A method for producing ergothioneine by mixed culture fermentation

Abstract

The application discloses a method for producing ergothioneine through mixed fermentation, and the method is to produce ergothioneine through mixed fermentation of Saccharomyces cerevisiae and Escherichia coli, wherein the Saccharomyces cerevisiae is Saccharomyces cerevisiae G7 with a preservation number of CCTCC NO: M 20231232, and the Escherichia coli is Escherichia coli G5 with a preservation number of CCTCC NO: M 2023894. The application utilizes mixed fermentation, adjusts the suitable cell amount of different strains by controlling the suitable inoculation ratio of seed liquid, so that the culture medium and the metabolic by-products of Saccharomyces cerevisiae are fully consumed and utilized by the two kinds of bacteria, more ergothioneine is accumulated, the conductivity of the fermentation liquor is reduced, and the extraction difficulty is reduced.

Description

Technical Field

[0001] This invention relates to the field of microbial fermentation technology, specifically to a method for producing ergothioneine through mixed-culture fermentation. Background Technology

[0002] Ergothioneine is a natural antioxidant that can scavenge intracellular free radicals, integrate copper ions, and inhibit the activity of some oxidases and many intracellular oxidation reactions. Therefore, ergothioneine is widely used in the fields of medical aesthetics, pharmaceuticals, health products, and biotechnology.

[0003] In existing ergothioneine fermentation processes, a single ergothioneine-producing strain is generally used for cultivation and fermentation. When brewer's yeast is used as the producing strain, the fermentation time is long, the feeding process is complex, the yield is low, the conductivity of the fermentation broth is high (>45ms / cm), extraction is difficult, and the wastewater volume is large. Summary of the Invention

[0004] To address the problems of complex procedures, low yield, difficult extraction, and high cost in existing processes, this invention provides a simple method for producing ergothioneine through mixed-culture fermentation by improving the fermentation technology.

[0005] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0006] A method for producing ergothioneine by mixed fermentation of brewer's yeast and Escherichia coli.

[0007] The brewing yeast is *Saccharomyces cerevisiae* G7, with accession number CCTCC NO: M 20231232, accession date July 7, 2023, and accession address Wuhan University, Wuhan, China. The *Escherichia coli* is *Escherichia coli* G5, with accession number CCTCC NO: M 2023894, accession date May 31, 2023, and accession address Wuhan University, Wuhan, China. The fermentation products accumulated by both *Saccharomyces cerevisiae* G7 and *Escherichia coli* G5 during fermentation are ergothioneine.

[0008] A method for producing ergothioneine by mixed-culture fermentation includes the following steps:

[0009] (1) Culture seed culture of Saccharomyces cerevisiae G7 and Escherichia coli G5 separately;

[0010] (2) Inoculate the Saccharomyces cerevisiae G7 seed culture into the fermentation medium and ferment until the OD of the fermentation broth reaches a certain level. 600 Once the value reaches 80, the E. coli G5 seed culture is inoculated into the fermentation broth for co-culture fermentation to produce ergothioneine.

[0011] In step (1), the seed culture medium formula for Saccharomyces cerevisiae G7 is as follows: peptone 5-10 g / L, yeast extract 5-10 g / L, anhydrous glucose 10-40 g / L, ammonium sulfate 1-10 g / L, potassium dihydrogen phosphate 5-15 g / L, and magnesium sulfate 0.1-2 g / L. Preferably, the formula is: peptone 10 g / L, yeast extract 5 g / L, anhydrous glucose 15 g / L, ammonium sulfate 5 g / L, potassium dihydrogen phosphate 7.6 g / L, and magnesium sulfate 1 g / L.

[0012] In step (1), Saccharomyces cerevisiae G7 is cultured in seed culture medium at 25-30℃ for 18-36 hours.

[0013] In step (1), the seed culture medium formula for Escherichia coli G5 is: 5-10 g / L peptone, 5-10 g / L yeast extract, 5-10 g / L sodium chloride, and 5-10 g / L glycerol. Preferably, the formula is 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, and 5 g / L glycerol.

[0014] In step (1), Escherichia coli G5 is cultured in seed culture medium at 34-37℃ for 12-16 hours.

