Method for producing cytidine through fermentation
By adding IPTG inducer and glucose in three times during the cytidine fermentation process, the fermentation conditions are optimized, and the problems of high costs and difficult to control strain mutagenesis in the existing technology are solved, and efficient and low-cost cytidine production is achieved. .
Patent Information
- Application Number
- CN202411560766.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-04
- Publication Date
- 2024-12-13
AI Technical Summary
In the existing cytidine fermentation production methods, the addition of precursor substances increases the cost, and the mutagenesis and breeding of strains is difficult to control, resulting in high production costs and is not suitable for industrial production.
During the fermentation process, the IPTG inducer was added in three times, and the amount was gradually reduced, and glucose was added at the same time, the dissolved oxygen amount was controlled, and the fermentation conditions were optimized to improve the sugar-acid conversion rate and bacterial density, and reduce the fermentation cost.
It improves the yield, yield and purity of cytidine finished products, reduces the consumption of fermentation raw materials, reduces production costs, and is suitable for industrial production.
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Figure SMS_1
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of fermentation engineering, and in particular relates to a method for producing cytidine by fermentation. Background Art
[0002] Cytosine nucleoside, abbreviated as cytidine, is a precursor for the synthesis of ribonucleic acid and deoxyribonucleic acid, and is involved in many reactions in life. At the same time, cytidine is also an important pharmaceutical synthesis intermediate, which can be used to synthesize antiviral and anti-tumor drugs such as zalcitabine, capecitabine, cyclocytidine, and cytarabine. Therefore, in order to meet the demand for cytidine raw materials in the field of pharmaceutical production, it is critical to develop a batch, industrialized, low-cost and efficient cytidine production method.
[0003] At present, most methods for producing cytidine use fermentation. In recent years, the technology of producing cytidine by fermentation has also made progress. For example, patent CN202410055604.X uses Escherichia coli as a production strain and glucose as a carbon source to ferment cytidine under certain fermentation conditions. During the fermentation process, the production of cytidine is improved by adding precursor substances and inducing twice. Although the production of cytidine is improved by this technical improvement, the addition of precursor substances increases the cost input of fermentation materials, and the production cost is high, which is not conducive to industrial production; Patent CN202010188550.6 obtains a mutant strain of Escherichia coli with high cytidine production through mutagenesis screening. After a series of fermentation production, the final production of cytidine is also improved, but due to the problems of difficult control of mutation direction, possible decline of strain vitality and excessive cost of strain cultivation in strain mutagenesis breeding, it is not suitable for industrial production of cytidine. Summary of the invention
[0004] In order to solve the deficiencies of the prior art, the present invention provides a method for producing cytidine by fermentation, which effectively improves the sugar-acid conversion rate of the fermentation broth by adding an IPTG inducer three times during the fermentation process, and the amount of the added IPTG inducer decreases gradually, thereby improving the output, yield and purity of the finished cytidine. At the same time, the consumption of fermentation raw materials is reduced, the fermentation cost is reduced, and it is more conducive to the industrial production of cytidine.
[0005] The technical effects to be achieved by the present invention are achieved through the following technical aspects:
[0006] The present invention provides a method for producing cytidine by fermentation, comprising the following steps:
[0007] S1, inoculating the cytidine-producing bacteria into the eggplant bottle culture medium for activation culture;
[0008] S2, inoculating the activated cytidine-producing strain into sterile water for shake flask culture to obtain a primary seed solution;
[0009] S3, inoculating the primary seed solution into the secondary seed culture medium for expansion, to obtain the secondary seed solution;
[0010] S4, inoculating the secondary seed liquid into the fermentation medium for fermentation, and adding IPTG inducer three times. When the expected optical density is 20-25 before fermentation, the IPTG inducer accounting for 45-55% of the total amount of IPTG inducer is added for the first time; when the fermentation is 15-20 hours, the IPTG inducer accounting for 25-35% of the total amount of IPTG inducer is added for the second time; when the fermentation is 30-36 hours, the IPTG inducer accounting for 15-25% of the total amount of IPTG inducer is added for the third time; at the same time, when the amount of reducing sugar in the fermentation liquid is 0.3-0.5%, glucose is added, and the dissolved oxygen content is controlled at 10-30% during the sugar addition process until the addition is completed, and the fermentation is terminated when the pH of the fermentation liquid rises;
[0011] S5. The fermentation broth is sterilized by heating, filtered, concentrated, cooled and crystallized to obtain a finished cytidine product.
