A high-rna-content strain of zygosaccharomyces bailii and uses thereof

Abstract

The application discloses a high-RNA-content strain of Zygosaccharomyces bailii and application thereof, and belongs to the technical field of microorganisms. Cyberlindnera jadinii The strain is named WB15 strain, and was preserved in the China Center for Type Culture Collection on March 21, 2023, with a preservation number of CCTCC NO: M 2023385. Cyberlindnera jadinii The WB15 strain has high RNA content and very high passage stability, and the RNA content is stable at 156-162 mg / g DCW on a slope for 10 generations, and the yield has no significant difference between the ten generations through single-factor variance analysis. Cyberlindnera jadinii The RNA content of the WB15 strain is further improved to be as high as 184 mg / g DCW through optimization of a fermentation medium.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a high RNA content strain of *G. jeldingerbergia lindnerella* and its applications. Background Technology

[0002] Ribonucleic acid (RNA) is a class of macromolecules that are extremely important to living organisms. It participates in gene expression and plays a crucial role in protein synthesis. RNA hydrolysis and its derivatives have a wide range of applications. 5'-inosinic acid (5'-IMP) and 5'-guanosine acid (5'-GMP), along with their disodium salts, can be used as food additives in the food industry to enhance flavor. The nucleotides, nucleosides, and bases produced by RNA degradation can serve as important pharmaceutical precursors, and their derivatives have significant effects in combating tumors and viruses. Adding RNA to food can also slow down aging and help enhance the body's resistance.

[0003] Currently, yeast fermentation is commonly used in industrial production to produce RNA. The RNA content of yeast cells accounts for 14%-15% of the dry weight of the cells, and Candida utilis is certified by the U.S. Food and Drug Administration (FDA). Candida albicans It is a GRAS (generally recognized as safe) microorganism, meaning it can be used in food production. Genome sequencing results indicate that it is *Gynostemma spp.* Cyberlindnera jadinii and C. utilis The two genomes are highly similar at the sequence level, with an overall sequence homology of 98%. C. jadinii It is considered Useful The sexual reproduction state of *G. selaginella* was later standardized under the name *G. selaginella sebifera*. C. jadinii . C. jadinii It is rich in protein, amino acids and ribonucleic acid, and can be used to produce a variety of biological products such as lactic acid, monetin, glucomannan and biotin. C. jadinii It has the following characteristics: high fermentation density; high tolerance to external environment; Crabtree-negative effect, does not produce ethanol under strict aerobic conditions; can grow in a broad temperature range of 19~37℃; can be grown using inexpensive molasses and industrial wastewater; and can utilize urea, nitrates and ammonium salts. C. jadinii Because of these excellent characteristics, it has a great advantage in industrial production.

[0004] The selection and breeding of high-RNA yeast strains plays a significant role in industrial production. Generally, physical or chemical mutagenesis is used to treat strains, followed by selection of mutant strains to identify high-yielding strains. Atmospheric Room Temperature Plasma (ARTP), as a novel mutagenesis strategy, uses highly reactive particles generated at normal atmospheric pressure to damage cells, inducing the activation of the SOS repair mechanism and causing mutations in microorganisms, resulting in genetically stable mutant strains. ARTP mutagenesis is low-cost, simple to operate, and highly safe. Its DNA damage is greater than traditional mutagenesis methods, and it has been successfully applied to bacteria, fungi, actinomycetes, and microalgae. Early screening of mutagenic colonies mainly involved random selection on plates, followed by fermentation in shake flasks and result measurement, which was time-consuming, labor-intensive, and costly. Currently, high-throughput screening technology can be used for rapid screening. Most microorganisms can be cultured or reacted in deep-well plates, and combined with a microplate reader, the goal of rapidly screening large numbers of microorganisms in a short time can be achieved, greatly improving the efficiency of screening mutant strains after mutagenesis. However, yeast strains with high RNA content are still very scarce. Summary of the Invention

[0005] To solve the above technical problems, Cyberlindnera jadinii MT1903 was used as the starting strain. ARTP mutagenesis was performed on it to obtain a mutant strain, which was then cultured in 48-well deep-well plates and shake flasks. Finally, a yeast strain with high RNA content was successfully screened, thus completing the present invention.

