Cytidine fermentation medium and fermentation method thereof

Abstract

The invention provides a cytidine fermentation culture method which comprises the following steps: step 1, activating a cytidine strain, inoculating the activated cytidine strain into a seed culture medium, and culturing for 8-16 hours at the temperature of 35-40 DEG C and the rotating speed of 200-700 rpm to prepare a fermentation seed solution for later use; and 2, transferring the fermentation seed solution into a fermentation tank filled with a cytidine fermentation culture medium according to the inoculation amount of 10-15% by volume percent, and culturing for 24-45 hours under the conditions that the temperature is 35-40 DEG C, the dissolved oxygen content is 15-30% and the pH is 6.7-7.2 to obtain the cytidine. According to the method, isopropyl-beta-D-thiogalactoside is added, inhibition on downstream gene transcription is relieved, expression of a target gene is started, corresponding protein can be synthesized into metabolic pathways, nutrient substances are utilized in other pathways, an intermediate fed-batch sugar control strategy is provided, the proper glucose concentration in each stage is guaranteed, and the yield is increased. The influence of too high or too low glucose concentration on cytidine metabolism of thalli is reduced, and the yield of cytidine is increased.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms and fermentation engineering, in particular to a cytidine fermentation medium and a fermentation method thereof. Background Art

[0002] The molecular formula of cytidine (cytidine nucleoside; cytidine, cytidine ribonucleoside, Cytosineβ-D-riboside; Cytosine-1-β-D-ribofuranoside) is C9H13N3O5, and the molecular weight is 243.22.

[0003] Cytidine is a pyrimidine nucleoside composed of cytosine and ribose. In organisms, cytidine plays an important role in the synthesis of nucleic acids (DNA and RNA). It can be phosphorylated through a series of biochemical reactions to form substances such as cytidine monophosphate (CMP), cytidine diphosphate (CDP) and cytidine triphosphate (CTP), and participate in a variety of biological processes.

[0004] Cytidine is an important pharmaceutical synthesis intermediate; the role of cytidine in the fields of nervous system diseases, tumor treatment, cardiovascular diseases and immune diseases is constantly being explored and excavated, which may bring new breakthroughs in the treatment of these diseases. Therefore, it is an important issue to develop a large-scale, industrialized, low-cost and efficient cytidine production method to meet the demand for cytidine raw materials in the pharmaceutical field.

[0005] There are three methods for cytidine:

[0006] 1. RNA hydrolysis method: cytidine is obtained by hydrolyzing RNA. However, this method has high requirements for raw materials, complex process, low product yield, and high production cost. It has been basically replaced by other methods.

[0007] 2. Chemical synthesis: For example, using bis-p-nitrophenol phosphate as a catalyst to catalyze the reaction of N4-acetylcytosine and tetra-O-acetyl-β-D-furanose, and then removing the acetyl group to generate cytidine. The development of chemical synthesis has significantly reduced the price of cytidine, but due to its inherent long reaction route, harsh reaction conditions, expensive catalysts, many by-products and potential environmental pollution, it is unlikely to further reduce production costs;

[0008] 3. Microbial fermentation method: This method includes the fermentation method with added precursors and the direct fermentation method. The fermentation method with added precursors utilizes the salvage pathway of cytidine synthesis, adds uracil to the culture medium, and makes it react with 5-phosphoribosyl pyrophosphate in the microbial cells under the catalysis of uracil ribose phosphotransferase to directly generate UMP, and then generates cytidine through UDP, UTP, and CTP. However, this method requires uracil as a fermentation precursor, which will cause problems of high raw material cost and low production efficiency. The direct fermentation method mainly utilizes the UMP of microorganisms to generate UMP "from scratch", and then generates cytidine through UDP, UTP, and CTP. Through microbial genetic breeding technology, high-yield cytidine fermentation strains can be selected, and glucose is used as the main raw material. Under mild conditions, high-quality cytidine can be produced through a relatively simple process. This method can further reduce the production cost of cytidine, and the production process is relatively environmentally friendly, with a large room for technical improvement. Summary of the invention

[0009] To solve the above problems, the present invention provides a cytidine fermentation medium and a fermentation method thereof. The fermentation method utilizes a biosafety bacterium-subtilis spore mutant strain, which knocks out or inhibits the cytidine deaminase gene (CytidineDeaminase), reduces the conversion of cytidine to uracil, and increases the accumulation of cytidine.

