Heat-resistant protective agents for live swine erysipelas vaccine, their preparation methods and applications

Abstract

This invention discloses a heat-resistant protectant for a live swine erysipelas vaccine, its preparation method, and its application. The heat-resistant protectant comprises the following components in the indicated weight-volume percentages: maltodextrin: 8%–12%, gelatin: 3%–5%, L-arginine hydrochloride: 1%–2%, polyvinylpyrrolidone: 1%–3%, L-histidine hydrochloride: 1%–3%, enzymatically hydrolyzed casein: 6%–8%, D-sorbitol: 3%–5%, sodium thiosulfate: 1%–2%, tert-butanol: 3%–6%, polyethyleneimine: 1%–3%, with the remainder being water for injection. The heat-resistant protectant provided by this invention ensures the stable long-term storage of the live swine erysipelas vaccine and effectively inhibits the decrease in the titer of each live bacterium, thereby ensuring the immunogenicity of swine erysipelas. Simultaneously, it yields a live swine erysipelas vaccine product with a good freeze-dried appearance, low moisture content, and good solubility.

Description

Technical Field

[0001] This invention relates to the field of heat-resistant protective agents for live vaccines, and particularly to a heat-resistant protective agent for a live swine erysipelas vaccine, its preparation method, and its application. Background Technology

[0002] Swine erysipelas is a febrile, acute infectious disease caused by infection with *Erysipelothrix rhusiopathiae*. The bacterium was first isolated from infected pigs in 1882, and cases have since occurred in various countries and regions. Sichuan Province in my country was the first province to report swine erysipelas, followed by its spread to other provinces and cities. This disease is classified as a Class II animal disease in my country and is a zoonotic disease. Livestock such as pigs, sheep, dogs, cattle, and horses, as well as birds such as geese and ducks, can be infected. Human infection mainly occurs through wounds, primarily affecting farmers, veterinarians, and slaughterhouse workers. Piglets are the most susceptible animals, especially those aged 3-6 months. Carrier pigs and infected pigs are the main sources of infection. Bacteria survive in the bone marrow, ileocecal valve, and tonsils of infected pigs and are excreted through the mouth, nose, feces, and urine, contaminating pens, bedding, and equipment, thus spreading the disease. Healthy pigs will exhibit obvious abnormalities after contracting the disease, including lethargy, reluctance to stand, cessation of appetite, chills, and high fever reaching around 42°C. Forcing a sick pig to stand will result in lameness. Based on different clinical symptoms, swine erysipelas can be classified into acute septicemic, subacute, and chronic forms, with the acute septicemic form being the most severe. Acute septicemic erysipelas is common in the early stages of an outbreak, with septicemia as the primary clinical symptom. Affected pigs exhibit fever, lethargy, depression, and abortion in sows, and may even die acutely. The skin of affected pigs develops red or purple lesions, which may become necrotic and slough off as the disease progresses. Pigs that survive the acute form of swine erysipelas may develop the subacute or chronic form. Swine erysipelas is widespread in nature. Pigs with low immunity are most susceptible to infection. The subacute form can also cause septicemia, but the condition is milder than the acute form, lasting 1-2 weeks. Symptoms include lethargy, decreased or absent appetite, fever, constipation, and pink or dark brown rhomboid rashes on the skin. Initially, the rashes are congested, later becoming ecchymic. After the rashes form, body temperature decreases and symptoms lessen, usually healing spontaneously after a few days. Severe cases may persist for a long time, with skin necrosis forming leathery scabs. Typical symptoms include chronic arthritis, endocarditis, and skin necrosis. Pigs with chronic erysipelas become emaciated and grow slowly. Pigs with chronic arthritis become lame and unable to stand. Pigs with chronic endocarditis often die suddenly from cardiac arrest. In pigs with skin necrosis, the affected skin resembles leather and may slough off after 2-3 months, forming scar tissue. Different strains of *Erysipelothrix rhusiopathiae* exhibit significant differences in virulence, a phenomenon regulated by various virulence factors such as neuraminidase, capsular polysaccharides, and surface proteins. Studies have reported that neuraminidase is present only in pathogenic *Erysipelothrix rhusiopathiae*, and its secretion level is positively correlated with the pathogenicity of the strain. Different *Erysipelothrix rhusiopathiae* strains exhibit significant differences in virulence, a phenomenon regulated by various virulence factors such as neuraminidase, capsular polysaccharides, and surface proteins. *Erysipelothrix rhusiopathiae* enters the body through the digestive tract, or through damaged skin or insect bites. In infected pigs with weakened immune systems, bacteremia can develop within 24 hours, followed by septicemia. In the early stages of septicemia, capillaries and venules in most organs of the host are damaged.Fibrin thrombi and bacterial emboli can form within blood vessels, leading to the leakage of blood cells and activation of connective tissue in joints, heart, skin, and other areas. In severe cases, hemolysis or ischemic necrosis may occur.

[0003] Swine erysipelas often spreads to a specific region, exhibiting recurrent outbreaks and causing varying degrees of economic losses to pig farmers. It is characterized by strong sporadic and localized transmission. Controlling water sources, implementing effective feeding and management practices, and improving the cleanliness and hygiene of feed and the environment are all effective ways to prevent the disease. Currently, my country's prevention and control of swine erysipelas mainly relies on vaccines and antibiotics. However, due to antibiotic overuse leading to drug tolerance and drug residues affecting meat safety, the use of antibiotics for swine erysipelas control is becoming increasingly impractical. Vaccination is considered the most economical and effective means of controlling swine erysipelas, and the widespread application of commercial vaccines has played a crucial role in the effective control of swine erysipelas outbreaks in my country. To ensure the quality of vaccine antigens, especially to reduce losses caused by rapid temperature changes during production and transportation, the development of heat-resistant protection technology is key to ensuring stable vaccine quality. Using suitable heat-resistant protectants is a crucial step in ensuring the proper preservation of vaccines under refrigeration conditions. Vaccine heat-resistant protectants not only facilitate cold chain transportation and storage of vaccines but, more importantly, enhance vaccine efficacy.

[0004] Currently, the main heat-resistant protective agents used in my country for live swine erysipelas vaccine consist of gelatin, lactose, casein, and water. These heat-resistant protective agents are simple in composition and easy to prepare. However, as the temperature of the vaccine storage environment increases, the vaccine efficacy often decreases or becomes ineffective, leading to immunization failure. Therefore, proposing a new heat-resistant protective agent for live swine erysipelas vaccine to ensure the efficacy of the vaccine is a very important research topic. Summary of the Invention

[0005] This application proposes a heat-resistant protective agent for swine erysipelas live vaccine, its preparation method, and its application, which solves the problem of poor protective effect of heat-resistant protective agents for swine erysipelas live vaccine in the prior art.