[0006] The first aspect of this invention provides a high RNA content strain of *G. selaginella* 'J. Seberlindner', named... Cyberlindnera jadinii strain WB15 was deposited at the China Center for Type Culture Collection (CCTCC) on March 21, 2023, with accession number CCTCC NO: M 2023385; deposit address: Wuhan University, Wuhan, China. A second aspect of the present invention provides a fermentation broth comprising the components described in the first aspect of the present invention. Cyberlindnera the gardeners Live or dead cells of strain WB15.

[0007] In some embodiments of the present invention, the fermentation broth is prepared using the... Cyberlindnera jadinii The fermentation broth was obtained by fermentation using strain WB15. Furthermore, the fermentation broth was treated to kill the bacteria; the fermentation broth at this point also falls within the scope of protection of this invention. A third aspect of this invention provides the application of the fermentation broth described in the second aspect of this invention in the preparation of RNA products. The RNA products include RNA itself and its hydrolysis and derivatives, including but not limited to 5'-inosinic acid (5'-IMP) and 5'-guanosine acid (5'-GMP) and their disodium salts, nucleotides, nucleosides, and bases produced by RNA degradation. The fourth aspect of the present invention provides a method for cultivating the [material described in the first aspect of the present invention]. Cyberlindnera the gardeners The culture medium composition for strain WB15 includes a seed medium for strain activation and / or a fermentation medium for strain fermentation.

[0008] In some embodiments of the present invention, the seed culture medium is composed of: 40 g / L glucose, 12-18 g / L corn steep liquor, 2.0-2.5 g / L KH2PO4, 2.0-3.0 g / L MgSO4, and pH 6-7.

[0009] In some preferred embodiments of the present invention, the seed culture medium is composed of: 40 g / L glucose, 15 g / L corn steep liquor, 2.3 g / L KH2PO4, 2.5 g / L MgSO4, and pH 6.5.

[0010] In some embodiments of the present invention, the fermentation medium comprises: 50 g / L sucrose, 8-15 g / L yeast extract, 8-15 g / L soybean peptone, 1.8-2.9 g / L KH₂PO₄, 2.0-3.0 g / L MgSO₄, 0.005-0.02 g / L FeSO₄, 0.005-0.02 g / L ZnSO₄, and pH 5-6.

[0011] In some preferred embodiments of the present invention, the fermentation medium is composed of: 50 g / L sucrose, 10 g / L yeast extract, 10 g / L soybean peptone, 2.3 g / L KH2PO4, 2.5 g / L MgSO4, 0.01 g / L FeSO4, 0.01 g / L ZnSO4, and pH 5.5.

[0012] In some other preferred embodiments of the present invention, the fermentation medium is composed of: 50 g / L sucrose, 13.4 g / L yeast extract, 12.1 g / L soybean peptone, 2.8 g / L KH2PO4, 2.5 g / L MgSO4, 0.01 g / L FeSO4, 0.01 g / L ZnSO4, and pH 5.5. The fifth aspect of the present invention provides the invention described in the first aspect. Cyberlindnera jadinii The fermentation method for strain WB15 includes the following steps: S1, the inclined plane is saved as described above. Cyberlindnera jadiniiThe WB15 strain was inoculated into a shake flask containing seed culture medium and cultured at 28-35℃ and 200-250 rpm for 16-24 hours to obtain the seed solution. S2, inoculate the seed culture at an inoculation rate of 1-5% into a shake flask containing fermentation medium, and incubate at a temperature of 28-35℃ and a shaker speed of 200-250 rpm for 6-10 hours. The seed culture medium comprises: 40 g / L glucose, 12-18 g / L corn steep liquor, 2.0-2.5 g / L KH₂PO₄, 2.0-3.0 g / L MgSO₄, and pH 6-7. In some preferred embodiments of the present invention, the seed culture medium comprises: 40 g / L glucose, 15 g / L corn steep liquor, 2.3 g / L KH₂PO₄, 2.5 g / L MgSO₄, and pH 6.5.

[0013] The fermentation medium consists of: 50 g / L sucrose, 8-15 g / L yeast extract, 8-15 g / L soybean peptone, 1.8-2.9 g / L KH₂PO₄, 2.0-3.0 g / L MgSO₄, 0.005-0.02 g / L FeSO₄, 0.005-0.02 g / L ZnSO₄, and pH 5-6. In some preferred embodiments of the present invention, the fermentation medium consists of: 50 g / L sucrose, 10 g / L yeast extract, 10 g / L soybean peptone, 2.3 g / L KH₂PO₄, 2.5 g / L MgSO₄, 0.01 g / L FeSO₄, 0.01 g / L ZnSO₄, and pH 5.5. In some other preferred embodiments of the present invention, the fermentation medium is composed of: 50 g / L sucrose, 13.4 g / L yeast extract, 12.1 g / L soybean peptone, 2.8 g / L KH2PO4, 2.5 g / L MgSO4, 0.01 g / L FeSO4, 0.01 g / L ZnSO4, and pH 5.5.

