Compound enzymolysis-continuous countercurrent extraction coupled cytidine extraction method and system

Abstract

The invention relates to the field of biochemical engineering separation and purification, and discloses a compound enzymolysis-continuous countercurrent extraction coupled cytidine extraction method and system.The method comprises the following steps that after cytidine fermentation liquor is pretreated, compound enzyme composed of cellulase, lysozyme and protease according to the mass ratio of 2: 1: 1 is added for enzymolysis, and cytidine is fully released; filtering the enzymatic hydrolysate through a microporous filter membrane to remove insoluble impurities, and performing continuous counter-current extraction by using an extracting agent formed by mixing n-butyl alcohol and isopropanol according to a volume ratio of 3: 2; and finally, carrying out water back extraction, concentration crystallization, washing and drying on the organic phase containing cytidine to obtain a high-purity cytidine product. The invention further discloses a special system for implementing the method. By coupling specific composite enzymatic hydrolysis with an efficient continuous countercurrent extraction process, the method solves the problems of low extraction rate and low product purity of a traditional cytidine extraction method, and is stable in process flow and suitable for industrial production.

Description

Technical Field

[0001] This invention relates to the field of biochemical separation and purification technology, specifically to a method and system for cytidine extraction coupled with a complex enzymatic hydrolysis-continuous countercurrent extraction process. Background Technology

[0002] Cytidine is an important bioactive substance with wide applications in medicine, health products, and other fields. Currently, cytidine is mainly produced through microbial fermentation, and efficient extraction of cytidine from the fermentation broth is a key step in achieving its industrial production.

[0003] However, traditional cytidine extraction methods have technical limitations and cannot meet the demands of large-scale production. The fermentation broth has a complex composition, still containing a large amount of bacterial fragments and extracellular polysaccharides even after pretreatment. Traditional processes struggle to break down the encapsulation or adsorption of cytidine by these substances, resulting in insufficient cytidine release, a major reason for low extraction efficiency. Simultaneously, in the separation and purification stage, the extraction or adsorption methods used in existing processes have limited selectivity. Impurities in the fermentation broth with properties similar to cytidine are easily extracted along with it, leading to difficulties in separation and purification and resulting in low product purity. Furthermore, insoluble impurities and colloidal substances in the fermentation broth accumulate at the phase interface and cause emulsification or equipment blockage during liquid-liquid extraction and other separation operations, severely affecting the continuous and stable operation of the process.

[0004] Therefore, developing an efficient, energy-saving, and environmentally friendly method for cytidine extraction is of great practical significance. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a cytidine extraction method and its supporting system that is coupled with a complex enzymatic hydrolysis-continuous countercurrent extraction method, which has high extraction efficiency and good product purity.

[0006] The first aspect of this invention provides a method for cytidine extraction coupled with a combined enzymatic hydrolysis and continuous countercurrent extraction, comprising the following steps: S1. Fermentation broth pretreatment: Centrifuge the cytidine fermentation broth, collect the supernatant, adjust the pH to 4.5-5.5, and control the temperature at 40-50℃; S2. Compound enzymatic hydrolysis: Add a compound enzyme to the supernatant. The compound enzyme is composed of cellulase, lysozyme and protease in a mass ratio of 2:1:1. The amount of enzyme added is 0.8%-1.2% of the fermentation broth mass. Enzymatic hydrolysis is carried out at 40-50℃ for 3-5 hours with a stirring speed of 120-180 rpm. S3. Enzyme hydrolysate filtration: The enzyme hydrolysate is filtered through a microporous membrane to obtain crude cytidine extract; S4. Continuous countercurrent extraction: The crude cytidine extract and the extractant are pumped into a continuous countercurrent extraction device. The extractant is a mixture of n-butanol and isopropanol at a volume ratio of 3:2. The material-liquid ratio is 1:1.5-2.5. The extraction temperature is 35-45℃ and the extraction time is 40-70 minutes. S5. Back-extraction: Transfer the organic phase containing cytidine to the back-extraction device, add water as the back-extraction agent, the volume ratio of back-extraction agent to organic phase is 1:1-1.5, adjust the pH to 8.5-9.5, control the temperature at 35-45℃, and stir the reaction for 20-40 minutes. S6. Concentration and Crystallization: The cytidine back-extraction solution is concentrated under reduced pressure, with the concentration temperature controlled at 45-55℃ and the vacuum degree at -0.07 to -0.09 MPa, until it is concentrated to 1 / 8 to 1 / 12 of the original volume. Then it is cooled to 2-8℃ and allowed to stand for crystallization for 15-30 hours. The crystals are collected by filtration, washed and dried to obtain the cytidine product.

