CN108865941B

CN108865941B - High-density fermentation method of duck escherichia coli

Info

Publication number: CN108865941B
Application number: CN201810782440.5A
Authority: CN
Inventors: 龚凤平; 袁建丰; 马海彬; 罗梦萍
Original assignee: Guangdong Haid Animal Husbandry And Veterinary Research Institute Co ltd
Current assignee: Guangdong Haid Animal Husbandry And Veterinary Research Institute Co ltd
Priority date: 2018-07-17
Filing date: 2018-07-17
Publication date: 2021-08-03
Anticipated expiration: 2038-07-17
Also published as: CN108865941A

Abstract

The invention discloses a high-density fermentation method of duck escherichia coli, which is based on a high-density fermentation culture medium,and (3) performing high-density fermentation on duck escherichia coli by using a fermentation tank. In the fermentation process, the pH value is 7.0-7.5, the temperature is 36.5-37.5 ℃, the rotating speed is 90-420 rpm, and the air ventilation volume is 1.2-5.2L/min. Adding appropriate amount of defoaming agent before inoculation, and waiting for bacterial OD after inoculation₆₀₀When the nm reaches 4.0-6.0, feeding 45-55 (w/v)% of glucose solution at a flow rate of 10-25 mL/h/L, wherein the fed-in amount of glucose is 50-100 g per liter of high-density fermentation medium. The high-density fermentation method of duck escherichia coli is based on a high-density fermentation culture medium, is rich in nutrition, can realize high-density fermentation of duck escherichia coli by feeding glucose, and can be used for large-scale fermentation production of duck escherichia coli vaccines.

Description

High-density fermentation method of duck escherichia coli

Technical Field

The invention belongs to the technical field of microbial fermentation, and particularly relates to a high-density fermentation method of duck escherichia coli.

Background

The duck colibacillosis is caused by pathogenic escherichia coli, and the pathological manifestations mainly include acute septicemia, air sacculitis, panophthalmitis, joint synovitis, salpingitis, peritonitis and the like. With the continuous expansion of the culture scale and the continuous deterioration of the water area environment for raising the waterfowls, colibacillosis becomes one of the more serious bacterial infectious diseases which harm the waterfowl culture at present. Because the antibiotics are widely used clinically at present to treat bacterial diseases such as escherichia coli and the like, a large number of drug-resistant strains are generated, and therefore, the vaccination in the escherichia coli prevention and treatment is an effective prevention way.

For bacterial vaccines, how to increase the number of bacterial cells (i.e., antigen content) is one of the key techniques for bacterial vaccine research. Generally, vaccines with high antigen content produce higher antibody levels and longer antibody duration after immunization of the body. The bacterial high-density fermentation technology has become a common culture technology means in the preparation of bacterial vaccines, and the technology can improve the concentration of thalli in a unit volume of culture solution and further improve the volume yield.

Pathogenic escherichia coli is facultative anaerobe, the requirement on culture conditions is not high, the pathogenic escherichia coli can normally grow in a common LB broth culture medium, however, high-density fermentation of the pathogenic escherichia coli is difficult to realize, and the high-density fermentation of pathogenic duck escherichia coli is more rarely reported.

Disclosure of Invention

The invention aims to provide a high-density fermentation method of duck escherichia coli, which can realize large-scale high-density fermentation of pathogenic duck escherichia coli, effectively improve the volume yield and reduce the production cost.

The technical scheme adopted by the invention is as follows:

a high-density fermentation method of duck escherichia coli comprises the following steps: inoculating duck escherichia coli into a high-density fermentation medium for fermentation, wherein in the fermentation process, the pH of fermentation liquor is adjusted to 7.0-7.5, the fermentation temperature is adjusted to 36.5-37.5 ℃, the stirring speed is 90-420 rpm, and the air ventilation of the fermentation liquor is 1.2-5.2L/min; when fermentation broth OD₆₀₀Feeding glucose solution when the nm reaches 4.0-6.0, and when the OD of the fermentation liquor is₆₀₀When the nm value stops increasing, the fermentation is finished;

the formula of the high-density fermentation medium is as follows: the water per liter contains 30.0-40.0 g of yeast extract, 5.0-15.0 g of tryptone, 5.0-15.0 g of soybean peptone, 5.0-10.0 g of hydrolyzed milk protein, 5.0-10.0 g of casein peptone, 4.0-7.0 g of gastric peptone, 1.0-3.0 g of malt extract powder, 2.0-5.0 g of dipotassium hydrogen phosphate, 1.0-3.0 mL of glycerol, 2.0-10.0 mg of iron salt, 2.0-10.0 mg of manganese salt and 2.0-10.0 mg of aluminum salt.

