Fermentation production method of glucaric acid

Abstract

The invention discloses a fermentation process for high-yield production of glucaric acid by saccharomyces cerevisiae engineering bacteria. According to the process, a fermentation culture medium is improved and dynamically regulated; the yield of glucaric acid in a 50 L fermentation tank reaches 18 g / L, the production cost is greatly reduced, and industrial production is facilitated.

Description

Technical Field

[0001] This invention belongs to the field of bio-fermentation technology, specifically relating to the fermentation process of engineered Saccharomyces cerevisiae for the production of gluconic acid. Background Technology

[0002] Glucaric acid is widely found in cruciferous plants and the excrement of mammals. It has broad applications in healthcare, chemical materials, and food additives, such as in the synthesis of detergents, corrosion inhibitors, adipic acid, esters, and polyamide derivatives, and in applications like lowering cholesterol, treating diabetes, and cancer. Therefore, glucocoric acid is considered "one of the most valuable biorefined products."

[0003] Traditionally, gluconic acid is mainly produced on a large scale in industry through the nitric acid oxidation process. This method uses nitric acid as a catalyst and glucose as a substrate to produce gluconic acid and a large number of byproducts. While relatively simple, this method involves numerous side reactions and results in a low yield of gluconic acid (below 50%). In recent years, research has reported the use of novel catalysts to oxidize glucose and other carbon sources to produce gluconic acid. Although this method yields higher results, its reaction conditions and product separation and extraction processes do not align with the principles of green and sustainable development. Developing low-carbon and environmentally friendly microbial fermentation methods can improve reaction selectivity and reduce environmental pollution, showing great promise for future applications.

[0004] The glucuronic acid research group at Jiangnan University has successfully constructed a glucuronic acid synthesis pathway by expressing the inositol-1-phosphate synthase gene (Inol) and the inositol oxygenase gene (MIOX) in Saccharomyces cerevisiae, and simultaneously expressing aldehyde dehydrogenase (Udh) from Pseudomonas putida. Through metabolic network optimization and culture medium optimization, they have successfully prepared glucuronic acid by biosynthesis in Saccharomyces cerevisiae, achieving a fermentation yield of 12 g / L. However, the high fermentation cost and the high fermentation process have greatly limited the industrialization of this project. Summary of the Invention

[0005] To address the problems of low yield, high cost, and unstable process in current fermentation processes for gluconic acid production, this invention provides a method for fermenting gluconic acid with brewing yeast that increases fermentation yield and significantly reduces costs by improving the fermentation formula and controlling the fermentation process.

[0006] This invention is achieved through the following technical solution:

[0007] A method for producing gluconic acid by fermentation with brewer's yeast includes the following steps:

[0008] 1) Activation of microbial strains;

[0009] 2) Seed liquid culture;

[0010] 3) Seed culture tank;

[0011] 4) Fermentation liquid culture: Inoculate 5-10% (V / V) into the fermentation medium, culture at 28-32℃ and 200-500 rpm, add an appropriate amount of 40% glucose daily until the sugar concentration reaches 8g / L, and add 400-800ml of 40% yeast extract. Control dissolved oxygen above 40% and pH 5.0-5.5. Ferment for 6 to 7 days.

[0012] The brewing yeast is brewing yeast Bga, which has been published in patent application document with publication number CN108220176A.

[0013] The fermentation medium contains glucose, peptone, yeast extract, inorganic salts, amino acids, and other components.

[0014] Specifically, it includes: 20-60 g / L peptone, 10-30 g / L yeast extract, 20-40 g / L glucose, 0.1-0.2 mM magnesium chloride, 10-13 g / L inositol, 0.01-0.05 g / L ferrous sulfate, 1-3 g / L arginine, and 1-3 g / L lysine, preferably with 3 g / L arginine and 1 g / L lysine added simultaneously; the final concentration of TF Composite nonionic surfactant is 0.1%-0.2% (V / V).