[0015] In step (2), the fermentation medium formula is as follows: methanol 0.3-0.9 g / L, ammonium sulfate 1-10 g / L, dipotassium hydrogen phosphate 5-30 g / L, magnesium sulfate 1-10 g / L, trace element mixture 1-10 mL / L, vitamin solution 1-10 mL / L, histidine 5-20 g / L, methionine 5-20 g / L, cysteine ​​5-20 g / L. Preferably, the formula is: methanol 0.5 g / L, ammonium sulfate 5 g / L, dipotassium hydrogen phosphate 8 g / L, magnesium sulfate 1 g / L, trace element mixture 7.5 mL / L, vitamin solution 5 mL / L, histidine 19 g / L, methionine 19 g / L, cysteine ​​19 g / L.

[0016] The formula for the trace element mixture is as follows: CaCl2 1-10g / L, ZnSO4 1-10g / L, FeSO4 1-10g / L, H3BO3 1-6g / L, MnCl2 1-5g / L, Na2MoO4 0.1-1g / L, CoCl2 0.1-1g / L, CuSO4 0.1-1g / L, KI 0.1-1g / L. Preferably, CaCl2·2H2O 1-10g / L, ZnSO4·7H2O 1-10g / L, FeSO4·7H2O 1-10g / L, H3BO3 1-6g / L, MnCl2·4H2O 1-5g / L, Na2MoO4·2H2O 0.1-1g / L, CoCl2·6H2O 0.1-1g / L, CuSO4·5H2O 0.1-1g / L, KI 0.1-1g / L. Most preferably, CaCl2·2H2O 6g / L, ZnSO4·7H2O 7.5g / L, FeSO4·7H2O 6.5g / L, H3BO3 5.3g / L, MnCl2·4H2O 4.1g / L, Na2MoO4·2H2O 0.4g / L, CoCl2·6H2O 0.4g / L, CuSO4·5H2O 0.3g / L, KI 0.1g / L.

[0017] The vitamin solution formula is as follows: Vitamin B1 20-70 mg / L, Vitamin B6 3-5 mg / L, Vitamin B... 12 10-50 mg / L. Preferably, vitamin B1 23 mg / L, vitamin B6 4 mg / L, and vitamin B... 12 20 mg / L.

[0018] In step (2), the *Saccharomyces cerevisiae* G7 seed culture is inoculated into the fermentation medium at an inoculation rate of 5%-30% v / v, and the *Escherichia coli* G5 seed culture is inoculated into the fermentation broth at an inoculation rate of 1%-10% v / v. Preferably, the *Saccharomyces cerevisiae* G7 seed culture is inoculated into the fermentation medium at an inoculation rate of 10%-20% v / v, and the *Escherichia coli* G5 seed culture is inoculated into the fermentation broth at an inoculation rate of 5%-7% v / v. Most preferably, the *Saccharomyces cerevisiae* G7 seed culture is inoculated into the fermentation medium at an inoculation rate of 20% v / v, and the *Escherichia coli* G5 seed culture is inoculated into the fermentation broth at an inoculation rate of 5% v / v.

[0019] In step (2), typically, after 24-48 hours of single-strain fermentation with *Saccharomyces cerevisiae* G7, the OD of the fermentation broth... 600 The value can reach 80.

[0020] In step (2), the tank pressure is 0.03-0.06 MPa and the aeration rate is 4-6 m³ / s throughout the fermentation process.3 The stirring rate is 100-450 rpm, the pH is 6.5-7.0, and the dissolved oxygen is maintained at 10%-40% by adjusting the stirring rate, aeration rate and tank pressure.

[0021] In step (2), the temperature during single-strain fermentation of *Saccharomyces cerevisiae* G7 is controlled at 28-32℃; during co-culture fermentation, when the OD of the fermentation broth... 600 When the concentration of organic matter in the fermentation broth is ≤100, the temperature should be controlled between 28-32℃. 600 When the concentration is >100, the temperature should be controlled between 22-28℃. Preferably, the temperature for single-strain fermentation of *Saccharomyces cerevisiae* G7 should be controlled at 30℃; during co-culture fermentation, when the OD of the fermentation broth... 600 When the concentration is ≤100, the temperature is controlled at 30℃. When the OD of the fermentation broth is... 600 When the temperature is >100, the temperature should be controlled at 26℃.

[0022] In step (2), the fermentation cycle of the two bacteria co-culture is 5-7 days, preferably 5-6 days, and most preferably 5 days.

