CN112725231A - Fermentation method for large-scale efficient expression of supercoiled plasmid DNA by escherichia coli - Google Patents
Fermentation method for large-scale efficient expression of supercoiled plasmid DNA by escherichia coli Download PDFInfo
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Abstract
The invention discloses a fermentation method for large-scale and high-efficiency expression of supercoiled plasmid DNA of escherichia coli, which changes the metabolic mode of the escherichia coli through temperature regulation and control transformation in the fermentation process, reduces the accumulation of biomass of thalli, and simultaneously improves the expression quantity of the supercoiled plasmid DNA so as to meet the huge demand on the supercoiled plasmid DNA in gene therapy. Compared with the yield of 100-1000 mg/L obtained by the conventional process for fermenting the supercoiled plasmid DNA by escherichia coli, the yield level of 500-1000mg/L can be obtained by adopting the fermentation method.
Description
Technical Field
The invention belongs to the technical field of biological engineering, relates to a fermentation method of supercoiled plasmid DNA for gene therapy in the technical field of biology, and particularly relates to a high-yield fermentation method for changing a thallus metabolism mode through temperature conversion regulation.
Background
Gene therapy (gene therapy) refers to the introduction of exogenous normal genes into target cells to correct or compensate for diseases caused by defective and abnormal genes, so as to achieve the therapeutic goal. The method also includes the technical application of transgenosis and the like, namely, the exogenous gene is inserted into a proper receptor cell of a patient through a gene transfer technology, so that a product made of the exogenous gene can treat a certain disease. In a broad sense, gene therapy may also include measures and new technologies taken from the DNA level to treat certain diseases.
Gene therapy mainly takes the form of both viral and non-viral vectors for transferring a gene of interest into a recipient cell of a patient. The current predominant mode of gene therapy is the use of viral vectors. The most commonly used viral vectors at present include Adeno-associated virus (AVV), Lentivirus (LV), Adenovirus (AdV), Retrovirus (Retrovirus, RV), and the like. These viral vectors can be packaged as supercoiled plasmid DNA, which is the basis for gene therapy and is therefore in great demand.
Supercoiled plasmid DNA is used as a biological product for human, and has strict requirements on production and quality control. The one-time totally-enclosed production process is a development trend of the gene therapy industry, the process is more stable and controllable, and the product quality is safer and more effective. The current mainstream production of supercoiled plasmid DNA is achieved by fermentation of E.coli using NTC3018 (batch) and NTC3019 (fed-batch) media, which are optimized for the specific components of both batch and fed-batch processes. For example, in which glycerol is used instead of glucose as a carbon source, the concentrations of trace elements and magnesium sulfate are also optimized.
However, based on the influence of the molecular weight of strains and plasmid vectors in the prior art for producing supercoiled plasmid DNA, the final yield is generally 50-400mg/L, and few processes can reach 500-600mg/L, but cannot maintain stable high-level expression, wherein the yield of the strains per unit weight obtained by a large proportion of fermentation processes is 0.5-2 mg/g. The problem with the existing technology is that the fermentation yield is low, and the yield of the bacteria per weight is low, and such expression level causes considerable workload for downstream purification and separation, because the purification and separation of the supercoiled plasmid DNA unit batch is still by weight. Thalli obtained by fermenting and collecting supercoiled plasmid DNA at a low expression level necessarily comprises highly expressed host DNA, host protein and RNA, which are impurities required to be removed in the purification and separation process, and the pressure of separation and purification is also increased. Therefore, how to effectively increase the total yield and the expression of the bacteria per unit weight is particularly important, how to increase the total yield and reduce the replication production of the bacteria to reduce the weight of the received bacteria is a technical problem to be solved urgently by the technical staff in the field.
Disclosure of Invention
The invention aims to solve the problems and provide a fermentation method for efficiently expressing supercoiled plasmid DNA in large scale by escherichia coli, which changes the metabolic mode of the escherichia coli through temperature regulation and control transformation in the fermentation process, reduces the biomass accumulation of thalli and simultaneously improves the expression quantity of the supercoiled plasmid DNA so as to meet the huge demand of the supercoiled plasmid DNA in gene therapy.