[0015] The peptone in the above-mentioned culture medium is selected from one or more of Angel peptone FP108, FP401, and FP402. The peptone content is preferably 20-40 g / L, and more preferably 30-40 g / L.

[0016] The yeast extract in the above-mentioned culture medium is selected from one or both of Angel FM885 and FM902. The content of yeast extract is preferably 10-30 g / L, more preferably 20-30 g / L.

[0017] Preferably, the fermentation medium comprises a combination of peptone FP108 and yeast extract FM885, or a combination of peptone FP401 and yeast extract FM902.

[0018] Dissolve each component except glucose in 900 ml of water, leave the pH at rest, and sterilize at 121°C for 30 min; dissolve glucose separately in 100 ml of water and sterilize at 115°C for 20 min.

[0019] More specifically, the method for producing gluconic acid by fermentation includes:

[0020] 1) Strain activation: Streaking the brewer's yeast stored at -80℃ into YPD solid medium and activating it for 2-3 days, then culturing at 28-32℃;

[0021] 2) Seed liquid culture: 2YPD medium, inoculate with an appropriate amount of plate cells, culture at 28-32℃ and 250rpm for 16 hours until OD600 reaches 10 or higher;

[0022] 3) Seed culture: Inoculate the seed culture into 2YPD medium (8L / 15L tank) at an inoculation rate of 5-10% (V / V), and culture at 28-32℃ and 200-400rpm, with dissolved oxygen controlled above 40%, for 9-12 hours until OD600 reaches above 10.

[0023] 4) Fermentation liquid culture: Inoculate 5-10% (V / V) into the fermentation medium (30L / 50L tank), culture at 28-32℃ and 200-500rpm, add an appropriate amount of 40% glucose every day until the sugar concentration reaches 8g / L, control dissolved oxygen above 40%, and add 400-800ml of 40% yeast extract powder. Ferment for 6 to 7 days.

[0024] Beneficial effects: Through research and exploration of different types of peptone, yeast extract, iron ions, amino acids and other components, as well as different feeding methods and aeration rates, the inventors have achieved a gluconic acid yield of 18g / L in a 50L fermenter, while significantly reducing production costs by more than 60-80%, which is conducive to the industrial production of gluconic acid. Attached Figure Description

[0025] Figure 1 This is the liquid chromatography chromatogram of the fermentation product (gluconic acid: RT 7.727 min).

[0026] Figure 2 The curves show the changes in bacterial concentration and gluconic acid content during the fermentation process for producing gluconic acid: bacterial concentration (OD) and yield increase synchronously. Detailed Implementation

[0027] The present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0028] The experimental strain was *Saccharomyces cerevisiae* Bga, as described in patent CN108220176A, constructed by Jiangnan University; the method for detecting the product gluconic acid is as follows:

[0029] HPLC detection conditions:

[0030] Water high performance liquid chromatograph 2695-2414; chromatographic column: BIO RAD brand AMINEX HPX-87 HOrganic Acid Analysis Column, 300*7.8mm;

[0031] Mobile phase: 5mM H2SO4, 30ml concentrated sulfuric acid, cooled and diluted to 1000ml volumetric flask to obtain 0.5M sulfuric acid, take 40ml of 0.5M sulfuric acid + 3960ml of water to prepare the mobile phase;

[0032] Detection conditions: The mobile phase used was 5 mM H2SO4, the flow rate was 0.6 ml / min, the internal temperature was 35 ℃, the column temperature was 35 ℃, the injection volume was 10 μl, and the injection time was 30 min. The metabolites in the fermentation broth were quantified by a differential detector.

[0033] Gluconic acid reference standard, purchased from Maclean's, purity 98%;

[0034] Standard sample preparation: Weigh a certain mass of potassium gluconate reference standard and dissolve it in ultrapure water to prepare gluconate solutions with final concentrations of 2 g / L, 1 g / L, 0.5 g / L, 0.25 g / L, 0.125 g / L and 0.0625 g / L.

