Method for separating and purifying citicoline sodium from bacillus subtilis fermentation liquor

Abstract

The invention belongs to the field of downstream treatment of synthetic biology technology, and provides a method for separating and purifying citicoline sodium from bacillus subtilis fermentation liquor. According to the method provided by the invention, the bacillus subtilis fermentation liquor for producing citicoline sodium is used as a raw material, and separation and purification optimization is performed through calcium salt coordination and graded alcohol precipitation, so that small molecular protein, amino acid and salt are effectively removed, the yield of citicoline sodium is increased by 6-11% compared with that of a traditional process, the production cost of citicoline sodium is reduced in a unified manner, and the method is suitable for large-scale production of citicoline sodium. And the product quality is improved.

Description

Technical Field

[0001] The present invention belongs to the field of downstream processing of synthetic biology technology, and particularly relates to a method for separating and purifying cytidine-5'-diphosphate choline sodium from Bacillus subtilis fermentation broth. Background Art

[0002] As a nucleoside derivative, the separation and purification process of cytidine-5'-diphosphate choline has long been restricted by its high water solubility and similar properties to intracellular components, resulting in significant defects in traditional separation methods. In existing production processes, the downstream separation steps of chemical synthesis, fermentation, and enzymatic synthesis methods all rely on anion-cation exchange resins. This process not only generates a large amount of industrial wastewater, prolongs the production cycle, but also requires a complex wastewater treatment system. Although some studies have attempted to achieve cyclic separation through ultrafiltration membrane / nanofiltration membrane pore size screening, the problem of insufficient recovery rate seriously weakens the product competitiveness.

[0003] In recent years, the breakthrough development of synthetic biology technology has provided a revolutionary solution for the production of cytidine-5'-diphosphate choline. By constructing a cell factory for enzymatic conversion, not only the production process is simplified and the conversion cost is reduced, but also it better conforms to the trend of green and low-carbon industry transformation. Aiming at the separation problem of cytidine-5'-diphosphate choline, the present invention innovatively proposes an efficient purification method, establishing a separation window by using the differences in specific physical and chemical properties between it and impurities, effectively breaking through the technical bottlenecks of low recovery rate and high pollution in traditional processes. This method not only significantly improves the product purity and yield of cytidine-5'-diphosphate choline sodium, but also provides a general technical paradigm for the downstream processing of synthetic biology products, opening up a new path for solving similar separation dilemmas in the field of biomanufacturing. Summary of the Invention

[0004] In order to solve the above technical problems, the present invention provides a method for separating and purifying cytidine-5'-diphosphate choline sodium from Bacillus subtilis fermentation broth. This method has high product recovery rate, is simple to operate, suitable for industrialization, has low requirements for equipment, and can effectively solve the problem of difficult separation of impurities with similar physical and chemical properties such as nucleotides, amino acids, and small molecular proteins during the separation and purification of cytidine-5'-diphosphate choline sodium, improving product quality and reducing production costs.

[0005] The technical solution of the present invention is as follows: A method for separating and purifying cytidine-5'-diphosphate choline sodium from Bacillus subtilis fermentation broth, comprising the following steps: (1) Adding a filter aid to the Bacillus subtilis fermentation broth, stirring for 1.5 - 2 h, performing solid-liquid separation to collect the thalli, adding purified water to the thalli to resuspend, and after cell wall breaking treatment, filtering to obtain a first microfiltrate; (2) Adding a calcium salt to the first microfiltrate for reaction, then filtering to obtain a second microfiltrate, and subjecting the second microfiltrate to ultrafiltration to obtain a first concentrate; (3) Under acidic conditions, the first concentrated solution is decolorized and then filtered to obtain a decolorized solution, which is concentrated under reduced pressure to obtain a second concentrated solution; The second concentrated solution is subjected to one-time ethanol precipitation, stirred for 1.5 - 2 h, allowed to stand and then filtered to collect the first supernatant; (4) After the first supernatant is concentrated under reduced pressure, under alkaline conditions, it is subjected to secondary ethanol precipitation, stirred for 1.5 - 2 h, allowed to stand and then filtered to obtain a second supernatant. The pH of the second supernatant is adjusted to neutral, and then filtered to obtain a nanofiltration concentrated solution; (5) The nanofiltration concentrated solution is concentrated under reduced pressure, crystallized, and dried to obtain pure sodium cytidine diphosphate choline.

