阅读说明：本技术 一种从发酵液中分离提取胞苷的方法 (Method for separating and extracting cytidine from fermentation liquor ) 是由 应汉杰 陈勇 温庆仕 魏荷芬 周精卫 杨玉晨 于 2021-08-17 设计创作，主要内容包括：本发明公开了一种从发酵液中分离提取胞苷的方法,将含有胞苷的液体经阳离子交换树脂吸附洗脱,所得洗脱液浓缩结晶,即得胞苷晶体；其中,所述阳离子交换树脂为NH-1树脂经硫酸银和/或硫酸改性所得。本发明中使用的阳离子交换柱为改性后的NH-1树脂,其胞苷吸附量达到0.3g/g,减少了树脂用量,间接节省了酸碱用量和操作时间。(The invention discloses a method for separating and extracting cytidine from fermentation liquor, which comprises the steps of adsorbing and eluting liquid containing cytidine by cation exchange resin, and concentrating and crystallizing the obtained eluent to obtain cytidine crystals; wherein the cation exchange resin is obtained by modifying NH-1 resin with silver sulfate and/or sulfuric acid. The cation exchange column used in the invention is modified NH-1 resin, and the cytidine adsorption capacity of the cation exchange column reaches 0.3g/g, so that the resin dosage is reduced, and the acid-base dosage and the operation time are indirectly saved.)

1. A method for separating and extracting cytidine is characterized in that liquid containing cytidine is adsorbed and eluted by cation exchange resin, and the obtained eluent is concentrated and crystallized to obtain cytidine crystals;

wherein the cation exchange resin is obtained by modifying NH-1 resin with silver sulfate and/or sulfuric acid; the adsorption capacity of the cation exchange resin is 0.2-0.4 g/g.

2. The method according to claim 1, wherein the amount of silver sulfate is 2-8% of the mass of the NH-1 resin; the dosage of the sulfuric acid is 10-20 times of the volume of the resin.

3. The method according to claim 1, characterized in that the solvent in the modification process is dichloroethane and/or trichloroethane; the volume ratio of the NH-1 resin to the solvent is 1: (6-20).

4. The method of claim 1, wherein the NH-1 resin is mixed with a solvent and maintained at 50-70 ℃ for 0.5-2 h; adding silver sulfate and/or sulfuric acid, maintaining at 70-90 deg.C for 2-4 hr, maintaining at 90-100 deg.C for 2-3 hr, filtering, and washing.

5. The method of claim 1, comprising the steps of:

(1) filtering the cytidine fermentation liquor by a ceramic membrane to obtain a first permeate;

(2) adsorbing the first permeate obtained in the step (1) by using cation exchange resin, washing impurities by using water and/or ammonia water, and eluting by using ammonia water to obtain an eluent;

(3) nanofiltration of the eluent obtained in the step (2) by a nanofiltration membrane to obtain a second permeate;

(4) and (4) concentrating the second permeate obtained in the step (3) through a nanofiltration membrane, adjusting the pH value, stirring, cooling and crystallizing to obtain a cytidine crystal.

6. The method according to claim 5, wherein in the step (2), the concentration of the ammonia water in the impurity washing process is 0-0.05 mol/L; in the elution process, the concentration of ammonia water is 0.05-0.1 mol/L.

7. The method as claimed in claim 5, wherein in step (3), the nanofiltration membrane has a molecular weight cut-off of 300-800 Da.

8. The method as claimed in claim 5, wherein in the step (4), the molecular weight cut-off of the nanofiltration membrane is 60-200 Da.

9. The method as claimed in claim 5, wherein in step (4), the concentration is up to 800g/L at 400-.

10. The method according to claim 5, wherein in the step (4), the pH is 5 to 10; the temperature is reduced to 0-10 ℃.

Technical Field

The invention belongs to the technical field of biochemical separation, and particularly relates to a method for separating and extracting cytidine from fermentation liquor.

Background

Cytidine is pyrimidine nucleoside, an intermediate of citicoline, and an intermediate of a plurality of antiviral, antitumor, anti-AIDS drugs and the like. Cytidine is widely used in various industries such as food, health products, cosmetics, and medicines. Cytidine is especially an important intermediate for drug synthesis in modern medicine, and as the application range of cytidine is continuously expanded, the demand for cytidine is increased, and the preparation research of cytidine is naturally deepened. At present, the development of a low-cost cytosine nucleoside production separation and purification process which can be applied in a large scale is urgently needed.

