A method for reducing acetic acid in a succinic acid fermentation process

Abstract

The application provides a method for reducing acetic acid in a succinic acid fermentation process, and belongs to the technical field of fermentation.The fermentation culture is characterized in that: during the fermentation culture process, fermentation aids are fed, specifically, at the 10th hour of fermentation, fermentation aid one is fed into the fermentation culture tank at a feeding speed of 0.07-0.09 ml / h / L until the end of fermentation; at the 24th hour of fermentation, fermentation aid two is fed into the fermentation culture tank at a feeding speed of 0.12-0.14 ml / h / L until the end of fermentation; the fermentation aid one comprises cobalt chloride hexahydrate, zinc sulfate heptahydrate, folic acid and biotin; the fermentation aid two comprises nicotinamide, cysteine and tyrosine; the fermentation culture liquid prepared by the method has a content as high as 59.1-60.4 g / L, an acetic acid content as low as 0.30-0.65 g / L and a malic acid content of 0.20-0.23 g / L.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of fermentation, and particularly relates to a method for reducing acetic acid in a succinic acid fermentation process. BACKGROUND

[0002] Succinic acid is a binary organic carboxylic acid containing four carbons, and is an important chemical raw material; succinic acid can produce an acid taste, and can be used in the fields of flavorings, soy sauce and the like, can be used as a preservative, a pH value regulator, a cosolvent, and can be used to synthesize antidotes, diuretics, sedatives and the like, and has a wide application field.

[0003] Succinic acid can be produced by a chemical synthesis method, but the environmental pollution is relatively serious, and the production cost is relatively high; with the development of society, the fermentation method has become a new succinic acid preparation method, the fermentation method uses bacteria, yeast and the like microorganisms, takes sugar as a raw material, and produces through microorganisms at normal temperature and pressure, the fermentation method avoids the dependence on petrochemical raw materials, reduces the pollution of the chemical synthesis process to the environment, and has the advantages of environmental protection, simple equipment, low cost and the like;

[0004] Therefore, the fermentation method is a main method for producing succinic acid at present, and Escherichia coli is widely applied due to the characteristics that the genetic characteristics of Escherichia coli are clear, Escherichia coli is easy to cultivate, and high-efficiency expression of a target gene can be realized; however, acetic acid is produced when Escherichia coli grows, and the production and accumulation of acetic acid not only affect the growth of the bacterial body, but also inhibit the synthesis of the product.

[0005] CN117004658A discloses a production process for producing succinic acid by a fermentation method, and specifically discloses that Escherichia coli is used as a production strain, micro-aerobic fermentation is first performed, and then anaerobic fermentation is performed, carbon dioxide is introduced as a supplementary carbon source in the anaerobic fermentation process, and finally succinic acid is obtained;

[0006] The patent improves the yield of succinic acid, but the acetic acid content is relatively high, and the yield of succinic acid is still at a low level. SUMMARY

[0007] In order to solve the technical problems existing in the prior art, the application provides a method for reducing acetic acid in a succinic acid fermentation process, which can effectively reduce the content of acetic acid in the succinic acid fermentation process, and the yield of succinic acid is high.

[0008] In order to solve the above technical problems, the application adopts the following technical solutions:

[0009] A method for reducing acetic acid in a succinic acid fermentation process, including shake flask culture, seed culture and fermentation culture, and specifically as follows:

[0010] 1. Shake flask culture

[0011] The yeast on the flat plate is inoculated into a shake flask culture medium for culture, the culture temperature is 35-37 DEG C, the culture time is 14-16h, the shake flask frequency is 174-185 rpm, and the shake flask culture liquid is prepared after the culture is finished;

[0012] The shake flask culture medium is composed of the following components: tryptone 5.7-6.2 g / L, NaCl 4.3-4.7 g / L, yeast powder 4.7-5.3 g / L, and the pH value is 7.0.

