A high-density fermentation culture process for Mycoplasma gallisepticum

Abstract

The present invention relates to the technical field of vaccine preparation, and in particular to a high-density fermentation and culture process of Mycoplasma gallisepticum. The present invention provides a high-density fermentation and culture method of Mycoplasma gallisepticum, comprising the following steps: a. inoculating Mycoplasma gallisepticum seeds in a culture medium for culture, and then inoculating and subculturing once to obtain a culture solution; b. inoculating the culture solution obtained in step a in a culture medium for culture for 44 to 50 hours, and adjusting the pH of the fermentation solution to 7.5 to 7.6 when the culture reaches 20 to 24 hours and 32 to 36 hours, respectively; after the culture is completed, the fermentation is stopped when the pH value of the fermentation solution drops to 6.5. The method of the present invention can carry out high-density culture of Mycoplasma gallisepticum, the immunogenicity of Mycoplasma gallisepticum is high, the protective effect of the prepared vaccine is good, and the protective effect of the vaccine is improved.

Description

Technical Field

[0001] The invention relates to the technical field of vaccine preparation, and in particular to a high-density fermentation and culture process of Mycoplasma gallisepticum. Background Art

[0002] Mycoplasma galliscepticum (MG) is a mycoplasma that causes respiratory infections in chickens. The main clinical manifestations after infection are runny nose, tears, and coughing. In severe cases, it manifests as dyspnea or open-mouth breathing, and moist rales can be heard. With the increase in the scale of chicken farming, changes in feeding methods, and increased feeding density, the incidence of Mycoplasma galliscepticum infection is getting higher and higher, which mainly affects the growth of chicks and reduces the egg production of adult chickens, causing significant economic losses to the poultry industry. The harmfulness is becoming increasingly prominent and has attracted widespread attention from poultry farmers. Generally, once infected with Mycoplasma galliscepticum, the infection will remain in a persistently infected carrier state for life. Its main mode of transmission is horizontal contact transmission between chickens, that is, it is discharged with respiratory secretions and infected through droplets and dust through the respiratory tract. In addition, vertical transmission is also its main mode of transmission.

[0003] At present, the state requires original breeder farms to complete the purification of mycoplasma, Marek's disease, avian leukosis and other diseases as soon as possible. In recent years, with the continuous improvement of vaccine application technology and the successful promotion of seed source avian leukosis purification demonstration, avian influenza, Newcastle disease, infectious bursal disease, Marek's disease and avian leukosis have been effectively controlled. However, the problem of mycoplasma infection in chicken flocks has become increasingly prominent. Although various farms have taken various precautions in biosafety isolation and other control measures, vaccine immunization is chaotic. The commonly used MG vaccines include TS-11, 6 / 85, and F strains. In addition, there is another R-type inactivated vaccine. Due to the lack of accurate detection for each vaccine strain, many testing agencies and the testing teams within the breeding group have no idea where to start when it comes to the occurrence of Mycoplasma gallisepticum in chicken farms or the detection of whether the breeder farms carry Mycoplasma gallisepticum pathogens and distinguishing whether it is vaccine or wild virus infection.

[0004] Most importantly, high-density culture of Mycoplasma gallisepticum is very difficult, for example, it has high nutritional requirements, especially strict pH requirements. In the prior art, the commonly used culture medium is modified Frey's medium, but it cannot meet the needs of large-scale culture.

[0005] Therefore, how to provide a method for large-scale cultivation of Mycoplasma gallisepticum to solve the above technical problems is an urgent problem to be solved by those skilled in the art. Summary of the invention

[0006] The purpose of the present invention is to provide a high-density fermentation and cultivation process for Mycoplasma gallisepticum.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides a high-density fermentation and culture method for Mycoplasma gallisepticum, comprising the following steps:

[0009] a. Inoculate Mycoplasma gallisepticum seeds in a culture medium for cultivation, and then inoculate and subculture once to obtain a culture solution;

[0010] b. Inoculate the culture solution obtained in step a into the culture medium and culture for 44 to 50 hours. Adjust the pH of the fermentation solution to 7.5 to 7.6 at 20 to 24 hours and 32 to 36 hours respectively. After the culture is completed, stop the fermentation when the pH value of the fermentation solution drops to 6.5.