[0010] Bacillus subtilis has been evaluated and verified as a biosafety bacterium, and its antibacterial activity and environmental adaptability are relatively outstanding; and isopropyl-β-D-thiogalactoside (IPTG) is added to the formula. Isopropyl-β-D-thiogalactoside can bind to the repressor protein and dissociate it from the operon, thereby relieving the inhibition of downstream gene transcription, starting the expression of the target gene, and allowing the corresponding protein to be synthesized. Configure a suitable culture medium to eliminate the waste of raw materials in the glycolysis pathway, promote the metabolic intensity of the HMP pathway, and facilitate the efficient synthesis of metabolic target products; and provide an intermediate flow sugar addition control strategy to ensure the appropriate glucose concentration at each stage and reduce the impact of excessively high or low glucose concentration on the bacterial metabolism of cytidine.

[0011] To achieve the above-mentioned purpose, the present invention provides the following technical scheme: a cytidine fermentation medium, which comprises the following components in parts by weight: 30-40 parts of glucose, 5-20 parts of corn steep liquor powder, 1-4 parts of yeast extract powder, 2-6 parts of peptone, 10-30 parts of beet molasses, 0.2-0.7 parts of potassium dihydrogen phosphate, 1-3 parts of dipotassium hydrogen phosphate, 1-5 parts of ammonium sulfate, 0.4-0.8 parts of magnesium sulfate, 8-25 parts of green vitriol, 2-7 parts of manganese sulfate, and 2-4 parts of vitamin B11; the fermentation medium also comprises 0.8-1.2 parts of a defoamer; the content of biotin in the fermentation medium is 2 parts; the content of isopropyl-β-D-thiogalactoside in the fermentation medium is 80 parts.

[0012] A cytidine fermentation method, using the above-mentioned cytidine fermentation medium, comprises the following steps: step 1, activating the cytidine strain, inoculating the activated cytidine strain into a seed culture medium, culturing at a temperature of 35-40°C, a rotation speed of 200-700rpm, culturing for 8-16h, obtaining a fermentation seed liquid, and keeping it for use;

[0013] Furthermore, the seed culture medium described in step 1 comprises the following components: 30-40 parts of glucose, 5-20 parts of corn steep liquor powder, 1-4 parts of yeast extract powder, 2-6 parts of peptone, 10-30 parts of beet molasses, 0.2-0.7 parts of potassium dihydrogen phosphate, 1-3 parts of dipotassium hydrogen phosphate, 1-5 parts of ammonium sulfate, 0.4-0.8 parts of magnesium sulfate, 8-25 parts of green vitriol O, 2-7 parts of manganese sulfate, and 2-4 parts of vitamin B11;

[0014] Furthermore, the specific steps of activating the cytidine strain are: inoculating the cytidine strain onto an activated slant solid culture medium, and culturing at 35-40° C. for 15-24 hours; the slant solid culture medium comprises the following components by weight: 5 parts of yeast extract powder, 10 parts of tryptone, 20 parts of glucose, 20 parts of agar, pH 7.0-7.2;

[0015] Furthermore, the cytidine bacterial species is of the genus Bacillus subtilis.

[0016] Step 2, transferring the fermentation seed liquid to a fermentation tank filled with cytidine fermentation medium according to an inoculum amount of 10-15% by volume, culturing for 24-45 hours at a temperature of 35-40° C., a dissolved oxygen content of 15-30%, and a pH of 6.7-7.2 to obtain cytidine;

[0017] Furthermore, in step 2, the glucose concentration in the fermentation broth is controlled in stages in the fermenter, maintaining the glucose concentration at 0.8-1.2% in the first 15 hours and maintaining the glucose concentration at 0.3-0.8% from 15 hours to the next tank.

