CN109836468A - A method of the purifying citicoline sodium from microbial fermentation solution - Google Patents
A method of the purifying citicoline sodium from microbial fermentation solution Download PDFInfo
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Abstract
The method of the invention discloses a kind of from microbial fermentation solution purifying citicoline sodium, include the following steps: (1) citicoline fermentation liquid, heated, flocculation, separation of solid and liquid obtains clear liquid, and clear liquid pre-processes to obtain pretreatment fluid through ultrafiltration, nanofiltration continous way；(2) strong-base anion-exchange resin pre-column on pretreatment fluid is pre-processed, efflux is adsorbed through strong-base anion-exchange resin purification column, it is eluted again through sodium chloride solution, after high-purity eluent is merged, by being concentrated under reduced pressure, alcohol precipitation crystallizes to obtain citicoline sodium crude product；(3) citicoline sodium crude product activity carbon decoloring, filtering, concentration, alcohol precipitation crystallization, vacuum drying are obtained into citicoline sodium finished product, quality is good, high income, at low cost, pollution is few.
Description
Technical field
The invention belongs to biochemistry separation technology fields, and in particular, to one kind separates pure from microbial fermentation solution
Change the method for citicoline sodium.
Background technique
Citicoline sodium (Fig. 1) is nucleoside derivates, can increase brain blood flow by reducing cerebral vascular resistance and promote
Metabolism of brain improves Brain circlulation.In addition, the function of reticular formation of brain stem ascending activating system can be enhanced, enhance pyramidal system
Function, improve motor paralysis, therefore to promote cerebral function recovery and promote revive, have certain effect.It is clinically used for acute
The disturbance of consciousness after craniocerebral trauma and brain surgery.
About the production of citicoline sodium, mainly there is (1) chemical synthesis；(2) enzyme' s catalysis；(3) microbial fermentation closes
It is several at conversion etc..For isolating and purifying for citicoline sodium, that reports both at home and abroad is fewer, traditional separation purifying technique
Not only process is many and diverse, yield is low, separation costs are relatively high, but also larger to the pollution of environment.Open (CN101130797A) such as swords
It proposes carbon column on conversion fluid, pure water rinsing carbon column, then eluted with ethyl alcohol-aqueous slkali, collects, then carry out macropore amberlite
Rouge purifying.Form of this carbon column in conjunction with exchange column, carbon column, which extremely adsorbs, seriously causes yield low, the elution of carbon column, resin regeneration
A large amount of waste water is generated, sewage load is very big.Hu Jianrong (CN102010454A) is proposed, is come using chromatographic column or silicagel column
Carry out the purifying of born of the same parents' phosphocholine sodium, higher cost, large-scale production is difficult to realize, and products obtained therefrom purity is relatively low, 98% with
Under, a large amount of organic solvent recycling are difficult in eluent.(Japanese Patent Publication 62-16497) such as the former good filial piety of rattan synthesizes chemical method
CDP-choline solution uses strong-acid ion exchange resin RHPK-25.4-60 (H-type) column and weak-base ion-exchange resin
RWA-30 (OH type) column two procedures isolate and purify, and up to 98%, yield has not been reported HPLC purity.
These methods all realize the separation of citicoline sodium, but exist complex process, product yield it is relatively low, generate
A large amount of waste water solid slags, environmental protection pressure is big, separation costs are high, etc. several disadvantages.
Citicoline sodium in the present invention is from specific Escherichia coli fermentation gained citicoline sodium fermentation liquid
(CDP-choline fermentation liquid).It is obtained by genetic engineering means transformation Escherichia coli (K12 system) with glucose and choline chloride
The industrial strain that citicoline is synthesized for substrate, by proliferation, induction, the stream of substrate adds, and control etc. ferments, is anti-in production concentration
It should obtain the fermentation liquid of citicoline sodium.In this fermentation liquid, there is a large amount of thallus and pigments, and have not been converted
Substrate choline chloride, inorganic phosphate, sulfate, ammonium salt, calcium, magnesium and glucose etc., in addition generated there are also aqtocytolysis
Impurity: the compounds such as albumen and nucleic acid.Impurity component will be far more than chemical synthesis or enzyme' s catalysis, and separating difficulty is very big.
