High-density fermentation method for improving immunogenicity of duck escherichia coli

Abstract

The invention relates to the technical field of microbial fermentation, and particularly provides a high-density fermentation method for improving immunogenicity of duck escherichia coli. The high-density basic culture medium is prepared from the following components: 2 to 15g / L of yeast extract, 5 to 20g / L of tryptone, 34 to 36g / L of KH2PO4, 1.6 to 2.0 g / L of (NH4) 2HPO4, 1.5 to 2.5 g / L of NaCl, 0.5 to 3g / L of citric acid, 0.5 to 2g / L of MgSO4, 2 to 20g / L of glucose and 0.01 to 0.1 g / L of VB1. And the high-density fed culture medium comprises 50-500g / L of glucose, 10-50g / L of yeast extract and 20-100g / L of tryptone. Compared with the prior art, large-scale high-density fermentation of duck Escherichia coli can be realized by using the two, and the immunogenicity of duck Escherichia coli can be improved.

Description

Technical Field

[0001] This application relates to the field of microbial fermentation technology, specifically providing a high-density fermentation method for improving the immunogenicity of duck Escherichia coli. Background Technology

[0002] Duck colibacillosis is a general term for acute septicemia, air sacculitis, panophthalmitis, synovitis, salpingitis, and peritonitis in ducks caused by pathogenic duck Escherichia coli (APEC). Ducks of all ages can be infected with APEC, and clinical manifestations include loss of appetite, diarrhea, emaciation, and reduced production performance. Once infected, APEC can spread rapidly throughout the flock, not only harming the health of the birds but also affecting egg production and the market time of broiler poultry, increasing feeding costs and causing serious economic losses to the poultry industry. Currently, the widespread use of antibiotics to treat duck colibacillosis has led to the emergence of many drug-resistant strains, causing APEC to become insensitive to antibiotics or even lose their effectiveness. Furthermore, antibiotics have withdrawal periods, thus antibiotic treatment for duck colibacillosis has certain limitations. Immunization with inactivated vaccines is an effective way to prevent duck colibacillosis.

[0003] The cultivation of *E. coli* in ducks is crucial in the inactivated vaccine preparation process. *E. coli* is a facultative anaerobe with relatively low requirements for culture conditions. While it can grow normally in ordinary LB medium, the culture density is low. High-density cultivation, on the other hand, offers advantages such as improved equipment utilization, reduced energy consumption, and lower wastewater volume, making it more suitable for industrial production. High-density cultivation processes have been reported, for example, using a self-made high-density fermentation medium as the basal medium and glucose as the supplemental medium. Through process optimization, the final OD of *E. coli* in ducks has been determined. 600 The OD value reached approximately 30.0 (Chinese Publication CN108865941A). Using modified Martin broth medium as the basal medium and glucose as the supplemental medium, the final OD value was achieved through process optimization. 600 The OD reached 30.32 (non-patent literature, "Establishment of Basic Seeds for Duck Escherichia coli Vaccination and High-Density Fermentation Culture Experiment Study," *Chinese Journal of Animal Husbandry and Veterinary Medicine*, 2011, Vol. 38, No. 6). Although both methods achieved high-density culture, no immunogenicity test was conducted. Furthermore, using modified Martin broth as the basal medium and culturing in an automated fermenter for 6-8 hours, the final OD... 600 The count reaches 30 or higher, and the viable count reaches 350 × 10⁻⁶. 8 Inactivated vaccines prepared with CFU / ml or higher have a protection rate of 70% for ducklings (Chinese publication CN104623650A), however, this method has a low protection rate.

[0004] In view of the above, this application is hereby submitted. Summary of the Invention

[0005] The purpose of this application is to provide a high-density fermentation method to improve the immunogenicity of duck Escherichia coli, so as to ensure its immunogenicity while culturing duck Escherichia coli at high density.