[0007] By adopting the above technical solution, this invention couples the composite enzyme system (a specific ratio of cellulase, lysozyme, and protease) introduced in step S2 with the continuous countercurrent extraction process in step S4. Its innovative principle lies in: In the S2 enzymatic hydrolysis stage, the synergistic action of three enzymes is utilized. Cellulase and lysozyme are responsible for breaking down cell walls (such as peptidoglycan) and other extracellular polysaccharide polymers that may be present in the fermentation broth; while protease hydrolyzes large protein molecules in cell debris or supernatant. This synergistic hydrolysis allows cytidine, which was previously encapsulated or adsorbed by biomolecules, to be fully released into the liquid phase, increasing the effective concentration of crude cytidine extract in step S3.

[0008] In the S3 filtration stage, insoluble impurities produced by enzymatic hydrolysis in S2 are retained through a microporous membrane to prepare a clear crude extract. This step is crucial for the subsequent S4 step, as it prevents solid particles from entering the continuous countercurrent extraction equipment, avoids emulsification, unclear interfaces, or equipment blockage during extraction, and ensures the stable operation of the S4 step.

[0009] In the S4 extraction stage, a composite extractant of n-butanol and isopropanol in a 3:2 ratio was used. This specific ratio of extractant exhibits excellent solubility selectivity and mass transfer performance for cytidine. Simultaneously, a continuous countercurrent extraction method was employed, allowing the fresh extractant and crude extract to come into multi-stage contact within the equipment. This maintained a high concentration gradient of cytidine transferring from the aqueous phase (crude extract) to the organic phase (extractant) throughout the process, achieving higher extraction efficiency than traditional batch extraction.

[0010] In the S5 back-extraction stage, the pH is adjusted to alkaline (8.5-9.5) by taking advantage of the difference in solubility of cytidine at different pH values. This reverses the partition coefficient of cytidine, allowing it to be efficiently transferred from the organic phase to the aqueous phase, which facilitates subsequent crystallization and purification.

[0011] In summary, this invention combines efficient bio-enzymatic decomposition and release with efficient continuous extraction and separation, resulting in a high degree of process matching and achieving high extraction rate and high purity of cytidine.

[0012] Preferably, in step S1, the centrifugation speed is 5500-6500 r / min and the centrifugation time is 18-22 minutes.

[0013] By adopting the above technical solution, optimized pretreatment parameters are provided, ensuring the efficient removal of bacteria and solid impurities in step S1.

[0014] Preferably, the pore size of the microporous filter membrane is 0.2-0.3 μm. By adopting the above technical solution, fine insoluble impurities generated by enzymatic hydrolysis can be reliably retained, resulting in a highly clear crude extract.

[0015] Preferably, in step S5, cytidine is transferred from the organic phase to the aqueous phase to obtain the cytidine back-extraction solution.

[0016] By adopting the above technical solution, the technical objective of step S5 is clarified, namely, to achieve the transfer of the target product from the extraction phase to the purification phase.

[0017] Preferably, the filtration in step S3 is used to remove insoluble impurities from the enzymatic hydrolysate to obtain a clear crude cytidine extract.

[0018] By adopting the above technical solution, it is clarified that step S3 serves as a bridge between S2 and S4, providing qualified liquid for step S4.

[0019] Preferably, in step S1, the cytidine fermentation broth is centrifuged to remove bacterial cells and solid impurities.

[0020] By adopting the above technical solution, the technical purpose of the centrifugation operation in step S1 is clarified, namely, to preliminarily purify the fermentation broth and reduce the load on subsequent enzymatic hydrolysis and extraction.

[0021] Preferably, step S2 is carried out in an enzymatic hydrolysis reactor equipped with a stirring device and a temperature control device.

[0022] By adopting the above technical solution, the specific implementation conditions of step S2 are defined, ensuring that the enzymatic hydrolysis reaction is carried out efficiently under controlled temperature and uniform mixing conditions.

[0023] Preferably, the acid used to adjust the pH in step S1 is hydrochloric acid, sulfuric acid, or phosphoric acid.

[0024] By adopting the above technical solution, specific reagents that can be used to adjust pH in step S1 are provided, all of which are industrially available and cost-controllable acids.