[0018] Compared with the existing known technologies, the technical solution provided by the present invention has the following significant effects:

[0019] (1) The microbial strain used in the present invention, Bacillus subtilis, has been evaluated and verified as a biosafety bacterium, and has outstanding antibacterial activity and environmental adaptability, and remains basically stable during production operation;

[0020] (2) The present invention adds isopropyl-β-D-thiogalactoside to the cytidine fermentation culture to release the inhibition of downstream gene transcription, start the expression of the target gene, and control the process to extend the time of metabolites and increase the yield of cytidine;

[0021] (3) Compared with the traditional chemical method and enzyme method, the present invention has simple equipment, small green pollution, conforms to the current low-carbon environment, low cost, and is suitable for large-scale;

[0022] (4) The method of the present invention can be used to ferment cytidine, which can reduce the metabolic flux to cytidine synthesis, thereby increasing the yield of cytidine to 86.4 parts, which is 33.4% higher than that of not using this method, and the fermentation cycle is shortened to 45 hours, thereby reducing costs.

[0023] (5) The present invention also provides a control strategy for adding sugar to the intermediate flow to ensure that the concentration of glucose is appropriate at each stage of operation, so as to reduce the impact of excessively high or low glucose concentration on the metabolism of cytidine by the bacteria. DETAILED DESCRIPTION

[0024] The technical solution of the present invention is further described in detail below in conjunction with specific embodiments.

[0025] The present invention will be further described below in conjunction with specific practical examples:

[0026] The content of cytidine in the fermentation product in the following implementation examples was determined using the following analytical method:

[0027] The chromatographic column is Agilent ZORBAXSB-C18 (150min×4.6mm, 3.5μm); the mobile phase is 2% acetonitrile; the flow rate is 1.0ml / min; the column temperature is 35℃, the detection wavelength is 270nm; the detection concentration is 0.01-0.05g / L, and the injection volume is 20μL. The standard of cytidine is accurately weighed and prepared into a standard solution with a mass concentration of 1g / L with deionized water; the pretreated fermentation broth sample to be tested is diluted with deionized water to an appropriate concentration and filtered with a 0.22μm microporous filter membrane as the fermentation sample solution to be tested, and the cytidine yield of the fermentation sample is calculated based on the peak area.

[0028] Embodiment 1:

[0029] (1) Using cytidine-producing bacteria Bacillus subtilis as the starting strain, the diluted Bacillus subtilis preserved bacterial solution is evenly applied to the activated slant solid culture medium and cultured at 37° C. for 18 hours to obtain the activated cytidine-producing bacteria Bacillus subtilis;

[0030] The activated slant solid culture medium includes: 5 g / L yeast extract powder, 10 g / L tryptone, 20 g / L glucose, and 20 g / L agar;

[0031] (2) scraping the activated cytidine-producing bacteria Bacillus subtilis obtained in step (1) with an inoculation loop and inoculating it into a seed culture medium at a temperature of 37° C., adjusting the rotation speed (200 to 700 rpm), tank pressure, and air volume to control the dissolved oxygen at 20 to 30%, and culturing for 15 hours to obtain a seed solution;

[0032] The seed culture medium includes: glucose 40g / L, corn steep liquor powder 20g / L, yeast extract powder 4g / L, peptone 8g / L, beet molasses 20ml / L, potassium dihydrogen phosphate (KH2PO4) 4g / L, dipotassium hydrogen phosphate (K2HPO4) 2g / L, ammonium sulfate ((NH4)2SO4) 2g / L, magnesium sulfate (MgSO4) 1g / L, green vitriol (FeSO4·7H2O) 12mg / L, manganese sulfate (MnSO4) 2mg / L, vitamin B1 12mg / L, defoamer 0.8mL / L;