[0006] To achieve the above objectives, this application adopts the following technical solution:

[0007] A high-density basal culture medium for culturing duck Escherichia coli, wherein the culture medium is formulated as follows: yeast extract 2-15 g / L, tryptone 5-20 g / L, KH2PO4 34-36 g / L, (NH4)2HPO4 1.6-2.0 g / L, NaCl 1.5-2.5 g / L, citric acid 0.5-3 g / L, MgSO4 0.5-2 g / L, glucose 2-20 g / L and VB1 0.01-0.1 g / L.

[0008] Further, the culture medium is formulated as follows: yeast extract 4-6 g / L, tryptone 9-11 g / L, KH2PO4 34-36 g / L, (NH4)2HPO4 1.7-1.9 g / L, NaCl 1.9-2.1 g / L, citric acid 0.9-1.1 g / L, MgSO4 0.9-1.1 g / L, glucose 9-11 g / L and VB1 0.01-0.03 g / L.

[0009] Preferably, the culture medium is formulated as follows: yeast extract 5 g / L, tryptone 10 g / L, KH2PO4 35 g / L, (NH4)2HPO4 1.8 g / L, NaCl 2 g / L, citric acid 1 g / L, MgSO4 1 g / L, glucose 10 g / L and VB1 0.02 g / L.

[0010] A high-density fed culture medium for culturing duck Escherichia coli, wherein the culture medium is formulated as follows: glucose 50-500 g / L, yeast extract 10-50 g / L and tryptone 20-100 g / L.

[0011] Furthermore, the formulation of the high-density fed culture medium is as follows: glucose 290-310 g / L, yeast extract 24-26 g / L and tryptone 49-51 g / L.

[0012] Preferably, the culture medium is formulated as follows: 300 g / L glucose, 25 g / L yeast extract and 50 g / L tryptone.

[0013] A high-density fermentation method for duck Escherichia coli includes the following steps: a secondary seed culture is inoculated into a fermenter containing the high-density basal medium at an inoculation rate of 1%-8%. In the initial stage of fermentation, when the dissolved oxygen (DO) is below 20%, the DO is increased to above 20% by increasing the rotation speed and aeration. After the glucose in the high-density basal medium is consumed and the DO recovers, the high-density feed medium is used to replenish the DO. The DO is adjusted to not be lower than 20% through feed replenishment. The pH is controlled at 6.8-7.0 and the fermentation temperature is 36-38℃ throughout the process.

[0014] Further, the preparation of the secondary seed culture includes the following steps: streak dried duck Escherichia coli on TSA solid medium, incubate at 37°C for 12-16 h, pick a single colony and inoculate it into TSB liquid medium, incubate at 37°C with shaking at 180 r / min for 10-16 h, as the primary seed culture; inoculate the primary seed culture at 1%-5% into a shake flask or fermenter containing TSB liquid medium, incubate at 37°C for 2-5 h, as the secondary seed culture.

[0015] Furthermore, the cells are harvested after 8-10 hours of fermentation.

[0016] The application of the above-mentioned high-density fermentation method in the preparation of duck Escherichia coli inactivated vaccine.

[0017] The duck Escherichia coli prepared by the above high-density fermentation method.

[0018] The above-mentioned application of duck Escherichia coli in duck Escherichia coli inactivated vaccine.

[0019] Compared with the prior art, the technical effects of this application are as follows:

[0020] The method of this application enables large-scale, high-density fermentation of duck Escherichia coli, with a viable count more than twice that of existing technologies. Furthermore, it was unexpectedly discovered that adding yeast extract and tryptone as nitrogen sources to the feed culture medium can enhance the immunogenicity of duck Escherichia coli. Vaccines made from antigens obtained using the process of this application can provide effective protection for ducks. Detailed Implementation

[0021] To enable those skilled in the art to better understand the technical solutions of this application, the technical solutions described in this application will be further described in detail below with reference to specific embodiments.

[0022] The inventors discovered that while using existing basal culture media and glucose as a feedstock increased cell density in high-density fermentation methods, it resulted in poor immunogenicity, low protection rates, and low levels of HI antibodies. To address these issues, a high-density culture method was developed, which includes both a high-density basal culture medium and a high-density feedstock.