At present, the production of cytidine at home and abroad mainly comprises a chemical synthesis method, an enzymatic synthesis method and a microbial fermentation synthesis method. The microbial fermentation synthesis method is mainly used, and many reports are reported on how to breed efficient and high-yield microbial strains, but the research reports on the large-scale separation and purification process of cytidine are less. Therefore, the present invention provides a method for separating and extracting cytidine from a fermentation broth.

Disclosure of Invention

The purpose of the invention is as follows: the technical problem to be solved by the invention is to provide a method for separating and extracting cytidine aiming at the defects of the prior art.

In order to solve the technical problems, the invention discloses a method for separating and extracting cytidine, which comprises the steps of adsorbing and eluting liquid containing cytidine by cation exchange resin, and concentrating and crystallizing the obtained eluent to obtain cytidine crystals.

Wherein the cation exchange resin is obtained by modifying NH-1 resin with silver sulfate and/or sulfuric acid.

Wherein the adsorption capacity of the cation exchange resin is 0.2-0.4 g/g; preferably, the adsorption amount of the cation exchange resin is 0.3 g/g.

Wherein the dosage of the silver sulfate is 2-8% of the mass of the NH-1 resin; preferably, the dosage of the silver sulfate is 4 to 6 percent of the mass of the NH-1 resin; preferably, the amount of silver sulfate used is 5% by mass of the NH-1 resin.

Wherein the dosage of the sulfuric acid is 10-20 times of the volume of the resin.

Wherein, the solvent in the modification process is dichloroethane and/or trichloroethane.

Wherein the volume ratio of the NH-1 resin to the solvent is 1: (6-20).

Wherein, NH-1 resin is mixed with solvent and kept for 0.5 to 2 hours at the temperature of between 50 and 70 ℃; adding silver sulfate and/or sulfuric acid, maintaining at 70-90 deg.C for 2-4 hr, maintaining at 90-100 deg.C for 2-3 hr, filtering, and washing.

Wherein the washing is to neutral.

Wherein the washing is washing with any one or more organic solvents selected from methanol, ethanol and acetone

The static adsorption experiment after the modification by the method shows that the adsorption capacity of the resin is improved to 0.3g/g from the original 0.09 g/g.

Preferably, the method for separating and extracting cytidine comprises the following steps:

(1) filtering the cytidine fermentation liquor by a ceramic membrane to remove solid impurities to obtain a first permeate;

(2) adsorbing the first permeate obtained in the step (1) by using cation exchange resin, washing impurities by using water and/or ammonia water, eluting by using ammonia water, continuously detecting by using HPLC (high performance liquid chromatography) in the process, and directly switching to cytidine elution when detecting that cytidine flows out to obtain eluent;

(3) synchronously performing nanofiltration and decoloration on the eluent obtained in the step (2) through a nanofiltration membrane to obtain a second permeate;

(4) and (4) concentrating the second permeate obtained in the step (3) through a nanofiltration membrane, carrying out vacuum evaporation concentration, adjusting the pH of the obtained concentrated solution, adding a small amount of seed crystals as an inducer, stirring, cooling, crystallizing, filtering, washing and drying to obtain cytidine crystals.

In the step (1), cytidine is derived from microbial fermentation liquor, uridine high-yield strains obtained through system metabolic engineering are cultured in a 50L fermentation tank, and the initial yield of cytidine is 30-60 g/L. The fermentation liquor contains a large amount of thalli, culture medium, pigment and other impurities, and the separation difficulty is high.

In the step (1), the cytidine fermentation liquor is filtered by a ceramic membrane after the pH is adjusted; preferably, the pH is 2-3.5.

In the step (1), the aperture of the ceramic membrane is 50-200nm, and the pressure of the ceramic membrane is 0.1-0.3 MPa.

In the step (1), the filtering temperature is normal temperature.

In the step (2), the sample loading speed of the first permeation solution to the cation exchange resin is 1-2 BV/h.