[0011] The culture medium in steps a and b comprises the following components: 10-20 g tryptone, 3-5 g soy peptone, 2-3 g sodium chloride, 3-4 g dipotassium hydrogen phosphate, 2.0-3.0 g glucose, 2-4 g yeast extract powder, 1-2 g silk peptide powder, 1-2 mg vitamin B12, 1-2 g L-glutamine, 6-10 g glutathione, 5-9 g coenzyme 1, 2-3 g ornithine, 2-3 g tyrosine, 5-7 mL fetal bovine serum, and water is added to make up to 1 L.

[0012] Preferably, the preparation method of the culture medium is: mixing the components except fetal bovine serum, sterilizing, adding fetal bovine serum, and adjusting the pH.

[0013] Preferably, the sterilization temperature is 115-123° C. and the time is 12-16 min.

[0014] Preferably, the pH is adjusted to 7.5-7.7.

[0015] Preferably, the culture temperature in step a is 36.5-37.5°C.

[0016] Preferably, the culturing time in step a is 20 to 24 hours.

[0017] Preferably, the inoculation ratio in step a is 8-12% (V:V).

[0018] Preferably, the inoculation ratio in step b is 4-6% (V:V); and the culture temperature in step b is 37-38°C.

[0019] Preferably, the pH regulator in step b is: calcium bicarbonate and aluminum sulfate; the mass ratio of calcium bicarbonate to aluminum sulfate is 10:5-7.

[0020] The present invention also provides the use of the Mycoplasma gallisepticum prepared by the Mycoplasma gallisepticum high-density fermentation culture method in the preparation of Mycoplasma gallisepticum vaccine.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The present invention does not select conventional culture medium during the culture process. The culture medium provided by the present invention also adds a growth activator of Mycoplasma gallisepticum, including coenzyme 1, ornithine and tyrosine. The addition of the above components activates the growth of Mycoplasma gallisepticum and promotes the expansion and propagation of Mycoplasma gallisepticum. The silk peptide powder added to the culture medium has a protective effect and can alleviate the influence of external stimulation on the growth of Mycoplasma. Glutathione can protect cells from oxidative stress and help to form a favorable redox environment inside and outside the cells, which is conducive to Mycoplasma gallisepticum to quickly pass the delayed period, accelerate the propagation speed of Mycoplasma gallisepticum, and thus achieve the purpose of high-density culture.

[0023] Specifically, coenzyme 1 (also known as NAD+ or nicotinamide adenine dinucleotide) is widely involved in redox reactions in organisms and is an essential cofactor for a variety of enzymatic reactions. In the metabolic process of Mycoplasma gallisepticum, coenzyme 1 promotes the smooth progress of certain key metabolic pathways, provides the necessary energy and intermediates for cell growth and division, thereby facilitating the increase in the number of bacteria and playing a role in growth activation.

[0024] Ornithine and tyrosine: These two amino acids are the basic building blocks of proteins and are essential for the growth of microorganisms. They not only serve as raw materials for protein synthesis, but also participate in the synthesis of other biologically active substances. In the culture process of Mycoplasma gallisepticum, the addition of ornithine and tyrosine supplements the deficiency of amino acids in the culture medium, meets the demand of bacterial growth for amino acids, and then promotes the growth and reproduction of bacteria, playing a role in the nutritional support of amino acids.

[0025] In the present invention, coenzyme 1, ornithine and tyrosine work together to optimize the growth environment of Mycoplasma gallisepticum. Coenzyme 1 promotes energy metabolism and the activity of key enzymes, and ornithine and tyrosine provide necessary amino acid nutrition. The three work together to enable Mycoplasma gallisepticum to grow and reproduce rapidly under high-density culture conditions.

[0026] Furthermore, the addition of the three components also has a positive effect on the physiological state of the bacteria: high-density culture requires not only an increase in the number of bacteria, but also requires the bacteria to maintain a good physiological state. The addition of coenzyme 1, ornithine and tyrosine helps maintain the membrane integrity, protein synthesis and energy metabolism of the bacteria, so that the bacteria can still maintain a high growth rate and vitality under high-density culture conditions. Coenzyme 1, ornithine and tyrosine help increase the bacterial density of Mycoplasma gallisepticum and achieve the goal of high-density culture by promoting energy metabolism, providing necessary amino acid nutrition and optimizing the growth environment.