The present invention is not only successfully realized impurity and separates difficult problem more, also mainly has that environmental protection pressure is small, inexpensive, high-quality, height
The advantages that yield.
Summary of the invention
Goal of the invention: the technical problem to be solved by the present invention is to overcome the deficiencies in the prior art, from quality, yield, at
Sheet, pollution level etc. comprehensively consider, provide a kind of good quality, high income, it is at low cost, pollution it is few from escherichia coli fermented broth
The method of purifying citicoline sodium.
Technical solution: the method for the present invention provides a kind of from microbial fermentation solution purifying citicoline sodium,
The following steps are included:
(1) citicoline escherichia coli fermented broth is heated, flocculation, and separation of solid and liquid obtains clear liquid, and clear liquid connects through ultrafiltration, nanofiltration
Continuous formula pre-processes to obtain pretreatment fluid；
(2) pretreatment fluid is pre-processed by pre-column, the purified column absorption of efflux, then is eluted through sodium chloride solution, high-purity
It spends after eluent merges and is crystallized through reduced pressure, alcohol precipitation, obtain citicoline sodium crude product；
(3) citicoline sodium crude product is dissolved, is filtered after active carbon decoloring, filtrate is true after alcohol precipitation crystallization through being concentrated under reduced pressure
Sky is dry, obtains citicoline sodium finished product.
The method of the purifying citicoline sodium of the present invention from microbial fermentation solution, method is reasonable, wherein
The citicoline fermentation liquid referred to is from Escherichia coli fermentation gained.Step (1) is separated by solid-liquid separation phase using heating, flocculation
In conjunction with mode, effective extraction citicoline and removed thallus solid slag, operated using ultrafiltration and nanofiltration continous way, removal is big
Partial pigment, albumen, partial salts alleviate the impurity in step (2) pretreatment fluid, substantially increase resin to two phosphorus gallbladder of born of the same parents
The adsorption capacity of alkali, reduces amount of resin, reduces wastewater flow rate caused by elution and resin regeneration.Step (2) is using pre-
Column processing, removes impurity anions and pigment, improves the adsorption capacity of purification column, extends resin service life, separation costs
Low, product is easy crystallization, and content can be obtained in the citicoline sodium crude product of 95-99%.It refines, can be obtained high-purity through step (3)
The citicoline sodium finished product of degree, high yield.This method period is relatively short, and total recovery is reported between 65-70% better than other
The citicoline sodium separating and purifying technology of (about 50% total recovery).
Further, the method for the above-mentioned purifying citicoline sodium from microbial fermentation solution, two phosphorus of born of the same parents
Flocculant flocculation is added after the heating of choline escherichia coli fermented broth, heating temperature is 70-100 DEG C, heating time 30-60min.
Further, the method for the above-mentioned purifying citicoline sodium from microbial fermentation solution, the flocculant
For acid-soluble chitosan or polypropyleneimine (PEI), dosage 0.05-1%.The most preferably acid-soluble chitosan of flocculant,
As a preference, acid-soluble chitosan dosage is 0.05-0.2%.
Further, above-mentioned solid-liquid separation method is plate-frame filtering or disk centrifugal separation or microfiltration of ceramic membrane, dish
It is 10000-11000G that formula, which separates centrifugal force, and ceramic membrane aperture is 100000-500000 dalton, and micro-filtration pressure is 0.2-
0.5Mpa, microfiltration of ceramic membrane is as a preference, all thallus and suspended impurity can be removed, and 200000 dalton are as preferred
Aperture.