[0006] The technical solution adopted by the invention is as follows: Firstly, the present invention proposes a heat-resistant protective agent for a live swine erysipelas vaccine, wherein the heat-resistant protective agent comprises the following components in weight-volume percentages:

[0007] Maltodextrin: 8%–12%;

[0008] Gelatin: 3%–5%;

[0009] L-arginine hydrochloride: 1%–2%;

[0010] Polyvinylpyrrolidone: 1%–3%;

[0011] L-histidine hydrochloride: 1%–3%;

[0012] Enzymatic hydrolysis of casein: 6%–8%;

[0013] D-sorbitol: 3%–5%;

[0014] Sodium thiosulfate: 1%–2%;

[0015] tert-Butanol: 3%–6%;

[0016] Polyethyleneimine: 1%–3%;

[0017] The remainder is: water for injection.

[0018] Preferably, in a preferred embodiment, the heat-resistant protective agent comprises the following components in weight-volume percentages:

[0019] Maltodextrin: 9%–10%;

[0020] Gelatin: 4%–5%;

[0021] L-arginine hydrochloride: 2%;

[0022] Polyvinylpyrrolidone: 2%–3%;

[0023] L-histidine hydrochloride: 1%–3%;

[0024] Enzymatic hydrolysis of casein: 6%–7%;

[0025] D-sorbitol: 5%;

[0026] Sodium thiosulfate: 1%–2%;

[0027] tert-Butanol: 5%–6%;

[0028] Polyethyleneimine: 1%–2%;

[0029] The remainder is: water for injection.

[0030] Secondly, the present invention also proposes a method for preparing a heat-resistant protective agent, which includes the following steps:

[0031] Step S1, Preparation of the first solution: Dissolve gelatin in water for injection to obtain the first solution. Add maltodextrin to the first solution until it is completely dissolved to obtain the second solution. Add polyethyleneimine to the second solution until it is completely dissolved to obtain the third solution. Then add enzymatically hydrolyzed casein to the third solution until it is completely dissolved to obtain the first solution.

[0032] Step S2, Preparation of the second solution: Dissolve L-histidine hydrochloride in water for injection to obtain the fourth solution. Add polyvinylpyrrolidone to the fourth solution until it is completely dissolved to obtain the fifth solution. Then add D-sorbitol, sodium thiosulfate, and tert-butanol to the fifth solution until they are completely dissolved to obtain the sixth solution. Subsequently, add L-arginine hydrochloride to the sixth solution until it is completely dissolved and adjust the pH to 7.2-7.4 to obtain the second solution.

[0033] Step S3: Mix the first solution and the second solution, adjust the volume, and sterilize to obtain the heat-resistant protective agent.

[0034] Thirdly, the present invention further proposes a live vaccine for swine erysipelas, which is formulated from swine erysipelas bacterial solution and the aforementioned heat-resistant protectant.

[0035] Fourthly, the present invention further proposes a method for preparing a live vaccine for swine erysipelas, comprising the following steps:

[0036] Step T1: Mix the swine erysipelas solution with the heat-resistant protective agent described in claim 1 or 2 to obtain the vaccine stock solution;

[0037] Step T2: Freeze-dry the vaccine stock solution to obtain the live swine erysipelas vaccine.

[0038] Preferably, the viable bacterial count of the swine erysipelas solution is 5×10⁹ CFU / ml to 8×10⁹ CFU / ml.

[0039] Preferably, the volume ratio of the swine erysipelas solution to the heat-resistant protective agent is in the range of 2:1 to 4:1.

[0040] Preferably, the volume ratio of the swine erysipelas solution to the heat-resistant protective agent is 3:1.

[0041] In conjunction with the fourth aspect, in some optional embodiments, the freeze-drying process in step T2 includes:

[0042] Step T2-1, Pre-freezing stage: First, cool the vaccine stock solution to -17°C within 20 min to 40 min, keep it at -17°C for 1 h, then cool it again to -40°C to -45°C within 20 min to 40 min, and keep it at -40°C to -45°C for 3 h to 5 h;

[0043] Step T2-2, Vacuuming stage: Reduce the vacuum level to 100mT~120mT within 10min~30min;

[0044] Step T2-3, Sublimation Drying Stage: First, raise the temperature to -22℃ within 2 hours and maintain it for 10 to 12 hours. Then, raise the temperature from -22℃ to -17℃ within 20 minutes and maintain it for 8 to 10 hours. Finally, raise the temperature from -17℃ to -5℃ within 20 to 30 minutes and maintain it for 3 to 5 hours.

[0045] Step T2-4, Drying stage: Adjust the vacuum to 10mT, raise the temperature to 28℃~30℃ in 1h~2h, and keep it at that temperature for 6h~8h.

[0046] Further, in step T2-1, the pre-freezing stage: the vaccine stock solution is cooled from room temperature to -17°C in 40 minutes, maintained at -17°C for 1 hour, then cooled to -45°C in 40 minutes, and maintained at -45°C for 4 hours; in step T2-2, the vacuuming stage: the vacuum level is reduced to 120 mT in 20 minutes; in step T2-3, the sublimation drying stage: heating begins when the vacuum level is reduced to 120 mT, the temperature is raised from -45°C to -22°C in 2 hours and maintained for 10 hours, then the temperature is raised from -22°C to -17°C in 20 minutes and maintained for 10 hours, and finally the temperature is raised from -17°C to -5°C in 20 minutes and maintained for 5 hours; in step T2-4, the desorption drying stage: the vacuum level is adjusted to 10 mT, the temperature is raised to 28°C in 2 hours, and held for 8 hours.