[0014] In some embodiments of the present invention, the volume of the shake flask in step S1 is 250 mL, of which the volume containing the seed culture medium is 25 mL.

[0015] In some embodiments of the present invention, the volume of the shake flask in step S1 is 250 mL, of which the volume of the fermentation medium is 50 mL.

[0016] This invention also provides a method for determining the RNA content of bacterial strains based on absorbance: After thoroughly shaking the cultured bacterial suspension, add 300 μL to a well plate or 1.5 mL EP tube. Centrifuge at 4000 rpm for 10 min, discard the supernatant, wash twice with 0.9% NaCl solution, resuspend in 800 μL of 0.25 mol / L HClO4 at 4℃, shake well, and incubate at 4℃ for 15 min without shaking. Centrifuge at 4000 rpm for 10 min, discard the supernatant, add 800 μL of 0.5 mol / L HClO4, and incubate in a 75℃ water bath for 15 min, shaking to mix every 4 min. After the water bath, centrifuge at 4000 rpm for 10 min, and use a microplate reader or UV-Vis spectrophotometer to analyze the OD values. 260 The determination.

[0017] After fermentation, the OD of the fermentation broth was measured in cells cultured in shake flasks. 600 Then, the RNA content of the strain is obtained according to the following formulas (2) and (3).

[0018] RNA content (mg / g DCW) = (A×D×0.03365×0.8) / (M×0.3) (2) M = 0.4333 × OD 600 +0.9011 (3) In formula (2): A is the OD of the sample supernatant. 260 D is the dilution factor; M is the dry weight of the cells (g / L); 0.8 is the volume (mL) of 0.5 mol / L HClO4 solution added; 0.3 is the volume (mL) of fermentation broth taken; 0.03365 is the RNA content (mg / 1mL) in the test solution corresponding to an absorbance of 1.00.

[0019] Formula (3) is the cell dry weight and fermentation broth OD 600 The regression equation between them.

[0020] Compared with the prior art, the present invention has the following beneficial effects: This invention is based on Cyberlindnera jadinii MT1903 was used as the starting strain. ARTP mutagenesis was induced to obtain mutant strains, which were then cultured in 48-well deep-well plates. Finally, strains with high RNA content were selected. Cyberlindnera the gardeners strain WB15.

[0021] The present invention Cyberlindnera jadinii The WB15 strain exhibits very high passage stability. After 10 passages on an agar slant, the RNA content remained stable between 156 and 162 mg / g DCW. One-way ANOVA showed no significant difference in yield among the ten passages.

[0022] This invention further improves the fermentation medium by optimizing it. Cyberlindnera jadinii The RNA content of strain WB15 can be as high as 184 mg / g DCW. Attached Figure Description

[0023] Figure 1 It shows Cyberlindnera jadinii Scanning electron microscope image of strain WB15.

[0024] Figure 2 It shows Cyberlindnera jadinii Stability diagram of strain WB15 after passage. Implementation

[0025] Unless otherwise stated, implied from the context, or as is customary in the art, all parts and percentages in this application are based on weight, and all testing and characterization methods used are concurrent with the filing date of this application. Where applicable, any patent, patent application, or disclosure relating to this application is incorporated herein by reference in its entirety, and its equivalent patent families are also incorporated herein by reference, in particular the definitions of relevant terms in the art disclosed in such documents. If any definition of a specific term disclosed in the prior art is inconsistent with any definition provided in this application, the definition provided in this application shall prevail.

[0026] The numerical ranges used in this application are approximate values ​​and therefore may include values ​​outside the range unless otherwise stated. The numerical range includes all values ​​from the lower limit to the upper limit, increasing by one unit, provided there is an interval of at least two units between any lower and any higher value. For ranges containing values ​​less than 1 or fractions greater than 1 (e.g., 1.1, 1.5, etc.), one unit is appropriately considered as 0.0001, 0.001, 0.01, or 0.1. For ranges containing single digits less than 10 (e.g., 1 to 5), one unit is generally considered as 0.1. These are merely specific examples of what is intended to be expressed, and all possible combinations of values ​​between the listed minimum and maximum values ​​are considered to be clearly described in this application.