[0033] (3) The seed liquid prepared in step (2) is transferred to a 50L fermentation tank filled with cytidine fermentation medium at an inoculum volume ratio of 15% (v / v), and fermented at a temperature of 37° C., a dissolved oxygen content of 15-30%, and a pH of 7.2 (adjusted by NH 3·H 2 O). During the fermentation process, the concentration of glucose in the fermentation liquid is controlled in stages according to the change of dissolved oxygen, and the glucose concentration is maintained at 0.8-1.2% in the first 15 hours, and the glucose concentration is maintained at 0.3-0.8% from 15 hours to the next tank. The fermentation cycle ends after 45 hours.

[0034] The cytidine fermentation medium includes: glucose 40g / L, corn steep liquor powder 20g / L, yeast extract powder 4g / L, peptone 8g / L, beet molasses 20ml / L, potassium dihydrogen phosphate (KH2PO4) 4g / L, dipotassium hydrogen phosphate (K2HPO4) 2g / L, ammonium sulfate ((NH4)2SO4) 2g / L, magnesium sulfate (MgSO4) 1g / L, green vitriol (FeSO4·7H2O) 12mg / L, manganese sulfate (MnSO4) 2mg / L, isopropyl-β-D-thiogalactoside 70mg / L, vitamin B1 12mg / L, defoamer 0.8mL / L,

[0035] According to HPLC detection, the cytidine yield in this example reached 73.42 g / L.

[0036] Embodiment 2:

[0037] (1) Using cytidine-producing bacteria Bacillus subtilis as the starting strain, the diluted Bacillus subtilis preserved bacterial solution is evenly applied to the activated slant solid culture medium and cultured at 37° C. for 18 hours to obtain the activated cytidine-producing bacteria Bacillus subtilis;

[0038] The activated slant solid culture medium includes: 5 g / L yeast extract powder, 10 g / L tryptone, 20 g / L glucose, and 20 g / L agar;

[0039] (2) scraping the activated cytidine-producing bacteria Bacillus subtilis obtained in step (1) with an inoculation loop and inoculating it into a seed culture medium at a temperature of 37° C., adjusting the rotation speed (200 to 700 rpm), tank pressure, and air volume to control the dissolved oxygen at 20 to 30%, and culturing for 15 hours to obtain a seed solution;

[0040] The seed culture medium includes: glucose 40g / L, corn steep liquor powder 20g / L, yeast extract powder 4g / L, peptone 8g / L, beet molasses 20ml / L, potassium dihydrogen phosphate (KH2PO4) 4g / L, dipotassium hydrogen phosphate (K2HPO4) 2g / L, ammonium sulfate ((NH4)2SO4) 2g / L, magnesium sulfate (MgSO4) 1g / L, green vitriol (FeSO4·7H2O) 12mg / L, manganese sulfate (MnSO4) 2mg / L., vitamin B1 12mg / L, defoamer 0.8mL / L;

[0041] (3) The seed liquid prepared in step (2) is transferred to a 50L fermentation tank filled with cytidine fermentation medium at an inoculum volume ratio of 15% (v / v), and fermented at a temperature of 37° C., a dissolved oxygen content of 15-30%, and a pH of 7.2 (adjusted by NH 3·H 2 O). During the fermentation process, the concentration of glucose in the fermentation liquid is controlled in stages according to the change of dissolved oxygen, and the glucose concentration is maintained at 0.8-1.2% in the first 15 hours, and the glucose concentration is maintained at 0.3-0.8% from 15 hours to the next tank. The fermentation cycle ends after 45 hours.

[0042] Among them, the cytidine fermentation medium includes: glucose 40g / L, corn steep liquor powder 20g / L, yeast extract powder 4g / L, peptone 8g / L, beet molasses 20ml / L, potassium dihydrogen phosphate (KH2PO4) 4g / L, dipotassium hydrogen phosphate (K2HPO4) 2g / L, ammonium sulfate ((NH4)2SO4) 2g / L, magnesium sulfate (MgSO4) 1g / L, green vitriol (FeSO4·7H2O) 12mg / L, manganese sulfate (MnSO4) 2mg / L, isopropyl-β-D-thiogalactoside 80mg / L, vitamin B1 12mg / L, and defoaming agent 0.8mL / L.