In the step (2), in the impurity washing process, the concentration of ammonia water is 0-0.05 mol/L; preferably, the concentration of ammonia is 0.03 mol/L.

In the step (2), the flow rate of the impurity washing is 1-2 BV/h.

In the step (2), the volume of the impurities is 4-14 BV.

In the step (2), the concentration of ammonia water is 0.05-0.1mol/L in the elution process; preferably, the concentration of ammonia is 0.06 mol/L.

In the step (2), the flow rate of elution is 1-2 BV/h.

In the step (2), the elution volume is 10-30 BV; preferably, elution is ended until the elution is spotted with no cytidine present.

In the step (3), the molecular weight cut-off of the nanofiltration membrane is 300-800 Da.

In the step (3), the pressure of the nanofiltration membrane is 1-3 MPa.

In the step (4), the molecular weight cut-off of the nanofiltration membrane is 60-200 Da; preferably, the molecular weight cut-off of the nanofiltration membrane is 60-120 Da; preferably, the nanofiltration membrane has a molecular weight cut-off of 90 Da.

In the step (4), the vacuum degree of the vacuum evaporation concentration is less than 100mbar, and the water bath temperature is 45-70 ℃; preferably, the bath temperature is 50 ℃.

In the step (4), the concentration is up to 800 g/L; preferably, the concentration is up to 400-; further preferably, the concentration is to 600 g/L.

In the step (4), the pH is 5-10; preferably, the pH is 6-8; further preferably, the pH is 7.

In the step (4), the rotation speed of the stirring is 100-400 rpm.

In the step (4), crystal precipitation occurs after the seed crystal is added and stirred for 1-2 h.

In the step (4), the temperature is reduced to 0-10 ℃; preferably, the temperature is reduced to 2-6 ℃; further preferably, the temperature is reduced to 4 ℃.

In the step (4), the temperature is reduced by adopting a gradient temperature reduction method; preferably, crystallization is completed within 6-12 h.

In the step (4), the crystal system is a water system, so that the workshop reconstruction and production cost is reduced.

In the step (4), the washing is to wash the crystals by using an ethanol solution with the concentration of 95-99%, and the crystals are collected by centrifugation or suction filtration, so that the purity of the crystals can be improved, and the influence of impurities in the crystallization mother liquor can be reduced.

In the step (4), the drying is forced air drying, double cone drying or vacuum drying; preferably, the drying is vacuum drying; preferably, the vacuum is 0 to 200 mbar; preferably, the vacuum is 10-100 mbar.

In the step (4), the drying temperature is 40-100 ℃; preferably, the temperature of the drying is 50-80 ℃.

In the step (4), preferably, the yield is improved by secondary crystallization or nested crystallization.

The purity of the cytidine obtained by the method is more than 99 percent, and the total yield is 90 to 95 percent.

Has the advantages that: compared with the prior art, the invention has the following advantages:

(1) compared with the existing crystallization method of cytidine, the cytidine hydrochloride is firstly formed and then crystallized for the second time to obtain cytidine, and the cytidine crystal is obtained by adopting a one-step separation direct crystallization method, so that the process of obtaining the secondary crystallization of a crude cytidine hydrochloride product is omitted.

(2) The cation exchange column used in the invention is modified NH-1 resin, and the cytidine adsorption capacity of the cation exchange column reaches 0.3g/g, so that the resin dosage is reduced, and the acid-base dosage and the operation time are indirectly saved.

(3) In the invention, cytidine is adsorbed on a cation exchange column NH-1, the impurity washing concentration of an ammonia water solution is determined to be 0.03mol/L and the elution concentration is determined to be 0.06mol/L, and the optimal process parameters are determined and stably operated.

(4) The invention adopts the 300-800Da nanofiltration membrane for decolorization, can obtain clear light yellow solution, and adopts the 60-200Da membrane for concentration, and the process has no solid waste, high speed, high yield, no pollution and low cost.

(5) The crystallization system in the invention is a water system, and no reagent is added, thereby greatly reducing the crystallization cost. The crystallization process of the invention is completed within 12h to obtain bright white crystals, the yield of one-time crystallization is more than 50%, the yield of crystals is 90-95% after multiple crystallization or nested crystallization, meanwhile, the purity of the crystals is more than 99%, and the product quality, yield and cost are higher than those of the prior art.