[0027] The applicant has found that during the expansion culture period, appropriately adjusting the pH value of the culture medium helps stimulate the growth of Mycoplasma gallisepticum and can significantly improve the culture effect. The selection of the pH regulator is also very important. Only by selecting the pH conditioner of the present invention can the best culture effect be achieved.

[0028] The method of the invention can carry out high-density culture of Mycoplasma gallisepticum, the immunogenicity of Mycoplasma gallisepticum is high, the prepared vaccine has good protective effect, and the protective effect of the vaccine is improved. DETAILED DESCRIPTION

[0029] The present invention provides a high-density fermentation and culture method for Mycoplasma gallisepticum, comprising the following steps:

[0030] a. Inoculate Mycoplasma gallisepticum seeds in a culture medium for cultivation, and then inoculate and subculture once to obtain a culture solution;

[0031] b. Inoculate the culture solution obtained in step a into the culture medium and culture for 44 to 50 hours. Adjust the pH of the fermentation solution to 7.5 to 7.6 at 20 to 24 hours and 32 to 36 hours respectively. After the culture is completed, stop the fermentation when the pH value of the fermentation solution drops to 6.5.

[0032] The culture medium in steps a and b comprises the following components: 10-20 g tryptone, 3-5 g soy peptone, 2-3 g sodium chloride, 3-4 g dipotassium hydrogen phosphate, 2.0-3.0 g glucose, 2-4 g yeast extract powder, 1-2 g silk peptide powder, 1-2 mg vitamin B12, 1-2 g L-glutamine, 6-10 g glutathione, 5-9 g coenzyme 1, 2-3 g ornithine, 2-3 g tyrosine, 5-7 mL fetal bovine serum, and water is added to make up to 1 L.

[0033] In the present invention, in step b, the culture fluid obtained in step a is preferably inoculated into the culture medium and cultured for 45 to 49 hours, and the pH of the fermentation fluid is adjusted to 7.5 at 21 to 23 hours and 33 to 35 hours, respectively; further preferably, the culture fluid obtained in step a is inoculated into the culture medium and cultured for 47 hours, and the pH of the fermentation fluid is adjusted to 7.5 at 22 hours and 34 hours, respectively.

[0034] In the present invention, the culture medium in steps a and b preferably comprises the following components: 12-18 g tryptone, 4 g soy peptone, 2.5 g sodium chloride, 3.5 g dipotassium hydrogen phosphate, 2.5 g glucose, 3 g yeast extract powder, 1.5 g silk peptide powder, 1.5 mg vitamin B12, 1.5 g L-glutamine, 7-9 g glutathione, 6-8 g coenzyme 1, 2.5 g ornithine, 2.5 g tyrosine, 6 mL fetal bovine serum, and water is added to 1 L; further preferably comprises the following components: 13-17 g tryptone, 4 g soy peptone, 2.5 g sodium chloride, 3.5 g dipotassium hydrogen phosphate, 2.5 g glucose, 3 g yeast extract powder, 1.5 g silk peptide powder, 1.5 mg vitamin B12, 1.5 g L-glutamine, 8g glutathione, 7g coenzyme 1, 2.5g ornithine, 2.5g tyrosine, 6mL fetal bovine serum, add water to 1L; more preferably, it includes the following components: 15g tryptone, 4g soy peptone, 2.5g sodium chloride, 3.5g dipotassium hydrogen phosphate, 2.5g glucose, 3g yeast extract powder, 1.5g silk peptide powder, 1.5mg vitamin B12, 1.5g L-glutamine, 8g glutathione, 7g coenzyme 1, 2.5g ornithine, 2.5g tyrosine, 6mL fetal bovine serum, add water to 1L.

[0035] In the present invention, the preparation method of the culture medium is: mixing the components except fetal bovine serum, sterilizing, adding fetal bovine serum, and adjusting the pH.

[0036] In the present invention, the sterilization temperature is 115-123°C and the time is 12-16 min; preferably, the sterilization temperature is 116-122°C and the time is 13-15 min; further preferably, the sterilization temperature is 117-121°C and the time is 14 min; more preferably, the temperature is 121°C and the time is 14 min.

[0037] In the present invention, the pH is adjusted to 7.5-7.7, preferably 7.6.