[0047] Compared with existing technologies, this application utilizes a heat-resistant protectant for the aforementioned live swine erysipelas vaccine. This heat-resistant protectant comprises appropriate amounts of maltodextrin, gelatin, L-arginine hydrochloride, polyvinylpyrrolidone, L-histidine hydrochloride, enzymatically hydrolyzed casein, D-sorbitol, sodium thiosulfate, tert-butanol, and polyethyleneimine. This protectant has a simple composition, readily available raw materials, and low cost. Experiments have shown that the live swine erysipelas vaccine prepared with this heat-resistant protectant retains its original physicochemical properties and physiological activity after freeze-drying, with minimal loss of the effective components of each viral antigen. After freeze-drying, the vaccine is porous and easily detaches from the vial wall, with a water content below 3.0%, allowing for rapid dissolution in water. Therefore, the resulting vaccine product exhibits a superior freeze-dried appearance, lower moisture content, and excellent solubility. Therefore, the heat-resistant protectant provided by this invention can effectively protect the live bacteria count of the swine erysipelas live vaccine used therein from degradation during freeze-drying, thereby ensuring the vaccine's immunogenicity and obtaining a swine erysipelas live vaccine product with good freeze-dried appearance, low moisture content, and good solubility properties.

[0048] Furthermore, the live erysipelas vaccine prepared with this heat-resistant protectant showed a decrease in the titer of each live bacteria number of no more than 1*10⁹ CFU / ml after being stored at 37°C for 7 days. The same applies to the vaccine after being stored at 2–8°C for 24 months. Therefore, the heat-resistant protectant provided by this invention can ensure that the live erysipelas vaccine can be stably stored for a long period of time and effectively inhibit the decrease in the titer of each live bacteria number, thereby ensuring the immunizing efficacy against erysipelas. Attached Figure Description

[0049] To more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0050] Figure 1 Photograph of the swine erysipelas live vaccine formulated with the protective agent in this invention after being repackaged and freeze-dried. Detailed Implementation

[0051] To make the technical problems to be solved, the technical solutions, and the beneficial effects of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present invention and are not intended to limit the present invention.

[0052] Unless otherwise specified, the methods used in the following embodiments are conventional methods. The methods for obtaining various biological materials described in the embodiments are merely to provide experimental methods for specific disclosure purposes and should not be construed as limiting the sources of biological materials for this invention. In fact, the sources of biological materials used are wide-ranging, and any biological material that can be obtained without violating laws and ethical standards can be substituted and used according to the suggestions in the embodiments.

[0053] The embodiments are implemented based on the technical solution of the present invention, and provide detailed implementation methods and specific operation processes. The embodiments will help to understand the present invention, but should not be regarded as limiting the content of the present invention.

[0054] This application discloses a heat-resistant protectant for a live swine erysipelas vaccine to ensure its long-term stable storage and maintain good immunogenicity and safety. Based on this heat-resistant protectant, a live swine erysipelas vaccine with good immunogenicity, high safety, and long-term stable storage is also provided.

[0055] The following specific examples illustrate the use of chemical substances including gelatin, maltodextrin, L-arginine hydrochloride, polyvinylpyrrolidone, L-histidine hydrochloride, enzymatically hydrolyzed casein, D-sorbitol, sodium thiosulfate, tert-butanol, and polyethyleneimine. Specifically, gelatin was purchased from Sigma-Aldrich; L-histidine hydrochloride from Sinopharm; maltodextrin from Roquette; enzymatically hydrolyzed casein from Sigma-Aldrich; polyvinylpyrrolidone from Wokai Chemical Co., Ltd.; tert-butanol from Tianjin Damao Chemical Reagent Factory; L-arginine hydrochloride from Sinopharm; polyethyleneimine from Yuanye Biotechnology Co., Ltd.; D-sorbitol from Sinopharm; and sodium thiosulfate from Tianjin Kemeng Chemical Co., Ltd. The instruments or bacterial strains used in the following examples include: an autoclave with a pressure range of 88 kPa to 114 kPa; and the bacterial strain G4T. 10 The attenuated strain is identified, stored, and supplied by the China Institute of Veterinary Drug Control.

[0056] Sample preparation: Serial dilution of bacterial suspension (10-fold): The dilution range is 10⁻¹ to 10⁻⁶. The procedure is as follows: Before dilution, vortex the bacterial suspension for 10 seconds. Then, take 1 ml of the bacterial suspension and add it to 9 ml of Martin agar medium containing 10% healthy animal serum. Vortex to mix for 10 seconds to complete the first dilution (10⁻¹). Repeat this process five or more times until the dilution reaches 10⁻⁶. When releasing the liquid, hold the pipette at a 30-degree angle to the inner wall of the test tube, allowing the liquid to fall naturally without the pipette touching the liquid surface. Replace the pipette each time liquid is transferred.

[0057] Solid culture medium culture: Select three suitable consecutive dilutions (generally 10⁻⁴, 10⁻⁵, and 10⁻⁶). Use a 1ml pipette to take 1.0ml of the diluted bacterial solution for each dilution, and add 0.1ml to each of three Martin agar plates containing 10% healthy animal serum. Tilt and rotate the plate to spread the bacterial solution, and air-dry the plates in a clean bench with the lids partially open for 15-30 minutes. Then, invert the plates and incubate them in a 37°C, 5% CO₂ incubator for 24-48 hours (the incubation time can be extended as needed).

[0058] Colony counting: Colony morphology should be observed as small, round, opaque, grayish-white colonies to the naked eye. Under a low-power microscope, they should appear pale yellow, with a visible nucleus-like structure in the center and short, hair-like branches growing outwards from the periphery. Count the colonies. Calculate the swine erysipelas bacterial solution content using the following formula:

[0059] Colony units per milliliter of bacterial culture (CFU / ml) = Average number of colonies in three replicates at the same dilution × 10 × dilution × M.

[0060] The live bacteria titer of the freeze-dried vaccine was determined by the agar plate counting method.

[0061] The freeze-dried vaccine was first diluted to 1 ml / dose using Martin agar medium containing 10% healthy animal serum, then diluted M times (M = 2-9). The diluted vaccine was vortexed for 10 seconds to disperse the swine erysipelas bacteria, and then further serially diluted 10-fold. Vortexing was performed again before each dilution. The remaining procedures followed the bacterial assay method described above.

[0062] In a specific embodiment:

[0063] Step 1: Prepare heat-resistant protective agent for swine erysipelas live vaccine.