[0023] Beneficial Effects: This invention provides a simple, low-cost, and easily extractable method for producing ergothioneine through mixed-culture fermentation. This invention utilizes mixed-culture fermentation, adjusting the appropriate inoculum ratio by controlling the seed culture ratio (5%-30% v / v for strain G7 and 1%-10% v / v for strain G5) to ensure sufficient consumption and utilization of the culture medium and the metabolic byproducts of *Saccharomyces cerevisiae* (yeast extracellular polysaccharides, glutamic acid, cysteine, and other amino acids) by both strains. This results in the accumulation of more ergothioneine while simultaneously reducing the conductivity of the fermentation broth, thereby simplifying extraction and reducing wastewater discharge. The final fermentation broth contains 8.5 g / L of ergothioneine with a conductivity ≤5 mS / cm. Attached Figure Description

[0024] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0025] Figure 1 The graph shows the changes in ergothionein content during single-strain fermentation and mixed-strain fermentation of Saccharomyces cerevisiae G7.

[0026] Figure 2 The graph shows the changes in conductivity during single-strain and mixed-strain fermentation of Saccharomyces cerevisiae G7.

[0027] Figure 3 The graph shows the changes in ergothionein content between single-strain fermentation and mixed-strain fermentation of Escherichia coli G5.

[0028] Figure 4 The graph shows the changes in conductivity during single-strain and mixed-strain fermentation of Escherichia coli G5. Detailed Implementation

[0029] The brewing yeast used in the following examples is Saccharomyces cerevisiae G7, with accession number CCTCC NO: M 20231232, accession date July 7, 2023, and accession address Wuhan University, Wuhan, China.

[0030] The Escherichia coli is Escherichia coli G5, with accession number CCTCC NO: M 2023894, accession date May 31, 2023, and accession address Wuhan University, Wuhan, China.

[0031] The culture medium for the Saccharomyces cerevisiae G7 seed culture used in the following examples consists of: 10 g / L peptone, 5 g / L yeast extract, 15 g / L anhydrous glucose, 5 g / L ammonium sulfate, 7.6 g / L potassium dihydrogen phosphate, and 1 g / L magnesium sulfate.

[0032] The Escherichia coli G5 seed culture medium consists of: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, and 5 g / L glycerol.

[0033] Fermentation medium composition: methanol 0.5 g / L, ammonium sulfate 5 g / L, dipotassium hydrogen phosphate 8 g / L, magnesium sulfate 1 g / L, trace element mixture 7.5 mL / L, vitamin solution 5 mL / L, histidine 19 g / L, methionine 19 g / L, cysteine ​​19 g / L.

[0034] The formula for the trace element mixed solution is as follows: CaCl2·2H2O 6g / L, ZnSO4·7H2O 7.5g / L, FeSO4·7H2O 6.5g / L, H3BO3 5.3g / L, MnCl2·4H2O 4.1g / L, Na2MoO4·2H2O 0.4g / L, CoCl2·6H2O 0.4g / L, CuSO4·5H2O 0.3g / L, KI 0.1g / L.

[0035] The vitamin solution formula is: Vitamin B1 23 mg / L, Vitamin B6 4 mg / L, Vitamin B... 12 20 mg / L.

[0036] Ergothionein detection method: HPLC method.

[0037] The fermentation broth was centrifuged to remove bacterial cells, and the ergothioneine content in the supernatant was tested. A HYPURITY C18 (250*4.6mm, 5um) column was used. The pH of the water was adjusted to 5.0 with formic acid. The mobile phase was formic acid aqueous solution (A) and methanol (B). The flow rate was 0.7 mL / min, the detection time was 20 min, and gradient elution was used: 0-10 min, A:B = 95:5; 10-15 min, A:B = 70:30; 15-20 min, A:B = 95:5. The column temperature was room temperature, and the detection wavelength was 254 nm.

[0038] Weigh 0.1 g of ergothioneine standard and dilute to 100 mL with pure water. Prepare standard solutions of different concentrations by measuring 0.01 mL, 0.05 mL, 0.1 mL, 0.5 mL, and 1 mL of each solution and diluting to 10 mL. Analyze the peak areas of the standard solutions at different concentrations under the analytical conditions for ergothioneine detection, and construct a standard curve with the concentration of each substance and the peak area as the x-axis and y-axis, respectively. After appropriate dilution, the fermented sample was analyzed under the same conditions, and the concentrations were calculated.

