A method for preparing vitamin B2 by fermentation using corn steep liquor as nitrogen source

Abstract

The present application belongs to the technical field of biological fermentation, and particularly relates to a method for preparing vitamin B2 by fermentation using corn syrup nitrogen source, and specifically comprises the following steps: evaluating the quality of corn syrup, determining the contents of total nitrogen, amino nitrogen, soluble phosphorus and glutamic acid, and dividing the corn syrup into three grades of A, B and C; preparing fermentation medium by directly using, blending or enzymatic pretreatment of corn syrup of different grades; inoculating bacillus subtilis seed liquid into the fermentation medium for fermentation culture; dynamically adjusting the formula of feeding medium according to the grade of corn syrup during the fermentation process, and dynamically feeding according to the concentration of amino nitrogen in the fermentation liquid. Through the establishment of the quality evaluation and dynamic blending system of corn syrup, the dynamic feeding strategy based on the key nutrient index during the fermentation process and the precise intervention of the metabolic regulation node, the batch fluctuation problem of corn syrup is effectively overcome, the fermentation yield is improved, the fermentation period is shortened, and the method is suitable for the industrialized and efficient and stable production of vitamin B2.

Description

Technical Field

[0001] This invention belongs to the field of bio-fermentation technology, specifically relating to a method for preparing vitamin B2 using corn steep liquor as a nitrogen source through fermentation. Background Technology

[0002] Vitamin B2 (riboflavin) is an essential vitamin for maintaining normal metabolism and is widely used in medicine, food, and feed additives. Currently, the mainstream production method for vitamin B2 is microbial fermentation, with commonly used microbial strains including Bacillus subtilis.

[0003] Nitrogen source is a core component of vitamin B2 fermentation medium, directly affecting strain growth, metabolic efficiency, and the yield of the target product. Corn steep liquor, a byproduct of wet corn starch production, is rich in soluble proteins, amino acids, peptides, and various growth factors, making it a cost-effective natural organic nitrogen source in the fermentation industry.

[0004] There are existing reports on the use of corn steep liquor as a nitrogen source for vitamin B2 fermentation. For example, CN109182438B discloses a culture medium and method for producing vitamin B2 using Bacillus fermentation, employing medium-efficiency carbon and nitrogen sources such as corn steep liquor, urea, and sucrose as the fermentation base. CN112280679A discloses a method for producing vitamin B2 by fermentation, wherein the fermentation medium contains 10-20 g / L of corn steep liquor. CN113817654B discloses a fermentation medium for producing riboflavin, wherein the amount of corn steep liquor used is 10-20 g / L.

[0005] However, the aforementioned existing technologies all have the following shortcomings: (i) The batch stability problem of corn steep liquor has not been effectively solved. The composition of corn steep liquor is easily affected by factors such as corn origin, processing technology, and storage and transportation conditions, resulting in large fluctuations in indicators such as total nitrogen, amino nitrogen, amino acid composition, and trace elements between batches. Existing technologies all use corn steep liquor directly as a fixed formula component without effectively controlling its batch differences, leading to problems such as yield fluctuations and unstable fermentation cycles during the fermentation process.

[0006] (ii) Lack of a dynamic control mechanism based on the actual composition of corn steep liquor. In existing technologies, the fermentation medium formulation and feeding strategy are fixed and cannot be dynamically adjusted according to the actual nutrient composition of the corn steep liquor used. When the key nutrient indicators of a batch of corn steep liquor deviate from the normal range, the fermentation efficiency of the fixed formulation will decrease significantly.

[0007] (III) The regulatory mechanism of nutrients in corn steep liquor on vitamin B2 synthesis has not been fully explored and utilized. Existing technologies only focus on the role of corn steep liquor as a nitrogen source substitute, without in-depth analysis of the effects of specific nutrients in corn steep liquor (such as specific amino acids, trace elements, growth factors, etc.) on the expression of key enzyme genes and metabolic flux in the vitamin B2 synthesis pathway. Therefore, precise fermentation based on metabolic regulation has not been achieved. Summary of the Invention

[0008] To address the aforementioned issues, this invention aims to provide a method for preparing vitamin B2 using corn steep liquor as a nitrogen source through fermentation. By establishing a quality evaluation and dynamic allocation system for corn steep liquor raw materials, a dynamic feeding strategy based on key nutrient indicators during fermentation, and precise intervention at metabolic regulation nodes, the method effectively overcomes the fermentation instability caused by batch fluctuations in corn steep liquor, and significantly improves the fermentation yield and batch consistency of vitamin B2.