(6) The method not only successfully realizes the problem of difficult separation of more impurities, but also has the advantages of high efficiency, low energy consumption, low cost, good quality, high yield and the like.

Drawings

The foregoing and/or other advantages of the invention will become further apparent from the following detailed description of the invention when taken in conjunction with the accompanying drawings.

FIG. 1 shows a crystalline product of cytidine.

FIG. 2 shows the cytidine crystal structure.

FIG. 3 is a liquid phase purity detection profile.

FIG. 4 is a liquid phase diagram of the original fermentation liquid.

Detailed Description

The experimental methods described in the following examples are all conventional methods unless otherwise specified; the reagents and materials are commercially available, unless otherwise specified.

The NH-1 resin described in the following examples was synthesized by Nanjing university of industry and was disclosed in Chinese patent CN106632519B, a process for separating nucleotides by continuous ion exchange chromatography, and other patents.

The cytidine-containing fermentation broth in the following examples is prepared by converting glucose, corn steep liquor, vitamins, inorganic salts, yeast extract and the like with escherichia coli to form cytidine-containing fermentation broth (with cytidine content of 32 g/L); the fermentation broth contains cytidine, and its components mainly comprise sugar, nucleic acid, salt, pigment, amino acids, protein, organic acid, etc., and its liquid phase is shown in FIG. 4.

The modified cation exchange resin NH-1 described in the following examples was prepared by mixing NH-1 resin with dichloroethane at a resin to solution volume ratio of 1: 6, maintaining the temperature at 70 ℃ for 1h, adding a silver sulfate catalyst accounting for 5 percent of the mass of the resin and 10 times of the volume of sulfuric acid, maintaining the temperature at 70 ℃ for 2h, heating to 90 ℃ for 3h, filtering to obtain modified cation resin, continuously washing the cation resin to be neutral by deionized water, then washing the cation resin by acetone, and washing the cation resin by water for later use. Static adsorption experiments show that the adsorption capacity of the modified resin is improved to 0.3g/g from the original 0.09g/g resin.

Example 1:

(1) adjusting pH of fermentation liquor containing cytidine to 2, filtering with 50nm ceramic membrane, collecting permeate, washing the retentate with tap water until the retentate contains no cytidine, and mixing to obtain ceramic membrane permeate.

(2) Loading the ceramic membrane permeate obtained in the step (1) to modified cation exchange resin NH-1215 g, adsorbing, washing impurities, eluting, and collecting eluent; the sampling rate is 1BV/h, and the sampling amount is 64g of cytidine fermentation broth clear liquid; in the impurity washing process, the concentration of ammonia water is 0.01mol/L, the flow rate is 1BV/h, and the impurity washing volume is 8 BV; in the elution process, the concentration of ammonia water is 0.05mol/L, the flow rate is 1BV/h, and the volume of elution is 14 BV.

(3) And (3) nano-filtering the eluent obtained in the step (2) by using a nano-filtration membrane of 300Da to remove macromolecular impurities such as pigments and the like, continuously washing the trapped fluid until the trapped fluid does not contain cytidine, and collecting the nano-filtration membrane permeate.

(4) Concentrating the permeate obtained in the step (3) to 50g/L through a nanofiltration membrane of 60Da, concentrating through evaporation to obtain cytidine with the concentration of 400g/L, adjusting the pH to 5.0 with hydrochloric acid, stirring at room temperature of 20 ℃ for 1h with seed crystal continuously at 200rpm, separating out crystals, and cooling to 4 ℃ in a gradient manner for 10 h.

(5) And (4) carrying out suction filtration on the crystals obtained in the step (4), removing the mother liquor, adding absolute ethyl alcohol, washing twice, and carrying out suction filtration to obtain the crystals.

(6) And (3) putting the crystals obtained in the step (5) into a vacuum drying oven at 40 ℃, and drying for 4h under 10mbar to obtain 35.6g of cytidine crystals, wherein the yield of cytidine once is 55.6%, the obtained cytidine product is shown in figure 1, the crystal structure is shown in figure 2, and the liquid phase is shown in figure 3.