[0039] Conductivity testing methods:

[0040] Use the Shanghai Leici DDB-303A portable conductivity meter. 1) Clean the electrode with high-purity water; 2) Place the electrode in the fermentation broth to be tested, and the displayed value is the actual conductivity.

[0041] Example 1:

[0042] Saccharomyces cerevisiae G7 and Escherichia coli G5 were cultured separately using seed culture medium. The seed culture conditions for Saccharomyces cerevisiae G7 were 28℃ for 24 h, and for Escherichia coli G5 were 37℃ for 16 h. Seed culture solution 1 and seed culture solution 2 were obtained accordingly.

[0043] Seed culture 1 was inoculated into the fermentation medium of the fermenter at an inoculation rate of 20% v / v and fermented for 26 hours until the OD of the fermentation broth reached a certain level. 600 After reaching 80%, seed liquid 2 was inoculated into a fermenter at 5% v / v for co-culture and fermentation for 5 days to produce ergothioneine with a content of 8.5 g / L and a conductivity of 4.6 mS / cm in the fermentation broth.

[0044] Throughout the fermentation process, the fermentation conditions were: tank pressure 0.03-0.06 MPa, and aeration rate of 4-6 m³ / h. 3 The stirring speed was 100-450 rpm (dissolved oxygen controlled at 20%-40%, with speed adjusted accordingly), and the pH was 6.8±0.2. Dissolved oxygen was maintained at 20%-40% by adjusting the stirring rate, aeration rate, and tank pressure. The temperature for single-strain fermentation of *Saccharomyces cerevisiae* G7 was controlled at 30℃. During co-culture fermentation, when the OD of the fermentation broth... 600When the concentration is ≤100, the temperature is controlled at 30℃. When the OD of the fermentation broth is... 600 When the temperature is >100, the temperature should be controlled at 26℃.

[0045] The curves showing the changes in ergothioneine content and conductivity of the fermentation broth over fermentation time are shown below. Figure 1 and Figure 2 .

[0046] Example 2:

[0047] Saccharomyces cerevisiae G7 and Escherichia coli G5 were cultured separately in seed culture medium. The seed culture conditions for Saccharomyces cerevisiae G7 were 28℃ for 24 h, and for Escherichia coli G5 were 37℃ for 16 h. Seed solution 1 and seed solution 2 were obtained accordingly.

[0048] Seed culture 1 was inoculated into the fermentation medium of the fermenter at inoculation rates of 5%, 10%, 15%, 20%, 25%, and 30% v / v, respectively, and fermented for 26 hours. The fermentation was carried out until the OD of the fermentation broth reached a certain level. 600 After reaching 80%, seed liquid 2 was inoculated into fermenters at 3%, 5%, 7%, and 9% v / v for co-culture and fermentation for 5 days to produce ergothioneine. The results are detailed in Table 1.

[0049] Throughout the fermentation process, the fermentation conditions were: tank pressure 0.03-0.06 MPa, and aeration rate of 4-6 m³ / h. 3 The stirring speed was 100-450 rpm (adjusted according to dissolved oxygen levels of 20%-40%), and the pH was 6.8±0.2. Dissolved oxygen was maintained at 20%-40% by adjusting the stirring rate, aeration rate, and tank pressure. The temperature for single-strain fermentation of *Saccharomyces cerevisiae* G7 was controlled at 30℃. During co-culture fermentation, when the OD of the fermentation broth... 600 When the concentration is ≤100, the temperature is controlled at 30℃. When the OD of the fermentation broth is... 600 When the temperature is >100, the temperature should be controlled at 26℃.

[0050] Table 1. Ergothionein content obtained from different inoculation ratios of *Escherichia coli* G5 and *Saccharomyces cerevisiae* G7.

[0051]

[0052] Example 3

[0053] Saccharomyces cerevisiae G7 and Escherichia coli G5 were cultured separately using seed culture medium. The seed culture conditions for Saccharomyces cerevisiae G7 were 26℃ for 34 h, and for Escherichia coli G5 were 37℃ for 14 h. Seed culture solution 1 and seed culture solution 2 were obtained accordingly.