[0006] Preferably, in (1), the fermentation broth source is the fermentation broth produced during the synthesis of sodium cytidine diphosphate choline by Bacillus subtilis fermentation; the filter aid is selected from any one of diatomaceous earth and perlite; the mass - volume ratio of the filter aid to the fermentation broth is (30 - 80) g: 1 L.

[0007] Preferably, in (1), the volume ratio of purified water to the fermentation broth is 0.5 - 1.0: 1; the operation of cell wall breaking treatment is to stir at 65 - 100 °C for 0.5 - 2.0 h.

[0008] Preferably, in (2), the calcium salt is selected from any one of calcium chloride, calcium carbonate, and calcium bicarbonate; the molar ratio of the calcium salt to cytidine diphosphate choline is 1.5 - 2.0: 1; the calcium salt reaction is carried out at pH 6.5 - 7.5 and 70 - 80 °C.

[0009] Preferably, in (2), the molecular weight cut - off of the ultrafiltration membrane used during ultrafiltration is 2k - 10 kDa; the material of the ultrafiltration membrane is a polyethersulfone composite containing sulfonic acid groups.

[0010] Preferably, in (3), oxalic acid is used to adjust the first concentrated solution to acidic conditions, with a pH of 3.0 - 4.5; the content of sodium cytidine diphosphate choline in the second concentrated solution is 55 - 150 g / L; during one - time ethanol precipitation, the volume ratio of ethanol to the second concentrated solution is 0.5 - 2: 1.

[0011] Preferably, in (4), after the first supernatant is concentrated under reduced pressure, the concentration of sodium cytidine diphosphate choline is 100 - 250 g / L; sodium hydroxide is used to adjust it to alkaline conditions, with a pH of 7 - 12.

[0012] Preferably, in (4), during secondary ethanol precipitation, the volume ratio of ethanol to the concentrated first supernatant is 0.5 - 2: 1.

[0013] Preferably, in (4), the pH of the second supernatant is adjusted to 6.0 - 7.5 with hydrochloric acid, and then nanofiltration is carried out through a 150 - 500 Da nanofiltration membrane to obtain a nanofiltration concentrated solution.

[0014] The present invention has the following advantages and effects compared with the prior art: (1) Compared with the processes reported in the existing relevant literature, the yield of the sodium cytidine diphosphate product separated and purified by the method mentioned in the present invention is 6 - 11% higher, and the product content and purity are both relatively high. This separation and purification method helps to reduce the production cost of sodium cytidine diphosphate and improve the product quality; (2) The method of the present invention utilizes the coordination ability of calcium ions. By controlling the pH value of the solution, the nucleotide phosphate groups are in an ionized state and carry negative charges, which can interact with the positively charged calcium ions to form stable coordination compounds. Before ultrafiltration, the present invention pretreats the filtrate by adding calcium salts to make cytidine diphosphate form stable calcium salt complexes. In addition, according to relevant research reports, for UF membranes with strongly acidic sulfonic acid groups, the strongly acidic sulfonic acid groups will be deprotonated and carry negative charges at neutral pH. The negatively charged groups of the membrane interact with positively charged cations (such as cation Ca 2+ , Cu 2+ ), and the poor retention of cations is caused by Coulomb interaction. Therefore, the ultrafiltration process involved in the present invention can retain the calcium salt of cytidine diphosphate while removing some small molecule proteins, amino acids, salts, etc., and can significantly increase the content of sodium cytidine diphosphate; (3) The present invention adopts the method of fractional alcohol precipitation. By adding ethanol to change the polarity of the solution, some impurity components with high solubility in water will precipitate due to the decrease in solubility. Compared with the traditional alcohol precipitation process, after concentrating the feed liquid by an appropriate multiple, this method utilizes the differences in the solubility and isoelectric point of different impurities in the ethanol solution, and removes different types of impurities by controlling the amount of ethanol added and different acid and alkalinity. This not only greatly reduces the amount of ethanol used, but also helps to remove impurities with similar physicochemical properties such as proteins, polypeptides, and oligonucleotides. Description of the Drawings