(7) After the mother liquor obtained in the step (5) is crystallized for the second time and the third time, 15.62g of cytidine crystals and 6.45g of cytidine crystals are obtained after drying respectively, the purity of the cytidine crystals is 99.9%, and the total yield is 90.1%.

Comparative example 1

(1) Adjusting pH of fermentation liquor containing cytidine to 2, filtering with 50nm ceramic membrane, collecting permeate, washing the retentate with tap water until the retentate contains no cytidine, and mixing to obtain ceramic membrane permeate.

(2) Loading the ceramic membrane permeate obtained in the step (1) to 7650 g of cation exchange resin 001 x, adsorbing, washing impurities, eluting, and collecting eluent; the sampling rate is 1.5BV/h, and the sampling amount is 64g of cytidine fermentation broth clear liquid; in the impurity washing process, the concentration of ammonia water is 0.01mol/L, the flow rate is 1.5BV/h, and the impurity washing volume is 10 BV; in the elution process, the concentration of ammonia water is 0.05mol/L, the flow rate is 1BV/h, and the volume of elution is 15 BV.

(3) And (3) nano-filtering the eluent obtained in the step (2) by using a nano-filtration membrane of 300Da to remove macromolecular impurities such as pigments and the like, continuously washing the trapped fluid until the trapped fluid does not contain cytidine, and collecting the nano-filtration membrane permeate.

(4) Concentrating the permeate obtained in the step (3) to 50g/L through a nanofiltration membrane of 60Da, concentrating through evaporation to obtain cytidine with the concentration of 400g/L, adjusting the pH to 5.0 with hydrochloric acid, stirring at room temperature of 20 ℃ for 1h with seed crystal continuously at 200rpm, separating out crystals, and cooling to 4 ℃ in a gradient manner for 10 h.

(5) And (4) carrying out suction filtration on the crystals obtained in the step (4), removing the mother liquor, adding absolute ethyl alcohol, washing twice, and carrying out suction filtration to obtain the crystals.

(6) And (3) putting the crystals obtained in the step (5) into a vacuum drying oven at 40 ℃, and drying for 4 hours at 10mbar to obtain 30.7g of cytidine crystals, wherein the primary yield of cytidine is 47.9%.

(7) And (3) crystallizing the mother liquor obtained in the step (5) for two times and three times, and drying the crystals to obtain 15.65g and 7.4g of cytidine crystals respectively, wherein the purity of the cytidine crystals is 98.8 percent, and the total yield of the cytidine crystals is 84 percent.

The change of the resin causes the reduction of the adsorption amount and the separation degree, the increase of the resin usage amount, the increase of the volume of the whole eluent, the longer the flow time, and the more the acid-base amount of the regenerated resin.

Comparative example 2

(1) Adjusting pH of fermentation liquor containing cytidine to 2, filtering with 50nm ceramic membrane, collecting permeate, washing the retentate with tap water until the retentate contains no cytidine, and mixing to obtain ceramic membrane permeate.

(2) Loading the ceramic membrane permeate obtained in the step (1) to original cation exchange resin NH-1600 g, adsorbing, washing impurities, eluting, and collecting eluent; the sampling rate is 1.5BV/h, and the sampling amount is 64g of cytidine fermentation broth clear liquid; in the impurity washing process, the concentration of ammonia water is 0.01mol/L, the flow rate is 1.5BV/h, and the impurity washing volume is 10 BV; in the elution process, the concentration of ammonia water is 0.05mol/L, the flow rate is 1BV/h, and the volume of elution is 15 BV.

(3) And (3) nano-filtering the eluent obtained in the step (2) by using a nano-filtration membrane of 300Da to remove macromolecular impurities such as pigments and the like, continuously washing the trapped fluid until the trapped fluid does not contain cytidine, and collecting the nano-filtration membrane permeate.

(4) Concentrating the permeate obtained in the step (3) to 50g/L through a nanofiltration membrane of 60Da, concentrating through evaporation to obtain cytidine with the concentration of 400g/L, adjusting the pH to 5.0 with hydrochloric acid, stirring at room temperature of 20 ℃ for 1h with seed crystal continuously at 200rpm, separating out crystals, and cooling to 4 ℃ in a gradient manner for 10 h.

(5) And (4) carrying out suction filtration on the crystals obtained in the step (4), removing the mother liquor, adding absolute ethyl alcohol, washing twice, and carrying out suction filtration to obtain the crystals.