Furthermore, before inoculation, a proper amount of antifoaming agent is added into the high-density fermentation medium.

Furthermore, the speed of feeding the glucose solution is 10-25 mL/h/L.

Further, the concentration of the glucose solution is 45-55 (w/v)%.

Further, the amount of the fed-batch glucose is 50.0-100.0 g of glucose fed-batch per liter of the high-density fermentation medium.

Further, the iron salt is selected from at least one of ferric nitrate, ferric chloride and ferric sulfate.

Further, the manganese salt is selected from at least one of manganese sulfate and manganese chloride.

Further, the aluminum salt is at least one selected from aluminum sulfate and aluminum chloride.

Furthermore, the inoculation amount of duck escherichia coli is 2% -10%.

Further, the initial stirring speed of fermentation is 90-120 rpm, and the air ventilation in the fermentation liquor is 1.2-1.7L/min; after fermenting for 0.4-0.6 h, stirring at a rotating speed of 130-170 rpm, and ventilating air in the fermentation liquor at a rate of 1.8-2.2L/min; after fermenting for 0.8-1.2 h, stirring at a rotating speed of 180-220 rpm, and ventilating air in the fermentation liquor at a rate of 2.8-3.2L/min; after fermentation is carried out for 1.8-2.2 h, the stirring speed is 280-320 rpm, and the air ventilation of the fermentation liquor is 3.8-4.2L/min; after fermenting for 2.8-3.2 h, the stirring speed is 380-420 rpm, and the air ventilation of the fermentation liquor is 4.8-5.2L/min.

The invention has the beneficial effects that:

(1) the duck escherichia coli high-density fermentation culture base used in the method is beneficial to the growth of pathogenic duck escherichia coli, and the preparation method is simple and is convenient for large-scale production and use.

(2) According to the high-density fermentation method for duck escherichia coli, a specific high-density fermentation culture medium is used as a fermentation culture medium, and glucose is fed in a specific mode in the fermentation process, so that high-density fermentation of duck escherichia coli can be realized, and the method can be used for large-scale production of duck escherichia coli vaccines. The high-density fermentation method of duck escherichia coli can effectively improve the concentration of thalli in a unit volume of culture solution, further improve the volume yield and reduce the production cost.

Drawings

FIG. 1 growth curves of duck E.coli in four examples. Example 1: namely the preferred embodiment of the high density fermentation process; comparative example 1: namely, on the basis of a high-density fermentation culture medium, no glucose is fed; comparative example 2: adding glucose in a fed-batch manner on the basis of LB broth culture medium; comparative example 3: that is, based on LB broth, no glucose was fed.

Detailed Description

The present invention will be further described with reference to the following examples.

The invention provides a high-density fermentation method of duck escherichia coli, which is used for performing high-density fermentation on duck escherichia coli by using a high-density fermentation culture medium. In the fermentation process, glucose is fed in a feeding manner at a constant speed, and the bacteria can realize high-density fermentation by controlling the air ventilation, pH, temperature, rotating speed and glucose flow rate.

The source and serotype of duck escherichia coli are not particularly limited, and in the specific embodiment of the invention, the duck escherichia coli adopted is O78 serotype duck escherichia coli virulent strain. The growth condition of duck escherichia coli is different from that of the existing laboratory engineering bacteria (such as recombinant escherichia coli), and the high-density fermentation of the duck escherichia coli virulent strain is difficult to realize under the existing culture conditions.

The invention has no special limitation on the types and preparation methods of pH adjusting reagents (acid and alkali) and defoaming agents, and can be prepared by adopting conventional reagents and preparation methods in the technical field.