[0012] As a further description of the technical solution of the present invention, in S4, the duration of the three additions of IPTG inducer is 25-35 min, 15-25 min and 5-15 min respectively, and the total addition time is 0.8-1.2 h.
[0013] As a further description of the technical solution of the present invention, in S4, the IPTG inducer added is an IPTG aqueous solution, the volume of the IPTG aqueous solution is 0.5-0.8% of the volume of the fermentation liquid before fermentation, and the concentration is 0.6-1.0%;
[0014] The added glucose is a glucose solution, the volume of which is 70-80% of the volume of the fermentation liquid before fermentation and the concentration is 55-65%.
[0015] As a further description of the technical solution of the present invention, in S4, the inoculation amount of the secondary seed liquid is 8-12%, the fermentation temperature is 36±1° C., and the fermentation time is 40-50 h.
[0016] As a further description of the technical solution of the present invention, the fermentation medium includes the following components by mass percentage: 2.5-3.5% glucose, 0.3-0.5% magnesium sulfate, 0.001-0.002% manganese sulfate, 0.001-0.002% ferrous sulfate, 0.8-1.2% yeast extract, 0.4-0.8% corn syrup, 0.2-0.4% citric acid, 0.01-0.05% defoaming agent, and the balance is water.
[0017] As a further description of the technical solution of the present invention, the cytidine-producing bacteria is Escherichia coli W3110 (ATCC27325).
[0018] As a further description of the technical solution of the present invention, in S1, the activation culture temperature is 36±1°C, and the culture time is 12 to 16 hours;
[0019] In S2, the temperature of the shake flask culture is 36±1°C and the culture time is 12 to 16 hours;
[0020] In the S3, the inoculation amount of the primary seed solution is 0.02-0.04%, the expansion temperature is 36±1° C., and the culture time is 16-20 h.
[0021] As a further description of the technical solution of the present invention, the eggplant bottle culture medium includes the following components by mass percentage: 2-5% beef extract, 0.3-0.6% peptone, 0.8-1.2% yeast powder, 2.5-3.5% agar, and the balance is water.
[0022] As a further description of the technical solution of the present invention, the secondary seed culture medium includes the following components by mass percentage: 2.0-4.0% glucose, 0.8-1.2% yeast extract, 0.2-0.5% potassium dihydrogen phosphate, 0.2-0.5% defoaming agent, 0.8-1.2% magnesium sulfate, 0.001-0.003% ferrous sulfate, 0.001-0.003% cobalt chloride, and the balance is water.
[0023] As a further description of the technical solution of the present invention, S5 specifically includes the following steps:
[0024] The fermentation broth after fermentation is sterilized by heating, and the pH of the fermentation broth is adjusted to 4.0±1, and filtered by an inorganic membrane. The filtrate is eluted by a cation exchange resin, concentrated, cooled and crystallized to obtain a cytidine product.
[0025] In summary, the present invention has at least the following benefits:
[0026] The method for producing cytidine by fermentation provided by the present invention reasonably controls the addition point of IPTG inducer during the fermentation process, supplements the IPTG inducer three times and controls the supplement amount to decrease successively, which not only makes up for the disadvantage of insufficient dissolved oxygen in the early stage of fermentation, but also increases the density of the thalli in the high-yield period of fermentation. At the same time, by additionally supplementing sugar, it is ensured that sufficient energy and nutrients are provided for high-concentration thalli, promotes the efficient synthesis of cytidine by the thalli, and effectively improves the output and yield of the finished product. By adopting the above-mentioned IPTG inducer supplementation and sugar supplementation mode, the sugar-acid conversion rate of the fermentation liquid is also correspondingly improved, thereby improving the yield and purity of the cytidine finished product, and at the same time, reducing the consumption of fermentation raw materials, reducing the fermentation production cost, and being more conducive to the industrial production of cytidine. DETAILED DESCRIPTION
[0027] For ease of understanding of the present invention, the present invention will be described more fully below in conjunction with specific embodiments. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present invention more thoroughly understood.
