A method for producing long-chain dibasic acids

Abstract

The application provides a long-chain dibasic acid production method, which comprises the following steps: (1) preparing a long-chain dibasic acid fermentation strain seed solution, wherein the fermentation strain is Candida viswanathii (ATCC 94310) Candida viswanathii ) DLY-230301; (2) transferring the seed solution to a fermentation medium taking alkanes and derivatives thereof as fermentation substrates, and supplementing at least one of fatty acids and derivatives thereof in an appropriate amount in the early fermentation stage until the fermentation is completed, and then extracting and refining to obtain a product. The application utilizes the high conversion characteristics of Candida viswanathii DLY-230301 to fatty acids / fatty acid esters, combines a long-period fermentation process, takes fatty acids / fatty acid esters as a metabolic regulator in the early stage of fermentation with alkanes as the fermentation substrate, optimizes the energy metabolism pathway of the strain, and improves the fermentation level.

Description

Technical Field

[0001] This invention belongs to the field of biochemical technology, specifically relating to a method for producing long-chain dicarboxylic acids. Background Technology

[0002] Long-chain dicarboxylic acids (DCn) are aliphatic dicarboxylic acids (DCn) containing more than 10 carbon atoms in their carbon chain. These include both saturated and unsaturated dicarboxylic acids and are a class of fine chemical products with important and wide-ranging industrial applications. They are also crucial raw materials in the chemical industry for synthesizing high-grade fragrances, high-performance nylon engineering plastics, high-grade nylon hot melt adhesives, high-temperature dielectrics, high-grade paints and coatings, high-grade lubricants, cold-resistant plasticizers, resins, pharmaceuticals, and pesticides.

[0003] The core of fermentation production of long-chain dicarboxylic acids lies in the selection of dominant fermentation strains. Currently, *Candida tropicalis* is the main microorganism used to convert n-alkanes or fatty acids and their derivatives into corresponding long-chain dicarboxylic acids, and breakthroughs have been made in the fermentation processes developed around it. Although *Candida tropicalis* has achieved good technical results in the field of long-chain dicarboxylic acid preparation, this strain has certain shortcomings in substrate suitability, fermentation conditions, and conversion efficiency. Strain improvement is generally achieved through mutagenesis in different ways and genetic engineering techniques, which can further improve the strain's suitability, stability, stress resistance, and ability to produce long-chain dicarboxylic acids, making it more suitable for industrial applications.

[0004] CN110616158A discloses a method for synthesizing long-chain dodecanoic acid using *Candida viswanathii* biosynthesis, comprising the following steps: a seed culture stage, a fermentation culture stage, and a dodecanoic acid separation and recovery stage. The *Candida viswanathii* strain is *Candida viswanathii* ws-1201, accession number CCTCC No. 2019076. The conversion is carried out at a pH of 5.5–9.0 and a temperature of 24–29°C for 48–52 hours. Starting from the 24th hour of fermentation, n-alkane is added daily to maintain a n-alkane concentration >20% (v / v) in the fermentation broth. In an industrial fermenter, when fermenting nC12 to produce DC12, 142 g / L of acid was produced after 52 hours of culture. At the end of 52 hours of fermentation, the average acid production rate was 2.73 g / h·L, with a conversion rate of 93.2%. The purity of DC12 reached over 99%, with a monocarboxylic acid content of less than 0.01%.

