then the toxin dose injected into the mice was exactly 1 MLD, and the toxin injected into the body was 1 ml / 10 times × 0.1 ml, that is, the actual injected toxin volume was 0.01 ml. That is, 0.01 ml of toxin contains 1 MLD titer, that is, the toxin titer is 100 MLD / ml).

[0073] The results showed that the concentration OD of the Clostridium perfringens bacterial liquid cultured in the self-made medium 4 600 value was about 1.5 times that of the meat liver gastric enzyme digestion broth recommended by the "Regulations for Veterinary Biological Products". The titer of the exotoxin of Clostridium perfringens type A cultured was 6 times that of the meat liver gastric enzyme digestion broth, and the titer of the exotoxin of Clostridium perfringens type C cultured was 5 times that of the meat liver gastric enzyme digestion broth. The toxin titer was significantly improved and could be further studied as a replacement medium.

[0074] Table 1 Comparison of the culture results of the self-made medium and meat liver gastric enzyme digestion broth for Clostridium perfringens

[0075]

[0076]

[0077] 2.5 Establishment of large-scale fermentation process for exotoxin

[0078] 2.5.1 Fermentation culture in a 10L bacterial fermenter. Connect the pH and dissolved oxygen electrodes to the 10L fermenter, fill it with an appropriate amount of PBS solution to submerge the electrode probes, and autoclave at 121°C for 30 min. Sterilely drain the PBS solution, and aseptically introduce the self-prepared medium 4 of Clostridium perfringens into the fermenter, with a volume of 7L (cultivation volume 60 - 80%). Connect the fermenter control system, and set the temperature at 37°C and the rotation speed at 60 r / min to operate the fermenter. When the temperature stabilizes at 37°C, add the seed liquid in 2.4.1 according to 2% of the medium volume, and add a sterile antifoaming agent according to 0.5‰ of the medium volume to start the cultivation. When the pH value drops to 7.2, start feeding and adding alkali to maintain the pH around 7.2. According to the previous screening results, tryptone can make the bacterial liquid concentration higher, while peptone and hydrolyzed milk protein can significantly improve the toxin titer. Combining the proportion of each component of the basal medium, the feeding medium is determined as follows: 25%, g / ml glucose solution, 20%, g / ml peptone solution, 20%, g / ml tryptone solution, 10%, g / ml yeast extract solution. After the four solutions are prepared, they are separately autoclaved at 116°C for 30 min, and after sterilization, they are mixed in a volume ratio of 2:2:3:3. The alkali solution is a sterilized 10% sodium hydroxide solution. Feeding method: fed-batch, with a speed of 3 - 5 ml / min. The total amount of the feeding medium used is 10% of the original cultivation volume. When the feeding medium is exhausted and the pH value no longer drops, harvest the bacterial liquid. The whole cultivation cycle is about 6 - 7 hours. The fermentation culture methods of Clostridium perfringens type A and C are the same.

[0079] The harvested bacterial liquid is respectively measured for OD 600 value and toxin titer according to the above method. The dilution multiples of Clostridium perfringens type A exotoxin are 20, 40, 50, 60, 70, 80 times; the dilution multiples of Clostridium perfringens type C exotoxin are 100, 200, 400, 600, 800, 1000 times. Take 16 - 20 g of SPF mice, and inject two mice for each dilution. Inject Clostridium perfringens type A exotoxin intraperitoneally into mice, 0.1 ml / mouse, and inject Clostridium perfringens type C exotoxin intravenously into mice, 0.1 ml / mouse. Observe the mice for 3 days after injection, count the death situations of each group of mice, and calculate the minimum lethal dose (MLD) titer of the exotoxin on mice with the highest dilution multiple that causes all 2 mice to die. The results show that the OD 600 values and toxin titers of the two bacterial liquids are significantly higher than those of static cultivation. The toxin titer of Clostridium perfringens type A is 600 MLD / ml, and the toxin titer of Clostridium perfringens type C is 6000 MLD / ml (Table 2).