[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.
[0029] The present invention provides a method for producing cytidine by fermentation, comprising the following steps:
[0030] S1, inoculating the cytidine-producing bacteria into the eggplant bottle culture medium for activation culture;
[0031] S2, inoculating the activated cytidine-producing strain into sterile water for shake flask culture to obtain a primary seed solution;
[0032] S3, inoculating the primary seed solution into the secondary seed culture medium for expansion, to obtain the secondary seed solution;
[0033] S4, inoculating the secondary seed liquid into the fermentation medium for fermentation, and supplementing the IPTG inducer three times. When the expected optical density is 20-25 before fermentation, the IPTG inducer accounting for 45-55% of the total amount of the IPTG inducer is supplemented for the first time, preferably 50%; when the fermentation is allowed to proceed for 15-20 hours, the IPTG inducer accounting for 25-35% of the total amount of the IPTG inducer is supplemented for the second time, preferably 30%; when the fermentation is allowed to proceed for 30-36 hours, the IPTG inducer accounting for 15-25% of the total amount of the IPTG inducer is supplemented for the third time, preferably 20%; at the same time, when the amount of reducing sugar in the fermentation liquid is 0.3-0.5%, glucose is supplemented, and the dissolved oxygen content is controlled at 10-30% during the sugar supplement process until the supplement is completed, and the fermentation is terminated when the pH of the fermented liquid rises;
[0034] S5. The fermentation broth is sterilized by heating, filtered, concentrated, cooled and crystallized to obtain a finished cytidine product.
[0035] Adding IPTG inducer during the fermentation process can induce the activity of β-galactosidase in the bacteria, effectively improving the efficiency of cytidine synthesis in the bacteria. IPTG inducer will not be metabolized by the bacteria, can produce a long-lasting and stable induction effect, and only a small amount is needed to achieve the ideal induction effect. Therefore, the investment cost of IPTG inducer is low, which can ensure the cytidine yield while controlling the production cost.
[0036] By reasonably controlling the IPTG inducer addition point, adding IPTG inducer three times and controlling the amount of addition to decrease gradually, it not only makes up for the disadvantage of insufficient dissolved oxygen in the early stage of fermentation, but also increases the density of the bacteria in the high-yield period of fermentation. At the same time, combined with additional sugar supplementation, it ensures that sufficient energy and nutrients are provided for high-concentration bacteria, promotes the efficient synthesis of cytidine by bacteria, and effectively improves the output and yield of cytidine finished products. Through the combination of the above-mentioned IPTG inducer supplementation mode and sugar supplementation mode, the sugar-acid conversion rate of the fermentation broth is also correspondingly improved, thereby improving the yield and purity of the cytidine finished product. At the same time, it reduces the consumption of fermentation raw materials, effectively reduces the fermentation production cost, and is conducive to the industrial production of cytidine.
[0037] In some embodiments, glucose can be supplemented by linkage feedback pulse, which is a new feeding technology. By reasonably controlling the opening and closing time of the feeder, the pressure waves at the material end and the feeding end are synchronized, thereby achieving accurate feeding. Compared with the traditional feeding method, it has higher accuracy and lower waste rate, which is more conducive to improving production quality and controlling production costs.
[0038] As a further optimization, in S4, the duration of the three times of replenishing the IPTG inducer is 25-35min, 15-25min and 5-15min, respectively, and the total replenishment time is 0.8-1.2h. Preferably, the duration of the three times of replenishing the IPTG inducer is 30min, 20min and 10min, respectively, and the total replenishment time is 1h. By controlling the IPTG inducer replenishment time to decrease successively, it is possible to better adapt to the mode of decreasing the amount of IPTG inducer replenishment, maintain the stability of the IPTG inducer replenishment rate, thereby stably controlling the dissolved oxygen content and bacterial density of the fermentation broth, making the fermentation production of the bacterial body more stable and efficient, and helping to improve the yield and conversion rate of the fermentation broth, thereby improving the product yield.