Further, above-mentioned ultrafiltration membrane and the preferred hollow fiber ultrafiltration membrane of nanofiltration membrane, membrane material are polyether sulfone, ultrafiltration pressure
General control is in 0.7-1.2MPa.Nanofiltration pressure general control is controlled in 30-45 in 1.0-2.0MPa, ultrafiltration, nanofiltration temperature
DEG C, by above-mentioned pretreatment can by CDP-choline fermentation liquid thallus and macro-molecular protein, macromolecular pigment,
Sulfate ion, most of monovalent salt, is removed phosphate anion.
Further, the method for the above-mentioned purifying citicoline sodium from microbial fermentation solution, the step (1)
Ultrafiltration membrane ultrafiltration of the clear liquid through 2000-20000 dalton obtained by middle separation of solid and liquid, the nanofiltration membrane nanofiltration of 100-500 dalton.Make
It is a kind of preferred, separation of solid and liquid gained ultrafiltration membrane ultrafiltration of the clear liquid through 8000-10000 dalton, the nanofiltration membrane of 200 dalton
Nanofiltration.
Further, the method for the above-mentioned purifying citicoline sodium from microbial fermentation solution, the step (2)
In be concentrated under reduced pressure into cytidine-5'-diphosphate choline na concn be 200-400g/L；Crystallization condition is: 3-6 times of material liquid volume of second is added
Alcohol stirs 12-24h under conditions of 4-25 DEG C, 80-120rpm；With 60-90% ethyl alcohol, 1:1 drenches by volume after crystal filtering
It washes.By step (2), residual pigment can largely be stayed in mother liquor, to obtain 99.9% or more HPLC purity, content 95-
99% citicoline sodium crude product.
Further, the method for the above-mentioned purifying citicoline sodium from microbial fermentation solution, the pre-column are
Strong-base anion-exchange resin, the purification column are strong-base anion-exchange resin.
Further, the method for the above-mentioned purifying citicoline sodium from microbial fermentation solution, the strong basicity
Anion exchange resin pre-column and strong-base anion-exchange resin purification column are one of 201*7,201*4,202 or several
Kind；Upper column flow rate 1-5BV/h, elution flow rate 0.5-3BV/h；The concentration of sodium chloride solution is 0.05-0.5mol/L.Preferably
It is: column flow rate 1.5-2.5BV/h on pre-column, column flow rate 1.5-2.5BV/h on purification column, elution flow rate 1-1.5BV/h, eluant, eluent
Sodium chloride concentration is 0.2mol/L.Pre-column and purification column are strongly basic anionic resin, for one in 201*7,201*4,202
Kind or several, preferably 201*4.
Further, the method for the above-mentioned purifying citicoline sodium from microbial fermentation solution, the step (2)
Middle pretreatment fluid adjusts pH2-14；The high-purity eluent adjusts pH5.5-7.5 after merging.
Further, the method for the above-mentioned purifying citicoline sodium from microbial fermentation solution, the step (3)
In, citicoline sodium crude product is redissolved after deionized water to 30-200g/L, active carbon room temperature decoloration 30min by 0.22 μ
M membrane filtration, is concentrated under reduced pressure into 200-400g/L；Crystallization condition is: 3-8 times of material liquid volume of ethyl alcohol is added, in 4-25 DEG C,
12-24h is stirred under conditions of 80-120rpm；With 60-90% ethyl alcohol, 1:1 is eluted by volume after crystal filtering；Vacuum drying temperature
Degree is 40-45 DEG C.By step (3), citicoline sodium can be further purified, 99.9% or more HPLC purity, content 99% with
On.
Further, the method for the above-mentioned purifying citicoline sodium from microbial fermentation solution, the active carbon
It is one or more of 723,767,769.Active carbon source is wide, and using conveniently, discoloration is thorough, and effect is good.
The utility model has the advantages that the invention has the following advantages that
1) present invention makes full use of membrane separation technique to carry out pretreated fermentation liquid, handles fermentation liquid by continuous UF membrane,
It is applied to next group, product loss late is reduced, improves separative efficiency and product yield.By with ion-exchange resin purification
Technology combines, through two strong alkalinity anion columns after CDP-choline fermentation liquid separation of solid and liquid, ultrafiltration, nanofiltration are handled
It is pre-processed and is purified, most impurity have been removed before purification column, substantially reduced later-period purification separating pressure, be
Crude, refining stage high yield provides guarantee.