[0080] Table 2 Results of cultivating Clostridium perfringens type A and C in a 10L bacterial fermenter with self-prepared medium No. 4

[0081]

[0082] 2.5.2 500L bacterial fermenter scale-up culture

[0083] According to the above-mentioned 10L fermenter culture process, scale up the culture volume by 50 times to 350L. The seed liquid is prepared by culturing in a 10L fermenter. The inoculation ratio is 2%, and fermentation culture is carried out at 37°C and a rotation speed of 60 r / min. The feeding medium and alkali supplementation are the same as above. Feeding method: fed-batch feeding, with a speed of 150 - 250 ml / min. The total amount of the feeding medium is 10% of the original culture volume. The whole culture cycle is about 6 - 7 hours. When the feeding medium is exhausted and the pH value no longer drops, the bacterial liquid is harvested.

[0084] The harvested bacterial liquid is respectively measured for OD 600 value and toxin titer according to the above method. The results show that the OD 600 values and toxin titers of the two bacterial liquids are slightly improved compared with those cultured in the 10L fermenter. The toxin titer of Clostridium perfringens type A is 700 MLD / ml, and the toxin titer of Clostridium perfringens type C is 8000 MLD / ml (Table 3).

[0085] Table 3 Results of culturing Clostridium perfringens type A and C in a 500L bacterial fermenter with self-made medium No. 4

[0086]

[0087] 2.6 Analysis of culture medium and culture process The traditional meat liver and gastric enzyme digestion broth medium is made of beef, beef liver, porcine pepsin, etc. The main components of the commonly used tryptone are similar to it. In this study, glucose is used as the carbon source, tryptone and yeast extract are used as the nitrogen source, tris(hydroxymethyl)aminomethane (Tris) provides buffering capacity, and sodium acetate inhibits the contamination of miscellaneous bacteria. On the basis of the components of this basic medium, by adding peptone, peptone, hydrolyzed milk protein, etc., the deficiency of the nutritional components of the basic medium is effectively made up. Through the improvement of the basic medium, the toxin-producing ability of the self-made medium No. 4 is significantly improved.

[0088] The traditional method for culturing Clostridium perfringens is static culture in a culture bottle, which is not conducive to monitoring the culture solution and feeding, etc. Since Clostridium perfringens is an anaerobic bacterium, when using a fermenter for culture in this study, stirring is carried out at a relatively low rotation speed as much as possible, and stirring can keep the overall temperature of the culture solution balanced. During the culture process, the bacteria consume glucose to produce acid, and the pH value of the bacterial solution is maintained at an appropriate level by adding alkali. The feeding medium ensures that the bacterial solution for fermentation has sufficient nutrients, further improving the concentration of the bacterial solution and the toxin titer. However, excessive feeding or too long culture time will also lead to an increase in osmotic pressure in the culture system and excessive accumulation of metabolites, etc., affecting the quality of the harvested toxin. According to the fermentation process established in this study, harvesting is carried out after 7 hours of culture. After centrifuging the bacterial solution, the toxin titer, purity, etc. in the supernatant are all at a good level. This process is suitable for short-term large-scale culture of qualified Clostridium perfringens type A and C toxins.

[0089] 2.7 Preparation of Clostridium perfringens type A and C bivalent toxoid vaccine

[0090] 2.7.1 Detoxification of toxin to prepare toxoid Take the bacterial solution obtained by fermentation in 2.5, centrifuge at 4°C and 8000 - 10000 r / min for 15 min, harvest the supernatant, add 0.6 - 1.0%, ml / ml formaldehyde solution to the collected toxin supernatant, place it at 37°C for 5 - 7 days for inactivation, and shake it once a day during the inactivation process. After inactivation, take the original solution and inject it into mice to observe whether the detoxification is complete. Inject Clostridium perfringens type A toxoid intraperitoneally into mice, 0.5 ml per mouse, and inject Clostridium perfringens type C toxoid into the tail vein of mice, 0.5 ml per mouse. Observe for 5 days, and the mice should not die. If there is death, continue to place it at 37°C for detoxification until the detoxification is complete.