[0039] In S4, the IPTG inducer added is an IPTG aqueous solution, the volume of the IPTG aqueous solution is 0.5-0.8% of the volume of the fermentation liquid before fermentation, and the concentration is 0.6-1.0%. Preferably, the volume of the IPTG aqueous solution is 0.67% of the volume of the fermentation liquid before fermentation, and the concentration is 0.8%. The glucose added is a glucose solution, the volume of the glucose solution is 70-80% of the volume of the fermentation liquid before fermentation, and the concentration is 55-65%. Preferably, the volume of the glucose solution is 75% of the volume of the fermentation liquid before fermentation, and the concentration is 60%.
[0040] In some embodiments, in S4, the inoculation amount of the secondary seed solution is 8-12%, preferably 10%, the fermentation temperature is 36±1° C., and the fermentation time is 40-50 h.
[0041] The fermentation medium comprises the following components by mass percentage: 2.5-3.5% glucose, 0.3-0.5% magnesium sulfate, 0.001-0.002% manganese sulfate, 0.001-0.002% ferrous sulfate, 0.8-1.2% yeast extract, 0.4-0.8% corn syrup, 0.2-0.4% citric acid, 0.01-0.05% defoamer, and the balance is water. Preferably, 3.0% glucose, 0.4% magnesium sulfate, 0.001% manganese sulfate, 0.001% ferrous sulfate, 1.0% yeast extract, 0.6% corn syrup, 0.3% citric acid, 0.03% defoamer, and the balance is water.
[0042] In some embodiments, the cytidine-producing bacterial species is Escherichia coli W3110 (ATCC 27325).
[0043] In some embodiments, in S1, the temperature of activation culture is 36±1°C, and the culture time is 12 to 16 hours, preferably 14 hours. In S2, the temperature of shake flask culture is 36±1°C, and the culture time is 12 to 16 hours, preferably 14 hours. In S3, the inoculation amount of the primary seed solution is 0.02 to 0.04%, preferably 0.03%, the temperature of expansion culture is 36±1°C, and the culture time is 16 to 20 hours, preferably 18 hours.
[0044] The eggplant bottle culture medium comprises the following components by mass percentage: 2-5% beef extract, 0.3-0.6% peptone, 0.8-1.2% yeast powder, 2.5-3.5% agar, and the balance is water. Preferably, 3% beef extract, 0.5% peptone, 1.0% yeast powder, 3.0% agar, and the balance is water.
[0045] The secondary seed culture medium comprises the following components by mass percentage: 2.0-4.0% glucose, 0.8-1.2% yeast extract, 0.2-0.5% potassium dihydrogen phosphate, 0.2-0.5% defoamer, 0.8-1.2% magnesium sulfate, 0.001-0.003% ferrous sulfate, 0.001-0.003% cobalt chloride, and the balance is water. Preferably, 3.0% glucose, 1.0% yeast extract, 0.4% potassium dihydrogen phosphate, 0.3% defoamer, 1.0% magnesium sulfate, 0.002% ferrous sulfate, 0.002% cobalt chloride, and the balance is water
[0046] In some embodiments, S5 specifically includes the following steps: heating and sterilizing the fermented broth, adjusting the pH of the fermented broth to 4.0±1, filtering with an inorganic membrane, and concentrating the filtrate after elution with a cation exchange resin, cooling and crystallizing to obtain a cytidine product. Filtration with an inorganic membrane has a good filtration effect and can reduce impurities in the filtrate. Then, elution with a cation exchange resin can further remove small molecular impurities in the filtrate, so that the prepared cytidine product has a higher purity.
[0047] Example 1
[0048] This embodiment provides a cytidine, which is produced by the following method steps:
[0049] Preliminary preparation:
[0050] Preparation of eggplant bottle medium: Add 30g beef extract, 5g peptone, 10g yeast powder, 30g agar into a sterile beaker, add water to 1L, and adjust the pH to 7.4;
[0051] Preparation of secondary seed culture medium: Add 30g glucose, 10g yeast extract, 4g potassium dihydrogen phosphate, 3g defoaming agent, 10g magnesium sulfate, 0.02g ferrous sulfate, and 0.02g cobalt chloride into a sterile beaker, and add water to make up to 1L.