In the specific embodiment of the invention, the seed bacterial liquid for fermenting the duck large intestine stems is cultured by adopting the corresponding fermentation culture medium in each embodiment. Firstly, recovering duck escherichia coli seed bacteria, taking out an O78 serotype duck escherichia coli virulent strain from a refrigerator at the temperature of-80 ℃, after thawing, streaking and inoculating the duck escherichia coli virulent strain on an LB (Langerhans Blume) common agar culture medium, and placing the duck escherichia coli strain in an incubator at the temperature of 37 ℃ for culturing for 12-16 hours. Secondly, preparing a seed bacterial liquid for duck escherichia coli fermentation, picking duck escherichia coli monoclonal colonies on an LB common agar plate by using an inoculating loop, respectively inoculating the duck escherichia coli monoclonal colonies in 5mL of corresponding fermentation culture media in respective embodiments or comparative examples, and culturing for 12-16 hours at 37 ℃ by using a shaking table at 180 rpm. According to the proportion of 1:100, 1mL of cultured monoclonal bacteria liquid is taken and respectively inoculated into 100mL of fermentation culture medium corresponding to each embodiment, the culture is carried out by a shaking table at 37 ℃ and under the condition of 180rpm until the logarithmic growth phase is reached, and the initial concentration OD of the seed bacteria is₆₀₀nm is controlled to be between 1.0 and 3.0, the seed bacteria is used as seed bacteria for fermentation, and the seed bacteria is inoculated into a fermentation tank containing fermentation culture media corresponding to the respective embodiments according to the proportion of 2 to 10 percent for fermentation.

The following is a detailed description of preferred embodiments of the invention (using a Saedolis 5L fermentor).

Example 1

The growth curves and the thallus morphology of duck escherichia coli (O78 serotype) under the conditions of four different fermentation methods are compared. The differences between the four fermentation processes are shown in the following table:

TABLE 1 Difference between the four fermentation processes

Example 1	On the basis of the high-density fermentation medium, glucose is fed
		Comparative example 1	Based on the high-density fermentation medium of the invention, no glucose is fed
Comparative example 2	Adding glucose in LB broth as base
		Comparative example 3	Based on LB broth culture medium, no glucose is fed

The specific fermentation method of example 1 was as follows (medium volume 1L):

(1) preparing an optimal duck escherichia coli high-density fermentation culture medium and related reagents, and performing tank extinguishing treatment and assembly of a fermentation tank:

a. preferred high density fermentation media: preparing a culture medium with the volume of 1L, weighing 40.0g of yeast extract, 10.0g of tryptone, 10.0g of soybean peptone, 5.0g of hydrolyzed milk protein, 10.0g of casein peptone, 5.0g of gastrone peptone, 2.0g of malt extract powder, 5.0g of dipotassium hydrogen phosphate, 2.0mL of glycerol, 5.0mg of ferric nitrate, 5.0mg of manganese sulfate and 5.0mg of aluminum sulfate, heating, dissolving and fixing the components in 1L of water, transferring the components into a 5L of sialus fermentation tank, and carrying out autoclaving at 121 ℃ for 15 min.

2M sulfuric acid solution: 109mL of 98% concentrated sulfuric acid is measured and slowly added into 800mL of water, after cooling, the water is added to a constant volume of 1L, the mixture is transferred into an acid pump feeding bottle of a fermentation tank, and autoclaving is carried out for 15min at 121 ℃.

c.6M aqueous Ammonia solution: measuring 230mL of pure ammonia water solution, adding water to a constant volume of 1L, filtering and sterilizing by a 0.22um filter, performing aseptic operation, and transferring into a sterilized alkaline pump feeding bottle.

d.1 (v/v)% 204 antifoam solution: measuring 1mL 204 defoamer, quantitatively adding into 1L water, transferring into a feeding bottle of a defoaming pump of a fermentation tank, and carrying out autoclaving at 121 ℃ for 15 min.

e.50 (w/v)% glucose solution: weighing 500g of glucose, heating to dissolve, dissolving in 1L of water, filtering with 0.22um filter for sterilization, performing aseptic operation, and transferring into sterilized glucose feeding bottle.

(2) Preparing a fermented seed solution, as described above, taking out the O78 serotype duck escherichia coli virulent strain which is separated, identified and stored in the laboratory from a refrigerator at the temperature of-80 ℃, thawing the virulent strain, streaking the virulent strain on an LB (Luria Bertani) common agar culture medium, and culturing the virulent strain in an incubator at the temperature of 37 ℃ for 12 to 16 hours. Then, a duck Escherichia coli monoclonal colony on an LB common agar plate is picked by using an inoculating loop, inoculated into 5mL of an optimized high-density fermentation medium, and cultured for 12-16 hours at 37 ℃ by a shaking table and 180 rpm. According to the proportion of 1:100, 1mL of cultured monoclonal bacteria liquid is inoculated into 100mL of optimized high-density fermentation medium, the culture is carried out in a shaker at 37 ℃ and under the condition of 180rpm until the logarithmic growth phase, and the optimized initial concentration OD of the seed bacteria₆₀₀The nm is 1.0, and the strain is used as a seed bacterium for fermentation.