[0013] 2. Seed culture

[0014] The shake flask culture liquid is inoculated into a seed culture medium for seed culture at an inoculation amount of 0.8-1.0% by volume, the culture time is 13-15h, the temperature is 35-37 DEG C, the rotation speed is 305-314 rpm, the pressure is 0.05-0.07 MPa, the air volume is 7-9 L / min, the pH value is controlled to be 6.5-6.7 by ammonia water during the culture, and the seed culture liquid is prepared;

[0015] The seed culture medium is composed of the following components: glucose 26-30 g / L, potassium dihydrogen phosphate 14-16 / L, yeast powder 11-13 g / L, ammonium sulfate 3.5-4.5 g / L, magnesium sulfate heptahydrate 1.4-2.0 g / L, citric acid 1.3-1.5 g / L, auxiliary mixed solution 2.2-2.6 ml / L, vitamin B1 1.2-1.4 mg / L, riboflavin 0.022-0.026 mg / L, and biotin 0.04-0.06 mg / L.

[0016] The components of the auxiliary mixed solution are: boric acid 1.4-1.6 g / L, sodium molybdate 2.4-2.8 g / L, nickel sulfate hexahydrate 3.1-3.3 g / L, and manganese sulfate monohydrate 4-6 g / L.

[0017] 3. Fermentation culture

[0018] The seed culture liquid is inoculated into a fermentation culture medium for fermentation culture at an inoculation amount of 4-6%, first aerobic fermentation is carried out, the fermentation temperature is controlled to be 35-37 DEG C, the aeration amount is 1.6-1.9 vvm, the rotation speed is 460-480 rpm, the dissolved oxygen content is 13-17%, the pH value is controlled to be 6.5-6.7, and a glucose solution with a mass concentration of 53-55% is added to control the glucose content to be 9-12 g / L, after the fermentation culture is carried out for 13-15h, anaerobic fermentation is carried out, carbon dioxide is introduced, the aeration amount is controlled to be 0.8-1.2 vvm, the fermentation period is 36.3-37.9h, and the fermentation culture liquid is prepared.

[0019] The fermentation medium is composed of the following components: glucose 19-21 g / L, yeast powder 3-5 g / L, potassium dihydrogen phosphate 10-12 g / L, citric acid 3.0-3.2 g / L, ammonium sulfate 2.6-3.0 g / L, magnesium sulfate heptahydrate 1.0-1.4 g / L, ferrous sulfate heptahydrate 58-63 mg / L, vitamin B1 0.7-0.9 mg / L;

[0020] The fermentation culture is characterized in that during the fermentation culture process, the fermentation aid is fed, specifically:

[0021] At the 10th hour of fermentation, the fermentation aid I is fed into the fermentation tank at a feeding rate of 0.07-0.09 ml / h / L until the end of fermentation;

[0022] At the 24th hour of fermentation, the fermentation aid II is fed into the fermentation tank at a feeding rate of 0.12-0.14 ml / h / L until the end of fermentation;

[0023] The fermentation aid I is composed of cobalt chloride hexahydrate 0.3-0.5 g / L, zinc sulfate heptahydrate 6-8 g / L, folic acid 1.6-1.8 mg / L, and biotin 0.03-0.06 g / L;

[0024] The fermentation aid II is composed of nicotinamide 5-7 g / L, cysteine 2.0-2.2 g / L, and tyrosine 1.8-2.3 g / L.

[0025] The yeast is Pichia pastoris, and the classification name is Pichia sp. , which is preserved in the Guangdong Microbial Culture Collection Center, located at No. 59, Building 5, 100, Martyrs' Road, Guangzhou, with the preservation number GDMCC No: 64917 and the preservation date of July 26, 2024.

[0026] Compared with the prior art, the present application has the following beneficial effects:

[0027] 1. The present application maintains a low glucose intake rate in the middle and late stages of fermentation by feeding glucose, which is beneficial to inhibit the formation of by-products such as acetic acid and malic acid, maintains the proliferation efficiency of the strain, and avoids the defects of "using high-concentration initial sugar fermentation, the growth of the strain is inhibited in the early stage, leading to the accumulation of a large amount of acetic acid, and the production of succinic acid is low; using low-concentration initial sugar fermentation is not conducive to the proliferation of the strain, which further affects the synthesis of the product, leading to the extension of the fermentation period";

[0028] The present application adds fermentation aid one in the early stage of fermentation culture, cobalt chloride hexahydrate and zinc sulfate heptahydrate in which can be used as enzyme activator, biotin as growth factor, which can promote the growth of bacteria and the synthesis of succinic acid, combined with folic acid, promote the growth of cell division and the synthesis of amino acid, so as to reduce the generation of by-products, improve the yield of succinic acid; fermentation aid two is added in the middle and late stage of fermentation, nicotinamide can provide nitrogen element for the growth of bacteria and the production of fermentation acid, promote the metabolism of microorganism, maintain the growth activity of strain, improve the acid production performance, combined with cysteine and tyrosine, improve the yield of succinic acid, and reduce the accumulation of acetic acid and malic acid.