[0052] Preparation of fermentation medium: Add 900 g of glucose, 120 g of magnesium sulfate, 0.3 g of manganese sulfate, 0.3 g of ferrous sulfate, 300 g of yeast extract, 180 g of corn syrup, 90 g of citric acid, and 9 g of defoaming agent into a sterile container, and add water to make up to 30 L.
[0053] Preparation of IPTG aqueous solution: Add 1.6 g IPTG to a sterile beaker and add water to make up to 200 mL.
[0054] Preparation of glucose solution: Add 13.5 kg of glucose into a sterile container and add water to make up to 22.5 L.
[0055] S1. Escherichia coli W3110 (ATCC27325) was inoculated into the eggplant bottle medium for activation culture at 36°C for 14 hours;
[0056] S2, pick a ring of mature bacteria from the eggplant bottle culture medium and inoculate it into a triangular flask filled with sterile water for shake flask culture at 36°C for 14 hours to obtain the first-level seed solution;
[0057] S3, inoculate the first-level seed liquid into the second-level seed culture medium at a volume ratio of 0.03% for expansion, the culture temperature is 36°C, pH is 6.9-7.1, the tank pressure is 0.03-0.05MPa, the dissolved oxygen is 15-50%, and the air volume is 30m 3 / h, rotation frequency 25Hz, culture time 18h, and obtain secondary seed solution;
[0058] S4. Add 30L fermentation medium into a 50L fermentation tank, inoculate the secondary seed liquid into the fermentation medium at a volume ratio of 10% for fermentation, the fermentation temperature is 36-36.5℃, pH is 7.0-7.2, the tank pressure is 0.03-0.05MPa, the dissolved oxygen is 15-50%, and the air volume is 500-600m3 / h, rotation frequency 50Hz, monitor dissolved oxygen, reducing sugar content, pH, optical density, fermentation liquid content and other parameters during the fermentation process. In the early stage of fermentation, when the optical density is 20, add 100mL of IPTG aqueous solution for 30min; when the fermentation is allowed to ferment for 18h, add 60mL of IPTG aqueous solution for 20min; when the fermentation is allowed to ferment for 34h, add 40mL of IPTG aqueous solution for 10min; at the same time, by monitoring the reducing sugar content in the fermentation liquid system, start to flow glucose solution when the reducing sugar content is 0.3%, control the dissolved oxygen content between 10% and 30% during the sugar addition process, until it is completed, and terminate the fermentation when the pH of the fermentation liquid rises;
[0059] S5. The fermentation broth was heated and sterilized, and the cytidine content in the fermentation broth was measured to be 78.05‰ and the cytidine purity was 88.39%. The pH of the fermentation broth was adjusted to 4.0±1, and the filtrate was filtered using an inorganic membrane. The filtrate was eluted with a cation exchange resin, concentrated, cooled and crystallized, and 1538.65 g of cytidine finished product was obtained.
[0060] Example 2
[0061] This embodiment provides a cytidine, which is produced by the following method steps:
[0062] Preliminary preparation: The preparation of the eggplant bottle medium, secondary seed medium, fermentation medium, IPTG aqueous solution and glucose solution is the same as that in Example 1.