[0043] The difference between Example 2 and Example 1 is that the amount of isopropyl-β-D-thiogalactoside added is increased.

[0044] According to HPLC detection, the cytidine yield in this example reached 85.6 g / L.

[0045] Embodiment 3:

[0046] (1) Using cytidine-producing bacteria Bacillus subtilis as the starting strain, the diluted Bacillus subtilis preserved bacterial solution is evenly applied to the activated slant solid culture medium and cultured at 37° C. for 18 hours to obtain the activated cytidine-producing bacteria Bacillus subtilis;

[0047] The activated slant solid culture medium includes: 5 g / L yeast extract powder, 10 g / L tryptone, 20 g / L glucose, and 20 g / L agar;

[0048] (2) scraping the activated cytidine-producing bacteria Bacillus subtilis obtained in step (1) with an inoculation loop and inoculating it into a seed culture medium at a temperature of 37° C., adjusting the rotation speed (200 to 700 rpm), tank pressure, and air volume to control the dissolved oxygen at 20 to 30%, and culturing for 15 hours to obtain a seed solution;

[0049] The seed culture medium includes: glucose 40g / L, corn steep liquor powder 20g / L, yeast extract powder 4g / L, peptone 8g / L, beet molasses 20ml / L, potassium dihydrogen phosphate (KH2PO4) 4g / L, dipotassium hydrogen phosphate (K2HPO4) 2g / L, ammonium sulfate ((NH4)2SO4) 2g / L, magnesium sulfate (MgSO4) 1g / L, green vitriol (FeSO4·7H2O) 12mg / L, manganese sulfate (MnSO4) 2mg / L, vitamin B1 12mg / L, defoamer 0.8mL / L;

[0050] (3) The seed liquid prepared in step (2) is transferred to a 50L fermentation tank filled with cytidine fermentation medium at an inoculum volume ratio of 15% (v / v), and fermented at a temperature of 37° C., a dissolved oxygen content of 15-30%, and a pH of 7.2 (adjusted by NH 3·H 2 O). During the fermentation process, the concentration of glucose in the fermentation liquid is controlled in stages according to the change of dissolved oxygen, and the glucose concentration is maintained at 0.8-1.2% in the first 15 hours, and the glucose concentration is maintained at 0.3-0.8% from 15 hours to the next tank. The fermentation cycle ends after 45 hours.

[0051] Among them, the cytidine fermentation medium includes: glucose 40g / L, corn steep liquor powder 20g / L, yeast extract powder 4g / L, peptone 8g / L, beet molasses 20ml / L, potassium dihydrogen phosphate (KH2PO4) 4g / L, dipotassium hydrogen phosphate (K2HPO4) 2g / L, ammonium sulfate ((NH4)2SO4) 2g / L, magnesium sulfate (MgSO4) 1g / L, green vitriol (FeSO4·7H2O) 12mg / L, manganese sulfate (MnSO4) 2mg / L, isopropyl-β-D-thiogalactoside 90mg / L, vitamin B1 12mg / L, and defoaming agent 0.8mL / L.

[0052] The difference between Example 3 and Example 2 is that the amount of isopropyl-β-D-thiogalactoside added is increased.

[0053] According to HPLC detection, the cytidine yield in this example reached 78.24 g / L.