[0091] 2.7.2 Vaccine preparation Take the two qualified detoxified toxoid antigens, and according to the toxin titer before detoxification, dilute them appropriately with sterile normal saline respectively, and mix them with sterile aluminum adjuvant to prepare the vaccine. Make the toxin titer of Clostridium perfringens type A in the vaccine be 50 MLD / ml, the toxin titer of Clostridium perfringens type C be 500 MLD / ml, and the final concentration of aluminum adjuvant be 15%. After mixing evenly, divide and store for later use.

[0092] 2.8 Vaccine inspection

[0093] 2.8.1 Conduct inspections on characters, sterility, formaldehyde residue, etc. according to the appendix of the Chinese Veterinary Pharmacopoeia (2020 edition), and all meet the regulations.

[0094] 2.8.2 Safety inspection Immunize 5 SPF mice weighing 18 - 22 g with the prepared vaccine, 0.5 ml per mouse, and observe for 14 days after immunization. All the mice are healthy and alive, with normal spirit and feeding, normal injection sites, and no hair loss or ulceration, etc.

[0095] 2.8.3 Potency test

[0096] 2.8.3.1 Experimental rabbits 20 rabbits weighing 1.5 - 2 kg were used for testing. Among them, 10 rabbits were immunized with a bivalent toxoid vaccine against Clostridium perfringens types A and C, 2 ml per rabbit, by subcutaneous injection. The remaining 10 rabbits were not immunized as controls. 21 days after immunization, 5 rabbits were randomly selected from both the immunized group and the control group and injected intravenously with a 2-fold lethal dose of Clostridium perfringens type A toxin; the remaining 5 rabbits in the immunized group and 5 rabbits in the control group were each injected intravenously with a 2-fold lethal dose of Clostridium perfringens type C toxin. Observation continued for 7 days. The results showed that within 48 hours after challenge, all 10 rabbits in the control group died. The experimental rabbits in the immunized group were all healthy and active, with normal mental state and food intake. This indicates that the experimental rabbits produced high levels of neutralizing antibodies against Clostridium perfringens types A and C toxins after immunization with the vaccine.

[0097] Table 4 Results of the potency test of the toxoid vaccine in immunized experimental rabbits

[0098]

[0099] 2.8.3.2 Potency test in pregnant sows 6 pregnant sows around 50 days before parturition were selected. Their sera were tested negative for Clostridium perfringens types A and C ELISA antibodies. Among them, 3 sows were immunized with a bivalent toxoid vaccine against Clostridium perfringens types A and C, 2 ml per sow, by intramuscular injection in the neck, and boosted once in the same way around 15 days before parturition. The remaining 3 sows were not immunized as controls. When the piglets born to the sows were around 7 days old, 10 piglets were randomly selected from both the immunized group and the control group, and their blood was collected to detect Clostridium perfringens types A and C ELISA antibodies respectively. At the same time, both the immunized group and the control group were randomly divided into 2 subgroups for challenge protection tests, with 5 pigs challenged with each of the type A and type C bacterial suspensions.

[0100] The results of the piglet antibody detection showed that the antibody levels of Clostridium perfringens types A and C in the immunized group were significantly higher than those in the control group, with a highly significant difference. ( Figure 1 )

[0101] After continuous observation for 14 days after challenge, all the piglets in the immunized group were healthy and active, with normal body temperature, and did not show symptoms such as decreased mental state, appetite, diarrhea, red dysentery, or death. When the piglets in the immunized group were dissected, the intestinal tissues were normal. All the piglets in the control group showed symptoms such as decreased mental state, food intake, emaciation, increased body temperature, diarrhea, and red dysentery. Among the piglets challenged with the type A bacterial suspension of Clostridium perfringens, 3 died within 7 days after challenge, and among the piglets challenged with the type C bacterial suspension of Clostridium perfringens, 4 died. When the non-dead pigs were dissected 14 days after challenge, the small intestinal tissues showed symptoms such as thinning, flatulence, and bleeding. The protection rates of the immunized group against challenges with Clostridium perfringens types A and C were both 5 / 5, while all 5 / 5 pigs in the control group developed the disease.

