Culture medium, preparation method and application of highly antigenic Pasteurella multocida in birds

Abstract

This invention discloses a high-antigenic-activity avian Pasteurella multocida culture medium, its preparation method, and its application. The high-antigenic-activity avian Pasteurella multocida culture medium consists of: 8-13 g / L bacteriological peptone, 3-8 g / L growth-promoting peptides, 18-23 g / L yeast extract, 2-7 g / L glucose, 4-9 g / L sodium chloride, 1-5 g / L dipotassium hydrogen phosphate, and the remainder being water. Using this high-antigenic-activity avian Pasteurella multocida vaccine culture medium promotes bacterial growth and reproduction, requires a shorter culture time, and achieves a high cell density, ultimately reaching 1.84 × 10¹⁰ CFU / mL. It significantly improves fermentation speed and the cell density of avian Pasteurella multocida in the fermentation broth, making it more suitable for culturing avian Pasteurella multocida. Furthermore, the vaccine prepared using the culture medium provided by this invention has a protection efficiency of up to 100% and excellent antigenic activity, laying the foundation for the production of avian Pasteurella multocida vaccines.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation, specifically relating to a culture medium for highly antigenic Pasteurella multocida, its preparation method, and its application. Background Technology

[0002] Pasteurella multocida (Pm) is a Gram-negative facultative anaerobic bacterium that can widely infect mammals, birds, and reptiles, causing a variety of diseases. Fowl cholera is one of the infectious diseases in poultry caused by Pasteurella multocida, resulting in significant economic losses to the global poultry industry. Capsular serotypes A (A:1, A:3, A:4) are the main serotypes causing fowl cholera.

[0003] Current research indicates that pathogens are becoming less sensitive to commonly used drugs, making drug treatment increasingly difficult. Vaccination is currently a crucial preventative measure against fowl cholera. Vaccination can significantly reduce the incidence of the disease in poultry and prevent its occurrence. In the vaccine preparation process, increasing bacterial density is a key factor in improving vaccine yield and reducing costs.

[0004] Currently, most cultures of Pasteurella multocida in avian birds use TSB and Martin broth, but there are also commercially available dedicated vaccine culture media. The peak viable count harvested is around 1×10⁻⁶. 10 The concentration of CFU / mL is around 100%, which results in poor enrichment and immunization of the prepared vaccine. Using Martin's broth also requires the addition of serum, leading to high culture costs.

[0005] CN110643522A discloses a culture medium, culture method, and application of Pasteurella multocida in birds. Although the cell density obtained by using the culture medium of this invention can reach 2.0–2.5 × 10⁻⁶ cells / day in 5 hours... 10 The concentration of CFU / mL was high, but the protective rate of the immunization vaccine prepared using this culture medium was only 83.3%. Summary of the Invention

[0006] The purpose of this invention is to overcome at least one deficiency of the prior art and to provide a culture medium for highly antigenic Pasteurella multocida, its preparation method, and its application.

[0007] The technical solution adopted in this invention is:

[0008] In a first aspect, the present invention provides a culture medium for *Pasteurella multocida* with high antigenic activity, comprising: 8-13 g / L bacteriological peptone, 3-8 g / L growth-promoting peptide, 18-23 g / L yeast extract, 2-7 g / L glucose, 4-9 g / L sodium chloride, 1-5 g / L dipotassium hydrogen phosphate, and the balance being water; wherein the growth-promoting peptide is prepared by: using soybean meal as raw material, cooking it, adding alkaline protease for enzymatic hydrolysis, the hydrolysis conditions being: temperature of 50-60℃, hydrolysis pH of 9.0-11.0, hydrolysis time of 6-8 h, enzyme mass percentage of 0.9-1.1%, soybean meal concentration of 4.0-5.0 g / 100 mL, and obtaining the culture medium after enzyme inactivation, centrifugation, and drying.

[0009] In some instances, the culture medium for the highly antigenic Pasteurella multocida comprises: 10 g / L bacteriological peptone, 5.95 g / L growth-promoting peptide, 21.04 g / L yeast extract, 3.95 g / L glucose, 7 g / L sodium chloride, 2 g / L dipotassium hydrogen phosphate, with the remainder being water.