[0054] Comparative Example 1:

[0055] (1) Using cytidine-producing bacteria Bacillus subtilis as the starting strain, the diluted Bacillus subtilis preserved bacterial solution is evenly applied to the activated slant solid culture medium and cultured at 37° C. for 18 hours to obtain the activated cytidine-producing bacteria Bacillus subtilis;

[0056] The activated slant solid culture medium includes: 5 g / L yeast extract powder, 10 g / L tryptone, 20 g / L glucose, and 20 g / L agar;

[0057] (2) scraping the activated cytidine-producing bacteria Bacillus subtilis obtained in step (1) with an inoculation loop and inoculating it into a seed culture medium at a temperature of 37° C., adjusting the rotation speed (200 to 700 rpm), tank pressure, and air volume to control the dissolved oxygen at 20 to 30%, and culturing for 15 hours to obtain a seed solution;

[0058] The seed culture medium includes: glucose 40g / L, corn steep liquor powder 20g / L, yeast extract powder 4g / L, peptone 8g / L, beet molasses 20ml / L, potassium dihydrogen phosphate (KH2PO4) 4g / L, dipotassium hydrogen phosphate (K2HPO4) 2g / L, ammonium sulfate ((NH4)2SO4) 2g / L, magnesium sulfate (MgSO4) 1g / L, green vitriol (FeSO4·7H2O) 12mg / L, manganese sulfate (MnSO4) 2mg / L, vitamin B1 12mg / L, defoamer 0.8mL / L;

[0059] (3) The seed liquid prepared in step (2) is transferred to a 50L fermentation tank filled with cytidine fermentation medium at an inoculum volume ratio of 15% (v / v), and fermented at a temperature of 37° C., a dissolved oxygen content of 15-30%, and a pH of 7.2 (adjusted by NH 3·H 2 O). During the fermentation process, the concentration of glucose in the fermentation liquid is controlled in stages according to the change of dissolved oxygen, and the glucose concentration is maintained at 0.8-1.2% in the first 15 hours, and the glucose concentration is maintained at 0.3-0.8% in the next 15 hours. The fermentation cycle ends after 45 hours.

[0060] Among them, the cytidine fermentation medium includes: glucose 40g / L, corn steep liquor powder 20g / L, yeast extract powder 4g / L, peptone 8g / L, beet molasses 20ml / L, potassium dihydrogen phosphate (KH2PO4) 4g / L, dipotassium hydrogen phosphate (K2HPO4) 2g / L, ammonium sulfate ((NH4)2SO4) 2g / L, magnesium sulfate (MgSO4) 1g / L, green vitriol (FeSO4·7H2O) 12mg / L, manganese sulfate (MnSO4) 2mg / L, isopropyl-β-D-thiogalactoside 80mg / L, vitamin B1 12mg / L, and defoaming agent 0.8mL / L.

[0061] The difference between Comparative Example 1 and Example 1 is that during the seed liquid fermentation process, the concentration of glucose in the fermentation liquid was not controlled in stages.

[0062] According to HPLC detection, the cytidine yield in this example reached 69.43 g / L.

[0063] Table 1 Cytidine production in different groups

[0064]

[0065] As can be seen from Table 1, the different amounts of isopropyl-β-D-thiogalactoside added have an impact on the accumulation of cytidine. Adding 80 mg / L of isopropyl-β-D-thiogalactoside to the fermentation medium (Example 2) can increase the yield of cytidine, which indicates that the addition of isopropyl-β-D-thiogalactoside relieves the inhibition of downstream gene transcription, activates the expression of the target gene, and enables the corresponding protein to synthesize the problem of the utilization of nutrients by other pathways in the metabolic pathway.

[0066] The present invention also provides an intermediate flow sugar addition control strategy to ensure appropriate glucose concentration in each stage, reduce the effect of excessively high or low glucose concentration on bacterial metabolism of cytidine, and increase the yield of cytidine.

[0067] It can be seen from Example 1 and Comparative Example 1 that the glucose concentration is maintained at 0.8% to 1.2% during the first 15 hours of the fermentation process, and the glucose concentration is maintained at 0.3% to 0.8% after 15 hours until the next tank, which can significantly increase the yield of cytidine.