[0029] 2. The fermentation culture solution prepared by the method of the present application has a high succinic acid content of 59.1-60.4 g / L, a low acetic acid content of 0.30-0.65 g / L, and a low malic acid content of 0.20-0.23 g / L. DETAILED DESCRIPTION

[0030] In order to more clearly understand the technical features, objectives and effects of the present application, the specific embodiments of the present application will be described.

[0031] The yeast involved in the examples is Pichia pastoris (P. pastoris) Pichia sp. , with the preservation number of GDMCC No:64917, collected and isolated and purified in Nanning, Guangxi, China in April 2024.

[0032] Example 1

[0033] 1. Shake flask culture

[0034] The yeast bacteria on the plate are inoculated in the shake flask culture medium for culture, the culture temperature is 36℃, the culture time is 15h, the shake flask frequency is 180rpm, and the shake flask culture solution is prepared after the culture is completed.

[0035] The shake flask culture medium is composed of the following components: tryptone 6g / L, NaCl 4.5g / L, yeast powder 5g / L, and pH value is 7.0.

[0036] 2. Seed culture

[0037] The shake flask culture solution is inoculated in the seed culture medium at an inoculation amount of 0.9% by volume percentage for seed culture, the culture time is 14h, the temperature is 36℃, the rotation speed is 310rpm, the pressure is 0.06MPa, the air volume is 8L / min, the pH value is controlled to be 6.6 by ammonia water during the culture process, and the seed culture solution is prepared.

[0038] The seed culture medium is composed of the following components: glucose 28 g / L, potassium dihydrogen phosphate 15 g / L, yeast powder 12 g / L, ammonium sulfate 4 g / L, magnesium sulfate heptahydrate 1.7 g / L, citric acid 1.4 g / L, auxiliary mixture 2.4 ml / L, vitamin B1 1.3 mg / L, riboflavin 0.024 mg / L, biotin 0.05 mg / L;

[0039] The components of the auxiliary mixture are as follows: boric acid 1.5 g / L, sodium molybdate 2.6 g / L, nickel sulfate hexahydrate 3.2 g / L, manganese sulfate monohydrate 5 g / L.

[0040] 3. Fermentation culture

[0041] The seed culture solution is inoculated into the fermentation culture medium at an inoculation amount of 5% for fermentation culture. First, aerobic fermentation is carried out, the fermentation temperature is controlled at 36℃, the aeration amount is 1.7vvm, the rotation speed is 470rpm, the dissolved oxygen amount is 15%, the pH value is controlled at 6.6, and the glucose solution with a mass concentration of 54% is added to control the glucose content at 11 g / L. After 14h of fermentation culture, anaerobic fermentation is carried out, carbon dioxide is introduced, the aeration amount is controlled at 1.0vvm, and the fermentation period is 36.3h to obtain the fermentation culture solution.

[0042] The fermentation culture medium is composed of the following components: glucose 20 g / L, yeast powder 4 g / L, potassium dihydrogen phosphate 11 g / L, citric acid 3.1 g / L, ammonium sulfate 2.8 g / L, magnesium sulfate heptahydrate 1.2 g / L, ferrous sulfate heptahydrate 61 mg / L, vitamin B1 0.8 mg / L;

[0043] The fermentation culture is carried out by adding fermentation aids during the fermentation culture process, specifically:

[0044] At the 10th hour of fermentation, fermentation aid one is added to the fermentation tank at an addition rate of 0.08 ml / h / L until the end of fermentation;

[0045] At the 24th hour of fermentation, fermentation aid two is added to the fermentation tank at an addition rate of 0.13 ml / h / L until the end of fermentation;

[0046] The components of the fermentation aid one are as follows: cobalt chloride hexahydrate 0.4 g / L, zinc sulfate heptahydrate 7 g / L, folic acid 1.7 mg / L, biotin 0.04 g / L;

[0047] The components of the fermentation aid two are as follows: nicotinamide 6 g / L, cysteine 2.1 g / L, tyrosine 2 g / L.