[0010] In a second aspect, the present invention provides a method for preparing a culture medium of highly antigenically active Pasteurella multocida described in the first aspect, comprising the following steps:

[0011] 1) Dissolve the bacteriological peptone, growth-promoting peptides, yeast powder, glucose, sodium chloride, and dipotassium hydrogen phosphate in water according to the formula to obtain a mixture;

[0012] 2) Then adjust the pH of the mixture to 7.2-7.4, sterilize at high temperature and cool to obtain the culture medium of the highly antigenic Pasteurella multocida avianis.

[0013] In some instances, the high-temperature sterilization temperature is 115–121°C, and the time is 15–30 min.

[0014] Thirdly, the present invention provides a method for high-density culture of Pasteurella multocida in birds, comprising the following steps:

[0015] 1) Preparation of primary seed: Pasteurella multocida was inoculated into TSA plates and cultured to obtain primary seed of Pasteurella multocida;

[0016] 2) Preparation of secondary seeds: Select round, translucent, smooth, moist, and intact primary seed colonies of Pasteurella multocida from birds and inoculate them into a culture medium for incubation until OD. 600 When the value reaches 1.2, secondary seed of Pasteurella multocida in birds is obtained;

[0017] 3) Fermentation culture: The culture medium is placed in the fermenter, and secondary seed of Pasteurella multocida is inoculated and cultured to obtain the fermentation culture of Pasteurella multocida.

[0018] The culture medium is the high antigenic activity Pasteurella multocida culture medium described in the first aspect.

[0019] In some instances, the preparation temperature of the primary seed of Pasteurella multocida is 36–38°C, and the preparation time is 16–30 h.

[0020] In some instances, the secondary seed of Pasteurella multocida was prepared at a temperature of 36–38°C for 7–9 hours.

[0021] In some instances, the fermentation conditions are as follows: the initial fermentation speed is 90–110 rpm / min, and the air flow rate per minute is 0.3–0.4 times the fermentation liquid volume; after 2–4 hours of fermentation, the fermentation speed is 240–260 rpm / min, and the air flow rate per minute is 0.9–1.1 times the fermentation liquid volume.

[0022] In some instances, the inoculation volume of secondary seed bacteria of Pasteurella multocida in the fermentation culture is 2-4% of the culture medium volume.

[0023] In some instances, the fermentation culture temperature is 36–38°C, and the fermentation culture time is 6–8 hours.

[0024] The above features can be combined arbitrarily as long as they do not conflict with each other.

[0025] The beneficial effects of this invention are:

[0026] 1) The high antigenic activity avian Pasteurella multocida vaccine culture medium of the present invention is conducive to the growth and reproduction of bacteria, with short culture time and high cell density, and is more suitable for the culture of avian Pasteurella multocida;

[0027] 2) The preparation method of the high antigenic activity avian Pasteurella multocida vaccine culture medium of the present invention is simple, the raw materials are readily available, and the cost is low, making it suitable for large-scale industrial production;

[0028] 3) The fermentation parameters provided by this invention can significantly improve the fermentation rate and the density of *Pasteurella multocida* cells in the fermentation broth, ultimately reaching 1.84 × 10⁻⁶. 10 CFU / mL;

[0029] 4) The vaccine prepared using the culture medium provided by this invention has a protection efficiency of up to 100% and excellent antigen activity, laying the foundation for the production of avian Pasteurella multocida vaccine. Attached Figure Description

[0030] Figure 1 The results of single-factor experiments on shake-flask fermentation with different addition amounts of each component in the culture medium;

[0031] Figure 2 Comparison of growth curves for culture media with added growth-promoting peptides and soybean peptone;

[0032] Figure 3 This is a response surface diagram showing the interactions of various components in the culture medium for the high antigenic activity avian Pasteurella multocida vaccine proposed in this invention.

[0033] Figure 4 This is a comparison of the growth curves of the high antigenic activity avian Pasteurella multocida vaccine culture medium and TSB culture medium described in this invention. Detailed Implementation

[0034] The following disclosure provides many different implementations or examples for different ways of implementing the present invention.