[0005] Fermentation of long-chain dicarboxylic acids is a typical four-phase system consisting of gas (oxygen), aqueous phase (fermentation broth), oil phase (alkane), and solid phase (microbial cells). Conventional pH adjustments, either neutral or alkaline, are required during fermentation. CN110669797A discloses a method for producing long-chain dicarboxylic acids via fermentation, where a 10%–40% (w / v) NaOH solution is used to control the pH at 5.0–8.5 during the later stages of fermentation. CN101698859A discloses a method for producing n-hexadecanedicarboxylic acid, where sodium hydroxide is used for pH gradient adjustment during fermentation, resulting in a final pH of 8.0. CN1292072C discloses a method for converting long-chain dicarboxylic acids via microbial fermentation, where the pH is 3.5–6.5 during the early stages of fermentation and 7.0–8.5 during the later stages. During fermentation, especially during the acid-producing phase, the pH of the fermentation broth needs to be adjusted to above 7.0. This is primarily because many Candida species exhibit higher enzyme activity and catalytic efficiency in a slightly alkaline environment. An alkaline environment also allows long-chain dicarboxylic acids to exist in the form of dicarboxylate salts, promoting mass transfer during fermentation. Therefore, a large amount of alkali is typically added during fermentation to neutralize the continuously generated long-chain dicarboxylic acids and maintain a slightly alkaline environment. After fermentation, a large amount of acid is needed to convert the salts of the long-chain dicarboxylic acids back into long-chain dicarboxylic acids, resulting in a large amount of high-concentration salt fermentation broth. The treatment of this high-salt wastewater severely impacts the development of the biological long-chain dicarboxylic acid industry. Furthermore, under alkaline conditions, the addition of fatty acids or fatty acid esters as substrates typically leads to severe saponification reactions, affecting substrate conversion efficiency. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a method for producing long-chain dicarboxylic acids. This invention utilizes the high fatty acid / fatty acid ester conversion characteristics of *Candida virescens* DLY-230301, combined with a long-cycle fermentation process, using fatty acids / fatty acid esters as metabolic regulators in the early stages of fermentation with alkane as the substrate, thereby optimizing the strain's energy metabolism pathway and improving fermentation efficiency.

[0007] This invention provides a method for producing long-chain dicarboxylic acids, comprising the following steps:

[0008] (1) Prepare seed culture of long-chain dicarboxylic acid fermentation bacteria, wherein the fermentation bacteria is Candida viswanathii DLY-230301;

[0009] (2) The seed culture is transferred to a fermentation medium with alkanes and their derivatives as fermentation substrates. At the same time, an appropriate amount of at least one of fatty acids and their derivatives is added in the early stage of fermentation until the fermentation is completed. The product is then extracted and purified.

[0010] In this invention, the general molecular formula of the long-chain dicarboxylic acid is HOOC(CH2)nCOOH, where 10≤n≤18.

[0011] In this invention, the *Candida viswanathii* DLY-230301 was deposited on March 9, 2023, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 26773, located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The main morphological characteristics of this bacterium are: white colonies with wrinkled surfaces and irregular edges; under a microscope, the individual strains are oval.

[0012] In this invention, the seed culture medium used in step (1) to prepare the fermentation bacterial seed liquid has the following formula: carbon source of 10-50 g / L, nitrogen source of 0.5-5 g / L, inorganic salt of 0.5-10 g / L, and vitamin B1 of 0.01-0.05 g / L. The carbon source is at least one of glucose, galactose, sucrose, maltose, trehalose, and mesotriose; the nitrogen source is at least one of yeast extract, corn steep liquor, urea, ammonium sulfate, and ammonia; and the inorganic salt is one or more of sodium chloride, potassium chloride, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, disodium hydrogen phosphate, sodium dihydrogen phosphate, magnesium sulfate, and ferric sulfate. Further preferably, the seed culture medium formula is as follows: sucrose 10–50 g / L, corn steep liquor 0.5–5 g / L, yeast extract 0.5–5 g / L, urea 0.5–5 g / L, sodium chloride 0.5–10 g / L, potassium dihydrogen phosphate 0.5–10 g / L, magnesium sulfate 0.5–10 g / L, and vitamin B1 0.01–0.05 g / L. Further preferably, one or more fatty acids and their derivatives are added to the seed culture medium in an amount of 2 wt%–10 wt%, preferably 3 wt%–6 wt%.

[0013] In this invention, the specific preparation process of the fermentation seed liquid in step (1) is as follows: the fermentation bacteria are inoculated into the seed culture medium and cultured at a temperature of 25-40℃, preferably 28-35℃, and a rotation speed of 200-400 rpm until OD. 620 It reaches 6.0 or above.