[0102] Table 5 Results of the challenge protection test of piglets born to pregnant sows immunized with the toxoid vaccine

[0103]

[0104] 2.8.3.3 Efficacy test in piglets Twenty piglets aged 7 - 14 days were selected, and their sera were tested negative for Clostridium perfringens type A and C ELISA antibodies. Ten of them were immunized with a bivalent toxoid vaccine against Clostridium perfringens type A and C, 2 ml per pig, by intramuscular injection. The remaining ten were not immunized as controls. Twenty - one days after immunization, the immunized group and the control group of pigs were tested for Clostridium perfringens type A and C ELISA antibodies. At the same time, the immunized group and the control group were each randomly divided into two subgroups for a challenge protection test, with 5 pigs challenged with type A and C bacterial solutions respectively.

[0105] The antibody test results showed that the antibody level in the immunized group was significantly higher 21 days after immunization than before immunization, and the difference was extremely significant compared with that before immunization; 21 days after immunization, the antibody level in the immunized group was significantly higher than that in the control group, and the difference between the two was extremely significant. ( Figure 2 )

[0106] The piglets in the immunized group were continuously observed for 14 days after challenge. All the piglets in the immunized group were healthy and alive, with normal body temperature, and did not show symptoms such as decreased mental state and appetite, diarrhea, red dysentery, or death. After autopsy of the piglets in the immunized group, the intestinal tissues were normal. The piglets in the control group all showed symptoms such as decreased mental state and food intake, emaciation, increased body temperature, diarrhea, and red dysentery. Two piglets died in the group challenged with Clostridium perfringens type A bacterial solution within 7 days after challenge, and three piglets died in the group challenged with Clostridium perfringens type C bacterial solution. The pigs that did not die were autopsied 14 days after challenge, and the small intestinal tissues showed symptoms such as thinning, flatulence, and bleeding. The protection rate of the immunized group against Clostridium perfringens type A and C challenges was 5 / 5, and the morbidity rate of the control group was 5 / 5.

[0107] Table 6 Results of the challenge protection test 21 days after immunizing piglets with the toxoid vaccine

[0108]

[0109] In summary, the culture media for Clostridium perfringens type A and C of the present invention enable Clostridium perfringens type A and C to adapt to fermentation in a fermenter. During the fermentation culture process, by controlling the stirring speed, the pH value of the fermentation broth, and the precise feeding method, the toxin - synthesizing ability of Clostridium perfringens type A and C is significantly improved. Using the supernatant obtained from its fermentation as the raw material for vaccine preparation can enable animals to produce high - level neutralizing antibodies against Clostridium perfringens type A and C toxins after immunization with the vaccine, avoiding the death of animals due to infection with Clostridium perfringens type A and C, and having practical application value for promotion.

Claims

1. A culture medium for type A and type C Clostridium perfringens, characterized in that: It is composed of component A and component B; The component A is composed of the following raw materials in parts by weight: 5-15 parts of tryptone, 5-15 parts of peptone, 5-15 parts of casein peptone, 5-15 parts of yeast extract, 3-8 parts of hydrolyzed milk protein, 2-6 parts of tris(hydroxymethyl)aminomethane, 1.5-3.5 parts of sodium acetate; The component B is 5 to 15 parts of glucose; The mass ratio of component A to component B is 26.5-77.5:5-15.

2. The culture medium for type A and type C Clostridium perfringens according to claim 1, characterized in that: The component A is composed of the following raw materials in parts by weight: Tryptone 10 parts, 10 parts of peptone, 10 parts of casein peptone, 10 parts of yeast extract, 5 parts of hydrolyzed milk protein, 4 parts of tris(hydroxymethyl)aminomethane, 2.5 parts of sodium acetate; The component B is 10 parts of glucose; The mass ratio of component A to component B is 51.5:

10.