[0023] The formula for high-density basal medium is as follows: yeast extract 2-15 g / L, tryptone 5-20 g / L, KH2PO4 34-36 g / L, (NH4)2HPO4 1.6-2.0 g / L, NaCl 1.5-2.5 g / L, citric acid 0.5-3 g / L, MgSO4 0.5-2 g / L, glucose 2-20 g / L and VB1 0.01-0.1 g / L.

[0024] It should be noted that yeast extract can be, but is not limited to, 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, 11 g / L, 12 g / L, 13 g / L, 14 g / L, or 15 g / L; tryptone can be, but is not limited to, 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, 11 g / L, 12 g / L, or 13 g / L. 14 g / L, 15 g / L, 16 g / L, 17 g / L, 18 g / L, 19 g / L, or 20 g / L; KH₂PO₄ can be, but is not limited to, 34 g / L, 34.5 g / L, 35 g / L, 35.5 g / L, or 36 g / L; (NH₄)₂HPO₄ can be, but is not limited to, 1.6 g / L, 1.7 g / L, 1.8 g / L, 1.9 g / L, or 2.0 g / L; NaCl can be, but is not limited to, 1.5 g / L. The concentrations of MgSO4 can be 1.6 g / L, 1.7 g / L, 1.8 g / L, 1.9 g / L, 2.0 g / L, 2.1 g / L, 2.2 g / L, 2.3 g / L, 2.4 g / L, or 2.5 g / L; citric acid can be, but is not limited to, 0.5 g / L, 1 g / L, 1.5 g / L, 2 g / L, 2.5 g / L, or 3 g / L; MgSO4 can be, but is not limited to, 0.5 g / L, 1 g / L, 1.5 g / L, or 2 g / L. The concentrations of glucose can be, but are not limited to, 2 g / L, 4 g / L, 6 g / L, 8 g / L, 10 g / L, 12 g / L, 14 g / L, 16 g / L, 18 g / L, or 20 g / L; the concentrations of vitamin B1 can be, but are not limited to, 0.01 g / L, 0.02 g / L, 0.03 g / L, 0.04 g / L, 0.05 g / L, 0.06 g / L, 0.07 g / L, 0.08 g / L, 0.09 g / L, or 0.1 g / L.

[0025] The formula for high-density fed culture medium is: glucose 50-500 g / L, yeast extract 10-50 g / L and tryptone 20-100 g / L.

[0026] It should be noted that glucose can be, but is not limited to, 50 g / L, 100 g / L, 150 g / L, 200 g / L, 250 g / L, 300 g / L, 350 g / L, 400 g / L, 450 g / L, or 500 g / L; yeast extract can be, but is not limited to, 10 g / L, 15 g / L, 20 g / L, 25 g / L, 30 g / L, 35 g / L, 40 g / L, 45 g / L, or 50 g / L; and tryptone can be, but is not limited to, 20 g / L, 30 g / L, 40 g / L, 50 g / L, 60 g / L, 70 g / L, 80 g / L, 90 g / L, or 100 g / L.

[0027] The inventors discovered that adding yeast extract and tryptone as nitrogen sources to a high-density fed culture medium can enhance the immunogenicity of duck *E. coli*. Furthermore, this fed culture medium, when combined with other commercially available basal media, can also improve the immunogenicity of duck *E. coli*, demonstrating its versatility.

[0028] A high-density fermentation method for duck Escherichia coli includes the following steps:

[0029] Step (1) Seed liquid preparation

[0030] Stranded duck *Escherichia coli* bacteria on TSA solid medium and incubated at 37°C for 12-16 hours. Single colonies were picked and inoculated into TSB liquid medium and cultured at 37°C with shaking at 180 rpm for 10-16 hours to obtain the primary seed culture. The primary seed culture was then inoculated at 1%-5% into shake flasks or fermenters containing TSB liquid medium and incubated at 37°C for 2-5 hours to obtain the secondary seed culture.