[0038] In the present invention, the culture temperature in step a is 36.5-37.5°C, preferably 37°C.

[0039] In the present invention, the culturing time in step a is 20 to 24 hours, preferably 21 to 23 hours, and more preferably 22 hours.

[0040] In the present invention, the inoculation ratio in step a is 8-12% (V:V), preferably 9-11% (V:V), and more preferably 10% (V:V).

[0041] In the present invention, the inoculation ratio in step b is 4-6% (V:V); preferably 5% (V:V).

[0042] In the present invention, the culture temperature in step b is 37-38°C, preferably 37.5°C.

[0043] In the present invention, the pH regulator in step b is: calcium bicarbonate and aluminum sulfate; the mass ratio of calcium bicarbonate to aluminum sulfate is 10:5-7; preferably 10:6.

[0044] The present invention also provides the use of the Mycoplasma gallisepticum prepared by the Mycoplasma gallisepticum high-density fermentation culture method in the preparation of Mycoplasma gallisepticum vaccine.

[0045] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0046] Example 1

[0047] A high-density fermentation and culture method for Mycoplasma gallisepticum comprises the following steps:

[0048] a. Inoculate the Mycoplasma gallisepticum seeds in a culture medium at a ratio of 8% (V:V) and culture at 36.5°C for 20 hours. Inoculate and subculture once more in the same manner to obtain a culture solution.

[0049] b. Inoculate 4% (V:V) of the culture solution obtained in step a into the culture medium in proportion and culture at 37°C for 44 hours. Adjust the pH of the fermentation solution to 7.5 at 20 hours and 3 hours, respectively. After the culture is completed, stop the fermentation when the pH value of the fermentation solution drops to 6.5, add formaldehyde solution to inactivate, end the continuous bacterial enrichment culture, keep samples for inspection, and store at 4°C for 1 day.

[0050] The culture medium in steps a and b includes the following components: 10g tryptone, 3g soy peptone, 2g sodium chloride, 3g dipotassium hydrogen phosphate, 2.0g glucose, 2g yeast extract powder, 1g silk peptide powder, 1mg vitamin B12, 1g L-glutamine, 6g glutathione, 5g coenzyme 1, 2g ornithine, 2g tyrosine, 5mL fetal bovine serum, and water is added to make up to 1L.

[0051] The preparation method of the culture medium is as follows: all components except fetal bovine serum are mixed, sterilized at 115° C. for 12 minutes, and then the fetal bovine serum is added and the pH is adjusted to 7.5.

[0052] The pH regulator in step b is: calcium bicarbonate and aluminum sulfate, with a mass ratio of 10:5.

[0053] Example 2

[0054] A high-density fermentation and culture method for Mycoplasma gallisepticum comprises the following steps:

[0055] a. Inoculate the Mycoplasma gallisepticum seeds in a culture medium at a ratio of 12% (V:V) and culture at 37.5°C for 24 hours. Inoculate and subculture once more in the same manner to obtain a culture solution;

[0056] b. Inoculate 6% (V:V) of the culture solution obtained in step a into the culture medium in proportion and culture at 38°C for 50h. Adjust the pH of the fermentation solution to 7.6 at 24h and 36h, respectively. After the culture is completed, stop the fermentation when the pH value of the fermentation solution drops to 6.5, add formaldehyde solution to inactivate, end the continuous bacterial enrichment culture, keep samples for inspection, and store at 4°C for 1 day.

[0057] The culture medium in steps a and b includes the following components: 20g tryptone, 5g soy peptone, 3g sodium chloride, 4g dipotassium hydrogen phosphate, 3.0g glucose, 4g yeast extract powder, 2g silk peptide powder, 2mg vitamin B12, 2g L-glutamine, 10g glutathione, 9g coenzyme 1, 3g ornithine, 3g tyrosine, 7mL fetal bovine serum, and water is added to make up to 1L.

[0058] The preparation method of the culture medium is as follows: all components except fetal bovine serum are mixed, sterilized at 123° C. for 16 minutes, and then the fetal bovine serum is added and the pH is adjusted to 7.7.

[0059] The pH regulator in step b is: calcium bicarbonate and aluminum sulfate, with a mass ratio of 10:7.