[0035] For ease of comparison, the methods for preparing growth-promoting peptides used in the following examples are as follows:

[0036] Soybean meal was used as raw material. The soybean meal was pulverized and passed through an 80-mesh sieve and collected. Water was added to make a soybean meal:water ratio of 1:30 (m:v). The mixture was stirred at room temperature for 15 minutes, then cooked for 30 minutes. After cooling to 55°C, alkaline protease was added to the cooking liquid for enzymatic hydrolysis. The hydrolysis conditions were: temperature 50–60°C, pH 9.0–11.0, hydrolysis time 6–8 hours, enzyme mass percentage 0.9–1.1%, and soybean meal concentration 4.0–5.0 g / 100 mL. After hydrolysis, enzyme inactivation was performed (water bath conditions: 100°C, 8 minutes). The hydrolysate was cooled to room temperature and centrifuged (4°C, 6000 rpm, 5 minutes). After centrifugation, the supernatant was collected and the pH adjusted to 7. Finally, the centrifuged liquid was desalted and dried to obtain the growth-promoting peptides. The alkaline protease used had an enzyme activity of approximately 6000 U / g.

[0037] Example 1

[0038] The comparison of single-factor experimental results in shake-flask fermentation includes the following steps:

[0039] The optimal addition amount of each component in the culture medium for high antigenic activity avian Pasteurella multocida vaccine must be determined by single-factor experiments, including single-factor experiments on six factors: bacteriological peptone, growth-promoting peptide, yeast extract, glucose, sodium chloride, and dipotassium hydrogen phosphate.

[0040] (1) The amount of bacteriological peptone added was 0 g / L, 5 g / L, 7.5 g / L, 10 g / L, 12.5 g / L and 15 g / L respectively, while the amount of other components remained unchanged. They were dissolved in purified water.

[0041] (2) The growth-promoting peptides were added at concentrations of 0 g / L, 2.5 g / L, 5 g / L, 7.5 g / L, 10 g / L, and 12.5 g / L, respectively, while the concentrations of other components remained unchanged. They were dissolved in purified water.

[0042] (3) The yeast powder was added at amounts of 0 g / L, 5 g / L, 10 g / L, 12.5 g / L, 15 g / L and 17.5 g / L, respectively, while the amounts of other components remained unchanged. The yeast powder was dissolved in purified water.

[0043] (4) The glucose addition amounts were 0 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L and 6 g / L, respectively, while the addition amounts of other components remained unchanged. They were dissolved in purified water.

[0044] (5) Sodium chloride was added at concentrations of 0 g / L, 3 g / L, 5 g / L, 7 g / L, 9 g / L, and 11 g / L, while the concentrations of other components remained unchanged. The solution was dissolved in purified water.

[0045] (6) The amounts of dipotassium hydrogen phosphate added were 0 g / L, 1 g / L, 2 g / L, 3 g / L and 4 g / L, respectively, while the amounts of other components remained unchanged. They were dissolved in purified water.

[0046] Take 50 mL of the above solutions and adjust the pH to 7.2–7.4 with 1 mol / L sodium carbonate solution. Place each solution into a 250 mL Erlenmeyer flask and autoclave at 121°C for 15 min. Inoculate with 3% (v / v) of secondary seed of *Pasteurella multocida* and incubate at 37°C on a shaker at 150 rpm. Measure the OD of the bacterial culture using a microplate reader. 600 The maximum values ​​are compared, and the results are as follows: Figure 1 As shown.

[0047] The analysis results showed that the cell density of Pasteurella multocida reached its highest value when the added amounts of bacteriological peptone, growth-promoting peptides, yeast extract, glucose, sodium chloride, and dipotassium hydrogen phosphate were 10 g / L, 5 g / L, 15 g / L, 3 g / L, 7 g / L, and 2 g / L, respectively.

[0048] Example 2

[0049] Culture medium A was prepared according to the optimal addition amounts of each component in the single-factor experiment of Example 1. Culture medium B was prepared by replacing the growth-promoting peptide in culture medium A with soybean peptone, while keeping other components unchanged. A comparative experiment was conducted to compare the enrichment effects of the growth-promoting peptide and soybean peptone on *Pasteurella multocida* in avian birds. In this example, *Pasteurella multocida* in avian birds was cultured using the fermentation culture method in Example 1, and growth curves were plotted. The results are as follows... Figure 2 As shown.

[0050] according to Figure 2It can be concluded that adding growth-promoting peptides to the culture medium has a significantly better enrichment effect on Pasteurella multocida in birds than adding soybean peptone. Therefore, it can be seen that growth-promoting peptides play a key role in enrichment culture medium, and growth-promoting peptides should be used for subsequent culture medium optimization experiments.