(6) And (3) putting the crystals obtained in the step (5) into a vacuum drying oven at 40 ℃, and drying for 4 hours at 10mbar to obtain 32.64g of cytidine crystals, wherein the primary yield of cytidine is 51%.

(7) And (3) crystallizing the mother liquor obtained in the step (5) for two times and three times, and drying the crystals to obtain 15.68g and 8g of cytidine crystals respectively, wherein the purity of the cytidine crystals is 99 percent, and the total yield of the cytidine crystals is 88 percent.

The adsorption amount is reduced due to the unmodified resin, the use amount of the resin is increased, the volume of the whole eluent is increased, the flow time is prolonged, and the acid-base amount of the regenerated resin is increased.

Example 2:

(1) adjusting pH of cytidine-containing fermentation liquor to 3.5, filtering with 200nm ceramic membrane, collecting permeate, washing the retentate with tap water until the retentate contains no cytidine, and mixing to obtain ceramic membrane permeate.

(2) Loading the ceramic membrane permeate obtained in the step (1) to modified cation exchange resin NH-1215 g, adsorbing, washing impurities, eluting, and collecting eluent; the sampling rate is 1.5BV/h, and the sampling amount is 64g of cytidine fermentation broth clear liquid; in the impurity washing process, the concentration of ammonia water is 0.05mol/L, the flow rate is 1.5BV/h, and the impurity washing volume is 10 BV; in the elution process, the concentration of ammonia water is 0.1mol/L, the flow rate is 2BV/h, and the volume of elution is 12 BV.

(3) And (3) nano-filtering the eluent obtained in the step (2) by using a nano-filtration membrane of 800Da to remove macromolecular impurities such as pigments and the like, continuously washing the trapped fluid until the trapped fluid does not contain cytidine, and collecting the nano-filtration membrane permeate.

(4) Concentrating the permeate obtained in the step (3) to 70g/L through a nanofiltration membrane of 200Da, concentrating through evaporation to obtain cytidine with concentration of 600g/L, adjusting pH to 10.0, adjusting the room temperature to 20 ℃, adding seed crystals, continuously stirring at 400rpm for 2h, separating out crystals, and cooling to 1 ℃ in a gradient manner for 10 h.

(5) And (4) carrying out suction filtration on the crystals obtained in the step (4), removing the mother liquor, adding absolute ethyl alcohol, washing twice, and carrying out suction filtration to obtain the crystals.

(6) And (3) putting the crystals obtained in the step (5) into a vacuum drying oven at 40 ℃, and drying for 4 hours at 10mbar to obtain 41.2g of cytidine crystals, wherein the primary yield of cytidine is 64.3%.

(7) And (3) crystallizing the mother liquor obtained in the step (5) for two times and three times, and drying the crystals to obtain 14.64g and 5g of cytidine crystals respectively, wherein the purity of the cytidine crystals is 99.1 percent, and the total yield of the cytidine crystals is 95 percent.

Example 3:

(1) adjusting pH of fermentation liquid containing cytidine to 2.5, filtering with 100nm ceramic membrane, collecting permeate, washing the retentate with tap water until the retentate contains no cytidine, and mixing to obtain ceramic membrane permeate.

(2) Loading the ceramic membrane permeate obtained in the step (1) to modified cation exchange resin NH-1215 g, adsorbing, washing impurities, eluting, and collecting eluent; the sampling rate is 2BV/h, and the sampling amount is 64g of cytidine to obtain fermentation liquor; in the impurity washing process, the concentration of ammonia water is 0.03mol/L, the flow rate is 2BV/h, and the impurity washing volume is 14 BV; in the elution process, the concentration of ammonia water is 0.06mol/L, the flow rate is 1.5BV/h, and the volume of elution is 13 BV.

(3) And (3) nano-filtering the eluent obtained in the step (2) by using a nano-filtration membrane of 500Da to remove macromolecular impurities such as pigments and the like, continuously washing the trapped fluid until the trapped fluid does not contain cytidine, and collecting the nano-filtration membrane permeate.