[0009] The present invention is implemented according to the following scheme: A method for preparing vitamin B2 using corn steep liquor as a nitrogen source through fermentation specifically includes the following steps: S1. Quality evaluation and classification of corn steep liquor raw materials, including the following sub-steps: S11. Collect corn steep liquor samples for use and determine the following key nutritional indicators: total nitrogen content (TN), amino nitrogen content (AN), soluble phosphorus content (P), reducing sugar content (RS), and the content of key amino acids (glutamic acid, aspartic acid, lysine). S12. Based on the test results, the corn steep liquor samples were divided into three grades: Grade A: TN ≥ 6.0%, AN / TN ≥ 35%, P ≥ 0.8%, Glutamic acid ≥ 0.8%; Grade B: 5.0%≤TN<6.0%, 30%≤AN / TN<35%, 0.5%≤P<0.8%, glutamic acid ≥0.5%; Grade C: TN < 5.0% or AN / TN < 30% or P < 0.5% or glutamate < 0.5%; Grade S13 and Grade A corn steep liquor are used directly for preparing fermentation media; Grade B corn steep liquor is used after supplementing total nitrogen with ammonium sulfate or urea and adjusting the amino acid composition by adding monosodium glutamate; Grade C corn steep liquor requires enzymatic pretreatment before use. The enzymatic pretreatment is as follows: adjust the pH of the corn steep liquor to 6.5-7.0, add a compound protease (the amount added is 0.05%-0.15% of the mass of the corn steep liquor), enzymatically hydrolyze at 50-55℃ for 2-4 hours, and use after enzyme inactivation.

[0010] S2. Preparation of fermentation medium: Using the corn steep liquor evaluated in step S1 as the main nitrogen source, the fermentation medium is prepared, and its components are as follows by weight-volume ratio: Carbon source: glucose 1.0%-3.0%, sucrose 2.0%-4.0%; Nitrogen source: corn steep liquor (dry matter) 2.0%-4.5%, ammonium sulfate 0.1%-0.5%; Inorganic salts: dipotassium hydrogen phosphate 0.1%-0.5%, magnesium sulfate 0.01%-0.15%, manganese sulfate 0.01%-0.05%; Growth factors: Biotin 0.5-2.0 mg / L, Glutamine 0.5-2.0 g / L; Light calcium carbonate 0.1%-0.5%; The rest is water; The pH of the above fermentation medium was adjusted to 6.8-7.2.

[0011] S3. Preparation of seed culture: The activated Bacillus subtilis seeds are inoculated into the seed culture medium and cultured for 15-22 hours at a temperature of 34-39℃ and an aeration rate of 1:0.4-0.7v / v / min to obtain the primary seed culture.

[0012] The seed culture medium consists of: 1.0%-4.0% sucrose, 0.2%-0.5% glucose, 0.5%-2.5% corn steep liquor, 0.1%-0.3% ammonium sulfate, 0.1%-0.5% light calcium carbonate, and 0.1%-0.5% magnesium sulfate.

[0013] S4. Fermentation culture: Inoculate the seed culture obtained in step S3 into the fermentation culture medium prepared in step S2 at an inoculation rate of 5%-15%. Fermentation culture is carried out at a temperature of 35-42℃ and an aeration rate of 1:0.8-1.3 v / v / min. The dissolved oxygen content of the fermentation broth is controlled to be above 25%-35%, and the pH is maintained at 6.6-7.2.