[0051] Example 3

[0052] This embodiment is a further optimization of Example 1. Specifically, based on the single-factor experimental screening, a Plackett-Burman (PB) experimental design is also required. The steps are as follows: bacteriological peptone A, growth-promoting peptide B, yeast extract C, glucose D, sodium chloride E, and dipotassium hydrogen phosphate F are selected as influencing factors, with cell density (OD) as the criterion. 600 Using PB experiments as evaluation indicators, Design-Expert software was used to design the experiments and screen out factors that significantly affect the growth of Pasteurella multocida. The design levels of each factor are shown in Table 1, and the combinations of PB experiments are shown in Table 2. Following the experimental scheme designed in the model, the OD of the bacterial suspension was measured under the culture conditions described in Example 1. 600 The values ​​were analyzed, and the results are shown in Table 3.

[0053] Table 1. Factors and Levels of the PB Experiment

[0054]

[0055] Table 2 PB Experimental Design and Results

[0056]

[0057] Table 3 Analysis of PB Experimental Design Results

[0058]

[0059] The analysis results show that the model is meaningful (F = 6.28, P < 0.05). Growth-promoting peptides, yeast extract, and glucose have significant effects on the growth of *Pasteurella multocida* (P < 0.05), and the estimated coefficients are all positive, indicating a positive correlation. Therefore, yeast extract, growth-promoting peptides, and glucose were selected for subsequent steepest climb experiments and response surface methodology experiments. The remaining factors were not significant (P > 0.05) and were maintained at their original levels in subsequent experiments.

[0060] Example 4

[0061] This embodiment is a further supplement and optimization of embodiment 2. Specifically, based on the PB experiment, a steepest climbing test design is required. The steps are as follows: According to the PB experiment results, the coefficients of growth-promoting peptide, yeast powder and glucose are all positive, indicating that their addition amount is positively correlated with the growth of Pasteurella multocida. Therefore, the climbing direction is upward. The steepest climbing test designed accordingly and the test results are shown in Table 4.

[0062] Table 4. Experimental Design and Results for the Steepest Climb

[0063]

[0064] The analysis results show that combination number 4 has the highest bacterial response value. Therefore, the amount of addition of combination number 4 was selected as the center point for subsequent response surface methodology experiments.

[0065] Example 5

[0066] This embodiment is a further supplement and optimization of Embodiment 3. Specifically, based on the steepest ramp test, a Box-Behnken experimental design (BBD) is also required. The steps are as follows: Based on the significant influencing factors obtained from the PB test, the center point of the response surface methodology is determined using the results of the steepest ramp test. Growth-promoting peptide A, yeast extract B, and glucose C are selected as influencing factors, with cell density (OD) as the influencing factor. 600 Using as the evaluation index, a response surface analysis experiment was designed using Design-Expert software. The levels of each factor are shown in Table 5, and the design scheme and results are shown in Table 6.

[0067] Table 5. BBD Factors and Levels

[0068]

[0069] Table 6 Response Surface Design Schemes and Results

[0070]

[0071] Analysis of the obtained response value (OD) 600 The relationship between the growth-promoting peptide (A), yeast extract (B), and glucose (C) is shown by a multiple quadratic regression equation:

[0072] OD 600 =1.33-0.005825A+0.031B-0.050C+0.002225AB+0.012AC+0.024BC-0.096A 2 -0.083B 2 -0.079C 2 .

[0073] The results of the analysis of variance for the response surface regression equation are shown in Table 7. The analysis results show that the F-value of the model is 89.59, which is highly significant (P<0.01), and the P-value for the lack-of-fit term is greater than 0.05, indicating an excellent fit. For individual factors, yeast extract (B) and glucose (C) both have highly significant effects on cell growth (P<0.01), with the influence of each factor in the order C>B>A. Regarding the interaction between two factors, the interaction between yeast extract and glucose has a highly significant effect on cell growth (P<0.01), while the others are not significant (P>0.05). For the quadratic term of the model, the effects of all three factors are highly significant (P<0.01).

[0074] Table 7. Analysis of variance of the response surface fitting regression equation

[0075]

[0076] Using Design-Expert software, draw a 3D response surface plot. (Refer to the attached diagram.) Figure 3 The response surface plots all open downwards, therefore the response value should have a maximum value. The optimal value can be predicted by taking the first partial derivative of the growth regression equation for *Pasteurella multocida*. The optimal conditions for prediction are: growth-promoting peptide 5.95 g / L, yeast extract 21.04 g / L, and glucose 3.95 g / L. Under these conditions, the model predicts the OD... 600 The value was 1.3416. Under these conditions, three parallel experiments were conducted, and the measured result was 1.334, which is similar to the predicted value, indicating that the equation fits well.