2) present invention has only used a small amount of active carbon decoloring in refining stage, and entire technique generation solid waste is seldom, meets ring
Factoring is read.
3) above-mentioned separating technology has process flow simple, cost compared with traditional Activated Carbon Adsorption Separation technique
It is cheap, the advantages of total recovery is high, superior product quality.
4) citicoline sodium obtained by this purifying process, purity reach 99.9% or more.Total recovery is 65-70%, significantly excellent
In other producer's total recoverys generally in the technological level of 50-55%.
Detailed description of the invention
Attached drawing 1: the structural schematic diagram of citicoline sodium；
Attached drawing 2: the test map of citicoline sodium.
Specific embodiment
Below will be by several specific embodiments, the present invention is furture elucidated, these embodiments simply to illustrate that problem,
It is not a kind of limitation.
Embodiment 1
CDP-choline fermentation liquid 8.4L is prepared according to the method described above, and wherein CDP-choline concentration is 19.5g/L, is amounted to
163.8g.Treatment process is as follows:
1) 0.2% acid-soluble flocculate with chitosan is added through 100 DEG C of heating 30min in 8.4L citicoline fermentation liquid, generates cotton-shaped
Precipitating obtains clear liquid through plate filter filters pressing, and clear liquid collects permeate through ultrafiltration, and permeate collects trapped fluid through nanofiltration, obtains
Final pretreatment fluid.Wherein: ultrafiltration membrane, nanofiltration membrane are polyether sulfone material；Ultrafiltration retaining molecular weight is 10000 dongles
, ultrafiltration operating pressure is 0.7-0.9MPa, and operating temperature is 30-35 DEG C；Nanofiltration retaining molecular weight is 500 dalton, is received
Filter operating pressure is 1.0-1.2MPa, and operating temperature is 30-35 DEG C.Pretreatment fluid amounts to 5.1L, wherein CDP-choline concentration
For 30.1g/L, pre-processing yield is 93.7%.
2) pretreatment fluid is adjusted into pH10, with strong-base anion-exchange resin 201*4 on the flow velocity of 2BV/h, efflux
It is adsorbed again with 201*4 chromatographic column on the flow velocity of 2BV/h.When CDP-choline concentration is concentration in upper prop liquid in absorption efflux
5% when, resin has been saturated, stop continue upper prop, then with 2BV deionized water wash column, washed with 0.2mol/L sodium chloride solution
De-, elution flow rate 1.5BV/h collects 95% or more high-purity component of HPLC purity, adjusts pH6.0 after merging, obtain 7.2L,
Middle CDP-choline concentration is 18.9g/L, and purifying yield is 88.6%.Through 60 DEG C of vacuum-concentrcteds to 600ml, wherein CDP-
Choline concentration is 220g/L, and 3L(5 times of material liquid volume is slowly added dropwise) dehydrated alcohol, at 4 DEG C, 120rpm stirred crystallization
16h, with 75% ethyl alcohol, 1:1 is eluted by volume after crystal filtering, obtains the two phosphorus gallbladder of born of the same parents of HPLC purity 99.9%, content 97.2%
Alkali sodium crude product 130.4g.Crude total recovery is 82.6%.
3) it is 2.4L by 130.4g citicoline sodium crude product dissolution constant volume, 1.25g(1% is added) SILVER REAGENT active carbon
It is filtered after 723, stirring at normal temperature 30min, cleaner liquid crosses 0.22 μm of filter membrane, 60 DEG C of vacuum-concentrcteds to 320ml, wherein CDP-
Choline concentration is 390.6g/L, and 2L(6 times of material liquid volume is slowly added dropwise) dehydrated alcohol, at 4 DEG C, 120rpm stirred crystallization
16h, with 75% ethyl alcohol, 1:1 is eluted by volume after crystal filtering, and 45 DEG C of vacuum drying obtain citicoline sodium finished product
115.2g.HPLC purity is 99.9%, content 99.5%.Refining yield is 90.4%.