[0014] A dynamic feedback control strategy based on key nutrient indicators of corn steep liquor was adopted during the fermentation process, which specifically included the following sub-steps: S41. Initial fermentation stage (0-12 hours): Take samples every 4 hours to measure the amino nitrogen concentration in the fermentation broth. When the amino nitrogen concentration is lower than 60% of the initial value, add Grade A corn steep liquor concentrate (dry matter content 15%-20%) evaluated in step one. The amount added is 0.5%-1.5% of the fermentation broth volume. S42. Mid-fermentation stage (12-36 hours): Samples are taken every 6 hours to determine the concentration of dissolved phosphorus and glutamate in the fermentation broth. When the dissolved phosphorus concentration is below 0.3 g / L, potassium dihydrogen phosphate solution is added; when the glutamate concentration is below 0.2 g / L, sodium glutamate solution is added. The amount added should be sufficient to maintain the glutamate concentration in the range of 0.2-0.5 g / L. S43. Late fermentation stage (36 hours to discharge): Take samples every 8 hours to measure the total sugar concentration in the fermentation broth. When the total sugar concentration drops below 2.0%, start adding feed medium to control the total sugar concentration to be maintained in the range of 1.0%-2.5%. The formulation of the above-mentioned feed medium is dynamically adjusted according to the evaluation results of corn steep liquor before fermentation: when the corn steep liquor used is grade A, the feed medium composition is 10%-20% sucrose, 20%-30% glucose, and 3%-6% corn steep liquor; when the corn steep liquor used is grade B, 0.2%-0.5% ammonium sulfate is added to the feed medium; when the corn steep liquor used is grade C, the feed medium uses enzymatically hydrolyzed corn steep liquor instead of ordinary corn steep liquor.

[0015] Preferably, the fermentation culture is carried out with temperature stepwise regulation between 12 and 24 hours: maintaining 37-39℃ for 12 hours, then raising the temperature to 40-42℃ and maintaining it until the fermentation tank is discharged.

[0016] Preferably, the feeding adopts a pulse feeding strategy, feeding once every 2 hours, and the amount of feeding each time is 0.3%-0.8% of the fermentation liquid volume.

[0017] S5. Fermentation endpoint determination and product extraction: When the vitamin B2 content in the fermentation broth no longer increases significantly (increase of <5% in two consecutive samplings) or the fermentation time reaches 60-80h, the broth is removed from the tank to obtain a fermentation mash rich in vitamin B2. After extraction and purification, vitamin B2 product is obtained.

[0018] The beneficial effects of this invention are: (1) By establishing a quality evaluation and grading system for corn steep liquor raw materials and adopting differentiated pretreatment and blending schemes according to different grades, the batch variation rate of key nutrient indicators (total nitrogen, amino nitrogen, dissolved phosphorus, etc.) of corn steep liquor entering the fermentation stage is controlled below 5%, which ensures the stability of fermentation raw materials from the source and effectively overcomes the problem of batch fluctuation of corn steep liquor.

[0019] (2) The present invention dynamically adjusts the fermentation medium formula and the feed medium formula based on the evaluation results of corn steep liquor before fermentation. At the same time, during the fermentation process, dynamic feeding is carried out based on the real-time monitoring results of key indicators such as amino nitrogen, soluble phosphorus, glutamic acid, and total sugar, which realizes precise fermentation control and significantly improves the batch consistency and yield stability of fermentation.

[0020] (3) This invention uses the glutamate content in corn steep liquor as one of the key indicators for raw material grading, and dynamically monitors and precisely replenishes the glutamate concentration during fermentation. Glutamate is not only a raw material for protein synthesis, but also an important precursor for the synthesis of guanosine triphosphate (GTP), which is a key precursor in the vitamin B2 (riboflavin) synthesis pathway. By precisely controlling the glutamate concentration, the biosynthesis of riboflavin can be directly promoted.

[0021] (4) By adopting the method of the present invention, the fermentation yield of vitamin B2 is increased by 8%-15% compared with the traditional fixed formula process, the fermentation cycle is shortened by 5%-10%, and the relative standard deviation (RSD) of batch yield is reduced from the traditional 12%-18% to below 5%, which significantly improves the stability and economy of industrial production.

[0022] (5) By establishing a corn steep liquor grading and utilization system, this invention enables low-grade corn steep liquor (Grade B and Grade C) to be effectively utilized through enzymatic pretreatment and dynamic blending, avoiding the waste of high-quality corn steep liquor and the disposal of low-grade corn steep liquor, and further reducing raw material costs. At the same time, the dynamic and precise feeding strategy reduces the excessive addition and residue of raw materials, and reduces the solid content in the fermentation liquid. Attached Figure Description

[0023] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0024] Figure 1 This is a flowchart of the method in Embodiment 1 of the present invention. Detailed Implementation

[0025] The present invention will now be clearly described with reference to specific embodiments. These descriptions are merely illustrative and are not intended to limit the scope of the invention. Any modifications, equivalent substitutions, or improvements made by those skilled in the art based on the embodiments of the present invention without inventive effort to obtain all other embodiments should be included within the scope of protection of the present invention.

[0026] Example 1 like Figure 1 As shown, this embodiment uses Grade A corn steep liquor for vitamin B2 fermentation production.