[0035] Fermentation sample processing: Take 1 ml of fermentation broth, centrifuge at 12000 rpm for 10 min, take the supernatant and dilute with 10 mmol / L H2SO4, centrifuge at 12000 rpm for 5 min, filter with a 0.22 μm aqueous filter membrane, and detect the content of metabolites by HPLC.

[0036] Example 1

[0037] Bga bacteria were streaked onto YPD plates for activation and cultured at 30±1℃ for 2 days. The bacterial cells were then inoculated into 2YPD liquid medium and cultured overnight at 30±1℃ and 250 rpm for 16 hours. Finally, 10% of the inoculum was inoculated into the seed tank 2YPD medium.

[0038] Seed tank process: aeration ratio of 1:1, initial stirring at 300 rpm, dissolved oxygen controlled at 40% or higher by stirring, 30±1℃, cultured for 9 hours; then inoculated into the fermentation medium at a 10% inoculum.

[0039] Fermentation process: inoculum 5% (v / v), fermentation volume 25 L / 50 L, initial rotation speed 300 rpm, aeration rate 25 L / min, culture temperature 30℃, pH controlled at 5.5, dissolved oxygen maintained above 40%; every 24 hours, add 40% yeast extract (FM885) in 400 ml.

[0040] Initial fermentation medium: peptone (FP108) 40 g / L, yeast extract (FM885) 30 g / L, glucose 40 g / L, inositol 10 g / L, magnesium chloride 0.1 mM, solvent: water; TF Composite nonionic surfactant 0.2% (V / V).

[0041] Replenish the culture medium every 24 hours: 40% glucose until the sugar concentration reaches 8 g / L;

[0042] pH self-controlled solution: KOH solution 200g / L, self-controlled pH 5.5;

[0043] Fermentation lasted 7 days, with an OD600 of 69. High-performance liquid chromatography (HPLC) analysis showed that the yield of gluconic acid in the supernatant of the fermentation broth was 14.1 g / L.

[0044] Example 2

[0045] Bga bacteria were streaked onto YPD plates for activation and cultured at 30±1℃ for 2 days. The bacterial cells were then inoculated into 2YPD liquid medium and cultured overnight at 30±1℃ and 250 rpm for 16 hours. Finally, 10% of the inoculum was inoculated into the seed tank 2YPD medium.

[0046] Seed tank process: aeration ratio of 1:1, initial stirring at 300 rpm, dissolved oxygen controlled at 40% or higher by stirring, 30±1℃, cultured for 9 hours; then inoculated into the fermentation medium at a 10% inoculum.

[0047] Fermentation process: inoculum 5% (v / v), fermentation volume 25 L / 50 L, initial rotation speed 300 rpm, aeration rate 25 L / min, culture temperature 30±1℃, pH control 5.5, maintain dissolved oxygen above 40%; add 600 ml of 40% yeast extract (FM902) every 24 hours.

[0048] Initial fermentation medium: peptone (FP401) 60 g / L, yeast extract (FM902) and (FM885) 5 g / L each, glucose 20 g / L, inositol 13 g / L, magnesium chloride 0.2 mM, solvent: water; TF Composite nonionic surfactant 0.1% (V / V).

[0049] Replenish the culture medium every 24 hours: glucose 400 g / L;

[0050] pH self-controlled solution: KOH solution 200g / L, self-controlled pH 5.5;

[0051] Fermentation lasted 7 days, with an OD600 of 65. High-performance liquid chromatography (HPLC) analysis showed that the yield of gluconic acid in the supernatant of the fermentation broth was 13.5 g / L.

[0052] Example 3

[0053] B. gibberellins were streaked onto YPD plates for activation and cultured at 30±1℃ for 2 days. The bacterial cells were then inoculated into 2 YPD liquid medium and cultured overnight at 30±1℃ and 250 rpm for 16 hours. A 10% inoculum was then added to the fermentation medium with the following inoculation process: aeration ratio of 1:1, initial stirring at 300 rpm, dissolved oxygen controlled to be above 40% by stirring, and cultured at 30±1℃ for 9 hours. Finally, a 10% inoculum was added to the fermentation medium.