[0014] In this invention, the fermentation culture medium in step (2) is formulated as follows: carbon source of 10-50 g / L, nitrogen source of 0.5-10 g / L, inorganic salt of 0.5-10 g / L, vitamin B1 of 0.01-0.05 g / L, and alkane substrate of 15 v%-40 v%. The carbon source is at least one of glucose, galactose, sucrose, maltose, trehalose, and mesotriose; the nitrogen source is at least one of yeast extract, corn steep liquor, urea, ammonium sulfate, and ammonia; and the inorganic salt is one or more of sodium chloride, potassium chloride, sodium acetate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, disodium hydrogen phosphate, sodium dihydrogen phosphate, magnesium sulfate, and ferric sulfate. Further optimization yielded the following fermentation medium formulation: sucrose 10–50 g / L, corn steep liquor 0.5–10 g / L, yeast extract 0.5–10 g / L, urea 0.5–10 g / L, ammonium sulfate 0.5–10 g / L, sodium acetate 0.5–10 g / L, sodium chloride 0.5–10 g / L, potassium dihydrogen phosphate 0.5–10 g / L, magnesium sulfate 0.5–10 g / L, vitamin B1 0.01–0.05 g / L, and alkane substrate 15 v%–30 v%.

[0015] In this invention, the alkane and its derivatives mentioned in step (2) are at least one of C10 to C18 alkane and its derivatives, preferably at least one of C12 to C16 n-alkanes.

[0016] In this invention, the inoculation amount of the seed liquid in step (2) is 5% to 20% of the fermentation medium volume.

[0017] In this invention, the early stage of fermentation in step (2) refers to the period within 24 hours of the start of fermentation, preferably within 0-12 hours of the start of fermentation.

[0018] In this invention, the fatty acids and their derivatives added in step (2) during the early stage of fermentation are one or more of C10-C18 fatty acids, fatty acid esters, fatty acid salts, etc., preferably one or more of C10-C18 straight-chain saturated fatty acids, straight-chain saturated fatty acid esters, etc. The amount added is 1wt% to 10wt%, preferably 3wt% to 6wt%.

[0019] In this invention, the fermentation culture conditions in step (2) are as follows: temperature is 25-40℃, preferably 28-35℃; rotation speed is 100-600rpm, preferably 300-600rpm; air flux is 0.3-1.5vvm, preferably 0.5-1.2vvm; fermentation time is 72-144h.

[0020] In this invention, during step (2) of fermentation, the pH of the fermentation system is controlled to 7.0-7.5 using an alkaline pH adjuster. The alkaline pH adjuster is at least one of sodium hydroxide, potassium hydroxide, ammonia, etc., with a mass concentration of 5% to 40%. More preferably, the pH is controlled in stages during fermentation. Specifically, from 0 to 24 hours, the pH of the system is controlled to be 4.0-7.0; after 24 hours, the pH is controlled to be 7.0-7.5. That is, starting from the 24th hour, the pH of the system is increased in stages according to the fermentation progress, and the final fermentation pH does not exceed 7.5.

[0021] In this invention, the extraction and purification are carried out using methods conventionally used in the art.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] (1) This invention targets the high conversion characteristics of fatty acids / fatty acid esters of the new functional strain Candida viswanathii DLY-230301, and combines it with a long-cycle fermentation process. While using alkanes as fermentation substrates, fatty acids / fatty acid esters are used as metabolic regulators in the early stage of fermentation, thereby optimizing the energy metabolism pathway of the strain and improving the alkane fermentation level.

[0024] (2) In the process of preparing seed liquid, the present invention adds fatty acids / fatty acid esters as metabolic regulators, which enhances the high biomass characteristics of the strain and helps to improve the fermentation level.

[0025] (3) In the early stage of fermentation, the present invention uses fatty acids / fatty acid esters as metabolic regulators and simultaneously regulates the pH of the system in the later stage of fermentation, so that the final pH is not higher than 7.5, thus overcoming the saponification problem that may occur during the fermentation process and ensuring the stable and efficient fermentation process. Detailed Implementation

[0026] The technical solution and its effects of the present invention will be further described in detail below through embodiments. These embodiments are implemented based on the technical solution of the present invention, providing detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments. In the present invention, v% is the volume fraction, and wt% is the mass fraction.

[0027] Unless otherwise specified, the experimental methods used in the following examples are conventional methods in the art. Unless otherwise specified, the experimental materials used in the following examples were purchased from conventional biochemical reagent stores.

[0028] In this embodiment of the invention, the concentration of long-chain dicarboxylic acids in the solution was determined by titration with a sodium hydroxide standard solution, and the purity of the long-chain dicarboxylic acids was analyzed by gas chromatography. The gas chromatography column stationary phase was bonded cross-linked polyethylene glycol-20M; the column temperature was initially 150℃, held for 2 min, then increased to 200℃ at a rate of 6℃ / min, and held for 30 min; the carrier gas N2 flow rate was 1.4 mL / min; the hydrogen flow rate was 40 mL / min; the air flow rate was 400 mL / min; and the injection volume was 1 μL.