[0048] The fermentation culture solution prepared by the method of Example 1 has a succinic acid content of 60.4 g / L, an acetic acid content of 0.41 g / L, and a malic acid content of 0.20 g / L.

[0049] Example 2

[0050] 1. Shake flask culture

[0051] The yeast on the plate was inoculated into a shake flask culture medium for culture, the culture temperature was 35℃, the culture time was 14h, the shake flask frequency was 174rpm, and the shake flask culture liquid was prepared after the culture was completed;

[0052] The shake flask culture medium is composed of the following components: tryptone 5.7g / L, NaCl 4.3g / L, yeast powder 4.7g / L, and the pH value is 7.0.

[0053] 2. Seed culture

[0054] The shake flask culture liquid was inoculated into a seed culture medium at an inoculation amount of 0.8% by volume for seed culture, the culture time was 13h, the temperature was 35℃, the rotation speed was 305rpm, the pressure was 0.05MPa, the air volume was 7L / min, and the pH value was controlled to be 6.5 by ammonia during the culture process, and the seed culture liquid was prepared;

[0055] The seed culture medium is composed of the following components: glucose 26g / L, potassium dihydrogen phosphate 14 / L, yeast powder 11g / L, ammonium sulfate 3.5g / L, magnesium sulfate heptahydrate 1.4g / L, citric acid 1.3g / L, auxiliary mixed solution 2.2ml / L, vitamin B1 1.2mg / L, riboflavin 0.022mg / L, and biotin 0.04mg / L;

[0056] The components of the auxiliary mixed solution are: boric acid 1.4g / L, sodium molybdate 2.4g / L, nickel sulfate hexahydrate 3.1g / L, and manganese sulfate monohydrate 4g / L.

[0057] 3. Fermentation culture

[0058] The seed culture liquid was inoculated into a fermentation culture medium at an inoculation amount of 4% for fermentation culture, first aerobic fermentation was carried out, the fermentation temperature was controlled to be 35℃, the aeration amount was 1.6vvm, the rotation speed was 460rpm, the dissolved oxygen content was 13%, the pH value was controlled to be 6.5, and the glucose content was controlled to be 9g / L by adding glucose solution with a mass concentration of 53%, and then anaerobic fermentation was carried out after 13h of fermentation culture, carbon dioxide was introduced, the aeration amount was controlled to be 0.8vvm, the fermentation period was 37.9h, and the fermentation culture liquid was prepared;

[0059] The fermentation culture medium is composed of the following components: glucose 19g / L, yeast powder 3g / L, potassium dihydrogen phosphate 12g / L, citric acid 3.0g / L, ammonium sulfate 2.6g / L, magnesium sulfate heptahydrate 1.4g / L, ferrous sulfate heptahydrate 58mg / L, and vitamin B1 0.9mg / L.

[0060] The fermentation culture, during the fermentation culture, the fermentation aid is added, specifically:

[0061] At the 10th hour of fermentation, the fermentation aid one is added to the fermentation tank at an addition rate of 0.07 ml / h / L until the end of fermentation;

[0062] At the 24th hour of fermentation, the fermentation aid two is added to the fermentation tank at an addition rate of 0.12 ml / h / L until the end of fermentation;

[0063] The fermentation aid one comprises cobalt chloride hexahydrate 0.3 g / L, zinc sulfate heptahydrate 8 g / L, folic acid 1.6 mg / L, and biotin 0.06 g / L;

[0064] The fermentation aid two comprises nicotinamide 5 g / L, cysteine 2.0 g / L, and tyrosine 2.3 g / L.

[0065] The fermentation culture liquid prepared by the method of Example 2 has succinic acid content of 59.1 g / L, acetic acid content of 0.65 g / L, and malic acid content of 0.23 g / L.