[0015] Figure 1 is the crystal form diagram after the crystallization of sodium cytidine diphosphate; Figure 2 is the HPLC chromatogram of the pure sodium cytidine diphosphate product in Example 1; Figure 3 is the HPLC chromatogram of the pure sodium cytidine diphosphate product in Example 2; Figure 4 is the HPLC chromatogram of the pure sodium cytidine diphosphate product in Example 3; Figure 5 is the HPLC chromatogram of the pure sodium cytidine diphosphate product in Example 4; Figure 6 is the HPLC chromatogram of the pure sodium cytidine diphosphate product in Example 5; Figure 7 is the HPLC chromatogram of the pure sodium cytidine diphosphate product in Example 6. Detailed Embodiments

[0016] To enable those skilled in the art to better understand the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0017] Example 1 In the embodiments of the present invention, the Bacillus subtilis fermentation broth refers to the fermentation broth produced during the fermentation of Bacillus subtilis to produce cytidine 5'-diphosphate choline sodium.

[0018] A method for separating and purifying cytidine 5'-diphosphate choline sodium from Bacillus subtilis fermentation broth, comprising the following steps: (1) Add 1.9 kg of diatomaceous earth to 37 L of Bacillus subtilis fermentation broth, stir for 2 h, perform solid-liquid separation with a chamber plate and frame filter press, collect the cells, add 19 L of purified water to the cells, control the temperature at 65 °C, keep stirring for 2.0 h for cell wall breaking, and obtain the first microfiltrate through ceramic membrane microfiltration; (2) Add 126.2 g of calcium chloride to the first filtrate, adjust the pH to 6.5, the temperature to 70 °C, stir and react for 1 h, filter with a 0.45 μm microfiltration membrane to obtain the second microfiltrate, and obtain the first concentrated solution after ultrafiltration with a 2 kDa ultrafiltration membrane; (3) Adjust the pH of the first concentrated solution to 3.0 with oxalic acid, add activated carbon, stir at 45 °C for 1.5 h and then filter to obtain the decolorized solution, concentrate under reduced pressure at 45 °C until the cytidine 5'-diphosphate choline sodium is 55 g / L and the volume is 5.4 L, stop concentration, and obtain the second concentrated solution; Add 2.8 L of ethanol to the second concentrated solution for the first ethanol precipitation, stir for 2 h, after standing, filter by vacuum with a 0.45 μm microfiltration membrane, and collect the first supernatant; (4) Concentrate the first supernatant under reduced pressure until the cytidine 5'-diphosphate choline sodium is 100 g / L and the volume is 3 L, stop concentration, adjust the pH to 9.5, add 3 L of ethanol for the second ethanol precipitation, stir for 2 h, after standing, filter by vacuum with a 0.45 μm microfiltration membrane, collect the second supernatant, adjust the pH of the second supernatant to 6.3, and obtain the nanofiltration concentrated solution through a 150 Da nanofiltration membrane; (5) Concentrate the nanofiltration concentrated solution under reduced pressure, and then through crystallization and drying, obtain the pure product of cytidine 5'-diphosphate choline sodium.

[0019] Detected by HPLC, the purity of cytidine 5'-diphosphate choline sodium is 99.900%, the content is 98.2% (dried), and the yield is 69.4%.

[0020] The pure product of cytidine 5'-diphosphate choline sodium obtained in the embodiments of the present invention is detected by HPLC, and the detection conditions of HPLC are as follows: Chromatographic column: C18 chromatographic column; Ultraviolet detector; Detection wavelength: 276 nm; Mobile phase: A mobile phase of phosphate buffer [a solution of potassium dihydrogen phosphate at 0.1 mol / L and tetrabutylammonium solution (a solution of tetrabutylammonium hydroxide at 0.01 mol / L adjusted to pH 4.5 with phosphate buffer) in equal amounts] - methanol (95∶5).