[0029] The YPD plate culture medium formula is: yeast extract 10g / L, peptone 20g / L, glucose 20g / L, and agar powder 20g / L.

[0030] Example 1

[0031] The seed culture medium formula is as follows: sucrose 30 g / L; corn steep liquor 0.5 g / L; yeast extract 0.5 g / L; urea 1.0 g / L; sodium chloride 2.0 g / L; potassium dihydrogen phosphate 4.0 g / L; magnesium sulfate 2.0 g / L; vitamin B1 0.03 g / L; lauric acid 5 wt%.

[0032] The fermentation medium formula is as follows: sucrose 20 g / L; corn steep liquor 0.5 g / L; yeast extract 0.5 g / L; urea 1.0 g / L; ammonium sulfate 1.0 g / L; sodium acetate 2.0 g / L; sodium chloride 2.0 g / L; potassium dihydrogen phosphate 4.0 g / L; magnesium sulfate 2.0 g / L; vitamin B1 0.03 g / L; n-dodecane 25 vol%.

[0033] The preserved *Candida viswanathii* strain DLY-230301 was inoculated into an Erlenmeyer flask containing seed culture medium and cultured on a shaker at 30°C and 200 rpm. The pH was not controlled during the culture process. The culture was continued until the OD value was reached. 620 The concentration was 7.8, and the seed culture of the fermentation bacteria was obtained.

[0034] The seed culture of the fermentation bacteria was transferred to a 5L fermenter containing fermentation medium, with an inoculum volume of 10% of the fermentation medium volume. 5wt% methyl lauryl acid was added at fermentation start-up (0h). Fermentation conditions were as follows: temperature controlled at 30℃, rotation speed at 500 rpm, air flow rate at 1.0 vvm, and pH of the fermentation system adjusted to 7.2-7.5 using a 30% sodium hydroxide solution.

[0035] Fermentation was completed after 144 hours. Samples were taken for analysis, and the concentration of long-chain dicarboxylic acids in the fermentation broth was 177.8 g / L, with a purity of 98.9% for monocarboxylic acids. Simultaneously, a parallel experiment was conducted using *Candida viride* CICC No. 33310 as the fermentation starter. After 144 hours of fermentation, the concentration of long-chain dicarboxylic acids in the fermentation broth was 48.2 g / L, with a purity of 98.2% for monocarboxylic acids.

[0036] Example 2

[0037] Same as Example 1, except that the fermentation substrate in the fermentation medium is n-tetradecane.

[0038] Fermentation was completed after 144 hours. Samples were taken for analysis, and the concentration of long-chain dicarboxylic acids in the fermentation broth was 96.7 g / L, with a purity of 98.6% for monocarboxylic acids. Simultaneously, a parallel experiment was conducted using *Candida viride* CICC No. 33310 as the fermentation starter. After 144 hours of fermentation, the concentration of long-chain dicarboxylic acids in the fermentation broth was 15.8 g / L, with a purity of 97.9% for monocarboxylic acids.

[0039] Example 3

[0040] Same as Example 1, except that the fermentation substrate in the fermentation medium is n-hexadecane.

[0041] Fermentation was completed after 144 hours. Samples were taken for analysis, and the concentration of long-chain dicarboxylic acids in the fermentation broth was 59.0 g / L, with a purity of 98.9% for monocarboxylic acids. Simultaneously, a parallel experiment was conducted using *Candida viride* CICC No. 33310 as the fermentation starter. After 144 hours of fermentation, the concentration of long-chain dicarboxylic acids in the fermentation broth was 9.6 g / L, with a purity of 97.8% for monocarboxylic acids.

[0042] Example 4

[0043] Same as Example 1, except that 5 wt% tetradecanoic acid was added to replace methyl laurate at the 4th hour of fermentation. Fermentation was completed after 144 hours, and samples were taken for analysis. The concentration of long-chain dicarboxylic acids in the fermentation broth was 164.2 g / L, and the purity of monocarboxylic acids was 98.0%.