3. Use of the culture medium for type A and type C Clostridium perfringens according to claim 1 or 2 in fermentation culture of type A and / or type C Clostridium perfringens.

4. A fermentation culture method for type A and type C Clostridium perfringens, characterized in that: The steps include: 1) taking activated A-type or C-type Clostridium perfringens, inoculating it into an anaerobic meat liver soup culture medium, and culturing it statically for 16 to 24 hours to obtain a seed solution; 2) taking the seed liquid and inoculating it into the liquid culture medium, fermenting it in a fermenter, and collecting the bacterial liquid; The liquid culture medium is prepared by dissolving and sterilizing the type A or type C Clostridium perfringens culture medium as described in claim 1 or 2 with water; The fermentation conditions of the fermentation tank are as follows: fermentation temperature of 35-38° C., liquid culture medium rotation speed of 50-100 r / min, seed liquid inoculation amount of 1-5%, defoamer addition amount of 0.1-1‰; when the pH value of the fermentation liquid drops to 7.0-7.4, supplementing the feed medium of 5-15% of the volume of the fermentation liquid, and maintaining the pH value of the fermentation liquid at 7-7.5 by supplementing alkali at the same time, and continuing to culture after supplementing until the pH value no longer decreases; The feed medium contains 4-6% glucose, 3-5% An aqueous solution of peptone, 5-7% tryptone, and 2-4% yeast extract.

5. The fermentation culture method according to claim 4, characterized in that: The temperature of the static culture in step 1) is 37°C; Step 2) the fermentation temperature is 37°C, the liquid culture medium speed is 60r / min, the seed liquid inoculation amount is 2%, and the defoaming agent addition amount is 0.5‰; when the pH value of the fermentation liquid drops to 7.2, 10% of the volume of the fermentation liquid is supplemented with a feed medium, and the pH value of the fermentation liquid is maintained at 7-7.5 by adding alkali; when the fermentation tank is a 10L fermentation tank, the feed medium is supplemented at a flow acceleration of 3-5ml / min, and when the fermentation tank is a 500L fermentation tank, the feed medium is supplemented at a flow acceleration of 150-250ml / min; The feed medium contains 5% glucose, 4% Peptone, 6% tryptone, 3% yeast extract in water.

6. The fermentation culture method according to claim 4, characterized in that: The preparation method of the liquid culture medium is: Take component A and add 18 to 19 times the amount of injection water to dissolve, sterilize, and obtain solution A; take component B and add 5 times the amount of injection water to dissolve, sterilize, and obtain solution B. Take solution A and solution B in a volume ratio of 19:1, mix, adjust the pH value to 8.2 to 8.4, and obtain.

7. Use of the bacterial solution obtained by the fermentation culture method according to any one of claims 4 to 6 in the preparation of Clostridium perfringens type A and / or type C toxoid vaccine.

8. A method for preparing a Clostridium perfringens type A and / or type C toxoid vaccine, characterized in that: The steps include: The Clostridium perfringens type A and / or type C bacterial solution obtained by the fermentation culture method according to any one of claims 4 to 6 is centrifuged, the supernatant is inactivated, and then an adjuvant is added and mixed to obtain the product.

9. The preparation method according to claim 8, characterized in that: The centrifugation temperature is 4°C, the speed is 8000-10000 r / min, and the time is 15 minutes; the inactivation is that the supernatant is added with formaldehyde and placed at 37°C until it is completely detoxified; the formaldehyde is added to a concentration of 0.6-1.0% in the solution; and the adjuvant is sterile aluminum gel.

10. The preparation method according to claim 8, characterized in that: When the vaccine is a Clostridium perfringens type A and C bivalent toxoid vaccine, the titer of Clostridium perfringens type A toxin is 50 MLD / ml, the titer of Clostridium perfringens type C toxin is 500 MLD / ml, and the final concentration of aluminum gel is 15%.

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