[0021] Example 2 A method for separating and purifying cytidine 5'-diphosphate choline sodium from Bacillus subtilis fermentation broth, comprising the following steps: (1) Add 2.4 kg of diatomaceous earth to 40 L of Bacillus subtilis fermentation broth, stir for 1.5 h, perform solid-liquid separation with a chamber filter press, collect the thalli, add 20 L of purified water to the thalli, control the temperature at 70 °C, keep stirring for 2.0 h for cell wall breaking, and obtain the first microfiltrate through ceramic membrane microfiltration; (2) Add 110.5 g of calcium chloride to the first filtrate, adjust the pH to 6.7, keep the temperature at 80 °C, stir and react for 1.5 h, filter with a 0.45 μm microfiltration membrane to obtain the second microfiltrate, and obtain the first concentrate after ultrafiltration treatment with a 3000 Da ultrafiltration membrane; (3) Adjust the pH of the first concentrate to 3.5 with oxalic acid, add activated carbon, stir at 50 °C for 1.5 h and then filter to obtain the decolorized solution, concentrate under reduced pressure at 45 °C to 100 g / L of cytidine 5'-diphosphate choline sodium and a volume of 3.2 L, stop concentration, and obtain the second concentrate; Add 3.2 L of ethanol to the second concentrate for the first ethanol precipitation, stir for 2 h, after standing, filter by vacuum using a 0.45 μm microfiltration membrane, and collect the first supernatant; (4) Concentrate the first supernatant under reduced pressure to 140 g / L of cytidine 5'-diphosphate choline sodium and a volume of 2.3 L, stop concentration, adjust the pH to 9.5, add 2.3 L of ethanol for the second ethanol precipitation, stir for 2 h, after standing, filter by vacuum using a 0.45 μm microfiltration membrane, collect the second supernatant, adjust the pH of the second supernatant to 6.1, and obtain the nanofiltration concentrate through a 300 Da nanofiltration membrane; (5) Concentrate the nanofiltration concentrate under reduced pressure, and then through crystallization and drying, obtain pure cytidine 5'-diphosphate choline sodium.

[0022] After detection, the purity of cytidine 5'-diphosphate choline sodium is 99.707%, the content is 97.8%, and the recovery rate is 70.4%.

[0023] Example 3 A method for separating and purifying cytidine 5'-diphosphate choline sodium from Bacillus subtilis fermentation broth, comprising the following steps: (1) Add 2.4 kg of diatomaceous earth to 40 L of Bacillus subtilis fermentation broth, stir for 1.5 h, perform solid-liquid separation with a chamber filter press, collect the thalli, add 20 L of purified water to the thalli, control the temperature at 70 °C, keep stirring for 2.0 h for cell wall breaking, and obtain the first microfiltrate through ceramic membrane microfiltration; (2) Add 132.6 g of calcium chloride to the first filtrate, adjust the pH to 6.7, the temperature to 70°C, stir and react for 1 hour, filter with a 0.45 μm microfiltration membrane to obtain a second microfiltrate, and then ultrafilter the second microfiltrate to obtain a first concentrated solution at 2500 Da. (3) The first concentrated solution was adjusted to pH 3.3 with oxalic acid, activated carbon was added, and the solution was stirred at 48°C for 1.5 h and filtered to obtain a decolorized solution, which was then concentrated at 45°C under reduced pressure to 90 g / L of citicoline sodium and a volume of 3.6 L. The concentration was stopped to obtain a second concentrated solution. Add 2.88 L of ethanol to the second concentrated solution for alcohol precipitation, stir for 1.5 h, let stand, and then vacuum filter with a 0.45 μm microfiltration membrane to collect the first supernatant; (4) The first supernatant was concentrated under reduced pressure to 125 g / L of citicoline sodium and a volume of 2.5 L. The concentration was stopped and the pH was adjusted to 9.5. 2 L of ethanol was added for secondary alcohol precipitation. The mixture was stirred for 2 h. After standing, the mixture was vacuum filtered using a 0.45 μm microfiltration membrane to collect the second supernatant. The pH of the second supernatant was adjusted to 6.5 and filtered through a 200 Da nanofiltration membrane to obtain a nanofiltration concentrate. (5) The nanofiltration concentrate is concentrated under reduced pressure, crystallized, and dried to obtain pure citicoline sodium.

[0024] After testing, the purity of citicoline sodium was 99.897%, the content was 99.1%, and the yield was 69.9%.