[0044] Example 5

[0045] Same as Example 1, except that 5 wt% palmitic acid was added to replace methyl laurate at the 8th hour of fermentation. Fermentation was completed after 144 hours, and samples were taken for analysis. The concentration of long-chain dicarboxylic acids in the fermentation broth was 168.6 g / L, and the purity of monocarboxylic acids was 97.8%.

[0046] Example 6

[0047] Same as Example 1, except that 5 wt% sodium lauryl precipitate was added at the 12th hour of fermentation. Fermentation was completed after 144 hours, and samples were taken for analysis. The concentration of long-chain dicarboxylic acids in the fermentation broth was 165.5 g / L, and the purity of monocarboxylic acids was 98.8%.

[0048] Example 7

[0049] Similar to Example 1, but with the following difference: the pH control method during fermentation was as follows: from 0 to 24 hours, the system pH was controlled at 5.5 to 6.5; after 24 hours, starting from the 24th hour, the pH was controlled at 7.0 to 7.5, with the system pH being increased periodically as needed, and the final fermentation pH not exceeding 7.5. Fermentation was completed after 144 hours, and samples were taken for analysis. The concentration of long-chain dicarboxylic acids in the fermentation broth was 179.2 g / L, and the purity of monocarboxylic acids was 99.2%.

[0050] Comparative Example 1

[0051] Same as Example 1, except that methyl lauryl ester was not added at the start of fermentation. After 144 hours of fermentation, samples were taken for analysis. The concentration of long-chain dicarboxylic acids in the fermentation broth was 154.5 g / L, and the purity of monocarboxylic acids was 98.9%.

[0052] Comparative Example 2

[0053] Same as Example 5, except that palmitic acid was not added at the 8th hour of fermentation. After 144 hours of fermentation, samples were taken for analysis. The concentration of long-chain dicarboxylic acids in the fermentation broth was 148.6 g / L, and the purity of monocarboxylic acids was 98.8%.

[0054] Comparative Example 3

[0055] Same as Example 1, except that the fermentation strain used was the Candida tropicalis mutant PF-UV-56, with preservation number CGMCC No. 0356. After 144 hours of fermentation, samples were taken for analysis. The concentration of long-chain dicarboxylic acids in the fermentation broth was 93.5 g / L, and the purity of monocarboxylic acids was 98.5%.

[0056] Comparative Example 4

[0057] Same as Example 1, except that methyl laurate was used instead of n-dodecane as the fermentation substrate. After 144 hours of fermentation, samples were taken for analysis. The concentration of long-chain dicarboxylic acids in the fermentation broth was 149.5 g / L, and the purity of the monoacids was 98.5%.

Claims

1. A method for producing long-chain dicarboxylic acids, characterized in that... Includes the following: (1) Preparation of seed culture for long-chain dicarboxylic acid fermentation bacteria, wherein the fermentation bacteria is Candida virescens ( Candida viswanathii DLY-230301; (2) Transfer the seed culture to a fermentation medium with alkanes and their derivatives as fermentation substrates, and add an appropriate amount of at least one of fatty acids and their derivatives in the early stage of fermentation until the end of fermentation, and then extract and purify to obtain the product.

2. The method according to claim 1, characterized in that: The general molecular formula of the long-chain dicarboxylic acid is HOOC(CH2)nCOOH, where 10≤n≤18.

3. The method according to claim 1, characterized in that: The aforementioned Candida virescens ( Candida viswanathii DLY-230301 was deposited on March 9, 2023, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 26773. The deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Its main morphological characteristics are: white colony color, wrinkled surface, irregular edges, and oval shape under a microscope.

4. The method according to claim 1, characterized in that: The seed culture medium used in step (1) to prepare the fermentation bacteria seed liquid has the following formula: carbon source of 10-50 g / L, nitrogen source of 0.5-5 g / L, inorganic salt of 0.5-10 g / L, and vitamin B1 of 0.01-0.05 g / L; wherein the carbon source is at least one of glucose, galactose, sucrose, maltose, trehalose, and mesotriose; the nitrogen source is at least one of yeast extract, corn steep liquor, urea, ammonium sulfate, and ammonia water; and the inorganic salt is one or more of sodium chloride, potassium chloride, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, disodium hydrogen phosphate, sodium dihydrogen phosphate, magnesium sulfate, and ferric sulfate.