[0064] This embodiment aims to prepare a heat-resistant protectant for a live swine erysipelas vaccine. The function of this heat-resistant protectant is to protect the active ingredients of the vaccine during the freeze-drying process and post-freeze-drying storage to ensure the vaccine's immunizing effect. The preparation method of this heat-resistant protectant specifically includes the following steps:

[0065] Step S1: Preparation of the first solution: According to the composition content of the heat-resistant protective agent shown in Table 1 below (where % represents the mass-volume percentage content, i.e., g / 100ml, the same below), accurately weigh the required amount of gelatin into quantitative injection water, heat in a 60℃ water bath and stir continuously until completely dissolved to obtain the first solution; then weigh maltodextrin and add it to the first solution until completely dissolved to obtain the second solution; then add the required amount of polyethyleneimine to the second solution until completely dissolved to obtain the third solution; subsequently, accurately weigh enzymatically hydrolyzed casein and add it to the third solution, heat in a 60℃ water bath and stir continuously to dissolve it to obtain the first solution.

[0066] Step S2, Preparation of the second solution: According to the component content of each heat-resistant protective agent shown in Table 1 below, accurately weigh L-histidine hydrochloride and dissolve it in water for injection. Place it in a 60°C water bath and shake continuously until it is completely dissolved to obtain the fourth solution. Then accurately weigh the required amount of polyvinylpyrrolidone and dissolve it completely in the fourth solution to obtain the fifth solution. Subsequently, accurately weigh L-arginine hydrochloride, sodium thiosulfate, tert-butanol, and D-sorbitol and add them to the fifth solution. Place it in a 60°C water bath and shake continuously to dissolve it. Adjust the pH to 7.2-7.4 using 7.5% sodium bicarbonate solution to obtain the second solution.

[0067] Step S3: After bringing the first solution to a fixed volume, sterilize it in an autoclave (116℃, 20-30 min). After bringing the second solution to a fixed volume, filter it for sterilization (0.22μm filter membrane). Mix the first and second solutions in a 1:1 ratio to obtain a heat-resistant protective agent.

[0068] As shown in Table 1 below, five heat-resistant protective agents were prepared in this embodiment and named heat-resistant protective agent 1, heat-resistant protective agent 2, heat-resistant protective agent 3, heat-resistant protective agent 4 and heat-resistant protective agent 5 respectively. The prepared heat-resistant protective agents were stored at 37°C for later use.

[0069] It should be noted that when preparing heat-resistant protective agents, each reagent should be dissolved individually, and the amount of solute should be gradually increased during the individual dissolution process to avoid supersaturation and incomplete dissolution.

[0070] Table 1: Composition and content of heat-resistant protective agent in live swine erysipelas vaccine

[0071]

[0072]

[0073] Step 2: Prepare live vaccine for swine erysipelas

[0074] In the second step, the heat-resistant protective agents prepared in the first step, namely protectant 1, protectant 2, protectant 3, protectant 4, and protectant 5, are used as heat-resistant protective agents for the swine erysipelas live vaccine. The specific method for preparing the swine erysipelas live vaccine includes the following steps:

[0075] Step T1: Mix the swine erysipelas bacterial solution with the five heat-resistant protective agents mentioned above. Specifically, add the vaccine bacterial solution evenly to the storage bottle containing the heat-resistant protective agents and mix slowly by hand for about 15 minutes (to avoid generating too much foam). This will give you the vaccine stock solution corresponding to the five heat-resistant protective agents.

[0076] Step T2: Freeze-dry each of the vaccine stock solutions to obtain the porcine erysipelas live vaccine.

[0077] More specifically, the specific operation method for step T1 above is as follows:

[0078] Step T1-1: Preparation of Swine Erysipelas Fluid

[0079] Step T1-1-1, Primary Seed Propagation and Identification: After opening the freeze-dried spawn, dilute with Martin broth, inoculate onto gelatin plates, and incubate at 15-18℃ for 3-5 days. The colony morphology should be observed as small, opaque, grayish-white, round colonies to the naked eye. Under a low-power microscope, they should appear pale yellow with a visible nucleus-like structure in the center and short, hair-like branches growing outwards from the periphery; G4T 10Take several colonies, mix them with a small amount of meat, liver, and gastric membrane digestion broth, and then streak them onto Martin agar plates. Incubate at 36–37°C for 24–36 hours. The colonies should be round and smooth. Under a low-power microscope, they should have tiny ripples around the edges and yellowish-brown dot-like particles in the center. Select several medium-sized smooth colonies, mix them with a small amount of meat, liver, and gastric membrane digestion broth, and then inoculate them onto several dried blood agar slants. Incubate at 36–37°C for 24 hours. The pure colonies tested are used as first-grade seed culture. Store at 2–8°C. The shelf life should not exceed one month (Ministry of Agriculture of the People's Republic of China. Regulations for Veterinary Biological Products of the People's Republic of China, 2000 Edition. Chemical Industry Press, 2001, hereinafter referred to as the "Regulations").

[0080] Step T1-1-2, Secondary Seed Propagation: Inoculate primary seeds into a meat, liver, and gastric membrane digestion broth, and incubate at 36–37°C for 20–22 hours. After testing for purity, take samples and store at 2–8°C. The shelf life should not exceed 3 days. The strain used for producing swine erysipelas should not exceed 5 generations.

[0081] Step T1-1-3: The culture medium for seedling preparation is meat liver gastric membrane digestion soup, and the pH value is adjusted to 7.6±0.2.

[0082] Step T1-1-4: Preparation of bacterial suspension for swine erysipelas vaccine production. Inoculate 1%–2% of the pure, qualified secondary seed culture into the digested broth containing dried liver and gastric membrane at the culture medium volume. Simultaneously add 2%–4% lysed hemoglobin and whole blood. Incubate at 36–37°C for 20–22 hours, shaking 2–3 times during the incubation period, or use enrichment culture methods. The bacterial suspension can be concentrated using high-speed centrifugation to prepare a bacterial suspension for vaccine preparation. It should be stored at 2–8°C for no more than 15 days.

[0083] Step T1-1-5, semi-finished product inspection, including purity testing and viable bacteria counting, shall be carried out in accordance with the methods specified in the Chinese Veterinary Pharmacopoeia.

[0084] Step T1-2, viable bacteria titer determination. The results of the viable bacteria determination are shown in Table 2 below:

[0085] Table 2: Results of viable bacteria assay

[0086]

[0087] Steps T1-3: Each dose of freeze-dried swine erysipelas live vaccine must contain ≥5 × 10⁻⁶ live swine erysipelas bacteria. 8 The required dosage of swine erysipelas solution for vaccine preparation was calculated based on the CFU / ml concentration. The solution was then mixed according to the calculated dosage (this invention incorporates freeze-drying losses from previous experiments to ensure vaccine quality). The calculated viable count of the swine erysipelas solution required in this application is 5 × 10⁻⁶ CFU / ml. 9 CFU / ml ~8×109 CFU / ml.