[0027] The terms “comprising,” “including,” “having,” and their derivatives do not exclude the presence of any other components, steps, or processes, regardless of whether such other components, steps, or processes are disclosed in this application. To eliminate any doubt, unless expressly stated otherwise, all compositions using the terms “comprising,” “including,” or “having” in this application may contain any additional additives, excipients, or compounds. Conversely, except for those necessary for operational performance, the term “substantially constitutes…” excludes any other components, steps, or processes described below with respect to that term. The term “consisting of…” does not include any components, steps, or processes not specifically described or listed. Unless expressly stated otherwise, the term “or” refers to the individual members listed or any combination thereof.

[0028] To make the technical problems solved by the present invention, the technical solutions and the beneficial effects of the present invention clearer, the present invention will be further described in detail below with reference to the embodiments. Example

[0029] The following examples are used to illustrate preferred embodiments of the invention. Those skilled in the art will understand that the techniques disclosed in the examples represent techniques discovered by the inventors that can be used to implement the invention, and therefore can be considered preferred embodiments for implementing the invention. However, those skilled in the art should understand from this specification that many modifications can be made to the specific embodiments disclosed herein, still yielding the same or similar results, without departing from the spirit or scope of the invention.

[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains, and all materials publicly cited herein and referenced by them are incorporated herein by reference.

[0031] Those skilled in the art will recognize, or can learn through routine experimentation, many equivalents of the specific embodiments of the invention described herein. These equivalents will be included in the claims.

[0032] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the instruments and equipment used in the following examples are all conventional laboratory instruments and equipment; unless otherwise specified, the experimental materials used in the following examples were all purchased from conventional biochemical reagent stores.

[0033] The strains and culture media used in the following examples are as follows: Strain: Jedinger Seberlindnerella vaginalis MT1903 ( C. jadinii MT1903), is the strain preserved by the applicant.

[0034] Solid culture medium (plate culture medium): glucose 20g / L, yeast extract 10g / L, tryptone 20g / L, agar 15g / L.

[0035] The seed culture medium compositions in Examples 1-3 were the same: 40 g / L glucose, 15 g / L corn steep liquor, 2.3 g / L KH2PO4, 2.5 g / L MgSO4, and pH 6.5.

[0036] During the ARTP mutagenesis treatment described below, contact between helium and oxygen to form ozone should be avoided as much as possible, as this can be toxic to cells. The mutagenesis lethality rate is calculated using a mutagenesis time of 0 seconds as a reference, as shown in formula (1) below: Mortality rate (%) = ( T 0 - T 1 ) / T 0 × 100 (1) in, T 0 The total number of colonies when the mutagenesis time is 0s. T 1 This represents the total number of colonies after mutagenesis treatment.

[0037] The following OD 260 The supernatant was measured using an enzyme-linked immunosorbent assay (ELISA) reader.

[0038] The methods for nucleic acid extraction and RNA content determination are as follows: After thoroughly shaking the cultured bacterial suspension, add 300 μL to a well plate or 1.5 mL EP tube. Centrifuge at 4000 rpm for 10 min, discard the supernatant, wash twice with 0.9% NaCl solution, resuspend in 800 μL of 0.25 mol / L HClO4 at 4℃, shake well, and incubate at 4℃ for 15 min without shaking. Centrifuge at 4000 rpm for 10 min, discard the supernatant, add 800 μL of 0.5 mol / L HClO4, and incubate in a 75℃ water bath for 15 min, shaking to mix every 4 min. After the water bath, centrifuge at 4000 rpm for 10 min, and use a microplate reader or UV-Vis spectrophotometer to analyze the OD values. 260 The determination.

[0039] After fermentation, the OD of the fermentation broth was measured in cells cultured in shake flasks. 600 Then, the RNA content of the strain is obtained according to the following formulas (2) and (3).

[0040] RNA content (mg / g DCW) = (A×D×0.03365×0.8) / (M×0.3) (2) M = 0.4333 × OD 600 +0.9011 (3) In formula (2): A is the OD of the sample supernatant. 260 D is the dilution factor; M is the dry weight of the cells (g / L); 0.8 is the volume (mL) of 0.5 mol / L HClO4 solution added; 0.3 is the volume (mL) of fermentation broth taken; 0.03365 is the RNA content (mg / 1mL) in the test solution corresponding to an absorbance of 1.00.