[0025] Example 4 A method for separating and purifying citicoline sodium from a Bacillus subtilis fermentation broth comprises the following steps: (1) 3 kg of diatomaceous earth was added to 38 L of Bacillus subtilis fermentation liquid, stirred for 2 h, and solid-liquid separation was performed using a box-type plate and frame filter press to collect the bacterial cells. 20 L of purified water was added to the bacterial cells, the temperature was controlled at 80 °C, and the mixture was stirred for 1 h to break the bacterial cell wall. The first microfiltrate was obtained by microfiltration through a ceramic membrane; (2) Add 126.8 g of calcium chloride to the first filtrate, adjust the pH to 7.0, the temperature to 68°C, stir and react for 1 hour, filter with a 0.45 μm microfiltration membrane to obtain a second microfiltrate, and then ultrafilter the second microfiltrate to obtain a first concentrated solution at 2500 Da. (3) The first concentrated solution was adjusted to pH 4.0 with oxalic acid, activated carbon was added, and the solution was stirred at 48°C for 1.5 h and filtered to obtain a decolorized solution, which was then concentrated under reduced pressure at 49°C to 122 g / L of citicoline sodium and a volume of 2.6 L. The concentration was stopped to obtain a second concentrated solution. Add 2.8 L of ethanol to the second concentrated solution for alcohol precipitation, stir for 2 h, let stand, and then vacuum filter with a 0.45 μm microfiltration membrane to collect the first supernatant; (4) The first supernatant was concentrated under reduced pressure until the concentration of citicoline sodium reached 200 g / L and the volume was 1.6 L. The concentration was stopped, the pH was adjusted to 9.5, 1.4 L of ethanol was added for secondary alcohol precipitation, stirred for 2 h, and after standing, it was filtered by a 0.45 μm microfiltration membrane under vacuum to collect the second supernatant. The pH of the second supernatant was adjusted to 6.7, and a nanofiltration concentrate was obtained through a 200 Da nanofiltration membrane; (5) The nanofiltration concentrate was concentrated under reduced pressure, and then through crystallization and drying, pure citicoline sodium was obtained. After detection, the purity of citicoline sodium was 99.798%, the content was 97.5%, and the yield was 73.5%.

[0026] Example 5 A method for separating and purifying citicoline sodium from Bacillus subtilis fermentation broth, comprising the following steps: (1) 1.8 kg of diatomaceous earth was added to 5 L of Bacillus subtilis fermentation broth, stirred for 2 h, and solid-liquid separation was carried out with a chamber plate and frame filter press to collect the bacterial cells. 19 L of purified water was added to the bacterial cells, the temperature was controlled at 75 °C, and stirring was carried out for 1.4 h for cell wall breaking, and the first microfiltrate was obtained through ceramic membrane microfiltration; (2) 128.9 g of calcium chloride was added to the first microfiltrate, the pH was adjusted to 6.7, the temperature was 67 °C, and stirring reaction was carried out for 1.2 h, and then filtered through a 0.45 μm microfiltration membrane to obtain the second microfiltrate. The second microfiltrate was ultrafiltered through a 1 kDa ultrafiltration membrane to obtain the first concentrate; (3) The pH of the first concentrate was adjusted to 4.5 with oxalic acid, activated carbon was added, stirred for 2 h at 40 °C and then filtered to obtain a decolorized solution, and concentrated under reduced pressure to 112 g / L of citicoline sodium and a volume of 2.5 L at 49 °C. The concentration was stopped to obtain the second concentrate; 2.8 L of ethanol was added to the second concentrate for primary alcohol precipitation, stirred for 2 h, and after standing, it was filtered by a 0.45 μm microfiltration membrane under vacuum to collect the first supernatant; (4) The first supernatant was concentrated under reduced pressure until the concentration of citicoline sodium reached 190 g / L and the volume was 1.4 L. The concentration was stopped, the pH was adjusted to 10, 1 L of ethanol was added for secondary alcohol precipitation, stirred for 2 h, and after standing, it was filtered by a 0.45 μm microfiltration membrane under vacuum to collect the second supernatant. The pH of the second supernatant was adjusted to 6.8, and a nanofiltration concentrate was obtained through a 300 Da nanofiltration membrane; (5) The nanofiltration concentrate was concentrated under reduced pressure, and then through crystallization and drying, pure citicoline sodium was obtained. After detection, the purity of citicoline sodium was 99.839%, the content was 97.5%, and the yield was 71.1%.