[0066] Example 3

[0067] 1. Shake flask culture

[0068] The yeast on the plate is inoculated in the shake flask culture medium for culture, the culture temperature is 37℃, the culture time is 16h, the shake flask frequency is 185rpm, and the shake flask culture liquid is prepared after the end of culture;

[0069] The shake flask culture medium is composed of the following components: tryptone 6.2 g / L, NaCl 4.7 g / L, and yeast powder 5.3 g / L, and the pH value is 7.0.

[0070] 2. Seed culture

[0071] The shake flask culture liquid is inoculated in the seed culture medium for seed culture at an inoculation amount of 1.0% by volume, the culture time is 15h, the temperature is 37℃, the rotation speed is 314rpm, the pressure is 0.07MPa, the air volume is 9L / min, the pH value is controlled to be 6.7 by ammonia water during the culture process, and the seed culture liquid is prepared;

[0072] The seed culture medium is composed of the following components: glucose 30 g / L, potassium dihydrogen phosphate 16 / L, yeast powder 13 g / L, ammonium sulfate 4.5 g / L, magnesium sulfate heptahydrate 2.0 g / L, citric acid 1.5 g / L, auxiliary mixed solution 2.6 ml / L, vitamin B1 1.4 mg / L, riboflavin 0.026 mg / L, and biotin 0.06 mg / L.

[0073] The components of the auxiliary mixed solution are: boric acid 1.6 g / L, sodium molybdate 2.8 g / L, nickel sulfate hexahydrate 3.3 g / L, manganese sulfate monohydrate 6 g / L.

[0074] 3. Fermentation culture

[0075] The seed culture solution is inoculated into the fermentation medium at an inoculation amount of 6% for fermentation culture. First, aerobic fermentation is performed, the fermentation temperature is controlled at 37℃, the aeration amount is 1.9vvm, the rotation speed is 480rpm, the dissolved oxygen amount is 17%, the pH value is controlled at 6.7, and the glucose solution with a mass concentration of 55% is fed to control the glucose content at 12g / L. After 15h of fermentation culture, anaerobic fermentation is performed, carbon dioxide is introduced, the aeration amount is controlled at 1.2vvm, and the fermentation period is 36.8h to obtain the fermentation culture solution.

[0076] The fermentation medium is composed of the following components: glucose 21 g / L, yeast powder 5 g / L, potassium dihydrogen phosphate 10 g / L, citric acid 3.2 g / L, ammonium sulfate 3.0 g / L, magnesium sulfate heptahydrate 1.0 g / L, ferrous sulfate heptahydrate 63 mg / L, vitamin B1 0.7 mg / L.

[0077] During the fermentation culture, fermentation aids are fed, specifically:

[0078] At the 10th hour of fermentation, fermentation aid one is fed into the fermentation tank at a feeding rate of 0.09 ml / h / L until the end of fermentation.

[0079] At the 24th hour of fermentation, fermentation aid two is fed into the fermentation tank at a feeding rate of 0.14 ml / h / L until the end of fermentation.

[0080] The components of the fermentation aid one are: cobalt chloride hexahydrate 0.5 g / L, zinc sulfate heptahydrate 6 g / L, folic acid 1.8 mg / L, biotin 0.03 g / L.

[0081] The components of the fermentation aid two are: nicotinamide 7 g / L, cysteine 2.2 g / L, tyrosine 1.8 g / L.

[0082] The fermentation culture solution prepared by the method of Example 3 has a succinic acid content of 59.8 g / L, an acetic acid content of 0.30 g / L, and a malic acid content of 0.22 g / L.

[0083] Comparative Example 1

[0084] Based on Example 1, the difference is that the components of fermentation aid one and fermentation aid two are directly added to the fermentation medium.

[0085] The fermentation medium is composed of the following components: glucose 20 g / L, yeast powder 4 g / L, potassium dihydrogen phosphate 11 g / L, citric acid 3.1 g / L, ammonium sulfate 2.8 g / L, magnesium sulfate heptahydrate 1.2 g / L, ferrous sulfate heptahydrate 61 mg / L, vitamin B1 0.8 mg / L, fermentation aid one 2.1 ml / L, fermentation aid two 1.5 ml / L;

[0086] The components of the fermentation aid one are cobalt chloride hexahydrate 0.4 g / L, zinc sulfate heptahydrate 7 g / L, folic acid 1.7 mg / L, and biotin 0.04 g / L.