[0054] Fermentation process: inoculum 5% (v / v), fermentation volume 25 L / 50 L, initial rotation speed 300 rpm, aeration rate 25 L / min, culture temperature 30±1℃, pH control 5.5, maintain dissolved oxygen above 40%; replenish 40% yeast extract powder (FM902) every 24 hours in 400 ml.

[0055] Initial fermentation medium: peptone (FP402) 20 g / L, yeast extract (FM902) 20 g / L, glucose 30 g / L, inositol 11 g / L, magnesium chloride 0.1 mM, solvent: water; TF Composite nonionic surfactant 0.2% (V / V).

[0056] Replenish the culture medium every 24 hours: 40% glucose until the sugar concentration reaches 8 g / L;

[0057] pH self-controlled solution: KOH solution 200g / L, self-controlled pH 5.0;

[0058] Fermentation lasted 7 days, with an OD600 of 68. High-performance liquid chromatography (HPLC) analysis showed that the yield of gluconic acid in the supernatant of the fermentation broth was 13.9 g / L.

[0059] Example 4

[0060] Bga bacteria were streaked onto YPD plates for activation and cultured at 30±1℃ for 2 days. The bacterial cells were then inoculated into 2YPD liquid medium and cultured overnight at 30±1℃ and 250 rpm for 16 hours. Finally, 10% of the inoculum was inoculated into the seed tank 2YPD medium.

[0061] Seed tank process: aeration ratio of 1:2, initial stirring at 300 rpm, dissolved oxygen controlled at 40% or higher by stirring, 30±1℃, cultured for 9 hours; then inoculated into the fermentation medium at a 10% inoculum.

[0062] Fermentation process: inoculum 5% (v / v), fermentation volume 25 L / 50 L, initial rotation speed 300 rpm, aeration rate 50 L / min, culture temperature 30±1℃, pH controlled at 5, dissolved oxygen maintained above 40%; every 24 hours, add 40% yeast extract powder (FM902) in 400 ml.

[0063] Initial fermentation medium: peptone (FP401) 30 g / L, yeast extract (FM902) 20 g / L, glucose 30 g / L, inositol 13 g / L, magnesium chloride 0.1 mM, ferrous sulfate 0.01 g / L, solvent: water; TF Composite nonionic surfactant 0.2% (v / v)

[0064] Replenish the culture medium every 24 hours: 40% glucose until the sugar concentration reaches 8 g / L;

[0065] pH self-controlled solution: KOH solution 200g / L, self-controlled pH 5.0;

[0066] Fermentation lasted 7 days, with an OD600 of 80. High-performance liquid chromatography (HPLC) analysis showed that the yield of gluconic acid in the supernatant of the fermentation broth was 14.8 g / L.

[0067] Example 5

[0068] Bga bacteria were streaked onto YPD plates for activation and cultured at 30±1℃ for 2 days. The bacterial cells were then inoculated into 2YPD liquid medium and cultured overnight at 30±1℃ and 250 rpm for 16 hours. Finally, 10% of the inoculum was inoculated into the seed tank 2YPD medium.

[0069] Seed tank process: aeration ratio of 1:2, initial stirring at 300 rpm, dissolved oxygen controlled at 40% or higher by stirring, 30±1℃, cultured for 9 hours; then inoculated into the fermentation medium at a 10% inoculum.

[0070] Fermentation process: inoculum 5% (v / v), fermentation volume 25 L / 50 L, initial rotation speed 300 rpm, aeration rate 50 L / min, culture temperature 30±1℃, pH controlled at 5, dissolved oxygen maintained above 40%; every 24 hours, add 40% yeast extract powder (FM902) in 400 ml.