[0027] 1. Evaluation of corn steep liquor raw materials A batch of corn steep liquor sample was taken and key nutrient indicators were measured: total nitrogen 6.3%, amino nitrogen 2.3% (AN / TN=36.5%), lysed phosphorus 0.9%, and glutamic acid 0.85%. All indicators met the Grade A standard and can be directly used for the preparation of fermentation culture medium.

[0028] 2. Preparation of fermentation culture medium Prepare the fermentation medium (per 1L) according to the following formula: 20g glucose, 30g sucrose; Grade A corn steep liquor (on a dry matter basis) 35g; 3g of ammonium sulfate; 3g of dipotassium hydrogen phosphate; Magnesium sulfate 0.8g; 0.3g of manganese sulfate; Biotin 1.0 mg; Glutamine 1.0g; Sodium citrate 2.0g; 3g of light calcium carbonate; The remainder is water; Adjust the pH to 7.0.

[0029] 3. Seed liquid preparation After activation, Bacillus subtilis was inoculated into a seed culture medium (2.0% sucrose, 0.3% glucose, 1.5% corn steep liquor, 0.2% ammonium sulfate, 0.3% light calcium carbonate, and 0.3% magnesium sulfate) and cultured at 37°C with an aeration rate of 1:0.5 v / v / min for 18 hours to obtain the primary seed culture.

[0030] 4. Fermentation culture The seed culture was inoculated into the fermentation medium at a rate of 10%, and fermented at 39°C with an aeration rate of 1:1.0 v / v / min.

[0031] Dynamic control during fermentation: 0-12 hours: Amino nitrogen was measured every 4 hours. When amino nitrogen dropped to 55% of the initial value at 12 hours, 1.0% of Grade A corn steep liquor concentrate was added. 12-36 hours: Measure solubilized phosphorus and glutamate every 6 hours. When solubilized phosphorus drops to 0.28 g / L at 18 hours, add potassium dihydrogen phosphate solution. When glutamate drops to 0.18 g / L at 24 hours, add sodium glutamate solution to 0.3 g / L. 36 hours later: When the total sugar content drops to 1.8%, start adding fed culture medium (15% sucrose, 25% glucose, 5% Grade A corn steep liquor) to control the total sugar content at 1.5%-2.5%; After fermenting for 12 hours, raise the temperature to 41°C.

[0032] 5. Results After 68 hours of fermentation, the vitamin B2 yield was increased by 12.3% compared to the traditional fixed-formula process (control group), and the fermentation cycle was shortened by 8.1%. The RSD (relative standard deviation) of the yield was 3.8% across three consecutive fermentation batches.

[0033] Example 2 This embodiment uses Grade B corn steep liquor for vitamin B2 fermentation production.

[0034] 1. Evaluation and Blending of Corn Stew Raw Materials A batch of corn steep liquor sample was taken, and key nutrient indicators were measured: total nitrogen 5.4%, amino nitrogen 1.6% (AN / TN = 29.6%), lysed phosphorus 0.6%, and glutamic acid 0.55%. Total nitrogen and glutamic acid met the Grade B standard, but the AN / TN was slightly lower than the Grade B standard. By adding 0.15% ammonium sulfate and 0.1% monosodium glutamate, the adjusted indicators reached: total nitrogen 6.0%, AN / TN = 34.2%, and glutamic acid 0.62%, meeting the Grade A standard before use.

[0035] 2-4. Fermentation process The amount of corn steep liquor in the fermentation medium was adjusted to 40 g / L (dry matter), and the amount of ammonium sulfate was adjusted to 4 g / L. The remaining formulation and fermentation conditions were the same as in Example 1. An additional 0.3% ammonium sulfate was added to the fed-batch medium.

[0036] 5. Results After 70 hours of fermentation, the vitamin B2 yield increased by 10.1% compared to the control group, and the fermentation cycle was shortened by 6.5%. The RSD of the yield was 4.5% across three consecutive fermentation batches.

[0037] Example 3 This embodiment uses Grade C corn steep liquor for vitamin B2 fermentation production.