[0031] Step (2) Fermentation tank process control

[0032] The secondary seed culture was inoculated into a fermenter containing the aforementioned high-density basal medium at an inoculation rate of 1%-8%. The fermentation temperature was 37℃ and the pH was 6.9. In the initial stage of fermentation, when the dissolved oxygen (DO) was below 20%, the DO was increased to above 20% by increasing the turbine rotation speed and aeration. After the glucose in the high-density basal medium was depleted and the DO recovered, the aforementioned high-density fed-batch medium was used to replenish the DO. The DO was kept above 20% through feeding and regulation. Throughout the process, the pH was controlled at 6.8-7.0 using acid and ammonia.

[0033] Step (3) Harvesting: Determine the harvesting time based on the bacterial growth rate, i.e., harvest aseptically at 8-10 hours and temporarily store at 4℃.

[0034] The fermentation tank can be either 10L or 100L. For a 10L fermentation tank, the feeding rate is 2.0-2.5ml / min, and after 2 hours, the feeding rate is 2.8-3.2ml / min. For a 100L fermentation tank, the feeding rate is 20-25ml / min, and after 2 hours, the feeding rate is 28-32ml / min.

[0035] The duck Escherichia coli antigen prepared by the above technical solution can be used to prepare duck Escherichia coli inactivated vaccines and to prevent and control duck colibacillosis.

[0036] OD of duck Escherichia coli cultured by this method 600 The value reaches 60 or above, and the viable bacteria count reaches 800 × 10⁻⁶. 8 CFU / ml or above, OD 600 Both the IHA titer and the number of viable bacteria are more than twice that of existing technologies. Results of immunization of ducks with the inactivated E. coli vaccine produced using the process described in this application showed an IHA titer of 1:32 and a protection rate of 100%, providing effective protection for ducks.

[0037] The present application will be further described below with reference to specific embodiments, but is not limited to examples.

[0038] This application uses the O78 serotype SH strain as an example to describe specific embodiments, and the serotypes involved include, but are not limited to, the O78 serotype.

[0039] Example 1: Preparation and sterilization of high-density culture medium

[0040] High-density basal culture medium formulation: yeast extract 5 g / L, tryptone 10 g / L, KH₂PO₄ 35 g / L, (NH₄)₂HPO₄ 1.8 g / L, NaCl 2 g / L, citric acid 1 g / L, MgSO₄ 1 g / L, glucose 10 g / L, VB₁ 0.02 g / L. MgSO₄ and glucose were sterilized at 116℃ for 30 min, and VB₁ was sterilized by filtration. The remaining components were sterilized in a fermenter at 121℃ for 30 min.

[0041] High-density fed-batch culture medium formulation: yeast extract 25 g / L, tryptone 50 g / L, glucose 300 g / L. Glucose was sterilized separately at 116°C for 30 min, and the remaining components were sterilized at 121°C for 30 min.

[0042] Example 2: 10L High-Density Fermentation Method for Duck Escherichia coli

[0043] Seed culture preparation: Freeze-dried duck *Escherichia coli* O78 serotype SH strain was streaked onto TSA solid medium and incubated at 37℃ for 12-16 h. Single colonies were picked and inoculated into TSB liquid medium and cultured at 37℃ with shaking at 180 rpm for 10-16 h to obtain the primary seed culture. The primary seed culture was then inoculated at 1%-5% into 1L shake flasks containing 200 ml of TSB liquid medium and cultured at 37℃ with shaking at 180 rpm for 2-5 h to obtain the secondary seed culture.

[0044] Inoculation: Adjust the fermentation temperature to 37℃, pH to 6.9, stirring speed to 200 r / min, and aeration rate to 4 L / min. Inoculate the secondary seed culture into a 10 L fermenter containing 5 L of high-density basal medium at an inoculation rate of 1%-8%.