(3) After the tank of the fermentation tank is extinguished, after the assembly and connection are finished, 1L of high-density fermentation medium is added; adding 20-50 mL of 204 (v/v)% defoaming agent solution into a high-density fermentation medium by a peristaltic pump before inoculation, inoculating 10% of the prepared seed bacteria for fermentation into a fermentation tank containing the preferred high-density fermentation medium, and fermenting at the pH of 7.0 and the temperature of 37 ℃. The rotational speed setting was stepped up from 100rpmThe air aeration is gradually increased from 1.5L/min to 5L/min (the fermentation liquid OD is increased by time points)₆₀₀About 3-4 hours are needed when the nm reaches 5.0-8.0, the rotating speed is set to be 100rpm when bacteria are just inoculated, the ventilation rate is 1.5L/min, the rotating speed is set to be 150rpm and 2L/min for 0.5h, the rotating speed is set to be 200rpm and 3L/min for 1h, the rotating speed is set to be 300rpm and 4L/min for 2h, and the rotating speed is set to be 400rpm and 5L/min for 3 h. ). The pH value of the whole fermentation process is controlled by automatically feeding 6M ammonia water solution and 2M sulfuric acid solution by an acid-base pump, the whole duck escherichia coli fermentation process can produce acid, and the consumption of ammonia water is high.

(4) When fermentation broth OD₆₀₀When the nm reaches about 5.0, 200mL of 50 (w/v)% glucose solution is slowly added through a constant-speed feeding pump at the flow rate of 15 mL/h/L. The glucose is added automatically, the air ventilation is fixed at 5L/min, and the rotating speed is 400 rpm. When OD of fermentation broth₆₀₀When the nm value stops increasing, the fermentation is finished.

Samples were taken every 2 hours throughout the fermentation period and OD was determined₆₀₀And (5) nm value, and drawing a growth curve. And (4) taking the logarithmic growth phase bacterial liquid, and observing the bacterial form by gram staining.

Comparative example 1

This comparative example was based on the high-density fermentation medium of the invention, and no glucose was fed. Except when the fermentation broth OD is₆₀₀When the nm reached 5.0, no glucose was fed, and all the other operations were the same as in example 1.

Comparative example 2

The comparative example was based on LB broth, with glucose fed-through. The specific operation steps are as follows (the volume of the culture medium is 1L):

the method for preparing 1L of LB broth culture medium comprises the following steps: weighing yeast extract 5.0g, tryptone 10.0g, and sodium chloride 10.0g, dissolving and dissolving the above components in 1L water, transferring into 5L Sadoris fermenter, and autoclaving at 121 deg.C for 15 min. And (3) the rest reagents: the preparation methods of 2M sulfuric acid solution, 6M ammonia solution, 1 (v/v)% 204 antifoaming agent solution, and 50 (w/v)% glucose solution were the same as in example 1.

Next, the preparation of the fermentation seed liquid was the same as in example 1 except for the difference in the fermentation medium used.

Finally, the whole fermentation process operation was identical to that of example 1, the only difference being when the OD of the fermentation broth was₆₀₀About 4.0 nm (OD without addition of glucose due to nutrient limitation of LB broth medium)₆₀₀The rise of nm to 5.0 takes a long time and the bacteria grow slowly. OD₆₀₀The bacteria are in logarithmic growth phase at nm around 4.0. ) 200mL of 50 (w/v)% glucose solution was slowly fed through a constant rate feed pump at a flow rate of 15 mL/h/L. When OD of fermentation broth₆₀₀When the nm value stops increasing, the fermentation is finished.

Comparative example 3

The comparative example was based on LB broth without feeding glucose. The specific operation steps are as follows:

the LB broth used was the same as in comparative example 2, with the remaining reagents: the preparation methods of the 2M sulfuric acid solution, the 6M ammonia solution and the 1 (v/v)% 204 antifoaming agent solution are the same as those in example 1, and a glucose solution is not required to be prepared. The preparation method of seed bacteria for fermentation is the same as that of comparative example 2. The whole fermentation process operation was identical to that of example 1, the only difference being that when the fermentation broth OD was reached₆₀₀When the nm reaches about 4.0, no glucose is fed. When OD of fermentation broth₆₀₀When the nm value stops increasing, the fermentation is finished.

Analysis of results

Comparing the shapes of the cells in the logarithmic growth phase of duck escherichia coli in the four fermentation methods (example 1 and comparative examples 1-3), gram staining is negative, corynebacterium parvum is not obvious, and the bacteria can normally grow by using the high-density fermentation method for fermenting duck escherichia coli.