The purpose of the invention is realized as follows:
the invention relates to a fermentation method for large-scale and high-efficiency expression of supercoiled plasmid DNA by escherichia coli, which comprises the following steps:
1) and (3) flask shaking inoculation: taking out the preserved working seeds from the strain library, recovering in a water bath, inoculating into a shake flask in a sterile environment, culturing at 30 ℃ for 6-10 hours, and preparing to inoculate a fermentation tank;
2) preparing a fermentation tank: fully dissolving a fermentation culture medium and a supplement culture medium, fixing the volume to a corresponding volume, filling the volume into a fermentation tank and a supplement bottle, and sterilizing by high-pressure steam sterilization;
3) cooling the tank body: after the sterilization is finished, cooling to room temperature, connecting cooling water and condensate water pipelines, connecting a pH electrode, an oxygen dissolving electrode and a temperature electrode, and keeping the fermentation temperature of the tank constant;
4) setting parameters: setting the stirring rotation speed of a fermentation tank at 200-300rpm, the ventilation capacity at 2-3L/min, setting the pH value at 7.0 +/-0.2, starting an automatic control alkali and acid supplementing bottle for adjustment, setting the pressure of the tank body at 0.5-0.6bar, setting the initial fermentation temperature at 100% point of dissolved oxygen electrode calibration under the conditions of maximum rotation speed and maximum ventilation, and setting the initial fermentation temperature at 30-35 ℃;
5) inoculation: under the protection of flame, inoculating the first-stage seeds in the shake flask into a fermentation tank body;
6) and (3) regulating and controlling the fermentation process: after inoculation, the DO is maintained at about 40% by increasing the rotating speed and the ventilation volume, and after sudden rising and rebounding of the DO, material supplement can be started, and the material supplement is linked with the DO to keep the DO at 40%;
7) temperature conversion: when DO parameter is stabilized within 0-2 hours after 40% +/-10%, the fermentation temperature conversion temperature of the tank body is 37-48 ℃;
8) and (3) collecting thalli: after the fermentation is finished, the thalli are collected by high-speed centrifugation and stored in a refrigerator at the temperature of minus 20 ℃.
The fermentation method for the escherichia coli large-scale high-efficiency expression supercoiled plasmid DNA is characterized in that the working seeds in the step 1) are stored in a refrigerator at the temperature of-80 ℃; the temperature range of the resuscitation water bath is 30-42 ℃; the inoculation can be arranged by culturing until OD600 reaches 0.8-1.1, and the selected competent cell is E.coli Stbl 3.
The fermentation method for large-scale efficient expression of supercoiled plasmid DNA by using escherichia coli is characterized in that in the step 2), a pH electrode and an oxygen dissolving electrode are arranged on a fermentation tank, an electrode joint is connected, the pH electrode and the oxygen dissolving electrode are calibrated, the calibration point of the pH electrode is 4.0 and 7.0, the zero point of the oxygen dissolving electrode is calibrated by saturated sodium sulfite, and the high-pressure steam sterilization condition is 121 ℃ and 20 min.
The fermentation method for the Escherichia coli large-scale high-efficiency expression of the supercoiled plasmid DNA is characterized in that in the step 4), the pH is adjusted by 25-28% of ammonium hydroxide and 5-10% of phosphoric acid.
The fermentation method for the escherichia coli large-scale high-efficiency expression of the supercoiled plasmid DNA is characterized in that the inoculation volume of the shake flask primary seed in the step 5) is 5-10% of the volume of the fermentation tank.
The fermentation method for the escherichia coli large-scale high-efficiency expression of the supercoiled plasmid DNA is characterized in that the rotating speed is increased in the step 6) to be 50-100 rpm.
The fermentation method for the Escherichia coli large-scale high-efficiency expression supercoiled plasmid DNA is characterized in that the feeding starting feeding speed in the step 7) is 0.5 mL/min.
The fermentation method for the escherichia coli large-scale efficient expression of the supercoiled plasmid DNA is characterized in that the shake flask culture medium in the step 1) is an LB culture medium, the components of the shake flask culture medium are peptone 10g/L, yeast extract 5g/L and NaCl 10 g/L.
The fermentation method for large-scale and high-efficiency expression of supercoiled plasmid DNA by escherichia coli is characterized in that in the step 2), every 1L of the fermentation medium comprises: 5-10g of soybean peptone, 8-12g of yeast powder, 4-8g of ammonium sulfate, 3-6g of monopotassium phosphate, 1-3g of disodium hydrogen phosphate, 5-15g of glycerol, 0.5-1mL of antifoaming agent, 0.5-2g of anhydrous magnesium sulfate and 1-3mL of trace elements.