[0087] The components of the fermentation aid two are nicotinamide 6 g / L, cysteine 2.1 g / L, and tyrosine 2 g / L.

[0088] The remaining operations are the same.

[0089] The fermentation culture solution prepared by the method of Comparative Example 1 has succinic acid content of 47.5 g / L, acetic acid content of 2.78 g / L, and malic acid content of 1.22 g / L.

[0090] Comparative Example 1 adds the components of the fermentation aid one and the fermentation aid two to the fermentation medium. In the early stage of fermentation, the bacteria grow rapidly and have high biomass. High specific growth rate can lead to an increase in acetic acid. Directly adding growth factors and other components to the fermentation medium can greatly increase the accumulation of acetic acid. In addition, adding zinc, cobalt, tyrosine, and cysteine to the fermentation medium can reduce the secretion of enzymes in the early stage of fermentation, which cannot effectively promote the fermentation reaction, thereby leading to an increase in by-products and a low yield of succinic acid. After a period of fermentation, the method of flow addition is used. In the main fermentation period, the bacteria grow weakly, the activity decreases, and the acid production level decreases. Nicotinamide can supplement nitrogen source in time, and it can combine with growth factors such as cobalt, zinc, tyrosine, and cysteine to promote the growth of bacteria and the activity of enzymes. Under the mutual influence and joint action, the yield of succinic acid is improved, and the accumulation of by-products such as acetic acid and malic acid is reduced, thereby improving the conversion rate.

[0091] Comparative Example 2

[0092] On the basis of Example 1, the change is that the step of flow addition of the fermentation aid is omitted, and the remaining operations are the same.

[0093] The fermentation culture solution prepared by the method of Comparative Example 2 has succinic acid content of 42.1 g / L, acetic acid content of 6.84 g / L, and malic acid content of 1.64 g / L.

[0094] Comparative Example 2 omits the fermentation aid component, when the strain growth is weak and the activity is decreased in the main fermentation period, it cannot effectively supplement the nutrient components in the fermentation middle and late period, and it cannot supplement the components such as zinc, cobalt, tyrosine and cysteine in time during the synthesis of aromatic amino acids in the fermentation process, thereby affecting the yield and synthesis rate of succinic acid, leading to incomplete fermentation, increase of by-products and low conversion rate.

[0095] The present application maintains a low glucose intake rate in the fermentation middle and late period by the method of glucose feeding, which is beneficial to inhibit the formation of by-products such as acetic acid and malic acid, maintain the proliferation efficiency of the strain, and avoid the defects of "using high-concentration initial sugar fermentation, the strain growth is inhibited in the early stage, leading to a large amount of acetic acid accumulation and low yield of succinic acid; using low-concentration initial sugar fermentation is not conducive to the proliferation of the strain, which further affects the synthesis of the product, leading to the extension of the fermentation period"; the present application feeds the fermentation aid I in the early stage of fermentation culture, the cobalt chloride hexahydrate and zinc sulfate heptahydrate in the fermentation aid I can be used as enzyme activators, and the biotin can be used as a growth factor, which can promote the growth of the strain and the synthesis of succinic acid, and the combination of the biotin and folic acid can promote the cell division and growth as well as the synthesis of amino acids, thereby reducing the generation of by-products and improving the yield of succinic acid; the fermentation aid II is added in the fermentation middle and late period, the nicotinamide in the fermentation aid II can provide nitrogen elements for the growth of the strain and the fermentation acid production, promote the metabolism of the microorganism, maintain the growth activity of the strain, and improve the acid production performance, and the combination of the nicotinamide, cysteine and tyrosine can improve the yield of succinic acid while reducing the accumulation amount of acetic acid and malic acid.

[0096] Unless otherwise specified, the percentages used in the present application are mass percentages.