Comparing the four fermentation methods (example 1 and comparative examples 1-3), the growth curves of all groups of duck escherichia coli are shown in figure 1, the growth speed of the duck escherichia coli by using the high-density fermentation method is obviously higher than that of the duck escherichia coli by using the other 3 fermentation methods, and the OD of the duck escherichia coli after 12h fermentation by using the method is shown in the invention₆₀₀The nm value reaches 26.36, while the OD of the fermentation method of comparative examples 1-3₆₀₀The nm values were 10.68, 13.78, 7.87, respectively, and the OD of the bacteria of comparative example 2 and comparative example 3₆₀₀The nm value has begun to decrease. The method of the present invention is to determine the cell density (OD) of the bacteria in the stationary phase₆₀₀The nm value is 30.51) is also obviously higher than that of the OD of the comparative examples 1 to 3 (the OD of the comparative examples 1 to 3)₆₀₀The nm values are respectively 11.87, 13.81 and 7.98).

In conclusion, the growth rate and the growth concentration of duck escherichia coli in the high-density fermentation method of the invention are higher than those in the comparative example 1 in the embodiment 1, and it is proved that the feeding of glucose in the fermentation process by using the fermentation tank is very important for the fermentation of duck escherichia coli.

The growth speed and the growth concentration of duck escherichia coli in the embodiment 1 of the high-density fermentation method are higher than those in the comparative example 2, which shows that the high-density fermentation medium is very suitable for the growth of duck escherichia coli.

The method combines the high-density fermentation culture medium with the glucose fed in the fermentation process, so that the OD of the bacteria concentration in the stable period of the bacteria₆₀₀The nm value can reach 30.51 which is higher than the OD of the thalli concentration in the stationary phase of the comparative example 1₆₀₀nm value (11.87) and OD of comparative example 2₆₀₀nm value (13.81) and comparative example 3OD₆₀₀The nm value (7.98) shows that in the invention, unexpected synergistic effect is generated between the high-density fermentation medium and the specific 'fed-batch glucose' method, the fermentation density of duck escherichia coli is obviously improved, and the method can be applied to large-scale production of duck escherichia coli vaccines.

Example 2 high-density fermentation method of duck escherichia coli

Inoculating duck Escherichia coli to high-density fermented soybean with the inoculation amount of 2%Fermenting in a fermentation culture medium (adding a proper amount of sterilized defoaming agent into the high-density fermentation culture medium before inoculation), and adjusting the pH value of the fermentation liquor to 7.0 and the fermentation temperature to 37.5 ℃ by using a sulfuric acid solution and ammonia water in the fermentation process; the rotation speed is gradually increased from 100rpm to 400rpm, the air aeration is gradually increased from 1.5L/min to 5L/min (the fermentation liquid OD is increased at different time points)₆₀₀About 3-4 hours are needed when nm reaches 5.0-8.0, the rotating speed is set to be 100rpm when bacteria are just inoculated, the ventilation rate is 1.5L/min, 0.5h is set to be 150rpm and 2L/min, 1h is set to be 200rpm and 3L/min, 2h is set to be 300rpm and 4L/min, and after 3h, 400rpm and 5L/min are set; when fermentation broth OD₆₀₀When the nm reaches 6.0, feeding sterile 55 (w/v)% glucose solution at a speed of 25 mL/h/L; the amount of glucose fed-in was 100.0g of glucose per liter of the high-density fermentation medium. The glucose is added automatically, the air ventilation is fixed at 5L/min, and the rotating speed is 400 rpm. When OD of fermentation broth₆₀₀When the nm value stops increasing, the fermentation is finished.

The formula of the high-density fermentation medium is as follows: the water per liter contained 40.0g of yeast extract, 15.0g of tryptone, 5.0g of soyabean peptone, 10.0g of hydrolyzed milk protein, 5.0g of casein peptone, 7.0g of gastric peptone, 3.0g of malt extract powder, 2.0g of dipotassium hydrogen phosphate, 3.0mL of glycerol, 2.0mg of ferric chloride, 10.0mg of manganese chloride and 2.0mg of aluminum chloride.