Total recovery is 70.0%.
Embodiment 2
CDP-choline fermentation liquid 8.0L is prepared according to the method described above, and wherein CDP-choline concentration is 21.3g/L, is amounted to
170.4g.Treatment process is as follows:
1) 0.15% acid-soluble flocculate with chitosan is added through 80 DEG C of heating 45min in 8.0L citicoline fermentation liquid, generates cotton-shaped
Precipitating is centrifuged to obtain clear liquid through tubular type, and clear liquid collects permeate through ultrafiltration, and permeate collects trapped fluid through nanofiltration, obtains final
Pretreatment fluid.Wherein: ultrafiltration membrane, nanofiltration membrane are polyether sulfone material；Ultrafiltration retaining molecular weight is 5000 dalton, ultrafiltration work
Making pressure is 0.9-1.1MPa, and operating temperature is 35-40 DEG C；Nanofiltration retaining molecular weight is 200 dalton, nanofiltration work pressure
Power is 1.2-1.5MPa, and operating temperature is 35-40 DEG C.Pretreatment fluid amounts to 5.0L, and wherein CDP-choline concentration is 31.5g/
L, pretreatment yield are 92.4%.
2) pretreatment fluid is adjusted into pH7.5, with strong-base anion-exchange resin 201*4 on the flow velocity of 3BV/h, efflux
It is adsorbed again with 201*4 chromatographic column on the flow velocity of 3BV/h.When CDP-choline concentration is concentration in upper prop liquid in absorption efflux
5% when, resin has been saturated, stop continue upper prop, then with 2BV deionized water wash column, with 0.3mol/L sodium chloride solution
Elution, elution flow rate 2BV/h collect 95% or more high-purity component of HPLC purity, adjust pH7.5 after merging, obtain 9.5L,
Middle CDP-choline concentration is 15.3g/L, and purifying yield is 92.3%.Through 60 DEG C of vacuum-concentrcteds to 420ml, wherein CDP-
Choline concentration is 338.8g/L, and 3.36L(8 times of material liquid volume is slowly added dropwise) dehydrated alcohol, at 10 DEG C, 100rpm stirring
16h is crystallized, with the 1:1 elution by volume of 60% ethyl alcohol after crystal filtering, obtains that HPLC purity is 99.5%, content is 96.5%
Citicoline sodium crude product 129.8g.Crude total recovery is 79.5%.
3) it is 1.2L by 129.8g citicoline sodium crude product dissolution constant volume, 1.2g(1% is added) SILVER REAGENT active carbon 767,
It is filtered after stirring at normal temperature 30min, cleaner liquid crosses 0.22 μm of filter membrane, 60 DEG C of vacuum-concentrcteds to 400ml, wherein CDP-
Choline concentration is 305.3g/L, and 2L(5 times of material liquid volume is slowly added dropwise) dehydrated alcohol, at 10 DEG C, 100rpm stirred crystallization
16h, with 80% ethyl alcohol, 1:1 is eluted by volume after crystal filtering, and 45 DEG C of vacuum drying obtain citicoline sodium finished product
111.8g.HPLC purity is 99.9%, content 99.2%.Refining yield is 88.5%.
Total recovery is 65.1%.