[0077] Example 6

[0078] This embodiment further demonstrates the results of Embodiment 4, specifically as follows:

[0079] (1) Prepare the culture medium according to the following ratio: 10 g / L bacteriological peptone, 5.95 g / L growth-promoting peptide, 21.04 g / L yeast extract, 3.95 g / L glucose, 7 g / L sodium chloride, 2 g / L dipotassium hydrogen phosphate, and the remainder is purified water. Adjust the pH to 7.2–7.4 using 1 mol / L sodium carbonate.

[0080] (2) Prepare TSB medium as a control.

[0081] (3) Add 20 mL of each of the two culture media (1) and (2) to a 100 mL Erlenmeyer flask. After autoclaving and cooling, inoculate with secondary seed of Pasteurella multocida at an inoculation rate of 3% (v / v). After shaking well, add 200 μL to a 96-well plate. Perform three replicates for each culture medium. Incubate at 37 °C and measure OD every hour. 600 Value, continuously shake and culture for more than 10 hours, plot growth curve, refer to Figure 4 .

[0082] (4) Add (1) and (2) culture media to the fermenter at 60% (v / v) respectively. After high temperature and high pressure sterilization, lower the temperature of the culture media to 37℃. Inoculate with secondary seed of Pasteurella multocida at an inoculation amount of 3% (v / v) based on the volume of the culture media. Incubate at 37℃ for 9 hours. Take samples every hour to measure OD. 600 pH value, and viable bacterial count were determined using the dilution plating method. Results are shown in Table 8.

[0083] Analysis results showed that, based on the growth curve, *Pasteurella multocida* reached the plateau phase in TSB medium after approximately 9 hours, with an OD... 600 The highest value was 1.184. In the culture medium for high-antigen-activity avian Pasteurella multocida vaccine, it reached the plateau phase after approximately 7 hours of growth. 600 The highest value was 1.338. The growth rate and enrichment effect of the bacteria in the high-antigen-activity avian Pasteurella multocida vaccine medium were superior to those in TSB medium. Fermentation data showed that the high-antigen-activity avian Pasteurella multocida vaccine medium not only reached the plateau phase earlier than TSB medium, but also had a higher cell density. The fermentation time was 6 hours, and the viable cell count reached 1.84 × 10⁸. 10 The CFU / mL concentration improved the enrichment effect by 2.7 times compared to TSB medium.

[0084] Table 8 Comparison of fermentation results between high antigenic activity Pasteurella multocida vaccine culture medium and TSB culture medium

[0085]

[0086] Example 7

[0087] This embodiment evaluates the immunization efficacy of a vaccine prepared using a high-antigen-activity avian Pasteurella multocida vaccine culture medium and measures the antigenic activity of fermentation products at different time points. Specifically:

[0088] (1) The strain was fermented and cultured using the above-mentioned avian Pasteurella multocida vaccine culture medium, and an inactivated vaccine was prepared by adding aluminum gel adjuvant. The vaccine was then used to conduct animal immunization protection tests. Balb / c mice of appropriate age were selected and randomly divided into 4 groups (n=10 per group): vaccine culture medium group, commercial inactivated vaccine group, PBS challenge group, and PBS blank group.

[0089] (2) Both the vaccine group and the PBS challenge group were immunized twice. Serum was collected 14 days after the second immunization to determine serum antibody titers for later use. 14 days after the second immunization, mice in both the vaccine group and the challenge group were injected with a lethal dose of 4 LD. 50 Live bacteria (2.4×10) 3 CFU was used to observe and record the clinical symptoms and mortality of mice within 7 days.

[0090] (3) Samples were taken at different time points during the fermentation of the above-mentioned high antigenic activity avian Pasteurella multocida vaccine culture medium. After centrifugation and resuspending, the samples were ultrasonically disrupted and adjusted to 10 μg / mL before the antigenic activity was determined by ELISA.

[0091] The challenge protection rate after immunization is shown in Table 9. It can be seen that the vaccine made by culturing Pasteurella multocida in avian culture medium of the present invention has a better challenge protection rate and serum titer than commercially available vaccines, and has no adverse reactions in immunized animals during the immunization process. This vaccine can serve as the basis for the production of this type of vaccine.