Example 3 high-density fermentation method of duck escherichia coli

Inoculating duck escherichia coli into a high-density fermentation culture medium according to the inoculation amount of 10% for fermentation (adding a proper amount of sterilized defoaming agent into the high-density fermentation culture medium before inoculation), and adjusting the pH value of a fermentation liquid to 7.5 and the fermentation temperature to 36.5 ℃ by using a sulfuric acid solution and ammonia water in the fermentation process; the rotation speed is gradually increased from 100rpm to 400rpm, the air aeration is gradually increased from 1.5L/min to 5L/min (the fermentation liquid OD is increased at different time points)₆₀₀About 3-4 hours are needed when nm reaches 5.0-8.0, the rotating speed is set to be 100rpm when bacteria are just inoculated, the ventilation rate is 1.5L/min, 0.5h is set to be 150rpm and 2L/min, 1h is set to be 200rpm and 3L/min, 2h is set to be 300rpm and 4L/min, and after 3h, 400rpm and 5L/min are set; when fermentation broth OD₆₀₀Feeding sterile concentrate when nm reaches 4.0The glucose solution with the degree of 45 (w/v)% is fed at the speed of 10 mL/h/L; the amount of glucose fed-in was 50.0g of glucose per liter of the high-density fermentation medium. The glucose is added automatically, the air ventilation is fixed at 5L/min, and the rotating speed is 400 rpm. When OD of fermentation broth₆₀₀When the nm value stops increasing, the fermentation is finished.

The formula of the high-density fermentation medium is as follows: each liter of water contains 30.0g of yeast extract, 5.0g of tryptone, 15.0g of soybean peptone, 5.0g of hydrolyzed milk protein, 10.0g of casein peptone, 4.0g of gastric peptone, 13.0g of malt extract powder, 5.0g of dipotassium hydrogen phosphate, 1.0mL of glycerol, 10.0mg of ferric chloride, 2.0mg of manganese chloride and 10.0mg of aluminum chloride.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims

1. A high-density fermentation method of duck Escherichia coli O78 is characterized by comprising the following steps: inoculating duck escherichia coli O78 into a high-density fermentation culture medium for fermentation, wherein in the fermentation process, the pH of fermentation liquor is adjusted to 7.0-7.5, the fermentation temperature is 36.5-37.5 ℃, the stirring speed is 90-420 rpm, and the air ventilation of the fermentation liquor is 1.2-5.2L/min; when fermentation broth OD_600nmFeeding glucose solution when the OD of the fermentation liquor reaches 4.0-6.0_600nmWhen the value stops increasing, the fermentation is finished;

the formula of the high-density fermentation medium is as follows: 30.0-40.0 g of yeast extract, 5.0-15.0 g of tryptone, 5.0-15.0 g of soybean peptone, 5.0-10.0 g of hydrolyzed milk protein, 5.0-10.0 g of casein peptone, 4.0-7.0 g of gastric peptone, 1.0-3.0 g of malt extract powder, 2.0-5.0 g of dipotassium hydrogen phosphate, 1.0-3.0 mL of glycerol, 2.0-10.0 mg of iron salt, 2.0-10.0 mg of manganese salt and 2.0-10.0 mg of aluminum salt in each liter of water;

the speed of feeding the glucose solution is 10-25 mL/h/L;

wherein the initial stirring speed of fermentation is 90-120 rpm, and the air ventilation in the fermentation liquid is 1.2-1.7L/min; after fermenting for 0.4-0.6 h, stirring at a rotating speed of 130-170 rpm, and ventilating air in the fermentation liquor at a rate of 1.8-2.2L/min; after fermenting for 0.8-1.2 h, stirring at a rotating speed of 180-220 rpm, and ventilating air in the fermentation liquor at a rate of 2.8-3.2L/min; after fermentation is carried out for 1.8-2.2 h, the stirring speed is 280-320 rpm, and the air ventilation of the fermentation liquor is 3.8-4.2L/min; after fermenting for 2.8-3.2 h, the stirring speed is 380-420 rpm, and the air ventilation of the fermentation liquor is 4.8-5.2L/min.

2. The method of claim 1, wherein an amount of antifoaming agent is added to the high-density fermentation medium prior to inoculation.

3. The method according to claim 1, wherein the concentration of the glucose solution is 45 to 55 (w/v)%.

4. The method according to claim 1, wherein the amount of fed-in glucose is 50.0 to 100.0g per liter of the high-density fermentation medium.

5. The method of claim 1, wherein the iron salt is selected from at least one of ferric nitrate, ferric chloride, and ferric sulfate.

6. The method according to claim 1, wherein the manganese salt is selected from at least one of manganese sulfate and manganese chloride.

7. The method according to claim 1, wherein the aluminum salt is selected from at least one of aluminum sulfate and aluminum chloride.

8. The method of claim 1, wherein the amount of inoculated duck E.coli is 2% to 10%.