[0038] 1. Evaluation and enzymatic pretreatment of corn steep liquor raw materials A batch of corn steep liquor samples was taken, and key nutrient indicators were measured: total nitrogen 4.6%, amino nitrogen 1.3% (AN / TN = 28.3%), soluble phosphorus 0.4%, and glutamic acid 0.42%. All indicators failed to meet the Grade A standard, so enzymatic pretreatment was performed: the pH of the corn steep liquor was adjusted to 6.8, and a complex protease was added (0.10% of the corn steep liquor mass). Enzymatic hydrolysis was carried out at 52℃ for 3 hours, followed by enzyme inactivation at 90℃ for 10 minutes. After enzymatic hydrolysis, the following results were obtained: total nitrogen 4.8%, amino nitrogen 2.0% (AN / TN = 41.7%), and glutamic acid 0.58%, with a significant increase in AN / TN.

[0039] 2-4. Fermentation process The amount of corn steep liquor in the fermentation medium was adjusted to 45 g / L (dry matter), and the amount of ammonium sulfate was adjusted to 5 g / L. The remaining formulation and fermentation conditions were the same as in Example 1. Enzymatically hydrolyzed corn steep liquor was used instead of ordinary corn steep liquor in the fed-batch medium.

[0040] 5. Results After 72 hours of fermentation, the vitamin B2 yield increased by 8.7% compared to the control group, and the fermentation cycle was shortened by 5.2%. The RSD of the yield was 4.9% across three consecutive fermentation batches.

[0041] Comparative Example Comparative fermentation was conducted using a method in existing technologies where "the fermentation medium formulation and feeding strategy are both fixed".

[0042] Fermentation medium composition: 4.0% sucrose, 1.0% glucose, 1.0% urea, 2.5% corn steep liquor, 0.5% ammonium sulfate, 0.08% magnesium sulfate, 0.3% light calcium carbonate, and 0.3% dipotassium hydrogen phosphate. No evaluation or grading of the corn steep liquor raw material was performed, and no dynamic feedback control was implemented. The feed medium was a fixed formula (15% sucrose, 25% glucose, and 8% corn steep liquor).

[0043] Results of five consecutive batches of fermentation: The average yield of vitamin B2 was 14.6% lower than that of Example 1 of this invention, the average fermentation cycle was 9.3% longer, and the yield RSD was 14.2%.

[0044] In summary, the method of this invention is simple to operate and suitable for large-scale industrial production. The established corn steep liquor raw material quality evaluation and grading system can be rapidly promoted among existing corn starch processing and fermentation enterprises; the dynamic feedback control strategy can be implemented based on existing automated control systems for fermentation processes (such as DCS and PLC systems), without the need for additional investment in large-scale equipment. This invention can be widely applied to the industrial production of vitamin B2 using Bacillus subtilis and other microorganisms, with significant economic and social benefits.

[0045] The embodiments of the present invention have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments.

Claims

1. A method for preparing vitamin B2 by fermentation using corn steep liquor as a nitrogen source, characterized in that: Includes the following steps: S1. Quality evaluation and classification of corn steep liquor raw materials: The total nitrogen content, amino nitrogen content, soluble phosphorus content and glutamic acid content of corn steep liquor are determined. Based on the test results, corn steep liquor is classified into three grades: Grade A, Grade B and Grade C. S2. Preparation of fermentation medium: Using the corn steep liquor evaluated in step S1 as the main nitrogen source, prepare the fermentation medium. S3. Preparation of seed solution: Bacillus subtilis is inoculated into seed culture medium and cultured to obtain seed solution; S4. Fermentation culture: The seed liquid obtained in step S3 is inoculated into the fermentation medium prepared in step S2 for fermentation culture; during the fermentation culture, the feed medium formula is dynamically adjusted according to the corn steep liquor evaluation results in step S1, and the concentrations of amino nitrogen, lysed phosphorus, glutamic acid and total sugar in the fermentation broth are monitored in real time during the fermentation process, and feed is dynamically added according to the monitoring results. S5. Fermentation endpoint determination and product extraction: When the vitamin B2 content in the fermentation broth no longer increases significantly or the fermentation time reaches 60-80 hours, the broth is removed from the tank to obtain a fermentation mash rich in vitamin B2. After extraction and purification, vitamin B2 products are obtained.

2. The method for preparing vitamin B2 by fermentation using corn steep liquor as a nitrogen source according to claim 1, characterized in that: In step S1, the criteria for classifying corn steep liquor are as follows: Grade A: Total nitrogen ≥ 6.0%, amino nitrogen / total nitrogen ≥ 35%, soluble phosphorus ≥ 0.8%, glutamic acid ≥ 0.8%; Grade B: 5.0% ≤ Total Nitrogen < 6.0%, 30% ≤ Amino Nitrogen / Total Nitrogen < 35%, 0.5% ≤ Soluble Phosphorus < 0.8%, Glutamic Acid ≥ 0.5%; Grade C: Total nitrogen <5.0% or amino nitrogen / total nitrogen <30% or lysed phosphorus <0.5% or glutamate <0.5%.