Embodiment 3
CDP-choline fermentation liquid 9.3L is prepared according to the method described above, and wherein CDP-choline concentration is 23.4g/L, is amounted to
217.6g.Treatment process is as follows:
1) 0.1% acid-soluble flocculate with chitosan is added through 90 DEG C of heating 40min in 9.3L citicoline fermentation liquid, and it is cotton-shaped heavy to generate
It forms sediment and obtains clear liquid through plate filter filters pressing, clear liquid collects permeate through ultrafiltration, and permeate collects trapped fluid through nanofiltration, obtains most
Whole pretreatment fluid.Wherein: ultrafiltration membrane, nanofiltration membrane are polyether sulfone material；Ultrafiltration retaining molecular weight is 2000 dalton, is surpassed
Filter operating pressure is 1.0-1.2 MPa, and operating temperature is 40-45 DEG C；Nanofiltration retaining molecular weight is 200 dalton, nanofiltration work
Making pressure is 1.5-2.0MPa, and operating temperature is 40-45 DEG C.Pretreatment fluid amounts to 6.1L, and wherein CDP-choline concentration is
33.2g/L, pretreatment yield are 93.1%.
2) pretreatment fluid is adjusted into pH13, with strong-base anion-exchange resin 201*7 on the flow velocity of 1BV/h, efflux
It is adsorbed again with 201*7 chromatographic column on the flow velocity of 1BV/h.When CDP-choline concentration is concentration in upper prop liquid in absorption efflux
5% when, resin has been saturated, stop continue upper prop, then with 2BV deionized water wash column, washed with 0.2mol/L sodium chloride solution
De-, elution flow rate 1BV/h collects 95% or more high-purity component of HPLC purity, adjusts pH5.5 after merging, obtain 10.4L, wherein
CDP-choline concentration is 18.1g/L, and purifying yield is 92.9%.Through 60 DEG C of vacuum-concentrcteds to 480ml, wherein CDP-
Choline concentration is 385.2g/L, and 2.4L(5 times of material liquid volume is slowly added dropwise) dehydrated alcohol, at 4 DEG C, 80rpm stirred crystallization
For 24 hours, with 85% ethyl alcohol, 1:1 is eluted by volume after crystal filtering, obtains the two phosphorus gallbladder of born of the same parents of HPLC purity 99.9%, content 95.3%
Alkali sodium crude product 183.9g.Crude total recovery is 86.5%.
3) it is 2.2L by 183.9g citicoline sodium crude product dissolution constant volume, 1.8g(1% is added) SILVER REAGENT active carbon 769,
It is filtered after stirring at normal temperature 30min, cleaner liquid crosses 0.22 μm of filter membrane, 60 DEG C of vacuum-concentrcteds to 680ml, wherein CDP-
Choline concentration is 249.7g/L, and 3.1L(4.5 times of material liquid volume is slowly added dropwise) dehydrated alcohol, at 4 DEG C, 80rpm stirring knot
For 24 hours, with 85% ethyl alcohol, 1:1 is eluted crystalline substance by volume after crystal filtering, and 42 DEG C of vacuum drying obtain citicoline sodium finished product
150.1g.HPLC purity is 99.9%, content 99.7%.Refining yield is 85.4%.
Total recovery is 68.8%.
Embodiment 4
CDP-choline fermentation liquid 10.2L is prepared according to the method described above, and wherein CDP-choline concentration is 14.2g/L, is amounted to
246.8g.Treatment process is as follows:
1) 10.2L citicoline fermentation liquid obtains clear liquid through microfiltration of ceramic membrane, clear liquid is received through ultrafiltration through 60 DEG C of heating 50min
Collect permeate, permeate collects trapped fluid through nanofiltration, obtains final pretreatment fluid.Wherein: microfiltration membranes are ceramic material, retention
Molecular weight is 200000 dalton, and operating pressure 0.3-0.4MPa, operating temperature is 50-55 DEG C；Ultrafiltration membrane, nanofiltration membrane are equal
For polyether sulfone material；Ultrafiltration retaining molecular weight is 10000 dalton, and ultrafiltration operating pressure is 0.9-1.0MPa, operating temperature
It is 35-40 DEG C；Nanofiltration retaining molecular weight is 100 dalton, and nanofiltration operating pressure is 1.5-2.0MPa, operating temperature 35-
40℃.Pretreatment fluid amounts to 6.4L, and wherein CDP-choline concentration is 35.8g/L, and pretreatment yield is 92.8%.