[0071] Initial fermentation medium: peptone (FP401) 40 g / L, yeast extract (FM902) 20 g / L, glucose 30 g / L, inositol 13 g / L, magnesium chloride 0.1 mM, ferrous sulfate 0.05 g / L, solvent: water; TF Composite nonionic surfactant 0.2% (v / v)

[0072] Replenish the culture medium every 24 hours: 40% glucose until the sugar concentration reaches 8 g / L;

[0073] pH self-controlled solution: KOH solution 200g / L, self-controlled pH 5.0;

[0074] Fermentation lasted 7 days, with an OD600 of 85. High-performance liquid chromatography (HPLC) analysis showed that the yield of gluconic acid in the supernatant of the fermentation broth was 15.4 g / L.

[0075] Example 6

[0076] Bga bacteria were streaked onto YPD plates for activation and cultured at 30±1℃ for 2 days. The bacterial cells were then inoculated into 2YPD liquid medium and cultured overnight at 30±1℃ and 250 rpm for 16 hours. Finally, 10% of the inoculum was inoculated into the seed tank 2YPD medium.

[0077] Seed tank process: aeration ratio of 1:2, initial stirring at 300 rpm, dissolved oxygen controlled at 40% or higher by stirring, 30±1℃, cultured for 9 hours; then inoculated into the fermentation medium at a 10% inoculum.

[0078] Fermentation process: inoculum 5% (v / v), fermentation volume 25 L / 50 L, initial rotation speed 300 rpm, aeration rate 50 L / min, culture temperature 30±1℃, pH controlled at 5, dissolved oxygen maintained above 40%; 600 ml of 40% yeast extract (FM902) is added every 24 hours.

[0079] Initial fermentation medium: peptone (FP401) 40 g / L, yeast extract (FM902) 20 g / L, glucose 30 g / L, inositol 13 g / L, magnesium chloride 0.1 mM, ferrous sulfate 0.05 g / L, arginine 1 g / L, lysine 3 g / L, solvent: water; T-FComposite nonionic surfactant 0.2% (V / V).

[0080] Replenish the culture medium every 24 hours: 40% glucose until the sugar concentration reaches 8 g / L;

[0081] pH self-controlled solution: KOH solution 200g / L, self-controlled pH 5.0;

[0082] Fermentation lasted 7 days, with an OD600 of 89. High-performance liquid chromatography (HPLC) analysis showed that the yield of gluconic acid in the supernatant of the fermentation broth was 16.5 g / L.

[0083] Example 7

[0084] Bga bacteria were streaked onto YPD plates for activation and cultured at 30±1℃ for 2 days. The bacterial cells were then inoculated into 2YPD liquid medium and cultured overnight at 30±1℃ and 250 rpm for 16 hours. Finally, 10% of the inoculum was inoculated into the seed tank 2YPD medium.

[0085] Seed tank process: aeration ratio of 1:2, initial stirring at 300 rpm, dissolved oxygen controlled at 40% or higher by stirring, 30±1℃, cultured for 9 hours; then inoculated into the fermentation medium at a 10% inoculum.

[0086] Fermentation process: inoculum 5% (v / v), fermentation volume 25 L / 50 L, initial rotation speed 300 rpm, aeration rate 50 L / min, culture temperature 30±1℃, pH controlled at 5, dissolved oxygen maintained above 40%; 600 ml of 40% yeast extract (FM902) is added every 24 hours.

[0087] Initial fermentation medium: peptone (FP401) 40 g / L, yeast extract (FM902) 20 g / L, glucose 40 g / L, inositol 11 g / L, magnesium chloride 0.1 mM, ferrous sulfate 0.05 g / L, arginine 3 g / L, lysine 1 g / L, solvent: water;

[0088] Replenish the culture medium every 24 hours: 40% glucose until the sugar concentration reaches 8 g / L;

[0089] pH self-controlled solution: KOH solution 200g / L, self-controlled pH 5.5;

[0090] Fermentation lasted 7 days, with an OD600 of 94. High-performance liquid chromatography (HPLC) analysis showed that the yield of gluconic acid in the supernatant of the fermentation broth was 18 g / L.