5. The method according to claim 4, characterized in that: The seed culture medium formula is as follows: sucrose 10-50 g / L, corn steep liquor 0.5-5 g / L, yeast extract 0.5-5 g / L, urea 0.5-5 g / L, sodium chloride 0.5-10 g / L, potassium dihydrogen phosphate 0.5-10 g / L, magnesium sulfate 0.5-10 g / L, and vitamin B1 0.01-0.05 g / L.

6. The method according to claim 4 or 5, characterized in that: One or more fatty acids and their derivatives are added to the seed culture medium, with an addition amount of 2wt% to 10wt%, preferably 3wt% to 6wt%.

7. The method according to claim 1, characterized in that: The specific preparation process of the fermentation seed culture in step (1) is as follows: the fermentation bacteria are inoculated into the seed culture medium and cultured at a temperature of 25-40℃, preferably 28-35℃, and a rotation speed of 200-400 rpm until OD. 620 It reaches 6.0 or above.

8. The method according to claim 1, characterized in that: The fermentation medium formula in step (2) is as follows: carbon source is 10-50 g / L, nitrogen source is 0.5-10 g / L, inorganic salt is 0.5-10 g / L, vitamin B1 is 0.01-0.05 g / L, and alkane substrate is 15 v%-40 v%; wherein the carbon source is at least one of glucose, galactose, sucrose, maltose, trehalose, and mesotriose; the nitrogen source is at least one of yeast extract, corn steep liquor, urea, ammonium sulfate, and ammonia water; and the inorganic salt is one or more of sodium chloride, potassium chloride, sodium acetate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, disodium hydrogen phosphate, sodium dihydrogen phosphate, magnesium sulfate, and ferric sulfate.

9. The method according to claim 8, characterized in that: The fermentation medium formula is as follows: sucrose 10-50 g / L, corn steep liquor 0.5-10 g / L, yeast extract 0.5-10 g / L, urea 0.5-10 g / L, ammonium sulfate 0.5-10 g / L, sodium acetate 0.5-10 g / L, sodium chloride 0.5-10 g / L, potassium dihydrogen phosphate 0.5-10 g / L, magnesium sulfate 0.5-10 g / L, vitamin B1 0.01-0.05 g / L, and alkane substrate 15 v%-30 v.

10. The method according to claim 1, characterized in that: The alkane and its derivatives mentioned in step (2) are at least one of C10 to C18 alkanes and their derivatives, preferably at least one of C12 to C16 n-alkanes.

11. The method according to claim 1, characterized in that: The inoculation amount of the seed liquid in step (2) is 5% to 20% of the fermentation medium volume.

12. The method according to claim 1, characterized in that: The early stage of fermentation mentioned in step (2) refers to the period within 24 hours of the start of fermentation, preferably within 0-12 hours of the start of fermentation.

13. The method according to claim 1, characterized in that: Step (2) The fatty acids and their derivatives added in the early stage of fermentation are one or more of C10-C18 fatty acids, fatty acid esters, and fatty acid salts, preferably one or more of C10-C18 straight-chain saturated fatty acids and straight-chain saturated fatty acid esters.

14. The method according to claim 1 or 13, characterized in that: Step (2) The amount of fatty acids and their derivatives added in the early stage of fermentation is 1wt% to 10wt%, preferably 3wt% to 6wt%.

15. The method according to claim 1, characterized in that: The fermentation conditions for step (2) are as follows: temperature is 25-40℃, preferably 28-35℃; rotation speed is 100-600rpm, preferably 300-600rpm; air flux is 0.3-1.5vvm, preferably 0.5-1.2vvm; fermentation time is 72-144h.

16. The method according to claim 1, characterized in that: In step (2), the pH of the fermentation system is adjusted to 7.0-7.5 by an alkaline pH adjuster. The alkaline pH adjuster is at least one of sodium hydroxide, potassium hydroxide, and ammonia water, with a mass concentration of 5% to 40%.

17. The method according to claim 1 or 16, characterized in that: During the fermentation process, the pH is regulated in stages. Specifically, from 0 to 24 hours, the pH of the system is controlled at 4.0 to 7.

0. After 24 hours, the pH is controlled at 7.0 to 7.

5. That is, starting from the 24th hour, the pH of the system is increased in stages as needed according to the fermentation situation, and the final fermentation pH does not exceed 7.5.