The fermentation method for large-scale and high-efficiency expression of supercoiled plasmid DNA by escherichia coli is characterized in that in the step 2), every 1L of the feed medium comprises: 5-20g of anhydrous magnesium sulfate, 600g of glycerol 200-.
Compared with the prior art, the fermentation method has the following advantages:
1. the glycerol is adopted to replace glucose as a carbon source, so that the acid production phenomenon in the fermentation process of the thalli is reduced, and the growth inhibition effect is reduced;
2. the accumulation of the biomass of the thalli is reduced through earlier-stage low-temperature culture, and the metabolic mode of the thalli is converted through subsequent temperature conversion, so that the thalli are stimulated to express and copy a large amount of supercoiled plasmid DNA;
3. the DO in the fermentation regulation and control process is always maintained at 40% through multi-parameter linkage, the parameters are clear and stable, the repeatability is high, and the thalli can be effectively stimulated to efficiently express and copy through multiple verification;
4. the feeding speed is in addition to the working speed, and the linked feeding is carried out according to DO, so that a starvation fermentation mode of thalli can be effectively formed, and the very high supercoiled plasmid DNA expression quantity is obtained under the condition of keeping the biomass of the thalli to be lower;
5. compared with the yield of 100-1000 mg/L obtained by the conventional process for fermenting the supercoiled plasmid DNA by escherichia coli, the yield level of the supercoiled plasmid DNA can reach 500-1000mg/L by adopting the fermentation method.
Drawings
FIG. 1 is a colony of E.Coli Stbl3 competent cells after transformation of pLP1(PUC) obtained in example 3 and overnight culture on LB plates.
FIG. 2 is a graph showing comparison of OD600 of E.coli fermented at a constant 37 ℃ with the expression amount of plasmid DNA in example 3;
FIG. 3 is a graph showing the comparison between OD600 and the expression level of plasmid DNA in the case of fermentation of E.coli at 30 ℃ after culturing to 37 ℃ in example 4.
Detailed Description
The present invention will be further described with reference to the following examples.
Example 1: working seeds were prepared by heat shock transformation of pLP1(PUC) into e.coli Stbl3 competent cells.
1. E.coli Stbl3(Thermo Fisher) competent cells were removed from the-80 ℃ freezer, quickly placed on ice and thawed slowly.
2. The pLP1(PUC) was removed and added to an EP tube containing competent cells, gently mixed and placed in an ice bath for 30 min.
3. After the ice bath is finished, the centrifuge tube is placed into a water bath with the temperature of 42 ℃ for heat shock for 90s, and then the centrifuge tube is quickly transferred into the ice bath for cooling for 2min, and the process prohibits shaking the centrifuge tube.
4. The centrifuge tube was filled with 500. mu.L of sterile LB medium without kanamycin antibiotic, mixed well and cultured at 37 ℃ for 60 min.
5. 100 μ L of the transformed bacterial suspension was dropped on an LB plate with kanamycin resistance (5 μ g/L) at 1% agarose concentration and spread evenly with a coating rod. The cells were cultured at 37 ℃ in an inverted manner overnight.
6. After overnight incubation, single colonies were picked to 100mL LB flasks with kanamycin resistance (5. mu.g/L) and incubated at 220rpm, 37 ℃ to an OD600 of 0.8-1.
7. Pipette 500. mu.L of the bacterial solution, add 500. mu.L of 50% glycerol solution, and store in a-80 ℃ refrigerator.
FIG. 1 is a colony of E.Coli Stbl3 competent cells after transformation of pLP1(PUC) and overnight culture on LB plates. After about 16 hours of culture, a monoclonal colony was cultured on an LB plate containing kanamycin resistance. These colonies were evenly distributed without ligation, indicating that pLP1(PUC) was successfully transformed into e.coli Stbl3 competent cells and efficiently expressed.
Example 2: basic steps of production process of 5L tank fermentation supercoiled plasmid DNA
1. And (3) flask shaking inoculation: and (3) taking out the preserved working seeds from the strain bank, resuscitating the seeds in water bath at 42 ℃ for 2min, inoculating the seeds into a shake flask, and culturing for 8 hours until OD600 reaches 1.2 to prepare for inoculating a fermentation tank.