[0091] Comparative Example 1

[0092] Bga bacteria were streaked onto YPD plates for activation and cultured at 30±1℃ for 2 days. The bacterial cells were then inoculated into 2YPD liquid medium and cultured overnight at 30±1℃ and 250 rpm for 16 hours. Finally, 10% of the inoculum was inoculated into the seed tank 2YPD medium.

[0093] Seed tank process: aeration ratio of 1:1, initial stirring at 300 rpm, dissolved oxygen controlled at 40% or higher by stirring, 30±1℃, cultured for 9 hours; then inoculated into the fermentation medium at a 10% inoculum.

[0094] Fermentation process: inoculum 5% (v / v), fermentation volume 25 L / 50 L, initial rotation speed 300 rpm, aeration rate 25 L / min, culture temperature 30±1℃, pH control 5, and dissolved oxygen maintained above 40%.

[0095] Initial fermentation medium: peptone (FP108) 40 g / L, yeast extract (FM885) 30 g / L, glucose 30 g / L, inositol 11 g / L, magnesium chloride 0.1 mM, solvent: water; TF Composite nonionic surfactant 0.2% (V / V).

[0096] Replenish the culture medium every 24 hours: 40% glucose until the sugar concentration reaches 8 g / L;

[0097] pH self-controlled solution: KOH solution 200g / L, self-controlled pH 5.0;

[0098] Fermentation lasted 7 days, with an OD600 of 61. High-performance liquid chromatography (HPLC) analysis showed that the yield of gluconic acid in the supernatant of the fermentation broth was 13.1 g / L.

[0099] Comparative Example 2

[0100] Bga bacteria were streaked onto YPD plates for activation and cultured at 30±1℃ for 2 days. The bacterial cells were then inoculated into 2YPD liquid medium and cultured overnight at 30±1℃ and 250 rpm for 16 hours. Finally, 10% of the inoculum was inoculated into the seed tank 2YPD medium.

[0101] Seed tank process: aeration ratio of 1:1, initial stirring at 300 rpm, dissolved oxygen controlled at 40% or higher by stirring, 30±1℃, cultured for 9 hours; then inoculated into the fermentation medium at a 10% inoculum.

[0102] Fermentation process: inoculum 5% (v / v), fermentation volume 25 L / 50 L, initial rotation speed 300 rpm, aeration rate 25 L / min, culture temperature 30±1℃, pH control 5, and dissolved oxygen maintained above 40%.

[0103] Initial fermentation medium: peptone (FP401) 40 g / L, yeast extract (FM902) 20 g / L, glucose 30 g / L, inositol 13 g / L, magnesium chloride 0.1 mM, solvent: water; TF Composite nonionic surfactant 0.2% (V / V).

[0104] Replenish the culture medium every 24 hours: glucose 400 g / L;

[0105] pH self-controlled solution: KOH solution 200g / L, self-controlled pH 5.0;

[0106] Fermentation lasted 7 days, with an OD600 of 58. High-performance liquid chromatography (HPLC) analysis showed that the yield of gluconic acid in the supernatant of the fermentation broth was 12.8 g / L.

[0107] Comparative Example 3

[0108] Bga bacteria were streaked onto YPD plates for activation and cultured at 30±1℃ for 2 days. The bacterial cells were then inoculated into 2YPD liquid medium and cultured overnight at 30±1℃ and 250 rpm for 16 hours. Finally, 10% of the inoculum was inoculated into the seed tank 2YPD medium.

[0109] Seed tank process: aeration ratio of 1:1, initial stirring at 300 rpm, dissolved oxygen controlled at 40% or higher by stirring, 30±1℃, cultured for 9 hours; then inoculated into the fermentation medium at a 10% inoculum.