[0063] S1. Escherichia coli W3110 (ATCC27325) was inoculated into the eggplant bottle medium for activation culture at 36°C for 14 hours;
[0064] S2, pick a ring of mature bacteria from the eggplant bottle culture medium and inoculate it into a triangular flask filled with sterile water for shake flask culture at 36°C for 14 hours to obtain the first-level seed solution;
[0065] S3, inoculate the first-level seed liquid into the second-level seed culture medium at a volume ratio of 0.03% for expansion, the culture temperature is 36°C, pH is 6.9-7.1, the tank pressure is 0.03-0.05MPa, the dissolved oxygen is 15-50%, and the air volume is 30m 3 / h, rotation frequency 25Hz, culture time 18h, and obtain secondary seed solution;
[0066] S4. Add 30L fermentation medium into a 50L fermentation tank, inoculate the secondary seed liquid into the fermentation medium at a volume ratio of 10% for fermentation, the fermentation temperature is 36-36.5℃, pH is 7.0-7.2, the tank pressure is 0.03-0.05MPa, the dissolved oxygen is 15-50%, and the air volume is 500-600m3 / h, rotation frequency 50Hz, monitor dissolved oxygen, reducing sugar content, pH, optical density, fermentation liquid content and other parameters during the fermentation process. In the early stage of fermentation, when the optical density is 25, add 100mL of IPTG aqueous solution for 30min; when the fermentation is allowed to ferment for 15h, add 60mL of IPTG aqueous solution for 20min; when the fermentation is allowed to ferment for 30h, add 40mL of IPTG aqueous solution for 10min; at the same time, by monitoring the reducing sugar content in the fermentation liquid system, start to add glucose solution when the reducing sugar content is 0.5%, control the dissolved oxygen content between 10% and 30% during the sugar addition process, until it is completed, and terminate the fermentation when the pH of the fermentation liquid rises;
[0067] S5. The fermentation broth was heated and sterilized, and the cytidine content in the fermentation broth was measured to be 80.10‰ and the cytidine purity was 89.60%. The pH of the fermentation broth was adjusted to 4.0±1, and the filtrate was filtered using an inorganic membrane. The filtrate was eluted with a cation exchange resin, concentrated, cooled and crystallized, and 1633.00 g of cytidine product was obtained.
[0068] Example 3
[0069] This embodiment provides a cytidine, which is produced by the following method steps:
[0070] Preliminary preparation: The preparation of the eggplant bottle medium, secondary seed medium, fermentation medium, IPTG aqueous solution and glucose solution is the same as that in Example 1.
[0071] S1. Escherichia coli W3110 (ATCC27325) was inoculated into the eggplant bottle medium for activation culture at 36°C for 14 hours;
[0072] S2, pick a ring of mature bacteria from the eggplant bottle culture medium and inoculate it into a triangular flask filled with sterile water for shake flask culture at 36°C for 14 hours to obtain the first-level seed solution;
[0073] S3, inoculate the first-level seed liquid into the second-level seed culture medium at a volume ratio of 0.03% for expansion, the culture temperature is 36°C, pH is 6.9-7.1, the tank pressure is 0.03-0.05MPa, the dissolved oxygen is 15-50%, and the air volume is 30m 3 / h, rotation frequency 25Hz, culture time 18h, and obtain secondary seed solution;
[0074] S4. Add 30L fermentation medium into a 50L fermentation tank, inoculate the secondary seed liquid into the fermentation medium at a volume ratio of 10% for fermentation, the fermentation temperature is 36-36.5℃, pH is 7.0-7.2, the tank pressure is 0.03-0.05MPa, the dissolved oxygen is 15-50%, and the air volume is 500-600m3 / h, rotation frequency 50Hz, monitor dissolved oxygen, reducing sugar content, pH, optical density, fermentation liquid content and other parameters during the fermentation process. In the early stage of fermentation, when the optical density is 22, add 100mL of IPTG aqueous solution for 30min; when the fermentation is allowed to ferment for 20h, add 60mL of IPTG aqueous solution for 20min; when the fermentation is allowed to ferment for 36h, add 40mL of IPTG aqueous solution for 10min; at the same time, by monitoring the reducing sugar content in the fermentation liquid system, start to flow glucose solution when the reducing sugar content is 0.5%, control the dissolved oxygen content between 10% and 30% during the sugar addition process, until it is completed, and terminate the fermentation when the pH of the fermentation liquid rises;
[0075] S5. The fermentation broth was heated and sterilized, and the cytidine content in the fermentation broth was measured to be 79.34‰ and the cytidine purity was 89.29%. The pH of the fermentation broth was adjusted to 4.0±1, and the filtrate was filtered using an inorganic membrane. The filtrate was eluted with a cation exchange resin, concentrated, cooled and crystallized, and 1593.43 g of cytidine finished product was obtained.