2. Preparing a fermentation tank: and (3) filling the fermentation medium and the feeding medium into a fermentation tank and a feeding bottle, installing a pH electrode and a dissolved oxygen electrode, connecting an electrode joint, and calibrating the pH electrode and the dissolved oxygen electrode. Ensuring that each sampling port and each vent are completely sealed, and sterilizing at 121 ℃ for 20min by high-pressure steam sterilization;
3. cooling the tank body: and after the sterilization is finished, cooling to room temperature, connecting cooling water, condensed water, an air inlet and exhaust pipeline, a pH electrode, an oxygen dissolving electrode and a temperature electrode, and keeping the fermentation temperature of the tank constant at the initial temperature.
4. Setting parameters: setting the stirring speed of a fermentation tank to be 200rpm, the ventilation capacity to be 2L/min, setting the pH to be 7.0, and starting an automatic control alkali and acid supplementing bottle for adjustment; setting the pressure of the tank body to be 0.5bar, and setting the initial fermentation temperature to be 100% point of calibration of the dissolved oxygen electrode under the conditions of maximum rotating speed and maximum ventilation;
5. inoculation: under the protection of flame, 100mL of first-class seeds cultured for 8 hours are inoculated into a tank body;
6. and (3) regulating and controlling the fermentation process: after inoculation, the DO is maintained at about 40% by increasing the rotating speed and the ventilation volume, and after sudden rising and rebounding of the DO, material supplement can be started, and the material supplement is linked with the DO to keep the DO at 40%;
7. temperature conversion: and setting the feeding speed to be linked with the DO parameter after a feeding signal appears, and setting the conversion of the fermentation temperature of the tank body after the DO parameter is stable.
8. And (3) collecting thalli: after fermentation, the cells were collected by high speed centrifugation at 8000g × 10min and stored in a-20 ℃ freezer.
Example 3: escherichia coli constant 37 ℃ fermentation expression supercoiled plasmid DNA
1. The basic fermentation medium has the formula per liter: 12g of soybean peptone, 20g of yeast powder, 6g of ammonium sulfate, 3g of monopotassium phosphate, 3.27g of disodium hydrogen phosphate, 7.5g of glycerol, 0.5mL of antifoaming agent, 1g of anhydrous magnesium sulfate and 1mL of trace elements. The formulation volume was 3L.
2. The formula of a supplemented medium per liter is as follows: 20g of anhydrous magnesium sulfate, 500g of glycerol, 30g of yeast extract and 50g of soybean peptone. The formulation volume was 300 mL.
3. The supplementary acid is 8% phosphoric acid solution.
4. The supplementary alkali is 28% ammonium hydroxide solution.
5. Fermentation conditions are as follows: the temperature is 37 ℃; the pH was 7.0.
6. The fermentation process regulation and control method comprises the following steps: after the first-stage seeds in the shake flask are inoculated into a fermentation tank, the culture is carried out for 20min, the DO is reduced to 30%, the stirring speed is started to be linked with the DO, and the DO is maintained at 40%. When the culture time is 8 hours, the stirring speed reaches the upper limit of 900rpm, the linkage of stirring and DO is closed, and the aeration volume is increased to maintain the DO at 40%. When the culture is carried out for 9 hours, the ventilation volume reaches the upper limit of 8L/min. The linkage of stirring speed and ventilation is cancelled, and the maximum upper limit is maintained. After 10 hours of incubation, the DO level began to ramp to 90% after 3.2%. Feeding is started, the feeding speed is set to be 0.5mL/min, feeding is linked with DO, and feeding is started when the DO is more than 40%.
7. Sampling in the process: OD600 was measured every 2 hours, and 1mL of sample 2 was stored for plasmid extraction and wet weight analysis starting at 8 hours.
8. And (3) collecting thalli: after the fermentation time reaches 28 hours, putting the strain into a tank to collect the strain, centrifuging the strain at 8000g for 10min, collecting the strain, and storing the strain in a refrigerator at the temperature of 20 ℃ below zero.
9. Yield: finally, the obtained thalli is put in a tank, and the content of supercoiled plasmid DNA in the thalli is 437 mg/L.
FIG. 2 is a graph showing the comparison of OD600 of E.coli fermented at a constant 37 ℃ with the expression level of plasmid DNA. As can be seen from fig. 1: OD600 reflects the accumulation of the bacterial organisms, OD600 steadily increases with time, and reaches the maximum value in 27 hours. The expression level of the plasmid reached a maximum of 540mg/L in 25 hours, and the expression level of the cells per unit weight was calculated to be 6.43mg/L, corresponding to a final weight of the cells placed in a jar of 252 g.