[0088] Step T1-4: Take the vaccine bacterial solution obtained in step T1-1 above and mix it with the five heat-resistant protective agents (protective agent 1, protective agent 2, protective agent 3, protective agent 4 and protective agent 5) obtained in the first step at volume ratios of 2:1, 3:1 and 4:1. Specifically, add the vaccine bacterial solution to the storage bottle containing the heat-resistant protective agent at a uniform speed and mix slowly by hand for about 15 minutes (to avoid generating too much foam) to obtain the vaccine stock solution.

[0089] The specific operation method for step T2 above is as follows:

[0090] The vaccine stock solution obtained in step T1-4 is dispensed into 3ml (30 doses) vials using a pipette, i.e., dispensed into 3ml vials. Then, it is freeze-dried to prepare the swine erysipelas live vaccine (freeze-dried preparation). The freeze-drying process is to select a slow freezing rate by cooling the freeze-drying chamber after the product is placed in the chamber. The minimum pre-freezing temperature should be selected based on the eutectic point temperature of the mixed vaccine, which is -17℃, and the minimum pre-freezing temperature should be lower than this temperature.

[0091] The freeze-drying curve is based on the freeze-drying of 3 ml of vaccine stock solution as an example. The specific freeze-drying process includes:

[0092] Step T2-1, Pre-freezing stage: First, the vaccine stock solution is cooled from room temperature to -17°C within 20 min to 40 min, and kept at -17°C for 1 hour. Then, it is cooled again to -40°C to -45°C within 20 min to 40 min, and kept at -40°C to -45°C for 3 hours to 5 hours. After this step, the sample is completely frozen and then proceeds to step T2-2.

[0093] Step T2-2, Vacuuming Stage: Immediately after pre-freezing the vaccine stock solution, the vacuuming and depressurization stage begins. Within 10 to 30 minutes, the vacuum level is reduced to 100 to 120 mT until the end.

[0094] Step T2-3, Sublimation Drying Stage: After the vacuum level is reduced to a specific value between 100mT and 120mT, heating begins. First, the temperature is raised to -22℃ within 2 hours and maintained for 10 to 12 hours. Then, the temperature is raised from -22℃ to -17℃ within 20 minutes and maintained for 8 to 10 hours. Finally, the temperature is raised from -17℃ to -5℃ within 20 to 30 minutes and maintained for 3 to 5 hours. After that, the process transitions to the desorption drying stage, causing the frozen product in the bottle to gradually sublimate from top to bottom, ultimately forming the final product.

[0095] Step T2-4, Drying stage: Adjust the vacuum to 10mT, raise the temperature to 28℃~30℃ in 1h~2h, and keep it at that temperature for 6h~8h.

[0096] In one specific embodiment, the freeze-drying process is as follows:

[0097] Step T2-1, Pre-freezing stage: Cool the vaccine stock solution from room temperature to -17°C in 40 minutes, keep it at -17°C for 1 hour, then cool it to -45°C in 40 minutes, and keep it at -45°C for 4 hours;

[0098] Step T2-2, Vacuuming stage: Reduce the vacuum level to 120mT within 20 minutes;

[0099] Step T2-3, Sublimation Drying Stage: When the vacuum degree drops to 120mT, heating begins. Within 2 hours, the temperature is raised from -45℃ to -22℃ and maintained for 10 hours. Then, within 20 minutes, the temperature is raised from -22℃ to -17℃ and maintained for 10 hours. Finally, within 20 minutes, the temperature is raised from -17℃ to -5℃ and maintained for 5 hours.

[0100] Step T2-4, Drying stage: Adjust the vacuum to 10mT, raise the temperature to 28℃ in 2 hours, and keep it at that temperature for 8 hours.

[0101] It should be noted that the maximum allowable temperature of the freeze-drying chamber plates is determined to be 30°C based on the product. Due to the difference in heat transfer, the plate temperature is often slightly lower than the maximum allowable temperature of the product; in this embodiment, it is set to 28°C. Based on the performance of the freeze dryer, the characteristics of the vaccine, and the dosage, the entire process of pre-freezing time, sublimation drying stage, and desorption drying time is determined to be approximately 44 hours.

[0102] Step 3: The number of live bacteria in each of the prepared swine erysipelas live vaccines before and after freeze-drying was tested. The test results are shown in Table 3 below.

[0103] Table 3: Results of viable bacterial count detection before and after freeze-drying of live swine erysipelas vaccine

[0104]

[0105] Step 4: The vaccine product prepared in this example was subjected to routine tests according to the methods described in the "Regulations for Veterinary Biological Products of the People's Republic of China" document, as shown in Table 4 below.

[0106] Table 4: Routine Test Results of Porcine Erysipelas Live Vaccine Finished Product

[0107]

[0108]

[0109] As shown in Tables 3 and 4 above, the live swine erysipelas vaccine prepared using heat protectant 4 and heat protectant 5 has a high viability rate and meets the appearance requirements before and after freeze-drying. The number (titer) of each live bacterium decreases less. Therefore, heat protectant 4 and heat protectant 5 can effectively protect the number of live bacteria in the live swine erysipelas vaccine from degradation during the freeze-drying process, resulting in a vaccine product with a high potency.

[0110] Compared to heat protectants 4 and 5, heat protectant 2 does not contain polyethyleneimine. The titer of viable bacteria in the swine erysipelas live vaccine decreased significantly before and after freeze-drying. This is because polyethyleneimine is a polymer, and the stabilizing effect of polymers typically depends on multiple properties, such as precipitation from macromolecular surfaces, surface activity, increasing the viscosity of macromolecular solutions to prevent the crystallization of other small molecules (such as sugars and polyhydroxy compounds), and inhibiting drastic pH changes during freezing. Heat protectant 1 does not contain L-histidine hydrochloride. The titer of viable bacteria in the swine erysipelas live vaccine also decreased significantly before and after freeze-drying. L-histidine hydrochloride can inhibit the oxidation and aggregation of macromolecules in protectants. Histidine, a classic and excellent protein stabilizer, effectively prevents adsorption on protein surfaces and is beneficial for freeze-drying. Most proteins in the process have a protective effect; the heat protectant 3 does not contain tert-butanol. Tert-butanol, as a freeze-drying protectant, has multiple advantages and has been widely used in freeze-dried preparations. It can be used alone as a solvent to dissolve insoluble drugs or drugs with poor stability in water; sodium thiosulfate is an antioxidant that prevents its own oxidation and consumes oxygen inside the freeze-dried sample and in the environment, so that the freeze-dried sample material is not oxidized; another way is to allow the antioxidant to donate electrons or hydrogen ions, blocking the oxidation chain reaction in the freeze-dried sample; yet another way is that the antioxidant prevents the freeze-dried sample from oxidizing and deteriorating by inhibiting the activity of oxidation. It can be seen that the appropriate proportion of each component in the heat protectant provided by this invention has a certain impact on the live swine erysipelas vaccine. According to the different functions of the components in the protectant, the number of live bacteria in the live swine erysipelas vaccine is protected from degradation, and a vaccine product with a high potency is obtained.