[0110] Fermentation process: inoculum 5% (v / v), fermentation volume 25 L / 50 L, initial rotation speed 300 rpm, aeration rate 25 L / min, culture temperature 30±1℃, pH controlled at 5, dissolved oxygen maintained above 40%; 40% yeast extract (OXOID) added every 24 hours in 400 ml.

[0111] Initial fermentation medium: tryptone (OXOID) 40 g / L, yeast extract (OXOID) 20 g / L, glucose 40 g / L, inositol 13 g / L, magnesium chloride 0.1 mM, solvent: water; TF Composite nonionic surfactant 0.2% (v / v)

[0112] Replenish the culture medium every 24 hours: glucose 400 g / L;

[0113] pH self-controlled solution: KOH solution 200g / L, self-controlled pH 5;

[0114] Fermentation ended after 7 days, with an OD600 of 70. High-performance liquid chromatography (HPLC) analysis showed that the yield of gluconic acid in the supernatant of the fermentation broth was 14 g / L.

[0115] Table 1 Summary of Fermentation Examples

[0116]

Claims

1. A method for producing gluconic acid by fermentation using engineered Saccharomyces cerevisiae, comprising the following steps: 1) Strain activation; 2) Seed liquid culture; 3) Seed tank culture; 4) Fermentation liquid culture, characterized in that, The fermentation liquid culture is as follows: inoculate 5-10% (V / V) into the fermentation medium, culture at 28-32℃ and 200-500 rpm, add an appropriate amount of 40% glucose every 24 hours until the sugar concentration reaches 8g / L, and add 400-600ml of 40% yeast extract. Control dissolved oxygen above 40%, pH 5.0-5.5, and ferment for 6 to 7 days.

2. The method as described in claim 1, characterized in that: The fermentation medium comprises: a nitrogen source consisting of a combination of 20-60 g / L peptone and 10-30 g / L yeast extract, 20-40 g / L glucose, 0.1-0.2 mM magnesium chloride, and 10-13 g / L inositol; the peptone is selected from one or more of Angel peptone FP108, FP401, and FP402; and the yeast extract is selected from one or two of Angel yeast extract FM885 and FM902.

3. The method as described in claim 2, characterized in that, The peptone concentration is 20-40 g / L.

4. The method as described in claim 2, characterized in that, The yeast extract concentration is 20-30 g / L.

5. The method as described in claim 2, characterized in that: The nitrogen source is a combination of peptone FP108 and yeast extract FM885, or a combination of peptone FP401 and yeast extract FM902.

6. The method as described in claim 2, characterized in that: The fermentation medium also contains 0.01-0.05 g / L of ferrous sulfate.

7. The method as described in claim 2, characterized in that: The fermentation medium also contains 1-3 g / L of arginine and 1-3 g / L of lysine.

8. The method as described in claim 2, characterized in that: The fermentation medium also contains 3 g / L of arginine and 1 g / L of lysine.

9. The method as described in claim 2, characterized in that: The fermentation medium also contains TF Composite nonionic surfactant at a final concentration of 0.1%-0.2% (V / V).

10. The method according to claim 1, wherein the fermentation liquid culture is as follows: inoculum size 5% (v / v), fermentation volume 25 L / 50 L, initial rotation speed 300 rpm, aeration rate 50 L / min, culture temperature 30±1℃, pH controlled at 5.5, dissolved oxygen maintained above 40%; 600 ml of 40% yeast extract (FM902) is added every 24 hours. Initial fermentation medium: peptone (FP401) 40 g / L, yeast extract (FM902) 20 g / L, glucose 40 g / L, inositol 11 g / L, magnesium chloride 0.1 mM, ferrous sulfate 0.05 g / L, arginine 3 g / L, lysine 1 g / L, TF Composite nonionic surfactant final concentration 0.1%-0.2% (V / V), solvent is water; every 24 hours, add an appropriate amount of 40% glucose until the sugar concentration reaches 8 g / L; pH self-regulating solution: KOH solution, 200g / L, self-regulating pH 5.0-5.5; Fermentation lasts for 7 days.

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