[0060] Example 3

[0061] A high-density fermentation and culture method for Mycoplasma gallisepticum comprises the following steps:

[0062] a. Inoculate the Mycoplasma gallisepticum seeds in a culture medium at a ratio of 10% (V:V) and culture at 37°C for 22 hours. Inoculate and subculture once more in the same manner to obtain a culture solution;

[0063] b. Inoculate 5% (V:V) of the culture solution obtained in step a into the culture medium in proportion and culture at 37.5°C for 48 hours. Adjust the pH of the fermentation solution to 7.6 at 22 hours and 34 hours respectively. After the culture is completed, stop the fermentation when the pH value of the fermentation solution drops to 6.5, add formaldehyde solution to inactivate, end the continuous bacterial enrichment culture, keep samples for inspection, and store at 4°C for 1 day.

[0064] The culture medium in steps a and b includes the following components: 15g tryptone, 4g soy peptone, 2.5g sodium chloride, 3.5g potassium dihydrogen phosphate, 2.5g glucose, 3g yeast extract powder, 1.5g silk peptide powder, 1.5mg vitamin B12, 1.5g L-glutamine, 8g glutathione, 7g coenzyme 1, 2.5g ornithine, 2.5g tyrosine, 6mL fetal bovine serum, and water is added to make up to 1L.

[0065] The preparation method of the culture medium is as follows: all components except fetal bovine serum are mixed, sterilized at 121° C. for 14 minutes, and then the fetal bovine serum is added and the pH is adjusted to 7.6.

[0066] The pH regulator in step b is: calcium bicarbonate and aluminum sulfate, with a mass ratio of 10:6.

[0067] Control group 1: other methods were the same as those in Example 3, except that tyrosine and ornithine were not added to the culture medium.

[0068] Control group 2: other methods were the same as those in Example 3, except that glutathione and coenzyme 1 were not added to the culture medium.

[0069] Control group 3: other methods were the same as those in Example 3, except that the pH of the fermentation liquid was not adjusted at 22 h and 34 h of culture.

[0070] Control group 4: other methods are the same as those of Example 3, except that the pH regulator in step b is sodium hydroxide.

[0071] Experimental Example 1

[0072] The concentration of the Mycoplasma gallisepticum bacterial solution after the culture was completed according to the methods of Examples 1 to 3 and Control Groups 1 to 4 was determined. Two parallel experiments were set up and the results were averaged, as shown in Table 1.

[0073] Table 1 Concentration of Mycoplasma gallisepticum liquid

[0074] Group Concentration CCU / mL Example 1 <![CDATA[5.2×10 10 ]]> Example 2 <![CDATA[3.4×10 10 ]]> Example 3 <![CDATA[8.6×10 10 ]]> Control group 1 <![CDATA[3.5×10 9 ]]> Control group 2 <![CDATA[4.4×10 9 ]]> Control group 3 <![CDATA[6.5×10 8 ]]> Control group 4 <![CDATA[1.6×10 9 ]]>

[0075] It is not difficult to see from the contents recorded in Table 1 that the maximum bacterial concentration of Mycoplasma gallisepticum obtained by the fermentation method of the present invention can reach 8.6×10 10 CCU / mL, which is significantly higher than that of other test groups. This shows that the method of the present invention can be used for high-density culture of Mycoplasma gallisepticum. From the data of control groups 1 and 2, it can be seen that only by adopting the complete formula of the present invention can the best use effect be achieved. For example, discarding one or more components therein may significantly affect the culture results of Mycoplasma gallisepticum. From the data of control groups 3 and 4, it can be seen that during the expansion culture period, appropriately adjusting the pH value of the culture solution helps to stimulate the growth of Mycoplasma gallisepticum and can significantly improve the culture effect. The selection of the pH regulator is also very important. Only by selecting the pH conditioner of the present invention can the best culture effect be achieved.

[0076] Experimental Example 2

[0077] The vaccine obtained in Example 3 was prepared as a vaccine, and the specific preparation method was as follows: the Mycoplasma gallisepticum obtained in Example 3 was mixed with Tween-80 at a ratio of 95:5 as the aqueous phase for use; white oil for injection was taken and mixed with Siben-80 at a ratio of 95:5 as the oil phase for use; the oil phase and the aqueous phase solution were mixed at a volume ratio of 2.6:1 to obtain the Mycoplasma gallisepticum inactivated vaccine.