[0111] According to the test results in Table 4, it can be seen that the live swine erysipelas vaccine obtained using heat-resistant protectant 4 and heat-resistant protectant 5 appears as a light yellow or light pink spongy, loose mass after freeze-drying, and it is easily detached from the vial wall. Figure 1 The image shows a photograph of the freeze-dried swine erysipelas vaccine obtained after dispensing (4 vials) and freeze-drying using heat protectant 4. The water content of all vials is below 3.0%, allowing for rapid dissolution in water. Therefore, the resulting vaccine product exhibits a superior freeze-dried appearance, lower moisture content, and better solubility. In summary, heat protectants 4 and 5 provide better heat protection, resulting in less loss of viable cell rate before and after freeze-drying, thus ensuring a higher potency of the swine erysipelas vaccine.

[0112] Step 5: In this embodiment, the porcine erysipelas live vaccine prepared with protectant 4 as the heat-resistant protectant of the vaccine was subjected to an aging resistance test according to the method described in the "Regulations for Veterinary Biological Products of the People's Republic of China". Specifically, the porcine erysipelas live vaccine was placed at 37°C for 7 days and stored at 2-8°C for 24 months. The changes in the number of live bacteria in the vaccine product were detected. The test results are shown in Table 5 below.

[0113] Table 5: Results of viable bacteria count in swine erysipelas during aging resistance test (CFU / ml)

[0114]

[0115] As shown in Table 5 above, the swine erysipelas live vaccine prepared using heat protectant 4, regardless of whether the bacterial solution to heat protectant ratio was 2:1, 3:1, or 4:1, showed no decrease in viable bacterial titers after 7 days of storage at 37°C, with the largest decrease being only 0.49*109 CFU / ml. Similarly, after 24 months of storage at 2–8°C, the decrease in viable bacterial titers also did not exceed 1*109 CFU / ml, with the largest decrease being only 0.39*109 CFU / ml. When heat protectant 5 was used to prepare the swine erysipelas live vaccine, it also exhibited good heat protection, similar to heat protectant 4.

[0116] Therefore, the heat-resistant protectant provided by this invention can ensure that the swine erysipelas live vaccine can be stably preserved for a long time and effectively inhibit the decrease in the titer of each live bacteria, thereby ensuring the immune efficacy of swine erysipelas.

[0117] Step 6: Preparation of heat-resistant protective agent for swine erysipelas live vaccine

[0118] Following steps S1 to S3, heat-resistant protective agents for swine erysipelas live vaccine were prepared according to the composition and content of the heat-resistant protective agents listed in Table 6 below, and named Protectant 6, Protectant 7, and Protectant 8, respectively. Swine erysipelas live vaccine was prepared using Protectant 6, Protectant 7, and Protectant 8 according to the same method as in step two, with a bacterial solution:protectant ratio of 3:1.

[0119] Table 6: Composition and content of heat-resistant protectant in live swine erysipelas vaccine

[0120]

[0121] Subsequently, following the same methods as in steps three and four, the decrease in viable bacterial count titers before and after freeze-drying of the porcine erysipelas live vaccine prepared using protectants 6, 7, and 8, as well as routine tests of the finished product, were performed. The test results are shown in Table 7 below.

[0122] Table 7: Decrease in viable bacterial count titer after freeze-drying of vaccines and results of routine tests on finished products

[0123]

[0124]

[0125] As shown in Tables 6 and 7 above, the live swine erysipelas vaccine prepared using protectant 6 and protectant 8 showed a smaller decrease in the viable bacterial count (titer) before and after freeze-drying, with the highest viable bacterial count reaching 91.3%. Therefore, protectant 6 and protectant 8 can effectively protect the viable bacterial count in the live swine erysipelas vaccine from degradation during the freeze-drying process, resulting in a vaccine product with a high potency.

[0126] According to the test results in Table 7, the porcine erysipelas live vaccine obtained using protectants 6-8 appears as a light yellow or light pink spongy, loose mass after freeze-drying, easily detaching from the vial wall. The water content is all below 3.0%, allowing for rapid dissolution in water. Therefore, the prepared vaccine product exhibits excellent freeze-dried appearance, low moisture content, and good solubility. Thus, the heat-resistant protectant provided by this invention can effectively protect the porcine erysipelas live vaccine from degradation during freeze-drying, thereby ensuring vaccine efficacy and yielding a porcine erysipelas live vaccine product with good freeze-dried appearance, low moisture content, and good solubility.

[0127] Step 7: Optimization of the freeze-drying curve for preparing live swine erysipelas vaccine

[0128] In this step, the protective agent 5 obtained in step 1 is used as a heat-resistant protective agent. The obtained vaccine bacterial solution and the protective agent 5 are mixed evenly at a volume ratio of 3:1 according to the same method as in step 2 to obtain the vaccine stock solution. Subsequently, the vaccine stock solution is dispensed into 3ml / bottles and then freeze-dried. The inventors designed a variety of freeze-drying curves to screen and determine the ideal freeze-drying curve suitable for swine erysipelas live vaccine. The following are three freeze-drying curves: freeze-drying curve 1, freeze-drying curve 2 and freeze-drying curve 3.