[0045] Fermentation process control method: When the DO value is below 20%, increase the rotation speed and aeration to make the DO value above 20%. After the glucose in the high-density basal medium is consumed and the DO value recovers, use high-density fed medium to feed the food at a rate of 2.0-2.5 ml / min. After 2 hours, the feeding rate is 2.8-3.2 ml / min. The DO value is kept above 20% by feeding regulation. The pH is controlled at 6.8-7.0 throughout the process by using acid and ammonia. Harvest after 8-10 hours of cultivation.

[0046] According to the method in Appendix 3405 of the Veterinary Pharmacopoeia of the People's Republic of China (2020 Edition), viable cell counts were performed on samples taken during the fermentation process. The OD values ​​of the samples taken during the fermentation process were... 600 The results of viable cell count are shown in Table 1. The results indicate that the OD (October Count) of the process in this application is at the 10L fermenter level. 600 The value reaches 60 or above, and the viable bacteria count reaches 800 × 10⁻⁶. 8 The concentrations of CFU / ml and above are approximately twice that of existing technologies.

[0047] Table 1: Oxygen Species of Duck Escherichia coli cultured in Example 2 600 Results of viable bacteria count

[0048] Example 3: Inactivation of duck Escherichia coli and preparation of vaccine

[0049] Centrifuge the fermentation broth at 8000 r / min and 4℃ for 30 min. Calculate the resuspending volume based on the viable cell count. Resuspend the bacterial sludge in sterile physiological saline. The viable cell content after resuspending should not be less than 250 × 10⁻⁶. 8 CFU / ml. Formaldehyde solution was added at 0.5% of the resuspended bacterial culture volume, and the mixture was inactivated at 37°C for 24 hours, with thorough stirring every 2 hours. After inactivation, purity testing was performed. The inactivated antigen was emulsified with mineral oil adjuvant at a 1:3 ratio to prepare the inactivated vaccine.

[0050] Example 4 Immunogenicity Test of Inactivated Duck Escherichia coli Vaccine

[0051] Select healthy and susceptible ducks aged 3 - 7 days, with 10 ducks in each group. The immunized group was subcutaneously injected with 0.3 ml of the vaccine in the neck. At the same time, 10 ducks were taken as the non - immunized control group. Blood was collected 14 days later to detect the HI titer and the ducks were challenged. For the immunized ducks, the HI titer of at least 8 ducks was not lower than 1:16, and for the control ducks, all were not higher than 1:4, which was considered qualified. Each duck was subcutaneously injected with 8×10 8 CFU / ml of SH strain bacterial solution, observed for 10 days. For the control ducks, at least 9 ducks got sick and at least 5 ducks died, and for the immunized ducks, at least 8 ducks did not get sick, which was considered qualified.

[0052] Example 5 10 - L Scale Fermentation Method with Glucose as Feed Medium

[0053] Different from the 10 - L fermentation method in Example 2 were the formulations of the basal medium and the feed medium. The basal medium was Modified Martin Broth Medium or TSB Liquid Medium or Rabbit Pasteurella Medium or the high - density basal medium of the present application, and the feed medium was 300 g / L glucose. The formulation of Modified Martin Broth Medium was peptone 5 g / L, yeast extract powder 2 g / L, glucose 20 g / L, K2HPO4 1 g / L. The formulation of TSB Medium was tryptone 17.0 g / L, soy peptone 3.0 g / L, NaCl 5.0 g / L, K2HPO4 2.5 g / L, glucose 2.5 g / L. The preparation method of the commercial medium was based on the instructions.

[0054] The results showed that when glucose was used as the feed, processes 1 - 4 could all achieve high - density culture of duck Escherichia coli. Among them, the OD 600 and viable cell count of duck Escherichia coli cultured by process 4 at harvest were significantly higher than those of other processes, indicating that when glucose was used as the feed, the high - density basal medium had the best bacteria - increasing effect. The results of the immunogenicity experiment showed that the antigens cultured in TSB Liquid Medium and Modified Martin Broth Medium were unqualified, and the HI antibody titer and protection rate did not meet the standards. The HI antibodies of the antigens cultured in Rabbit Pasteurella Medium and high - density basal medium were all qualified, and the protection rate was 8 / 10, and the antigen immunogenicity met the standards.