[0076] Comparative Example 1
[0077] This comparative example provides a cytidine, which is produced by the following method steps:
[0078] Preliminary preparation: The preparation of the eggplant bottle medium, secondary seed medium, fermentation medium, IPTG aqueous solution and glucose solution is the same as that in Example 1.
[0079] S1. Escherichia coli W3110 (ATCC27325) was inoculated into the eggplant bottle medium for activation culture at 36°C for 14 hours;
[0080] S2, pick a ring of mature bacteria from the eggplant bottle culture medium and inoculate it into a triangular flask filled with sterile water for shake flask culture at 36°C for 14 hours to obtain the first-level seed solution;
[0081] S3, inoculate the first-level seed liquid into the second-level seed culture medium at a volume ratio of 0.03% for expansion, the culture temperature is 36°C, pH is 6.9-7.1, the tank pressure is 0.03-0.05MPa, the dissolved oxygen is 15-50%, and the air volume is 30m 3 / h, rotation frequency 25Hz, culture time 18h, and obtain secondary seed solution;
[0082] S4. Add 30L fermentation medium into a 50L fermentation tank, inoculate the secondary seed liquid into the fermentation medium at a volume ratio of 10% for fermentation, the fermentation temperature is 36-36.5℃, pH is 7.0-7.2, the tank pressure is 0.03-0.05MPa, the dissolved oxygen is 15-50%, and the air volume is 500-600m3 / h, rotation frequency 50Hz, monitor dissolved oxygen, reducing sugar content, pH, optical density, fermentation liquid content and other parameters during the fermentation process, when the optical density in the fermentation system is 20, add 200mL of IPTG aqueous solution at a time, and start to add glucose solution when the reducing sugar content is 0.3%. During the sugar addition process, control the dissolved oxygen content between 10% and 30% until it is completed, and terminate the fermentation when the pH of the fermentation liquid rises;
[0083] S5. The fermentation broth was heated and sterilized, and the cytidine content in the fermentation broth was measured to be 70.00‰ and the cytidine purity was 84.25%. The pH of the fermentation broth was adjusted to 4.0±1, and the filtrate was filtered using an inorganic membrane. The filtrate was eluted with a cation exchange resin, concentrated, cooled and crystallized, and 1277.39 g of cytidine finished product was obtained.
[0084] Test Example 1
[0085] In this test example, the sugar-acid conversion rate of the fermented broth in Examples 1-3 and Comparative Example 1 was calculated. At the same time, the purity of the cytidine product was determined and the yield was calculated. The sugar-acid conversion rate, purity and yield of the cytidine product are shown in Table 1.
[0086] Table 1 Sugar acid conversion rate, purity and yield of cytidine finished product in Examples 1 to 3 and Comparative Example 1
[0087]
[0088] By comparing the data of Examples 1 to 3 and Comparative Example 1 in Table 1, it can be seen that the sugar-acid conversion rate, the purity of the cytidine finished product and the yield of the cytidine finished product of Examples 1 to 3 are all higher than those of Comparative Example 1, which proves that Examples 1 to 3 can maximize the efficiency of cytidine synthesis by the bacterial cell by reasonably controlling the replenishment node, replenishment amount and replenishment time of the IPTG inducer, and the sugar-acid conversion rate of the fermentation broth and the yield of the cytidine finished product are significantly improved, and the purity of the cytidine finished product is also improved accordingly. At the same time, the improvement of the sugar-acid conversion rate of the fermentation broth also improves the utilization rate of the fermentation raw materials, thereby reducing the consumption of the fermentation raw materials, reducing the fermentation production cost, and being more conducive to industrial production.
[0089] The above contents are merely examples and illustrations of the structure of the present invention, and the description is relatively specific and detailed, but it cannot be understood as limiting the scope of the present invention. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, and these obvious replacement forms all belong to the protection scope of the present invention.