[0129] Freeze-drying curve 1: The same freeze-drying curve used in the second step to prepare the live swine erysipelas vaccine; the live swine erysipelas vaccine obtained based on this freeze-drying curve 1 is named live swine erysipelas vaccine 1;

[0130] Freeze-drying curve 2 (as a control freeze-drying curve): The freeze-drying curve is calculated based on 3 ml of freeze-dried vaccine stock solution:

[0131] Pre-freezing stage: The vaccine stock solution is cooled from room temperature to -45°C in 1 hour, kept at -45°C for 1 hour, then cooled to -45°C in 40 minutes, and kept at -45°C for 4 hours;

[0132] Vacuuming phase: Reduce the vacuum level to 120mT within 25 minutes;

[0133] Sublimation drying stage: heating begins when the vacuum level drops to 120mT. The temperature is raised from -45℃ to -20℃ within 2 hours and maintained for 10 hours. Then, the temperature is raised from -20℃ to -10℃ within 20 minutes and maintained for 10 hours. Finally, the temperature is raised from -10℃ to -3℃ within 20 minutes and maintained for 4 hours.

[0134] Analysis and drying stage: The temperature is raised to 28℃ in 2 hours and kept at that temperature for 8 hours.

[0135] The live erysipelas vaccine obtained based on this freeze-drying curve 2 is named Live Erysipelas Vaccine 2.

[0136] Freeze-drying curve 3 (as a control freeze-drying curve): The freeze-drying curve is calculated based on 3 ml of freeze-dried vaccine stock solution:

[0137] Pre-freezing stage: The vaccine stock solution is cooled from room temperature to -25°C in 30 minutes, kept at -25°C for 1 hour, and then cooled to -45°C in 30 minutes, kept at -45°C for 4 hours;

[0138] Vacuuming phase: Reduce the vacuum level to 100 to 120 mT within 20 minutes;

[0139] Sublimation drying stage: The temperature is raised from -45℃ to -30℃ within 2 hours and maintained for 5 to 8 hours. Then, the temperature is raised from -30℃ to -10℃ within 40 minutes and maintained for 10 hours. Finally, the temperature is raised from -10℃ to 2℃ within 20 minutes and maintained for 8 hours.

[0140] Analysis and drying stage: The temperature is raised to 28℃ in 2 hours and kept at that temperature for 8 hours.

[0141] The live erysipelas vaccine obtained based on the freeze-drying curve 3 is named Live Erysipelas Vaccine 3.

[0142] Following the same method as in step two, the decrease in the number of live bacteria before and after freeze-drying of swine erysipelas live vaccine 1, swine erysipelas live vaccine 2, and swine erysipelas live vaccine 3, as well as routine items of the finished vaccine products, were tested. The test results are shown in Table 8 below.

[0143] Table 8: Decrease in viable cell rate after freeze-drying of vaccines and results of routine tests on finished products

[0144]

[0145]

[0146] According to the test results in Table 8 above, the viable bacterial rate of swine erysipelas in live swine erysipelas vaccine 1 was 91.4%, and the physical properties, solubility, and residual moisture of the vaccine after freeze-drying were all good. The viable bacterial rate of swine erysipelas in live swine erysipelas vaccine 2 was 82.8%, and the physical properties after freeze-drying were slightly worse, with slight melting at the bottom and shrinkage. The viable bacterial rate of swine erysipelas in live swine erysipelas vaccine 3 was 68.4%, and after freeze-drying, it exhibited stratification, collapse, and difficulty in detaching from the vial wall. For the same bacterial solution in the live swine erysipelas vaccines, the viable bacterial rate decrease was relatively greater in live swine erysipelas vaccine 3 compared to live swine erysipelas vaccine 1, while the decrease was less in live swine erysipelas vaccine 2. By determining the eutectic point of the samples, basic freeze-drying parameters were established, and different factors affecting the freeze-drying effect were adjusted to finally determine the freeze-drying curve. Different plate temperatures at different stages had different effects on freeze-drying efficiency. Therefore, the freeze-drying curve 1 used in this invention can not only reduce the loss of viable bacterial titers during the freeze-drying process of swine erysipelas live vaccine, but also improve product quality.

[0147] Step 8: Safety Trial of Live Vaccine for Swine Erysipelas

[0148] 1) Using the above-mentioned protectant 4 as a heat-resistant protectant, the live swine erysipelas vaccine was prepared according to the method in step 2 with a volume ratio of bacterial solution to protectant of 2:1, 3:1 and 4:1, and was named live swine erysipelas vaccine 3, live swine erysipelas vaccine 4 and live swine erysipelas vaccine 5, respectively.

[0149] 2) Using protectant 5 as a heat-resistant protectant, and following the method in step 2, prepare live swine erysipelas vaccines with a bacterial solution to protectant volume ratio of 2:1, 3:1 and 4:1, respectively, and name them live swine erysipelas vaccine 6, live swine erysipelas vaccine 7 and live swine erysipelas vaccine 8.

[0150] 3) Using protectant 6 as a heat-resistant protectant, and following the method in step 2, prepare live swine erysipelas vaccines with a bacterial solution to protectant volume ratio of 2:1, 3:1 and 4:1, respectively, and name them live swine erysipelas vaccine 9, live swine erysipelas vaccine 10 and live swine erysipelas vaccine 11.

[0151] 4) Using protectant 7 as a heat-resistant protectant, and following the method in step 2, prepare live swine erysipelas vaccines with a bacterial solution to protectant volume ratio of 2:1, 3:1 and 4:1, respectively, and name them live swine erysipelas vaccine 12, live swine erysipelas vaccine 13 and live swine erysipelas vaccine 14, as the immunization groups;

[0152] Sterile saline solution was used as the control group. Healthy, susceptible pigs weighing 20-25 kg and negative for swine erysipelas antibodies were selected as experimental animals. Each pig in the immunization group was injected subcutaneously with 20 doses of vaccine, while each pig in the control group was injected with saline solution using the same method and dosage. The pigs were observed for 14 days, and the results are shown in Table 9 below. All experimental pigs in each group showed no abnormal reactions, and their mental state and appetite were normal. No symptoms of swine erysipelas were observed. Specifically, 2 / 5 of the experimental pigs using swine erysipelas vaccine 3 and 7 developed a fever 6 hours after immunization, with a maximum temperature reaching 40.4℃; 1 / 5 of the experimental pigs using swine erysipelas vaccine 6 and swine erysipelas vaccine 12 developed a fever 6 hours after immunization, with a maximum temperature reaching 40.2℃. The fever was transient, but the body temperature of all experimental pigs returned to normal 12 hours after immunization. This indicates that the live swine erysipelas vaccine formulated in this invention has good safety.