[0027] Example 6 A method for separating and purifying citicoline sodium from Bacillus subtilis fermentation broth, comprising the following steps: (1) 1.7 kg of diatomaceous earth was added to 22 L of Bacillus subtilis fermentation broth, and the mixture was stirred for 2 h. Solid-liquid separation was carried out using a box-type plate and frame filter press to collect the bacterial cells. 22 L of purified water was added to the bacterial cells, the temperature was controlled at 79 °C, and the mixture was stirred for 1 h for cell wall breaking. The first microfiltrate was obtained by ceramic membrane microfiltration. (2) 72.9 g of calcium chloride was added to the first microfiltrate, the pH was adjusted to 7, the temperature was 67 °C, and the mixture was stirred and reacted for 1 h. The mixture was filtered through a 0.45 μm microfiltration membrane to obtain the second microfiltrate. The second microfiltrate was ultrafiltered through a 1 kDa ultrafiltration membrane to obtain the first concentrate. (3) The pH of the first concentrate was adjusted to 4.1 with oxalic acid, activated carbon was added, and the mixture was stirred for 2 h at 38 °C and then filtered to obtain the decolorized solution. The solution was concentrated under reduced pressure at 50 °C until the concentration of cytidine 5'-diphosphate choline sodium was 75 g / L and the volume was 2.3 L. The concentration was stopped to obtain the second concentrate. 2.5 L of ethanol was added to the second concentrate for the first ethanol precipitation, and the mixture was stirred for 2 h. After standing, the mixture was filtered by vacuum using a 0.45 μm microfiltration membrane to collect the first supernatant. (4) The first supernatant was concentrated under reduced pressure until the concentration of cytidine 5'-diphosphate choline sodium was 150 g / L and the volume was 1.2 L. The concentration was stopped, the pH was adjusted to 10, 1 L of ethanol was added for the second ethanol precipitation, and the mixture was stirred for 2 h. After standing, the mixture was filtered by vacuum using a 0.45 μm microfiltration membrane to collect the second supernatant. The pH of the second supernatant was adjusted to 6.8, and the nanofiltration concentrate was obtained through a 300 Da nanofiltration membrane. (5) The nanofiltration concentrate was concentrated under reduced pressure, and then crystallized and dried to obtain pure cytidine 5'-diphosphate choline sodium. After detection, the purity of cytidine 5'-diphosphate choline sodium was 99.946%, the content was 99.2%, and the yield was 70.5%.

[0028] In summary, the method for separating and purifying cytidine 5'-diphosphate choline sodium from Bacillus subtilis fermentation broth provided by the present invention uses the Bacillus subtilis fermentation broth producing cytidine 5'-diphosphate choline sodium as the raw material, and through the optimization of calcium salt coordination and fractional ethanol precipitation for separation and purification, effectively removes small molecule proteins, amino acids and salts. Finally, the obtained cytidine 5'-diphosphate choline sodium has a purity as high as 99.839%, a content of 97.5%, and a yield of 71.1%.

[0029] The above are only the preferred embodiments of the present invention, and do not limit the patent scope of the present invention accordingly. All equivalent changes and modifications made according to the scope of the present invention should still fall within the scope covered by the present invention.

Claims

1. A method for separating and purifying citicoline sodium from Bacillus subtilis fermentation broth, characterized in that, It includes the following steps: (1) Add a filter aid to the Bacillus subtilis fermentation broth, stir for 1.5 - 2 h, perform solid-liquid separation to collect the thalli, add purified water to the thalli for resuspension, and after cell wall breaking treatment, filter to obtain the first microfiltrate; (2) Add a calcium salt to the first microfiltrate and react, then filter to obtain the second microfiltrate, and the second microfiltrate is ultrafiltered to obtain the first concentrate; (3) Under acidic conditions, the first concentrate is decolorized and then filtered to obtain the decolorized solution, and it is concentrated under reduced pressure to obtain the second concentrate; Perform primary ethanol precipitation on the second concentrate, stir for 1.5 - 2 h, let it stand and then filter to collect the first supernatant; (4) After the first supernatant is concentrated under reduced pressure, under alkaline conditions, perform secondary ethanol precipitation, stir for 1.5 - 2 h, let it stand and then filter to obtain the second supernatant, adjust the pH of the second supernatant to neutral, and filter to obtain the nanofiltration concentrate; (5) Concentrate the nanofiltration concentrate under reduced pressure, crystallize, and dry to obtain pure cytidine 5'-diphosphate choline sodium.