Claims
1. A method for producing cytidine by fermentation, characterized in that: The following steps are involved: S1, inoculating the cytidine-producing bacteria into the eggplant bottle culture medium for activation culture; S2, inoculating the activated cytidine-producing strain into sterile water for shake flask culture to obtain a primary seed solution; S3, inoculating the primary seed solution into the secondary seed culture medium for expansion, to obtain the secondary seed solution; S4, inoculating the secondary seed liquid into the fermentation medium for fermentation, and adding IPTG inducer three times. When the expected optical density is 20-25 before fermentation, the IPTG inducer accounting for 45-55% of the total amount of IPTG inducer is added for the first time; when the fermentation is 15-20 hours, the IPTG inducer accounting for 25-35% of the total amount of IPTG inducer is added for the second time; when the fermentation is 30-36 hours, the IPTG inducer accounting for 15-25% of the total amount of IPTG inducer is added for the third time; at the same time, when the amount of reducing sugar in the fermentation liquid is 0.3-0.5%, glucose is added, and the dissolved oxygen content is controlled at 10-30% during the sugar addition process until the addition is completed, and the fermentation is terminated when the pH of the fermentation liquid rises; S5. The fermentation broth is sterilized by heating, filtered, concentrated, cooled and crystallized to obtain a finished cytidine product.
2. The method for producing cytidine by fermentation according to claim 1, characterized in that: In the S4, the duration of the three additions of IPTG inducer is 25-35 min, 15-25 min and 5-15 min respectively, and the total addition time is 0.8-1.2 h.
3. The method for producing cytidine by fermentation according to claim 1, characterized in that: In S4, the IPTG inducer added is an IPTG aqueous solution, the volume of the IPTG aqueous solution is 0.5-0.8% of the volume of the fermentation liquid before fermentation, and the concentration is 0.6-1.0%; The added glucose is a glucose solution, the volume of which is 70-80% of the volume of the fermentation liquid before fermentation and the concentration is 55-65%.
4. The method for producing cytidine by fermentation according to claim 1, characterized in that: In S4, the inoculation amount of the secondary seed liquid is 8-12%, the fermentation temperature is 36±1° C., and the fermentation time is 40-50 h.
5. The method for producing cytidine by fermentation according to claim 1, characterized in that: The fermentation medium comprises the following components by mass percentage: 2.5-3.5% glucose, 0.3-0.5% magnesium sulfate, 0.001-0.002% manganese sulfate, 0.001-0.002% ferrous sulfate, 0.8-1.2% yeast extract, 0.4-0.8% corn starch extract, 0.2-0.4% citric acid, 0.01-0.05% defoamer, and the balance is water.
6. The method for producing cytidine by fermentation according to claim 1, characterized in that: The cytidine-producing bacteria is Escherichia coli W3110 (ATCC27325).
7. The method for producing cytidine by fermentation according to claim 1, characterized in that: In S1, the activation culture temperature is 36±1°C and the culture time is 12 to 16 hours; In S2, the temperature of the shake flask culture is 36±1°C and the culture time is 12 to 16 hours; In the S3, the inoculation amount of the primary seed solution is 0.02-0.04%, the expansion temperature is 36±1° C., and the culture time is 16-20 h.
8. The method for producing cytidine by fermentation according to claim 1, characterized in that: The eggplant bottle culture medium comprises the following components by mass percentage: 2-5% beef extract, 0.3-0.6% peptone, 0.8-1.2% yeast powder, 2.5-3.5% agar, and the balance is water.
9. The method for producing cytidine by fermentation according to claim 1, characterized in that: The secondary seed culture medium comprises the following components by mass percentage: 2.0-4.0% glucose, 0.8-1.2% yeast extract, 0.2-0.5% potassium dihydrogen phosphate, 0.2-0.5% defoamer, 0.8-1.2% magnesium sulfate, 0.001-0.003% ferrous sulfate, 0.001-0.003% cobalt chloride, and the balance is water.
10. The method for producing cytidine by fermentation according to claim 1, characterized in that: The S5 specifically includes the following steps: The fermentation broth after fermentation is sterilized by heating, and the pH of the fermentation broth is adjusted to 4.0±1, and filtered by an inorganic membrane. The filtrate is eluted by a cation exchange resin, concentrated, cooled and crystallized to obtain a cytidine product.