[0025] Preferably, in step S6, the washing involves washing with cold water 3-5 times.

[0026] By adopting the above technical solution, a specific washing operation is provided for crystallization purification in step S6, which effectively removes mother liquor impurities attached to the crystal surface.

[0027] A second aspect of the present invention provides a cytidine extraction system coupled with a combined enzymatic hydrolysis-continuous countercurrent extraction, comprising: The centrifuge, pH adjustment device, enzymatic hydrolysis reactor, microporous filtration device, continuous countercurrent extraction equipment, back extraction device and concentration and crystallization device are connected in sequence. The centrifuge is used to centrifuge the cytidine fermentation broth to separate the bacterial cells and solid impurities; The pH adjustment device is used to adjust the pH value of the supernatant after centrifugation; The enzymatic hydrolysis reactor is equipped with a stirring device and a temperature control device for carrying out complex enzymatic hydrolysis reactions; The microporous filtration device is used to filter the enzymatic hydrolysate to obtain crude cytidine extract. The continuous countercurrent extraction equipment is used to achieve continuous countercurrent extraction of cytidine crude extract and extractant; The back-extraction device is used to transfer cytidine from the organic phase to the aqueous phase; The concentration and crystallization device is used to concentrate and crystallize the cytidine back-extract.

[0028] By adopting the above technical solution, the present invention provides a dedicated equipment system that matches the method described in the first aspect. This system integrates all functional units required for steps S1 to S6 in series, forming a complete cytidine extraction production line. The functions of each device (e.g., stirring and temperature control of the enzymatic hydrolysis reactor, solid-liquid separation of the microporous filtration device, and liquid-liquid extraction of the continuous countercurrent extraction device) precisely correspond to the requirements of the method steps. This systematic design ensures the stable execution of the method steps, enabling the entire process to operate continuously and automatically, providing hardware support for the efficient and stable industrial production of cytidine.

[0029] This invention provides a method and system for cytidine extraction coupled with a combined enzymatic hydrolysis and continuous countercurrent extraction. It has the following beneficial effects: 1. This invention utilizes a composite enzyme composed of cellulase, lysozyme, and protease in a specific ratio of 2:1:1 in step S2 to achieve the synergistic hydrolysis of cell walls, extracellular polysaccharides, and miscellaneous proteins in the fermentation broth. This enzymatic hydrolysis method can fully release cytidine that was previously encapsulated or adsorbed by biomolecules, increasing the effective concentration of the crude cytidine extract in step S3, laying a material foundation for subsequent efficient extraction, and achieving a higher product extraction rate.

[0030] 2. This invention couples the S2 complex enzymatic hydrolysis with the S4 continuous countercurrent extraction process, and removes insoluble impurities generated by enzymatic hydrolysis through the S3 microporous filtration step, avoiding solid impurities from entering the S4 extraction equipment. This effectively prevents the formation of an emulsion layer or blockage during the extraction process, ensuring the long-term stable operation of the continuous countercurrent extraction process and improving the industrial production feasibility of the entire process.

[0031] 3. In step S4, this invention uses a composite extractant of n-butanol and isopropanol for continuous countercurrent extraction, and in step S5, it uses pH adjustment for back-extraction. The specific extractant and continuous countercurrent method in step S4 ensure efficient mass transfer of cytidine from the aqueous phase to the organic phase. The pH adjustment in step S5 utilizes the physicochemical properties of cytidine to achieve selective separation from the organic phase back to the aqueous phase, ensuring that cytidine is efficiently enriched while being effectively separated from impurities, ultimately obtaining a high-purity cytidine product. Attached Figure Description

[0032] Figure 1 This is a flowchart of the cytidine extraction method provided in an embodiment of the present invention; Figure 2 This is a schematic diagram of the cytidine extraction system provided in an embodiment of the present invention. Detailed Implementation

[0033] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0034] See attached document Figure 1 and attached Figure 2 , Figure 1 This is a flowchart of the cytidine extraction method provided in an embodiment of the present invention. Figure 2 A schematic diagram of the cytidine extraction system provided in an embodiment of the present invention. Preparation Example 1: Preparation of complex enzymes: Accurately weigh cellulase, lysozyme, and protease (e.g., all commercially available industrial-grade enzyme preparations) according to a mass ratio of 2:1:1. Place the weighed enzyme powders in a mixing container and mix thoroughly to obtain the composite enzyme of this invention. Seal the prepared composite enzyme and store it under suitable conditions (e.g., dryness, low temperature) for later use.