2. The method according to claim 1, characterized in that, The source of the fermentation broth described in (1) is the fermentation broth produced during the synthesis of cytidine 5'-diphosphate choline sodium by Bacillus subtilis fermentation; The filter aid is selected from any one of diatomite and perlite; The mass-volume ratio of the filter aid to the fermentation broth is (30 - 80) g : 1 L.

3. The method according to claim 1, characterized in that The volume ratio of the purified water to the fermentation broth described in (1) is 0.5 - 1.0 : 1; The operation of cell wall breaking treatment is to stir at 65 - 100 °C for 0.5 - 2.0 h.

4. The method according to claim 1, wherein The calcium salt described in (2) is selected from any one of calcium chloride, calcium carbonate, and calcium bicarbonate; The molar ratio of the calcium salt to cytidine 5'-diphosphate choline is 1.5 - 2.0 : 1; The calcium salt reaction is carried out at pH 6.5 - 7.5 and 70 - 80 °C.

5. The method according to claim 1, characterized in that, The molecular weight cut-off of the ultrafiltration membrane used during ultrafiltration described in (2) is 2k - 10 kDa; The material of the ultrafiltration membrane is a polyethersulfone composite containing sulfonic acid groups.

6. The method according to claim 1, wherein In (3), oxalic acid is used to adjust the first concentrate to acidic conditions with a pH of 3.0 - 4.5; The content of cytidine 5'-diphosphate choline sodium in the second concentrate is 55 - 150 g / L; During primary ethanol precipitation, the volume ratio of ethanol to the second concentrate is 0.5 - 2 :

1.

7. The method according to claim 1, wherein After the first supernatant described in (4) is concentrated under reduced pressure, the concentration of cytidine 5'-diphosphate choline sodium is 100 - 250 g / L; It is adjusted to alkaline conditions with a pH of 7 - 12 using sodium hydroxide.

8. The method according to claim 1, characterized in that, During the secondary ethanol precipitation described in (4), the volume ratio of ethanol to the concentrated first supernatant is 0.5 - 2 :

1.

9. The method according to claim 1, characterized in that, The pH of the second supernatant described in (4) is adjusted to 6.0 - 7.5 using hydrochloric acid, and it is nanofiltrated through a 150 - 500 Da nanofiltration membrane to obtain the nanofiltration concentrate.

10. A method for separating and purifying citicoline sodium from Bacillus subtilis fermentation broth, characterized in that, It includes the following steps: (1) Add 3 kg of diatomite to 38 L of Bacillus subtilis fermentation broth, stir for 2 h, perform solid-liquid separation to collect the thalli, add 20 L of purified water to the thalli for resuspension, stir at 80 °C for 1.0 h, and use a 0.45 μm ceramic membrane for microfiltration to obtain the first microfiltrate; (2) Add 126.8 g of calcium chloride to the first microfiltrate, stir and react at pH 7.0 and 68 °C for 1 h, and filter with a 0.45 μm microfiltration membrane to obtain the second microfiltrate; The second microfiltrate is ultrafiltered through 2500 Da to obtain the first concentrate; (3) The pH of the first concentrated solution was adjusted to 4.0 with oxalic acid, activated carbon was added, and after stirring at 48 °C for 1.5 h, it was filtered to obtain a decolorized solution, which was concentrated under reduced pressure at 49 °C until the content of cytidine 5'-diphosphate choline sodium was 122 g / L to obtain a second concentrated solution; 2.8 L of ethanol was added to the second concentrated solution for primary alcohol precipitation, and it was stirred for 2 h. After standing, it was filtered by vacuum using a 0.45 μm microfiltration membrane, and the first supernatant was collected; (4) The first supernatant was concentrated under reduced pressure until the content of cytidine 5'-diphosphate choline sodium was 200 g / L, the pH was adjusted to 9.5, 1.4 L of ethanol was added for secondary alcohol precipitation, and it was stirred for 2 h. After standing, it was filtered by vacuum using a 0.45 μm microfiltration membrane to obtain a second supernatant. The pH was adjusted to 6.7, and a nanofiltration concentrated solution was obtained through a 200 Da nanofiltration membrane; (5) After the nanofiltration concentrated solution was concentrated under reduced pressure, crystallized, and dried, pure cytidine 5'-diphosphate choline sodium was obtained.

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