[0055] Table 2 Effects of Different Basal Media with Glucose as Feed on Biomass and Immunogenicity of Duck Escherichia coli at Harvest

[0056] Example 6 10 - L Scale Fermentation Method Using High - Density Feed Medium

[0057] The difference between the 10L fermentation method in Example 2 and the basal culture medium is that the basal culture medium is modified Martin broth medium, TSB liquid medium, rabbit pasteurellosis medium, or high-density basal culture medium. The commercial culture medium is prepared according to the instructions.

[0058] The results showed that compared with processes 1-4, processes 5-8 improved the viable bacterial count, HI antibody titer, and / or protection rate. Among them, the duck E. coli cultured by process 8 had the highest viable bacterial count at harvest and the vaccine produced had the best immunization effect, with a viable bacterial count reaching 800 × 10⁶. 8 With CFU / ml or higher, the HI antibody titer reached 1:32, achieving a 100% protection rate. Combining the viable cell count, HI antibody titer, and protection rate results in Tables 2 and 3, it can be concluded that compared to glucose as a feed, high-density fed-batch culture medium not only increases the viable cell count but also enhances the immunogenicity of *E. coli* in ducks. Considering the cost, source, culture effect, and immunogenicity of the culture medium, the high-density fermentation method of this application is optimal.

[0059] Table 3. Effects of different basal media as supplemental high-density fed media on biomass and immunogenicity of *Escherichia coli* at harvest.

[0060] Example 7: 100L High-Density Fermentation Method for Duck Escherichia coli

[0061] Seed culture preparation: Freeze-dried duck *Escherichia coli* O78 serotype SH strain was streaked onto TSA solid medium and incubated at 37℃ for 12-16 h. Single colonies were picked and inoculated into TSB liquid medium and cultured at 37℃ with shaking at 180 rpm for 10-16 h to obtain primary seed culture. 200 ml of primary seed culture was inoculated into a 10 L fermenter containing 5 L of TSB medium and incubated at 37℃ for 2-5 h to obtain secondary seed culture.

[0062] Inoculation: Adjust the fermentation temperature to 37℃, pH to 6.9, stirring speed to 100 r / min, and aeration rate to 20 L / min. Inoculate the secondary seed culture into the high-density basal medium in a 100 L fermenter at an inoculation rate of 1%-8%.

[0063] Fermentation process control method: When the DO value is below 20%, increase the rotation speed and aeration to make the DO value above 20%. After the glucose in the high-density basal medium is consumed and the DO value recovers, use high-density fed medium to feed the food at a feeding rate of 20-25 ml / min. After 2 hours, the feeding rate is increased to 28-32 ml / min. The DO value is maintained at no less than 20% by feeding regulation. The pH is controlled at 6.8-7.0 throughout the process by using acid and ammonia. Harvest after 8-10 hours of cultivation.

[0064] OD of samples taken during fermentation600 The results for viable cell count are shown in Table 4. The results indicate that the OD at harvest from a 100L fermenter... 600 The viable bacterial counts were 66.0 and 827 × 10⁻⁶, respectively. 8 The CFU / ml value showed no significant difference from the biomass in a 10L fermenter, indicating that the duck Escherichia coli culture process described in this application is easy to scale up and suitable for industrial production.

[0065] Table 4 OD of Escherichia coli in ducks at a fermentation scale of 100L 600 Results of viable bacteria count

[0066] Unless otherwise defined, all technical and scientific terms used throughout this application have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. In case of any inconsistency, the meaning as stated in this application or derived from the content described herein shall prevail. Furthermore, the terminology used in this description is for the purpose of describing embodiments of this application only and is not intended to limit this application.