[0035] Examples 1-3: Example 1: This embodiment provides a cytidine extraction method coupled with a combined enzymatic hydrolysis and continuous countercurrent extraction, including the following steps: Fermentation broth pretreatment: 100L of cytidine fermentation broth was pumped into a centrifuge and centrifuged at 6000r / min for 20 minutes to remove bacterial cells and solid impurities. The collected supernatant was transferred to a pH adjustment device, the temperature control was activated to maintain the temperature of the broth at 45℃, and the pH of the supernatant was precisely adjusted to 5.0 with hydrochloric acid.

[0036] Compound enzymatic hydrolysis: The pretreated supernatant is fed into the enzymatic hydrolysis reactor. The temperature control device inside the reactor is activated to maintain the temperature at 45℃, and the stirring device is turned on to stabilize the rotation speed at 150 rpm. A compound enzyme (cellulase, lysozyme, and protease in a mass ratio of 2:1:1) is added to the supernatant at a mass ratio of 1.0% of the fermentation broth, and the enzymatic hydrolysis reaction is carried out for 4 hours.

[0037] Enzymatic hydrolysis solution filtration: After the enzymatic hydrolysis reaction is completed, the solution is pumped into a microporous filtration device and filtered through a microporous membrane with a pore size of 0.22 μm to remove insoluble impurities and obtain a clear crude cytidine extract.

[0038] Continuous countercurrent extraction: The obtained crude cytidine extract and the extractant (n-butanol to isopropanol volume ratio 3:2) were continuously pumped into the continuous countercurrent extraction equipment at a feed ratio of 1:2. The equipment operating temperature was set to 40℃, and continuous countercurrent extraction was carried out under these conditions for 50 minutes.

[0039] Back-extraction: The cytidine-rich organic phase flowing out of the continuous countercurrent extraction unit is collected and transferred to the back-extraction device. Water is added as the back-extraction agent, making the volume ratio of back-extraction agent to organic phase 1:1.2. The pH of the feed solution in the device is adjusted to 9.0, and the temperature is controlled at 40°C. The reaction is stirred for 30 minutes to obtain the cytidine back-extraction solution.

[0040] Concentration and Crystallization: The obtained cytidine back-extraction solution was fed into a concentration and crystallization apparatus. First, it was concentrated under reduced pressure at 50°C and a vacuum of -0.08 MPa until the liquid volume was 1 / 10 of its original volume. After concentration, the solution was cooled to 5°C in the apparatus and allowed to crystallize for 20 hours. Finally, the precipitated crystals were filtered, washed four times with cold water, and dried to obtain the cytidine product.

[0041] Example 2: This embodiment provides a cytidine extraction method coupled with a combined enzymatic hydrolysis and continuous countercurrent extraction, including the following steps: Fermentation broth pretreatment: 150 L of cytidine fermentation broth was pumped into a centrifuge and centrifuged at 5500 rpm for 18 minutes. The collected supernatant was sent to a pH adjustment device, the temperature was controlled at 42°C, and the pH was adjusted to 4.5 with sulfuric acid.

[0042] Compound enzymatic hydrolysis: The feed solution is fed into the enzymatic hydrolysis reactor. The reactor temperature is set to 42℃ and the stirring speed to 120 rpm. Add a compound enzyme at 0.8% of the fermentation broth mass and react for 3.5 hours.

[0043] Enzyme hydrolysate filtration: Same as in Example 1, the enzyme hydrolysate is pumped into a microporous filter for filtration.

[0044] Continuous countercurrent extraction: The crude extract and extractant are pumped into the continuous countercurrent extraction equipment at a feed-to-liquid ratio of 1:1.5. The operating temperature is set to 35℃ and the extraction time is 40 minutes.

[0045] Back-extraction: Collect the organic phase into the back-extraction apparatus. Add water as the back-extraction agent, making the volume ratio of back-extraction agent to organic phase 1:1. Adjust the pH to 8.5, control the temperature at 35°C, and stir the reaction for 20 minutes.

[0046] Concentration and Crystallization: The back-extraction solution was fed into a concentration and crystallization apparatus. It was concentrated to 1 / 8 of its original volume under a vacuum of -0.07 MPa at 45°C. It was then cooled to 2°C and allowed to crystallize for 15 hours. After filtration, washing, and drying, the cytidine product was obtained.