[0092] The titers of the fermentation products at different time points are shown in Table 10. The results indicate that the immunogenicity of the bacterial antigen protein is relatively weak during the logarithmic growth phase, but reaches its peak after the stationary phase and remains relatively stable in subsequent stages. Therefore, when using this culture medium to prepare avian Pasteurella multocida vaccine, the bacteria can be harvested at the stationary phase, when both immunogenicity and cell density reach their peak, resulting in a vaccine with good efficacy and low cost.

[0093] Table 9 Results of Vaccine Protection Trials

[0094]

[0095] Table 10 Results of immunogenicity assay for bacterial antigen proteins at different time points

[0096]

[0097] The above is a further detailed description of the present invention and should not be considered as a limitation on the specific implementation of the present invention. For those skilled in the art, simple deductions or substitutions without departing from the concept of the present invention are all within the protection scope of the present invention.

Claims

1. A culture of avian Pasteurella multocida having high antigenic activity, characterized in that, The composition is: bacteriological peptone 10 g / L, growth promoting peptide 5.95 g / L, yeast powder 21.04 g / L, glucose 3.95 g / L, sodium chloride 7 g / L, potassium phosphate 2 g / L, and the rest is water; wherein the preparation method of the growth promoting peptide is: taking soybean meal as raw material, after cooking, adding alkaline protease for enzymolysis, the enzymolysis conditions are: temperature is 50-60 DEG C, enzymolysis pH value is 9.0-11.0, enzymolysis time is 6-8 h, the mass percentage of enzyme is 0.9-1.1%, the concentration of soybean meal is 4.0-5.0 g / 100 mL, after enzyme inactivation, centrifugation, drying, the growth promoting peptide is obtained.

2. The method of claim 1, wherein the culture medium for avian Pasteurella multocida having high antigenicity is prepared by the steps of: Comprising the following steps: ​ 1) according to the formula amount of bacteriological peptone, growth promoting peptide, yeast powder, glucose, sodium chloride, potassium phosphate is dissolved with water, and the mixed solution is obtained; 2) then adjust the pH value of the mixed solution to 7.2-7.4, after high temperature sterilization and cooling, the culture medium of high antigenic activity of avian Pasteurella multocida is obtained; The culture medium is as claimed in claim 1.

3. The process for the preparation of Avian Pasteurella multocida culture medium as claimed in claim 2, wherein, The temperature of high temperature sterilization is 115-121 DEG C, and the time is 15-30 min.

4. A method for high density culture of avian Pasteurella multocida, characterized by, Comprising the following steps: 1) preparation of primary seed: avian Pasteurella multocida is inoculated into TSA plate for culture, and the primary seed of avian Pasteurella multocida is prepared; 2) Preparation of secondary seed: select round, translucent, smooth, moist, intact Avian Pasteurella multocida primary seed single colony to inoculate in culture medium for culture until OD 600 value reaches 1.2, to obtain Avian Pasteurella multocida secondary seed; 3) fermentation culture: the culture medium is placed in the fermentation tank, and the secondary seed of avian Pasteurella multocida is inoculated for culture, and the fermentation culture of avian Pasteurella multocida is prepared; The culture medium is the high antigenic activity avian Pasteurella multocida culture medium according to claim 1.

5. The high density culture method according to claim 4, characterized by, The preparation temperature of the primary seed of avian Pasteurella multocida is 36-38 DEG C, and the preparation time is 16-30 h.

6. The high density culture method according to claim 4, characterized by, The preparation temperature of the secondary seed of avian Pasteurella multocida is 36-38 DEG C, and the preparation time is 7-9 h.

7. The high density culturing method according to any one of claims 4 to 6, characterized by, The fermentation culture conditions are: the initial rotation speed of fermentation is 90-110 rpm / min, the air ventilation amount per minute is 0.3-0.4 times the volume of fermentation liquid; after 2-4 h of fermentation, the rotation speed of fermentation is 240-260 rpm / min, and the air ventilation amount per minute is 0.9-1.1 times the volume of fermentation liquid.

8. The high density culturing method according to any one of claims 4 to 6, characterized by, The inoculation volume of the secondary seed of avian Pasteurella multocida in the fermentation culture is 2-4% based on the volume of the culture medium.

9. The high density culturing method according to any one of claims 4 to 6, characterized by, The fermentation culture temperature is 36-38 DEG C, and the fermentation culture time is 6-8 h.

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