Example 4: the fermentation method of the invention is that the Escherichia coli is transformed to 37 ℃ by 30 ℃ to express supercoiled plasmid DNA by fermentation
1. The basic fermentation medium has the formula per liter: 12g of soybean peptone, 20g of yeast powder, 6g of ammonium sulfate, 5.33g of monopotassium phosphate, 1g of disodium hydrogen phosphate, 7.5g of glycerol, 0.5mL of antifoaming agent, 2g of anhydrous magnesium sulfate and 1mL of trace elements. The formulation volume was 3L.
2. The formula of a supplemented medium per liter is as follows: 10g of anhydrous magnesium sulfate, 500g of glycerol, 30g of yeast extract and 50g of soybean peptone. The formulation volume was 300 mL.
3. The supplementary acid is 10% phosphoric acid solution.
4. The supplementary alkali is 25% ammonium hydroxide solution.
5. Fermentation conditions are as follows: the initial fermentation temperature is 30 ℃, and is further determined to be 37 ℃ after feeding; the pH was 7.0.
6. The fermentation process regulation and control method comprises the following steps: after the first-stage seeds in the shake flask are inoculated into a fermentation tank, the culture is carried out for 40min, the DO is reduced to 39%, the stirring speed is started to be linked with the DO, and the DO is maintained at 40%. When the culture is carried out for 12 hours, the stirring speed reaches 850rpm, and the DO is always maintained at 40 percent, so that the stirring speed can not be increased any more. Culture to 14 hours, DO from 40% to 89% sudden rise. Feeding is started, the feeding speed is set to be 0.5mL/min, feeding is linked with DO, and feeding is started when the DO is more than 60%.
7. Temperature conversion: 1 hour after the feed signal response was determined, the pot temperature was set to 37 ℃. And continuing culturing for 15 hours, changing the DO and feeding linkage point to 40%, and continuing fermenting after the DO fluctuation is confirmed to be about 40%.
8. Sampling in the process: OD600 was measured every 2 hours, and 1mL of sample 2 was stored for plasmid extraction and wet weight analysis starting at 8 hours.
9. And (3) collecting thalli: after the fermentation time reaches 28 hours, putting the strain into a tank to collect the strain, centrifuging the strain at 8000g for 10min, collecting the strain, and storing the strain in a refrigerator at the temperature of 20 ℃ below zero.
10. Yield: finally, the obtained bacteria are put in a tank to measure the DNA content of the supercoiled plasmid to be 734 mg/L.
FIG. 3 is a graph showing the comparison between OD600 and the expression level of plasmid DNA in E.coli cultured at 30 ℃ and then transformed to 37 ℃. As can be seen from fig. 2: OD600 reflects the accumulation of the thallus organisms, OD600 steadily increases with time, and reaches the maximum value in 28 hours. The expression level of the plasmid reached a maximum of 801mg/L at 26 hours. It can be seen that the expression level of plasmid DNA was only 77.75mg/L up to 14 hours, which is far less than 419mg/L for constant expression at 37 ℃. However, after the culture is carried out for 18 hours, the expression level begins to increase in a doubling way, and finally reaches 801mg/L in 26 hours, so that the fermentation yield is effectively improved by 48 percent. The expression level of the cells per unit weight was calculated to be 11.33mg/L, corresponding to 212g of the final weight of the cells placed in the tank. Therefore, the total yield can be effectively improved, and the weight of the received bacteria can be reduced, so that the expression amount of the bacteria per unit weight is improved by 76.21 percent.
The above embodiments are provided only for illustrating the present invention and not for limiting the present invention, and those skilled in the art can make various changes and modifications without departing from the spirit and scope of the present invention, and therefore all equivalent technical solutions should also fall within the scope of the present invention, and should be defined by the claims.