[0047] Example 3: This embodiment provides a cytidine extraction method coupled with a combined enzymatic hydrolysis and continuous countercurrent extraction, including the following steps: Fermentation broth pretreatment: 200 L of cytidine fermentation broth was pumped into a centrifuge and centrifuged at 6500 rpm for 22 minutes. The supernatant was then transferred to a pH adjustment device, the temperature was controlled at 48 °C, and the pH was adjusted to 5.5 with phosphoric acid.

[0048] Compound enzymatic hydrolysis: The feed solution is fed into the enzymatic hydrolysis reactor. The reactor temperature is set to 48℃ and the stirring speed to 180 rpm. A compound enzyme at 1.2% of the fermentation broth mass is added, and the reaction is carried out for 5 hours.

[0049] Enzyme hydrolysate filtration: The enzyme hydrolysate is filtered through a microporous filter to obtain crude cytidine extract.

[0050] Continuous countercurrent extraction: The crude extract and extractant are pumped into the continuous countercurrent extraction equipment at a feed-to-liquid ratio of 1:2.5. The operating temperature is set to 45℃ and the extraction time to 70 minutes.

[0051] Back-extraction: Collect the organic phase into the back-extraction apparatus. Add water as the back-extraction agent, making the volume ratio of back-extraction agent to organic phase 1:1.5. Adjust the pH to 9.5, control the temperature at 45℃, and stir the reaction for 40 minutes.

[0052] Concentration and Crystallization: The back-extraction solution was fed into a concentration and crystallization apparatus. It was concentrated to 1 / 12 of its original volume under a vacuum of 55°C and -0.09 MPa. It was then cooled to 8°C and allowed to crystallize for 30 hours. After filtration, washing, and drying, the cytidine product was obtained.

[0053] Comparative Examples 1-5: Comparative Example 1: Compared with Example 1, the difference is that after the fermentation broth pretreatment, the compound enzymatic hydrolysis step is not performed, and it directly enters the filtration step; all other steps are the same.

[0054] Comparative Example 2: Compared with Example 1, the difference is that in the complex enzymatic hydrolysis step, instead of using a complex enzyme composed of cellulase, lysozyme and protease, a single lysozyme of equal mass (accounting for 1.0% of the fermentation broth mass) is used, while the rest are the same.

[0055] Comparative Example 3: Compared with Example 1, the difference is that in the complex enzymatic hydrolysis step, a single protease of equal mass (1.0% of the fermentation broth mass) is used instead of a complex enzyme. All other steps are the same.

[0056] Comparative Example 4: Compared with Example 1, the difference is that in the compound enzymatic hydrolysis step, a single cellulase of equal mass (1.0% of the fermentation broth mass) is used instead of a compound enzyme. All other steps are the same.

[0057] Comparative Example 5: Compared with Example 1, the difference is that the extraction step does not use continuous countercurrent extraction, but instead uses the traditional batch extraction method (for example, adding equal amounts of extractant to the extraction vessel three times for stirring extraction), while the rest are the same.

[0058] Test Example 1-2: Test Example 1: Experimental description: This test case aims to verify the feasibility and practical effectiveness of the technical solution proposed in this invention during the cytidine extraction process. The test subjects were the final cytidine products prepared in Examples 1, 2, and 3, and their corresponding initial fermentation broths. The test indicators were the cytidine extraction rate and the purity of the final product.

[0059] Experimental steps: Initial cytidine content determination: The initial cytidine fermentation broth used in Examples 1-3 was taken, appropriately diluted, and the mass concentration (C0) of cytidine was determined by high performance liquid chromatography (HPLC). The volume (V0) of the fermentation broth used in each example was recorded. The total mass of initial cytidine was calculated based on C0 × V0.

[0060] Final product quality determination: The dried cytidine products obtained in Examples 1-3 were accurately weighed using an analytical balance, and their final product quality (M) was recorded.

[0061] Product purity analysis: Weigh appropriate amounts of the final products from Examples 1-3 respectively, prepare standard concentration solutions, and use high performance liquid chromatography (HPLC) to calculate the percentage of the cytidine main peak area to the total peak area using the area normalization method, which is the product purity.