3. The method for preparing vitamin B2 by fermentation using corn steep liquor as a nitrogen source according to claim 2, characterized in that: Grade A corn steep liquor is used directly for the preparation of fermentation culture medium; Grade B corn steep liquor is used after adding ammonium sulfate or urea to supplement total nitrogen and adding monosodium glutamate to adjust the amino acid composition; Grade C corn steep liquor is used after enzymatic hydrolysis pretreatment.

4. The method for preparing vitamin B2 by fermentation using corn steep liquor as a nitrogen source according to claim 3, characterized in that: The enzymatic pretreatment is as follows: adjust the pH of corn steep liquor to 6.5-7.0, add compound protease, and enzymatically hydrolyze at 50-55℃ for 2-4 hours. Use after enzyme inactivation. The amount of compound protease added is 0.05%-0.15% of the mass of corn steep liquor.

5. The method for preparing vitamin B2 by fermentation using corn steep liquor as a nitrogen source according to claim 1, characterized in that: In step S2, the fermentation medium contains, by weight-volume ratio: glucose 1.0%-3.0%, sucrose 2.0%-4.0%, corn steep liquor 2.0%-4.5%, ammonium sulfate 0.1%-0.5%, dipotassium hydrogen phosphate 0.1%-0.5%, magnesium sulfate 0.01%-0.15%, manganese sulfate 0.01%-0.05%, biotin 0.5-2.0 mg / L, glutamine 0.5-2.0 g / L, light calcium carbonate 0.1%-0.5%, and the remainder is water.

6. The method for preparing vitamin B2 by fermentation using corn steep liquor as a nitrogen source according to claim 1, characterized in that: Step S4, dynamically adjusting the feeding process specifically includes the following sub-steps: S41. Fermentation 0-12 hours: Measure the amino nitrogen concentration every 4 hours. When the amino nitrogen concentration is lower than 60% of the initial value, add Grade A corn steep liquor concentrate. S42, Fermentation for 12-36 hours: Measure the dissolved phosphorus concentration and glutamic acid concentration every 6 hours. When the dissolved phosphorus concentration is lower than 0.3 g / L, add potassium dihydrogen phosphate solution. When the glutamic acid concentration is lower than 0.2 g / L, add sodium glutamate solution to 0.2-0.5 g / L. S43. After 36 hours of fermentation: Measure the total sugar concentration every 8 hours. When the total sugar concentration drops below 2.0%, add feed medium to control the total sugar concentration to be maintained between 1.0% and 2.5%.

7. The method for preparing vitamin B2 by fermentation using corn steep liquor as a nitrogen source according to claim 6, characterized in that: The formulation of the feed medium is dynamically adjusted based on the evaluation results of corn steep liquor: the feed medium for Grade A corn steep liquor consists of 10%-20% sucrose, 20%-30% glucose, and 3%-6% corn steep liquor; the feed medium for Grade B corn steep liquor is supplemented with 0.2%-0.5% ammonium sulfate; and the feed medium for Grade C corn steep liquor uses enzymatically hydrolyzed corn steep liquor instead of ordinary corn steep liquor.

8. The method for preparing vitamin B2 by fermentation using corn steep liquor as a nitrogen source according to claim 1, characterized in that: In step S4, the fermentation culture temperature is 35-42℃, the aeration rate is 1:0.8-1.3v / v / min, the dissolved oxygen content is controlled above 25%-35%, and the pH is maintained at 6.6-7.

2.

9. The method for preparing vitamin B2 by fermentation using corn steep liquor as a nitrogen source according to claim 1, characterized in that: In step S4, the fermentation culture is subjected to step temperature regulation between 12 and 24 hours: maintaining the temperature at 37-39℃ for 12 hours, then raising the temperature to 40-42℃ and maintaining it until the fermentation is completed.

10. The method for preparing vitamin B2 by fermentation using corn steep liquor as a nitrogen source according to claim 1, characterized in that: In step S4, a pulse feeding strategy is adopted, with feeding once every 2 hours and each feeding amount being 0.3%-0.8% of the fermentation liquid volume.

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