[0054] Seed liquid 1 was inoculated into the fermentation medium of the fermenter at a 20% v / v inoculation rate and fermented for 34 hours. After the OD600 of the fermentation liquid reached 80, seed liquid 2 was inoculated into the fermenter at a 5% v / v inoculation rate and co-cultured for 7 days to produce ergothioneine with a content of 5.3 g / L and a conductivity of 5.1 mS / cm in the fermentation liquid.

[0055] Throughout the fermentation process, the fermentation conditions were: tank pressure 0.03-0.06 MPa, and aeration rate of 4-6 m³ / h. 3 The stirring speed was 100-450 rpm (dissolved oxygen was controlled at 20%-40%, and the stirring speed was adjusted accordingly), and the pH was 6.8±0.2. The dissolved oxygen was maintained at 20%-40% by adjusting the stirring rate, aeration rate, and tank pressure. The temperature for single-strain fermentation of Saccharomyces cerevisiae G7 was controlled at 32℃. During co-culture fermentation, the temperature was controlled at 32℃ when the OD600 of the fermentation broth was ≤100, and at 24℃ when the OD600 of the fermentation broth was >100.

[0056] Example 4

[0057] Saccharomyces cerevisiae G7 and Escherichia coli G5 were cultured separately using seed culture medium. The seed culture conditions for Saccharomyces cerevisiae G7 were 30℃ for 20 h, and for Escherichia coli G5 were 34℃ for 16 h. Seed culture solution 1 and seed culture solution 2 were obtained accordingly.

[0058] Seed liquid 1 was inoculated into the fermentation medium of the fermenter at a 20% v / v inoculation rate and fermented for 34 hours. After the OD600 of the fermentation liquid reached 80, seed liquid 2 was inoculated into the fermenter at a 5% v / v inoculation rate and co-cultured for 6 days to produce ergothioneine with a content of 6.7 g / L and a conductivity of 5.0 mS / cm in the fermentation liquid.

[0059] Throughout the fermentation process, the fermentation conditions were: tank pressure 0.03-0.06 MPa, and aeration rate of 4-6 m³ / h. 3 The stirring speed was 100-450 rpm (dissolved oxygen was controlled at 20%-40%, and the stirring speed was adjusted accordingly), and the pH was 6.8±0.2. The dissolved oxygen was maintained at 20%-40% by adjusting the stirring rate, aeration rate, and tank pressure. The temperature for single-strain fermentation of Saccharomyces cerevisiae G7 was controlled at 28℃. During co-culture fermentation, the temperature was controlled at 28℃ when the OD600 of the fermentation broth was ≤100, and at 28℃ when the OD600 of the fermentation broth was >100.

[0060] Comparative Example 1: Brewing yeast G7 fermented alone

[0061] The formula and seed culture conditions for the Saccharomyces cerevisiae G7 seed culture medium are the same as in Example 1.

[0062] The fermentation medium formulation is the same as in Example 1.

[0063] The seed culture of Saccharomyces cerevisiae G7 was inoculated into the fermenter at an inoculation rate of 16% v / v for pure fermentation culture.

[0064] Fermentation conditions were: tank pressure 0.03-0.06 MPa, temperature 28℃, and aeration rate of 4-6 m³ / h. 3 The stirring speed was 100-450 rpm, and the pH was 6.8. Dissolved oxygen was maintained between 10% and 40% by adjusting the stirring speed, aeration rate, and tank pressure. Fermentation lasted 120 hours. The ergothioneine content in the fermentation broth was determined to be 3.2 g / L by HPLC, and the conductivity of the fermentation broth was 45 mS / cm. The curves showing the changes in ergothioneine content and conductivity of the fermentation broth over fermentation time are shown below. Figure 1 and Figure 2 .

[0065] Comparative Example 2: Fermentation of Escherichia coli G5 strain alone

[0066] The formulation and seed culture conditions for Escherichia coli G5 seed culture medium are the same as in Example 1.

[0067] The fermentation medium formulation is the same as in Example 1.

[0068] The cultured Escherichia coli G5 seed culture was inoculated into a fermenter at an inoculation rate of 2% v / v for pure fermentation culture.