[0062] Extraction rate calculation: The cytidine extraction rate is calculated using the following formula: Cytidine extraction rate (%) = [(M × product purity) / (C0 × V0)] × 100% Experimental data: Table 1. Data on cytidine extraction rate and product purity in Examples 1-3 in conclusion: The test data in Table 1 show that the technical solution of the present invention is technically feasible.

[0063] The cytidine extraction rate ranged from 88.56% to 95.25%. This result validates the effectiveness of the combined enzymatic hydrolysis step. The synergistic effect of cellulase, lysozyme, and protease effectively degrades the cellular structures and other macromolecules encapsulating cytidine in the fermentation broth, allowing cytidine to be fully released into the liquid phase. This is the technical basis for obtaining a high extraction rate.

[0064] The final product purity was consistently above 99.1%. This metric confirms the highly efficient separation capability of the continuous countercurrent extraction coupled with a multi-stage purification process for impurities. The continuous countercurrent extraction technology, by establishing a continuous concentration gradient, enhances the mass transfer efficiency of cytidine from the aqueous phase to the organic phase, effectively separating water-soluble impurities such as proteins and pigments. Subsequent back-extraction, concentration, and crystallization steps further remove lipid-soluble impurities and other trace interfering substances, ultimately yielding high-purity cytidine crystals.

[0065] In summary, the test data confirms that the technical route of coupling compound enzymatic hydrolysis and continuous countercurrent extraction can achieve the goal of efficiently extracting high-purity cytidine from fermentation broth.

[0066] Test Example 2: Experimental description: This test case aims to quantitatively evaluate the contribution and technical advantages of key technical steps (compound enzymatic hydrolysis and continuous countercurrent extraction) in the present invention to the extraction efficiency of cytidine by comparing with multiple comparative examples. The test objects are the final cytidine products prepared in Example 1 and Comparative Examples 1-5 and their corresponding initial fermentation broths.

[0067] Experimental steps: Using the same experimental procedures and calculation methods as in Test Example 1, the extraction rate and purity of the cytidine products obtained in each group of experiments in Example 1 and Comparative Examples 1-5 were determined. All comparative experiments used the same initial fermentation broth batch to ensure comparability.

[0068] Experimental data: Table 2. Comparison of test data between Example 1 and each comparative example in conclusion: The comparative test data in Table 2 quantitatively confirms the technical superiority of the present invention. The effect analysis of its core innovation points is as follows: Synergistic effect of compound enzymatic hydrolysis: Compared with Comparative Example 1, the extraction rate jumped from 57.34% to 92.18%, directly proving that enzymatic hydrolysis is a key prerequisite for achieving efficient extraction. Its role is to destroy cell structure, allowing cytidine to be released. Further comparison of Example 1 with Comparative Examples 2, 3, and 4 shows that the extraction rate of the compound enzyme scheme is higher than any single enzyme scheme. This indicates that the compound system composed of cellulase, lysozyme, and protease has a clear synergistic effect. The mechanism lies in the fact that different enzymes act on different macromolecular substrates in the fermentation broth, achieving a more thorough breakdown of the cytidine release barrier, thereby maximizing the amount of free cytidine, an effect that a single enzyme preparation cannot achieve.

[0069] The high efficiency of continuous countercurrent extraction in mass transfer: Compared with Comparative Example 5, Example 1, which uses continuous countercurrent extraction, achieved an extraction rate (92.18%) higher than Comparative Example 5, which uses conventional batch extraction (85.92%). This result verifies the superiority of continuous countercurrent extraction technology. The mechanism lies in the fact that continuous countercurrent operation maintains a stable and high interphase concentration gradient, providing a continuous driving force for the mass transfer of cytidine from the aqueous phase to the organic phase, achieving a more complete and efficient interphase transfer than batch extraction (where the mass transfer driving force decays as equilibrium approaches).

[0070] Overall impact on product purity: The data also show that the product purity of Example 1 (99.23%) was the highest among all groups. This indicates that the overall process route of the present invention, namely, the combined enzymatic hydrolysis coupled with continuous countercurrent extraction, not only improves the extraction rate, but also creates favorable conditions for subsequent purification steps through more thorough impurity pretreatment (enzymatic hydrolysis) and more efficient phase separation (continuous extraction), ultimately achieving higher product purity.

[0071] In summary, the comparative data confirms that the combined enzymatic hydrolysis and continuous countercurrent extraction scheme adopted in this invention is superior to schemes that do not involve enzymatic hydrolysis, use single enzymatic hydrolysis, or use batch extraction in terms of both cytidine extraction rate and product purity.