(4) Concentrating the permeate obtained in the step (3) to 60g/L through a nanofiltration membrane of 90Da, concentrating through evaporation to obtain a cytidine concentration of 500g/L, adjusting the pH to 7.0, adjusting the room temperature to 20 ℃, adding seed crystals, continuously stirring at 400rpm for 1.5h, separating out crystals, and carrying out gradient cooling to 4 ℃ for 10 h.

(5) And (4) carrying out suction filtration on the crystals obtained in the step (4), removing the mother liquor, adding absolute ethyl alcohol, washing twice, and carrying out suction filtration to obtain the crystals.

(6) And (3) putting the crystals obtained in the step (5) into a vacuum drying oven at 40 ℃, and drying for 4 hours at 10mbar to obtain 38.1g of cytidine crystals, wherein the primary yield of cytidine is 59.5%.

(7) After the mother liquor obtained in the step (5) is crystallized for the second time and the third time, 15.28g of cytidine crystals and 6.27g of cytidine crystals are obtained after drying, the purity of the cytidine crystals is 99.6%, and the total yield is 93.2%.

Example 4:

(1) adjusting pH of fermentation liquid containing cytidine to 3.0, filtering with 150nm ceramic membrane, collecting permeated liquid, washing trapped liquid with tap water until the trapped liquid contains no cytidine, and mixing to obtain ceramic membrane permeated liquid.

(2) Loading the ceramic membrane permeate obtained in the step (1) to modified cation exchange resin NH-1215 g, adsorbing, washing impurities, eluting, and collecting eluent; the sampling rate is 1.5BV/h, and the sampling amount is 64g of cytidine fermentation broth clear liquid; in the impurity washing process, the concentration of ammonia water is 0.02mol/L, the flow rate is 1.5BV/h, and the impurity washing volume is 11 BV; in the elution process, the concentration of ammonia water is 0.07mol/L, the flow rate is 1BV/h, and the volume of elution is 12.5 BV.

(3) And (3) nano-filtering the eluent obtained in the step (2) by using a nano-filtration membrane of 400Da to remove macromolecular impurities such as pigments and the like, continuously washing the trapped fluid until the trapped fluid does not contain cytidine, and collecting the nano-filtration membrane permeate.

(4) Concentrating the permeate obtained in the step (3) to 65g/L through a nanofiltration membrane of 150Da, concentrating through evaporation to obtain cytidine with the concentration of 450g/L, adjusting the pH to 7.0, adjusting the room temperature to 20 ℃, adding seed crystals, continuously stirring at 400rpm for 0.5h, separating out crystals, and carrying out gradient cooling to 10 ℃ for 10 h.

(5) And (4) carrying out suction filtration on the crystals obtained in the step (4), removing the mother liquor, adding absolute ethyl alcohol, washing twice, and carrying out suction filtration to obtain the crystals.

(6) And (3) putting the crystals obtained in the step (5) into a vacuum drying oven at 40 ℃, and drying for 4 hours at 10mbar to obtain 36.1g of cytidine crystals, wherein the primary yield of the glycoside is 56.4%.

(7) After the mother liquor obtained in the step (5) is crystallized for the second time and the third time, 15.63g of cytidine crystals and 6.45g of cytidine crystals are obtained after drying respectively, the purity of the cytidine crystals is 99.4%, and the total yield is 90.9%.

The method innovatively adopts fermentation liquor to directly pass through a membrane, adopts three sets of membrane systems to carry out coarse separation to remove thalli and macromolecular impurities, and carries out one-step refined chromatographic separation by cation exchange resin synthesized in a laboratory, wherein the maximum cytidine adsorption amount is 0.3 g/g; and the optimal impurity concentration and elution concentration are determined: in the impurity washing process, the concentration of ammonia water is 0.03 mol/L; in the elution process, the concentration of ammonia water is 0.06 mol/L. Concentrating the eluent, adding a crystal seed water system for crystallization, and washing and drying crystals to obtain the product. The whole separation yield is more than 90 percent, the purity is higher than 99 percent, the yield is better than that of the prior art, and the separation cost is low.

The present invention provides a method and a concept for separating and extracting cytidine from fermentation broth, and a method and a way for implementing the technical scheme are numerous, and the above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, a plurality of improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be regarded as the protection scope of the present invention. All the components not specified in the present embodiment can be realized by the prior art.