[0069] Fermentation conditions were as follows: tank pressure 0.03-0.06 MPa, temperature 32℃ for 0-38 hours, temperature 28℃ for 39-120 hours, and aeration rate of 4-6 m³ / h. 3 The stirring speed was 100-450 rpm, and the pH was 6.8. The dissolved oxygen was maintained at 10%-40% by adjusting the stirring speed, aeration rate, and tank pressure. Fermentation lasted 120 hours. The ergothioneine content in the fermentation broth was determined to be 4.12 g / L by HPLC, and the conductivity of the fermentation broth was 13 mS / cm. The curves showing the changes in ergothioneine content and conductivity of the fermentation broth over fermentation time are shown below. Figure 3 and Figure 4 .

[0070] This invention provides a method and approach for producing ergothioneine through mixed-culture fermentation. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. A method for producing ergothioneine through mixed-culture fermentation, characterized in that, Ergothioneine is produced by mixed fermentation of brewer's yeast and Escherichia coli; the brewer's yeast is brewer's yeast ( Saccharomyces cerevisiae G7, with accession number CCTCC NO: M 20231232; the Escherichia coli mentioned is Escherichia coli ( Escherichia coli G5, accession number CCTCC NO: M 2023894; The method includes the following steps: (1) Culture seed culture of Saccharomyces cerevisiae G7 and Escherichia coli G5 respectively; (2) Inoculate the Saccharomyces cerevisiae G7 seed culture into the fermentation medium and ferment until the OD of the fermentation broth reaches a certain level. 600 Once the value reaches 80, the E. coli G5 seed culture is inoculated into the fermentation broth for co-culture fermentation to produce ergothioneine. In step (2), the Saccharomyces cerevisiae G7 seed liquid is inoculated into the fermentation medium at an inoculation rate of 10%-20% v / v, and the Escherichia coli G5 seed liquid is inoculated into the fermentation broth at an inoculation rate of 5%-7% v / v. In step (2), the temperature during the fermentation of single-strain brewer's yeast G7 is controlled at 30℃; during the co-culture fermentation process, when the OD of the fermentation broth... 600 When the concentration is ≤100, the temperature is controlled at 30℃. When the OD of the fermentation broth is... 600 When the temperature is >100, the temperature should be controlled at 26℃.

2. The method according to claim 1, characterized in that, In step (1), the seed culture medium formula for Saccharomyces cerevisiae G7 is as follows: peptone 5-10 g / L, yeast extract 5-10 g / L, anhydrous glucose 10-40 g / L, ammonium sulfate 1-10 g / L, potassium dihydrogen phosphate 5-15 g / L, and magnesium sulfate 0.1-2 g / L.

3. The method according to claim 1, characterized in that, In step (1), Saccharomyces cerevisiae G7 is cultured in seed culture medium at 25-30℃ for 18-36 h.

4. The method according to claim 1, characterized in that, In step (1), the seed culture medium formula for Escherichia coli G5 is: peptone 5-10 g / L, yeast extract 5-10 g / L, sodium chloride 5-10 g / L, and glycerol 5-10 g / L.

5. The method according to claim 1, characterized in that, In step (1), Escherichia coli G5 is cultured in seed culture medium at 34-37℃ for 12-16 h.

6. The method according to claim 1, characterized in that, In step (2), the fermentation medium formula is as follows: methanol 0.3-0.9 g / L, ammonium sulfate 1-10 g / L, dipotassium hydrogen phosphate 5-30 g / L, magnesium sulfate 1-10 g / L, trace element mixture 1-10 mL / L, vitamin solution 1-10 mL / L, histidine 5-20 g / L, methionine 5-20 g / L, cysteine ​​5-20 g / L.

7. The method according to claim 6, characterized in that, The formula for the trace element mixture is as follows: CaCl2 1-10 g / L, ZnSO4 1-10 g / L, FeSO4 1-10 g / L, H3BO3 1-6 g / L, MnCl2 1-5 g / L, Na2MoO4 0.1-1 g / L, CoCl2 0.1-1 g / L, CuSO4 0.1-1 g / L, KI 0.1-1 g / L; The vitamin solution formula is as follows: Vitamin B1 20-70 mg / L, Vitamin B6 3-5 mg / L, Vitamin B... 12 10-50 mg / L.

8. The method according to claim 1, characterized in that, In step (2), the tank pressure is 0.03-0.06 MPa, the aeration rate is 4-6 m³ / h, the stirring rate is 100-450 rpm, the pH is 6.5-7.0, and the dissolved oxygen is maintained at 10%-40% throughout the fermentation process.

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