Claims

1. A method for the extraction of cytidine by coupling enzymatic hydrolysis with continuous counter-current extraction, characterized in that, The method comprises the following steps: S1, fermentation broth pretreatment: centrifuging the cytidine fermentation broth, collecting the supernatant, adjusting the pH to 4.5-5.5, and controlling the temperature at 40-50℃; S2, complex enzymolysis: adding a complex enzyme to the supernatant, the complex enzyme being composed of cellulase, lysozyme and protease at a mass ratio of 2:1:1, the enzyme addition amount being 0.8%-1.2% of the mass of the fermentation broth, and carrying out enzymolysis at 40-50℃ for 3-5 hours with a stirring speed of 120-180rpm; S3, filtration of the enzymolysis liquid: filtering the enzymolysis liquid through a microporous filter membrane to obtain a cytidine crude extract; S4, continuous countercurrent extraction: pumping the cytidine crude extract and an extractant into a continuous countercurrent extraction device, the extractant being a mixture of n-butanol and isopropyl alcohol at a volume ratio of 3:2, the solid-liquid ratio being 1:1.5-2.5, the extraction temperature being 35-45℃, and the extraction time being 40-70 minutes; S5, back extraction: transferring the organic phase containing cytidine to a back extraction device, adding water as a back extraction agent, the volume ratio of the back extraction agent to the organic phase being 1:1-1.5, adjusting the pH to 8.5-9.5, controlling the temperature at 35-45℃, and stirring for 20-40 minutes; S6, concentration and crystallization: reducing the pressure to concentrate the cytidine back extraction liquid, the concentration temperature being controlled at 45-55℃, the vacuum degree being-0.07 to-0.09MPa, the concentration being carried out to 1 / 8-1 / 12 of the original volume, then cooling to 2-8℃, and standing for crystallization for 15-30 hours to collect the crystals by filtration, and washing and drying to obtain the cytidine product.

2. The method according to claim 1, wherein the method is characterized in that, In the S1 step, the centrifugation speed is 5500-6500r / min, and the centrifugation time is 18-22 minutes.

3. The method according to claim 1, wherein the method is characterized by, The microporous filter membrane has a pore size of 0.2-0.3μm.

4. The method according to claim 1, wherein the method is characterized by, In the S5 step, the cytidine is transferred from the organic phase to the aqueous phase to obtain the cytidine back extraction liquid.

5. The method of claim 1, wherein the method is a method of extracting cytidine by coupling enzymatic hydrolysis with continuous countercurrent extraction, characterized in that, The filtration in the S3 step is used to remove insoluble impurities in the enzymolysis liquid to obtain a clear cytidine crude extract.

6. The method of claim 1, wherein the method is a method of extracting cytidine by coupling enzymatic hydrolysis with continuous countercurrent extraction, characterized in that, In the S1 step, the cytidine fermentation broth is centrifuged to remove the bacteria and solid impurities.

7. The method according to claim 1, wherein the method is characterized by, The S2 step is carried out in an enzymolysis reactor with a stirring device and a temperature control device.

8. The method according to claim 1, wherein the method is characterized by, In the S1 step, the acid used to adjust the pH is hydrochloric acid, sulfuric acid or phosphoric acid.

9. The method according to claim 1, wherein the method is characterized by, In the S6 step, the washing is carried out with cold water for 3-5 times.

10. A system for the extraction of cytidine by coupling enzymatic hydrolysis with continuous counter-current extraction, characterized in that, The method comprises the following steps: a centrifugal separator, a pH adjusting device, an enzymolysis reactor, a microporous filtration device, a continuous countercurrent extraction device, a back extraction device and a concentration and crystallization device connected in sequence; The centrifugal separator is used to centrifuge the cytidine fermentation broth to separate the bacteria and solid impurities; the pH adjusting device is used to adjust the pH of the supernatant after centrifugation; The enzymolysis reactor is provided with a stirring device and a temperature control device and is used to carry out complex enzymolysis reaction; The microporous filtration device is used to filter the enzymolysis liquid to obtain a cytidine crude extract; The continuous countercurrent extraction device is used to realize continuous countercurrent extraction of the cytidine crude extract and an extractant; The back extraction device is used to transfer cytidine from the organic phase to the aqueous phase; The concentration and crystallization device is used to concentrate and crystallize the cytidine back extraction liquid.

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