[0068] Finally, in Example 2, 80 mg / L of isopropyl-β-D-thiogalactoside was added to the fermentation medium, and the glucose concentration was maintained at 0.8% to 1.2% in the first 15 h of the fermentation process, and the glucose concentration was maintained at 0.3% to 0.8% after 15 h until the next tank. Under the conditions, the ability of the cytidine-producing bacteria Bacillus subtilis to produce cytidine can be better maintained, and the cytidine yield can reach a higher level.

[0069] The above verification shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are only for illustrating the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various improvements and adjustments, which are all within the scope of protection of the present invention. The scope of protection required by the present invention is defined by the attached claims and their equivalents.

Claims

1. A cytidine fermentation medium, characterized in that: The fermentation medium comprises the following components in parts by weight: 30-40 parts of glucose, 5-20 parts of corn steep liquor powder, 1-4 parts of yeast extract powder, 2-6 parts of peptone, 10-30 parts of beet molasses, 0.2-0.7 parts of potassium dihydrogen phosphate, 1-3 parts of dipotassium hydrogen phosphate, 1-5 parts of ammonium sulfate, 0.4-0.8 parts of magnesium sulfate, 8-25 parts of green vitriol, 2-7 parts of manganese sulfate, and 2-4 parts of vitamin B11.

2. A cytidine fermentation medium according to claim 1, characterized in that: The fermentation medium also includes 0.8 to 1.2 parts of a defoaming agent.

3. A cytidine fermentation medium according to claim 2, characterized in that: The content of biotin in the fermentation medium is 2 parts.

4. A cytidine fermentation medium according to claim 3, characterized in that: The content of isopropyl-β-D-thiogalactoside in the fermentation medium is 80 parts.

5. A cytidine fermentation method, using the cytidine fermentation medium according to any one of claims 1 to 4, characterized in that: The following steps are involved: Step 1: activating the cytidine strain, inoculating the activated cytidine strain into a seed culture medium, culturing at a temperature of 35 to 40° C., a rotation speed of 200 to 700 rpm, and culturing for 8 to 16 hours to obtain a fermentation seed liquid, which is reserved for later use; Step 2: Transfer the fermentation seed liquid to a fermentation tank filled with cytidine fermentation medium at an inoculation rate of 10-15% by volume, and culture for 24-45 hours at a temperature of 35-40° C., a dissolved oxygen content of 15-30%, and a pH of 6.7-7.2 to obtain cytidine.

6. A cytidine fermentation method according to claim 5, characterized in that: The specific steps of activating the cytidine strain are: inoculating the cytidine strain onto an activated slant solid culture medium, and culturing at 35 to 40° C. for 15 to 24 hours; The slant solid culture medium comprises the following components by weight: 5 parts of yeast extract powder, 10 parts of tryptone, 20 parts of glucose, 20 parts of agar, and pH 7.0-7.

2.

7. A cytidine fermentation method according to claim 5, characterized in that: The cytidine bacterial species is of the genus Bacillus subtilis.

8. A cytidine fermentation method according to claim 5, characterized in that: The seed culture medium described in step one comprises the following components: 30-40 parts of glucose, 5-20 parts of corn steep liquor powder, 1-4 parts of yeast extract powder, 2-6 parts of peptone, 10-30 parts of beet molasses, 0.2-0.7 parts of potassium dihydrogen phosphate, 1-3 parts of dipotassium hydrogen phosphate, 1-5 parts of ammonium sulfate, 0.4-0.8 parts of magnesium sulfate, 8-25 parts of green vitriol O, 2-7 parts of manganese sulfate, and 2-4 parts of vitamin B11.

9. A cytidine fermentation method according to claim 8, characterized in that: The seed culture medium also includes 0.8 to 1.2 parts of a defoaming agent.

10. A cytidine fermentation method according to claim 5, characterized in that: In step 2, the glucose concentration in the fermentation liquid is controlled in stages in the fermenter, and the glucose concentration is maintained at 0.8-1.2% in the first 15 hours, and the glucose concentration is maintained at 0.3-0.8% from 15 hours to the next tank.

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