[0041] Formula (3) is the cell dry weight and fermentation broth OD 600 The regression equation between them.

[0042] This embodiment provides a method for selecting yeast strains with high RNA content, including the following steps: (1) The MT1903 yeast from the slant culture was placed on the slant culture. Cyberlindnera jadinii MT1903) Inoculate a piece the size of the tip of a bamboo stick into a 250 mL shake flask containing 25 mL of seed culture medium using a sterile bamboo stick, and incubate at 30 °C and 220 rpm on a shaker until the late logarithmic phase. (2) Centrifugation to collect the cells from step (1) of liquid culture, using a 0.9% NaCl aqueous solution to prepare a solution with a density of 10. 7 The bacterial suspension of cells / mL was subjected to ARTP mutagenesis. The specific mutagenesis process was as follows: 10µL of bacterial suspension was evenly spread on the slide, the power was adjusted to 120W, the helium flow rate was 10SLM, the distance between the nozzle and the slide was 2mm, the mutagenesis time was 30s, and the lethality rate was 90%-95% as the standard. (3) Dilute the bacterial suspension induced in step (2) and spread it on a plate. The bacterial suspensions of 0~20s, 30~90s, and 120~180s were diluted 1000 times, 100 times, and 10 times, respectively. Incubate at 30℃ for 48h. Select single colonies obtained by treatment with a mutagenesis time of 30s (lethality rate of 94.12%) and inoculate them into 48-well deep-well plates. Incubate at 30℃ and shaker speed of 220rpm for 18h. Perform RNA extraction and initially screen out high OD values ​​in each well plate. 260 For outliers, the corresponding mutant strains were inoculated onto slant plates and cultured at 30°C for 48 hours. (4) The mutant strains obtained from the initial screening after step (3) were inoculated from the slant into a 250 mL shake flask containing 25 mL of seed culture medium and cultured at 30 °C and 220 rpm for 18 h. (5) The seed culture prepared in step (4) was inoculated into a 250 mL baffle shaker flask containing 50 mL of fermentation medium at an inoculation rate of 4%. After culturing at 30 °C and 220 rpm for 8 h, the RNA content was measured to obtain a yeast strain with high RNA content. Cyberlindnera jadinii WB15.

[0043] The fermentation medium composition of step (5) in this embodiment is: sucrose 50 g / L, yeast extract 10 g / L, soybean peptone 10 g / L, KH2PO4 2.3 g / L, MgSO4 2.5 g / L, FeSO4 0.01 g / L, ZnSO4 0.01 g / L, pH 5.5.

[0044] The breeding obtained in this embodiment C. jadinii The RNA content of WB15 is 156 mg / g DCW, where DCW is the dry weight of the cells.

[0045] C. jadinii The scanning electron microscope image of strain WB15 is shown in Figure 1. As can be seen from the figure, C. jadinii The cells of strain WB15 are relatively plump, with a smooth and intact surface without wrinkles. The bud scars caused by proliferation on the surface of the yeast can be clearly seen, and there is almost no adhesion between the cells.

[0046] Example 2 C. jadinii WB15 passage stability test Will C. jadinii The WB15 strain was continuously passaged on slant for 10 generations, followed by shake-flask fermentation to determine its RNA content. The experimental results are as follows: Figure 2 As shown, the RNA content remained stable between 156 and 162 mg / g DCW. One-way ANOVA showed no significant difference in yield among the ten generations, indicating that... C. jadinii The WB15 strain exhibits good genetic stability.

[0047] Will C. jadinii WB15 is deposited at the China Center for Type Culture Collection (CCTCC) of Wuhan University, China, with accession number CCTCC NO: M2023385, and the deposit date is March 21, 2023.

[0048] Example 3 C. jadinii Culture medium optimization for WB15 The inventors discovered that when the fermentation medium composition is: sucrose 50 g / L, yeast extract 8-15 g / L, soybean peptone 8-15 g / L, KH₂PO₄ 1.8-2.9 g / L, MgSO₄ 2.0-3.0 g / L, FeSO₄ 0.005-0.02 g / L, ZnSO₄ 0.005-0.02 g / L, pH 5-6, C. jadinii The RNA content of WB15 was higher than 150 mg / g DCW.