[0078] The obtained inactivated Mycoplasma gallisepticum vaccine was tested for efficacy according to the "Manufacture and Inspection Procedure for Inactivated Mycoplasma Gallisepticum Vaccine" of the current "Chinese Veterinary Pharmacopoeia", and the results are shown in Table 2.

[0079] [Efficacy test] Six SPF chickens aged 40 to 60 days were injected with the vaccine subcutaneously in the back of the neck or intramuscularly in the thigh, 0.5 mL per chicken. After 30 days, six control chickens with the same conditions were placed in the same cage for 2 to 3 hours. 3 The R strain of Mycoplasma gallisepticum was challenged by spraying in a closed room. The spraying duration should be no less than 5 minutes, and the droplets should be about 2.0 μm. After 14 days of observation, the air sac lesions were observed and scored. At least 4 control chickens should have air sac lesions with a score of more than 2 points, and the average air sac protection rate of immunized chickens should be no less than 60%.

[0080] [Usage and Dosage] Subcutaneous injection at the back of the neck or intramuscular injection at the thigh. For chickens under 40 days old, 0.25 mL per chicken; for chickens over 40 days old, 0.5 mL per chicken; for laying hens, vaccinate once more before laying eggs, 0.5 mL per chicken.

[0081] Scoring criteria for airbag lesions and calculation method for average airbag protection rate

[0082] Scoring criteria for air sac lesions:

[0083] 0 points: The air sac is normal, clean, transparent and thin;

[0084] 1 point: The air sac is slightly thickened and slightly turbid, with a few gray or yellow exudate spots locally;

[0085] 2 points: gray and yellow exudates are visible in some air sac areas, accompanied by moderate thickening of the air sacs;

[0086] 3 points: large air sacs are covered with yellow cheesy exudate;

[0087] 4 points: The entire air sac is covered with yellow cheesy exudate and the air sac loses its elasticity.

[0088] Protection rate calculation formula:

[0089] Average air sac protection rate (%) = {{average air sac lesion score of control chickens}-{average air sac lesion score of experimental chickens}}\{average air sac lesion score of control chickens}*100%

[0090] Table 2 Vaccine protection efficiency test questionnaire

[0091] Inspection Chicken Number Attack score Control chicken number Attack score 1 1 1 0 2 0 2 3 3 0 3 3 4 1 4 4 5 1 5 2 6 0 6 4

[0092] It can be calculated from the records in Table 2 that the vaccine protection efficiency of the vaccine prepared by the method of Example 3 is (2.67-0.5) / 2.67=81.27%. This shows that the Mycoplasma gallisepticum prepared by the present invention has high immunogenicity and can be prepared into a vaccine with good protection effect.

[0093] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for high-density fermentation and cultivation of Mycoplasma gallisepticum, comprising the following steps: a. Inoculate the Mycoplasma gallisepticum seeds in a 10% (V:V) ratio into the culture medium and culture at 37°C for 22 hours. Inoculate and subculture once more in the same manner to obtain the culture fluid. b. Inoculate 5% (V:V) of the culture solution obtained in step a into the culture medium in proportion and culture at 37.5°C for 48 hours. Adjust the pH of the fermentation solution to 7.6 at 22 hours and 34 hours respectively. After the culture is completed, stop the fermentation when the pH value of the fermentation solution drops to 6.5, add formaldehyde solution to inactivate, end the continuous bacterial enrichment culture, keep samples for inspection, and store at 4°C for 1 day. The culture medium in steps a and b comprises the following components: 15 g tryptone, 4 g soy peptone, 2.5 g sodium chloride, 3.5 g potassium dihydrogen phosphate, 2.5 g glucose, 3 g yeast extract powder, 1.5 g silk peptide powder, 1.5 mg vitamin B12, 1.5 g L-glutamine, 8 g glutathione, 7 g coenzyme 1, 2.5 g ornithine, 2.5 g tyrosine, 6 mL fetal bovine serum, and water is added to make up to 1 L; The preparation method of the culture medium is as follows: mixing all components except fetal bovine serum, sterilizing at 121° C. for 14 minutes, adding fetal bovine serum, and adjusting the pH to 7.6; The pH regulator in step b is: calcium bicarbonate and aluminum sulfate, with a mass ratio of 10:6.