Claims (10)
1. A fermentation method for large-scale high-efficiency expression of supercoiled plasmid DNA by escherichia coli is characterized by comprising the following steps:
1) and (3) flask shaking inoculation: taking out the preserved working seeds from the strain library, recovering in a water bath, inoculating into a shake flask in a sterile environment, culturing at 30 ℃ for 6-10 hours, and preparing to inoculate a fermentation tank;
2) preparing a fermentation tank: fully dissolving a fermentation culture medium and a supplement culture medium, fixing the volume to a corresponding volume, filling the volume into a fermentation tank and a supplement bottle, and sterilizing by high-pressure steam sterilization;
3) cooling the tank body: after the sterilization is finished, cooling to room temperature, connecting cooling water and condensate water pipelines, connecting a pH electrode, an oxygen dissolving electrode and a temperature electrode, and keeping the fermentation temperature of the tank constant;
4) setting parameters: setting the stirring rotation speed of a fermentation tank at 200-300rpm, the ventilation capacity at 2-3L/min, setting the pH value at 7.0 +/-0.2, starting an automatic control alkali and acid supplementing bottle for adjustment, setting the pressure of the tank body at 0.5-0.6bar, setting the initial fermentation temperature at 100% point of dissolved oxygen electrode calibration under the conditions of maximum rotation speed and maximum ventilation, and setting the initial fermentation temperature at 30-35 ℃;
5) inoculation: under the protection of flame, inoculating the first-stage seeds in the shake flask into a fermentation tank body;
6) and (3) regulating and controlling the fermentation process: after inoculation, the DO is maintained at about 40% by increasing the rotating speed and the ventilation volume, and after sudden rising and rebounding of the DO, material supplement can be started, and the material supplement is linked with the DO to keep the DO at 40%;
7) temperature conversion: when DO parameter is stabilized within 0-2 hours after 40% +/-10%, the fermentation temperature conversion temperature of the tank body is 37-48 ℃;
8) and (3) collecting thalli: after the fermentation is finished, the thalli are collected by high-speed centrifugation and stored in a refrigerator at the temperature of minus 20 ℃.
2. The fermentation method of Escherichia coli large-scale high-efficiency expression supercoiled plasmid DNA as claimed in claim 1, wherein the working seeds of step 1) are stored in a-80 ℃ refrigerator; the temperature range of the resuscitation water bath is 30-42 ℃; the inoculation can be arranged by culturing until OD600 reaches 0.8-1.1, and the selected competent cell is E.coli Stbl 3.
3. The fermentation method of Escherichia coli large-scale highly efficient expression supercoiled plasmid DNA as claimed in claim 1, wherein in step 2), the pH electrode and dissolved oxygen electrode are installed on the fermentation tank, the electrode joint is connected, the pH electrode and dissolved oxygen electrode are calibrated, the calibration points of the pH electrode are 4.0 and 7.0, the zero point of the dissolved oxygen electrode is calibrated by saturated sodium sulfite, and the conditions of autoclaving are 121 ℃ and 20 min.
4. The fermentation method of Escherichia coli for large-scale highly efficient expression of supercoiled plasmid DNA according to claim 1, wherein the pH in step 4) is adjusted by 25-28% ammonium hydroxide and 5-10% phosphoric acid.
5. The fermentation process of claim 1, wherein the inoculation volume of the shake flask primary seed in step 5) is 5-10% of the volume of the fermentor.
6. The fermentation method of Escherichia coli for large-scale highly efficient expression of supercoiled plasmid DNA according to claim 1, wherein the increase of the rotation speed in step 6) is in the range of 50-100 rpm.
7. The fermentation method of Escherichia coli for large-scale highly efficient expression of supercoiled plasmid DNA, according to claim 1, wherein the feed initiation and feed rate in step 7) is 0.5 mL/min.
8. The fermentation method of the supercoiled plasmid DNA expressed in E.coli in large scale and high efficiency according to claim 1, wherein the shake flask culture medium in step 1) is LB culture medium with the components of peptone 10g/L, yeast extract 5g/L, and NaCl 10 g/L.
9. The fermentation method of claim 1, wherein step 2) comprises the following steps for every 1L of fermentation medium: 5-10g of soybean peptone, 8-12g of yeast powder, 4-8g of ammonium sulfate, 3-6g of monopotassium phosphate, 1-3g of disodium hydrogen phosphate, 5-15g of glycerol, 0.5-1mL of antifoaming agent, 0.5-2g of anhydrous magnesium sulfate and 1-3mL of trace elements.
10. The fermentation method of claim 1, wherein step 2) comprises the following steps for every 1L of the feed medium: 5-20g of anhydrous magnesium sulfate, 600g of glycerol 200-.
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