[0067] Note that the above are merely preferred embodiments and the technical principles employed in this application. Those skilled in the art will understand that this application is not limited to the specific embodiments described herein, and various obvious changes, readjustments, and substitutions can be made without departing from the scope of protection of this application. Therefore, although this application has been described in detail through the above embodiments, this application is not limited to the above embodiments. Many other equivalent embodiments may be included without departing from the technical concept of this application, all of which fall within the scope of protection of this application.

Claims

1. A high-density basal culture medium for culturing duck Escherichia coli, characterized in that, The culture medium is formulated as follows: yeast extract 2-15 g / L, tryptone 5-20 g / L, KH2PO4 34-36 g / L, (NH4)2HPO4 1.6-2.0 g / L, NaCl 1.5-2.5 g / L, citric acid 0.5-3 g / L, MgSO4 0.5-2 g / L, glucose 2-20 g / L and VB1 0.01-0.1 g / L.

2. The high-density basal culture medium according to claim 1, characterized in that, The culture medium is formulated as follows: yeast extract 4-6 g / L, tryptone 9-11 g / L, KH2PO4 34-36 g / L, (NH4)2HPO4 1.7-1.9 g / L, NaCl 1.9-2.1 g / L, citric acid 0.9-1.1 g / L, MgSO4 0.9-1.1 g / L, glucose 9-11 g / L and VB1 0.01-0.03 g / L; Preferably, the culture medium is formulated as follows: yeast extract 5 g / L, tryptone 10 g / L, KH2PO4 35 g / L, (NH4)2HPO4 1.8 g / L, NaCl 2 g / L, citric acid 1 g / L, MgSO4 1 g / L, glucose 10 g / L and VB1 0.02 g / L.

3. A high-density fed culture medium for culturing duck Escherichia coli, characterized in that, The culture medium is formulated as follows: glucose 50-500 g / L, yeast extract 10-50 g / L and tryptone 20-100 g / L.

4. The high-density fed culture medium according to claim 3, characterized in that, The culture medium is formulated as follows: glucose 290-310 g / L, yeast extract 24-26 g / L and tryptone 49-51 g / L; Preferably, the culture medium is formulated as follows: 300 g / L glucose, 25 g / L yeast extract and 50 g / L tryptone.

5. A high-density fermentation method for duck Escherichia coli, characterized in that, Includes the following steps: The secondary seed culture is inoculated into a fermenter containing the high-density basal medium as described in claim 1 or 2 at an inoculation rate of 1%-8%. In the initial stage of fermentation, when the dissolved oxygen (DO) is below 20%, the DO is increased to above 20% by increasing the rotation speed and aeration. After the glucose in the high-density basal medium is consumed and the DO recovers, the high-density fed medium as described in claim 3 or 4 is used to feed the culture. The DO is kept above 20% by feeding the culture. The pH is controlled at 6.8-7.0 and the fermentation temperature is 36-38℃ throughout the process.

6. The high-density fermentation method according to claim 5, characterized in that, The preparation of the secondary seed solution includes the following steps: Streaking of freeze-dried duck Escherichia coli onto TSA solid medium and incubating at 37°C for 12-16 hours; picking single colonies and inoculating them into TSB liquid medium and incubating at 37°C with shaking at 180 rpm for 10-16 hours to obtain the primary seed culture; inoculating the primary seed culture at 1%-5% into shake flasks or fermenters containing TSB liquid medium and incubating at 37°C for 2-5 hours to obtain the secondary seed culture.

7. The high-density fermentation method according to claim 5 or 6, characterized in that, The cells are harvested after 8-10 hours of fermentation.

8. The application of the high-density fermentation method according to any one of claims 5 to 7 in the preparation of duck Escherichia coli inactivated vaccine.

9. The duck Escherichia coli prepared by the high-density fermentation method according to any one of claims 5 to 7.

10. The use of the duck Escherichia coli according to claim 9 in duck Escherichia coli inactivated vaccine.

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