[0049] C. jadinii When the fermentation medium for WB15 is prepared as follows: sucrose 50 g / L, yeast extract 13.4 g / L, soybean peptone 12.1 g / L, KH2PO4 2.8 g / L, MgSO4 2.5 g / L, FeSO4 0.01 g / L, ZnSO4 0.01 g / L, pH 5.5, its RNA content is the highest, reaching 184 mg / g DCW.

[0050] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A type of Cyber ​​indnera jadinii yeast (Cyber ​​indnera jadinii) WB15, characterized in that, It was deposited at the China Center for Type Culture Collection on March 21, 2023, with accession number CCTCCNO: M2023385; the deposit address is Wuhan University, Wuhan, China.

2. A fermentation broth, characterized in that, This includes live or dead cells of Cyberindnera jadinii WB15 as described in claim 1.

3. A culture medium composition for culturing Cyber ​​indnera jadinii WB15 as described in claim 1, characterized in that, This includes a seed culture medium for strain activation and / or a fermentation culture medium for strain fermentation. The seed culture medium consists of: 40 g / L glucose, 12-18 g / L corn steep liquor, 2.0-2.5 g / L KH₂PO₄, 2.0-3.0 g / L MgSO₄, and pH 6-7. The fermentation culture medium consists of: 50 g / L sucrose, 8-15 g / L yeast extract, and 8-15 g / L soybean peptone. KH 2PO41.8~2.9g / L, MgSO42.0~3.0g / L, FeSO40.005~ 0.02g / L, ZnSO 40.005~0.02g / L, pH 5~6.

4. The culture medium combination according to claim 3, characterized in that, The seed culture medium consists of: 40 g / L glucose and 15 g / L corn steep liquor. KH 2PO42.3g / L, MgSO42.5g / L, pH6.

5.

5. The culture medium combination according to claim 3 or 4, characterized in that, The fermentation medium consisted of: 50 g / L sucrose, 8-15 g / L yeast extract, and 8-15 g / L soybean peptone. KH 2PO41.8~2.9g / L, MgSO42.0~3.0g / L, FeSO 40.005~0.02g / L, ZnSO 40.005~0.02g / L, pH 5~6.

6. The culture medium combination according to claim 5, characterized in that, The fermentation medium consisted of: 50 g / L sucrose, 10 g / L yeast extract, 10 g / L soybean peptone, 2.3 g / L KH2PO4, 2.5 g / L MgSO4, 0.01 g / L FeSO4, 0.01 g / L ZnSO4, and pH 5.

5.

7. The culture medium combination according to claim 5, characterized in that, The fermentation medium consisted of: 50 g / L sucrose, 13.4 g / L yeast extract, 12.1 g / L soybean peptone, 2.8 g / L KH₂PO₄, 2.5 g / L MgSO₄, and 0.01 g / L FeSO₄. ， ZnSO4 0.01 g / L, pH 5.

5.

8. The fermentation method of Cyber ​​indnera jadinii WB15 according to claim 1, characterized in that, Includes the following steps: S1, the Cyber ​​indnera jadinii WB15 preserved on the slant was inoculated into a shake flask containing seed culture medium and cultured at a temperature of 28~35℃ and a shaking speed of 200~250rpm for 16~24h to obtain seed liquid; S2. Inoculate the seed culture at an inoculation rate of 1-5% into a shake flask containing fermentation medium and culture at a temperature of 28-35℃ and a shaking speed of 200-250 rpm for 6-10 hours. The seed culture medium consists of: glucose 40 g / L, corn steep liquor 12-18 g / L, KH2PO4 2.0-2.5 g / L, MgSO4 2.0-3.0 g / L, pH 6-7. The fermentation medium consists of: sucrose 50 g / L, yeast extract 8-15 g / L, soybean peptone 8-15 g / L, KH2PO4 1.8-2.9 g / L, MgSO4 2.0-3.0 g / L, FeSO4 0.005-0.02 g / L, ZnSO4 0.005-0.02 g / L, pH 5-6.

9. The fermentation method according to claim 7, characterized in that, The fermentation medium consisted of: 50 g / L sucrose, 13.4 g / L yeast extract, 12.1 g / L soybean peptone, 2.8 g / L KH2PO4, 2.5 g / L MgSO4, 0.01 g / L FeSO4, 0.01 g / L ZnSO4, and pH 5.5.

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