[0097] Finally, it should be noted that: the above only describes the preferred embodiments of the present application and is not intended to limit the present application, although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions recorded in the foregoing embodiments or make equivalent replacement for some technical features. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A method for reducing acetic acid in a succinic acid fermentation process, characterized in that, The method comprises a shake flask culture, a seed culture and a fermentation culture; The shake flask culture step is to inoculate the yeast into a shake flask culture medium for culture; The yeast is Pichia sp., and the preservation number is GDMCC No:64917; The seed culture step is to inoculate the shake flask culture liquid into a seed culture medium at an inoculation amount of 0.8-1.0% by volume for seed culture, the culture time is 13-15 h, the temperature is 35-37℃, the rotation speed is 305-314 rpm, the pressure is 0.05-0.07 MPa, the air volume is 7-9 L / min, the pH value is controlled to be 6.5-6.7 by ammonia water during the culture process, and the seed culture liquid is prepared; The seed culture medium is composed of the following components: glucose 26-30 g / L, potassium dihydrogen phosphate 14-16 g / L, yeast powder 11-13 g / L, ammonium sulfate 3.5-4.5 g / L, magnesium sulfate heptahydrate 1.4-2.0 g / L, citric acid 1.3-1.5 g / L, auxiliary mixed liquid 2.2-2.6 ml / L, vitamin B1 1.2-1.4 mg / L, riboflavin 0.022-0.026 mg / L, and biotin 0.04-0.06 mg / L; The components of the auxiliary mixed liquid are as follows: boric acid 1.4-1.6 g / L, sodium molybdate 2.4-2.8 g / L, nickel sulfate hexahydrate 3.1-3.3 g / L, and manganese sulfate monohydrate 4-6 g / L; The fermentation culture step is to inoculate the seed culture liquid into a fermentation culture medium at an inoculation amount of 4-6% for fermentation culture, first aerobic fermentation is performed, the fermentation temperature is controlled to be 35-37℃, the aeration amount is 1.6-1.9 vvm, the rotation speed is 460-480 rpm, the dissolved oxygen content is 13-17%, the pH value is controlled to be 6.5-6.7, the glucose solution with a mass concentration of 53-55% is added to control the glucose content to be 9-12 g / L, after the fermentation culture for 13-15 h, anaerobic fermentation is performed, carbon dioxide is introduced, the aeration amount is controlled to be 0.8-1.2 vvm, the fermentation cycle is 36.3-37.9 h, and the fermentation culture liquid is prepared; The fermentation culture medium is composed of the following components: glucose 19-21 g / L, yeast powder 3-5 g / L, potassium dihydrogen phosphate 10-12 g / L, citric acid 3.0-3.2 g / L, ammonium sulfate 2.6-3.0 g / L, magnesium sulfate heptahydrate 1.0-1.4 g / L, ferrous sulfate heptahydrate 58-63 mg / L, and vitamin B1 0.7-0.9 mg / L; In the fermentation culture step, the fermentation aid is added during the fermentation culture process, specifically as follows: The fermentation aid one is added in the early stage of fermentation, and the fermentation aid two is added in the middle and late stages of fermentation; The fermentation aid one is added into the fermentation tank at the 10th hour of fermentation at an adding speed of 0.07-0.09 ml / h / L until the end of fermentation; At the 24th hour of fermentation, the fermentation aid two is added into the fermentation tank at a flow rate of 0.12-0.14 ml / h / L until the end of fermentation; The fermentation aid one comprises 0.3-0.5 g / L of cobalt chloride hexahydrate, 6-8 g / L of zinc sulfate heptahydrate, 1.6-1.8 mg / L of folic acid and 0.03-0.06 g / L of biotin; The fermentation aid two comprises 5-7 g / L of nicotinamide, 2.0-2.2 g / L of cysteine and 1.8-2.3 g / L of tyrosine; The fermentation broth prepared by the method has a succinic acid content of 59.1-60.4 g / L, an acetic acid content of 0.30-0.65 g / L and a malic acid content of 0.20-0.23 g / L.

2. The method according to claim 1, characterized in that, The shake flask culture step is to inoculate the yeast on the plate into a shake flask culture medium for culture, the culture temperature is 35-37 ℃, the culture time is 14-16 h, the shake flask frequency is 174-185 rpm, and the shake flask culture broth is prepared after the culture.

3. The method according to claim 2, characterized in that, The shake flask culture medium is composed of the following components: 5.7-6.2 g / L of tryptone, 4.3-4.7 g / L of NaCl and 4.7-5.3 g / L of yeast powder, and the pH value is 7.0.