[0153] Table 9: Safety test results of live swine erysipelas vaccine

[0154] vaccine Inoculation dosage (per dose) Weight(kg) Number of doses administered per group (heads) Test results swine erysipelas live vaccine 3 20 20-25 5 5 / 5 normal 4 live vaccine for swine erysipelas 20 20-25 5 5 / 5 normal 5 live vaccine for swine erysipelas 20 20-25 5 5 / 5 normal 6 live vaccine for swine erysipelas 20 20-25 5 5 / 5 normal swine erysipelas live vaccine 7 20 20-25 5 5 / 5 normal 8 live vaccine for swine erysipelas 20 20-25 5 5 / 5 normal 9 live vaccine for swine erysipelas 20 20-25 5 5 / 5 normal 10 live vaccine for swine erysipelas 20 20-25 5 5 / 5 normal Swine erysipelas live vaccine 11 20 20-25 5 5 / 5 normal swine erysipelas live vaccine 12 20 20-25 5 5 / 5 normal swine erysipelas live vaccine 13 20 20-25 5 5 / 5 normal swine erysipelas live vaccine 14 20 20-25 5 5 / 5 normal Sterile saline — 20-25 5 5 / 5 normal

[0155] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

[0156] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A heat-resistant protective agent for a live swine erysipelas vaccine, characterized in that, The heat-resistant protective agent comprises the following components in weight-volume percentages: Maltodextrin: 8%–12%; Gelatin: 3%–5%; L-arginine hydrochloride: 1%–2%; Polyvinylpyrrolidone: 1%–3%; L-histidine hydrochloride: 1%–3%; Enzymatic hydrolysis of casein: 6%–8%; D-sorbitol: 3%–5%; Sodium thiosulfate: 1%–2%; tert-Butanol: 3%–6%; Polyethyleneimine: 1%–3%; The remainder is: water for injection.

2. The heat-resistant protective agent according to claim 1, characterized in that, The heat-resistant protective agent comprises the following components in weight-volume percentages: Maltodextrin: 9%–10%; Gelatin: 4%–5%; L-arginine hydrochloride: 2%; Polyvinylpyrrolidone: 2%–3%; L-histidine hydrochloride: 1%–3%; Enzymatic hydrolysis of casein: 6%–7%; D-sorbitol: 5%; Sodium thiosulfate: 1%–2%; tert-Butanol: 5%–6%; Polyethyleneimine: 1%–2%; The remainder is: water for injection.

3. A method for preparing a heat-resistant protective agent as described in claim 1 or 2, characterized in that, Includes the following steps: Step S1, Preparation of the first solution: Dissolve gelatin in water for injection to obtain the first solution. Add maltodextrin to the first solution until it is completely dissolved to obtain the second solution. Then add polyethyleneimine to the second solution until it is completely dissolved to obtain the third solution. Subsequently, add enzymatically hydrolyzed casein to the third solution until it is completely dissolved to obtain the first solution. Step S2, Preparation of the second solution: Dissolve L-histidine hydrochloride in water for injection to obtain the fourth solution. Add polyvinylpyrrolidone to the fourth solution until it is completely dissolved to obtain the fifth solution. Then add D-sorbitol, sodium thiosulfate, and tert-butanol to the fifth solution until they are completely dissolved to obtain the sixth solution. Subsequently, add L-arginine hydrochloride to the sixth solution until it is completely dissolved and adjust the pH to 7.2-7.4 to obtain the second solution. Step S3: Mix the first solution and the second solution, adjust the volume, and sterilize to obtain the heat-resistant protective agent.

4. A live vaccine for swine erysipelas, characterized in that, The live swine erysipelas vaccine is formulated from swine erysipelas bacterial solution and the heat-resistant protectant as described in claim 1 or 2.

5. A method for preparing a live vaccine for swine erysipelas as described in claim 4, characterized in that, Including the following steps: Step T1: Mix the swine erysipelas solution with the heat-resistant protective agent described in claim 1 or 2 to obtain the vaccine stock solution; Step T2: Freeze-dry the vaccine stock solution to obtain the live swine erysipelas vaccine.

6. The method for preparing the live swine erysipelas vaccine according to claim 5, characterized in that, The viable count of the swine erysipelas solution was 5 × 10⁻⁶. 9 CFU / ml ~8×10 9 CFU / ml.

7. The method for preparing the live swine erysipelas vaccine according to claim 5, characterized in that, The volume ratio of the swine erysipelas solution to the heat-resistant protective agent ranges from 2:1 to 4:

1.

8. The method for preparing the live swine erysipelas vaccine according to claim 7, characterized in that, The volume ratio of the swine erysipelas solution to the heat-resistant protective agent is 3:

1.

9. The method for preparing the live swine erysipelas vaccine according to claim 5, characterized in that, The freeze-drying process in step T2 includes: Step T2-1, Pre-freezing stage: First, cool the vaccine stock solution to -17°C within 20 min to 40 min, keep it at -17°C for 1 h, then cool it again to -40°C to -45°C within 20 min to 40 min, and keep it at -40°C to -45°C for 3 h to 5 h; Step T2-2, Vacuuming stage: Reduce the vacuum level to 100mT~120mT within 10min~30min; Step T2-3, Sublimation Drying Stage: First, raise the temperature to -22℃ within 2 hours and maintain it for 10 to 12 hours. Then, raise the temperature from -22℃ to -17℃ within 20 minutes and maintain it for 8 to 10 hours. Finally, raise the temperature from -17℃ to -5℃ within 20 to 30 minutes and maintain it for 3 to 5 hours. Step T2-4, Drying stage: Adjust the vacuum to 10mT, raise the temperature to 28℃~30℃ in 1h~2h, and keep it at that temperature for 6h~8h.

10. The method for preparing the live swine erysipelas vaccine according to claim 8, characterized in that, Step T2-1, Pre-freezing stage: Cool the vaccine stock solution from room temperature to -17°C in 40 minutes, keep it at -17°C for 1 hour, then cool it to -45°C in 40 minutes, and keep it at -45°C for 4 hours; Step T2-2, Vacuuming stage: Reduce the vacuum level to 120mT within 20 minutes; Step T2-3, Sublimation Drying Stage: When the vacuum degree drops to 120mT, heating begins. Within 2 hours, the temperature is raised from -45℃ to -22℃ and maintained for 10 hours. Then, within 20 minutes, the temperature is raised from -22℃ to -17℃ and maintained for 10 hours. Finally, within 20 minutes, the temperature is raised from -17℃ to -5℃ and maintained for 5 hours. Step T2-4, Drying stage: Adjust the vacuum to 10mT, raise the temperature to 28℃ in 2 hours, and keep it at that temperature for 8 hours.

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