2) pretreatment fluid is adjusted into pH2, with strong-base anion-exchange resin 201*7 on the flow velocity of 2.5BV/h, efflux
It is adsorbed again with 201*7 chromatographic column on the flow velocity of 2.5BV/h.When CDP-choline concentration is dense in upper prop liquid in absorption efflux
Degree 5% when, resin has been saturated, stop continue upper prop, then with 2BV deionized water wash column, it is molten with 0.05mol/L sodium chloride
Liquid elution, elution flow rate 3BV/h collect 95% or more high-purity component of HPLC purity, adjust pH6.5 after merging, obtain 12.2L,
Wherein CDP-choline concentration is 16.7g/L, and purifying yield is 88.9%.Through 60 DEG C of vacuum-concentrcteds to 660ml, wherein
CDP-choline concentration be 300.6g/L, 3L(4.5 times of material liquid volume is slowly added dropwise) dehydrated alcohol, at 15 DEG C, 100rpm is stirred
Mix crystallization for 24 hours, crystal filtering after with 90% ethyl alcohol by volume 1:1 elution, obtain HPLC purity be 99.8%, content 95.9%
Citicoline sodium crude product 197.2g.Crude total recovery is 82.5%.
3) it is 1.3L by 197.2g citicoline sodium crude product dissolution constant volume, 1.9g(1% is added) SILVER REAGENT active carbon 767,
It is filtered after stirring at normal temperature 30min, cleaner liquid crosses 0.22 μm of filter membrane, 60 DEG C of vacuum-concentrcteds to 520ml, wherein CDP-
Choline concentration is 351.0g/L, and 2.1L(4 times of material liquid volume is slowly added dropwise) dehydrated alcohol, at 15 DEG C, 100rpm stirring knot
Brilliant 16h, with 80% ethyl alcohol, 1:1 is eluted by volume after crystal filtering, and 40 DEG C of vacuum drying obtain citicoline sodium finished product
168.2g.HPLC purity is 99.9%, content 99.6%.Refining yield is 88.6%.
Total recovery is 67.9%.
Embodiment 5
CDP-choline fermentation liquid 7.5L is prepared according to the method described above, and wherein CDP-choline concentration is 23.1g/L, is amounted to
173.3g.Treatment process is as follows:
1) 7.5L citicoline fermentation liquid is added 0.3% polyethyleneimine (PEI) flocculation, generates wadding through 70 DEG C of heating 50min
Shape precipitating is centrifuged to obtain clear liquid through tubular type, and clear liquid collects permeate through ultrafiltration, and permeate collects trapped fluid through nanofiltration, obtains final
Pretreatment fluid.Wherein: ultrafiltration membrane, nanofiltration membrane are polyether sulfone material；Ultrafiltration retaining molecular weight is 20000 dalton, is surpassed
Filter operating pressure is 0.7-0.9MPa, and operating temperature is 40-45 DEG C；Nanofiltration retaining molecular weight is 500 dalton, nanofiltration work
Making pressure is 1.2-1.5MPa, and operating temperature is 40-45 DEG C.Pretreatment fluid amounts to 5.5L, and wherein CDP-choline concentration is
29.5g/L, pretreatment yield are 93.7%.
2) pretreatment fluid is adjusted into pH10, with strong-base anion-exchange resin 201*4 on the flow velocity of 3BV/h, efflux
It is adsorbed again with 201*4 chromatographic column on the flow velocity of 2BV/h.When CDP-choline concentration is concentration in upper prop liquid in absorption efflux
5% when, resin has been saturated, stop continue upper prop, then with 2BV deionized water wash column, washed with 0.5mol/L sodium chloride solution
De-, elution flow rate 0.5BV/h collects 95% or more high-purity component of HPLC purity, adjusts pH6.0 after merging, obtain 9.3L,
Middle CDP-choline concentration 15.6g/L, purifying yield are 89.4%.Through 60 DEG C of vacuum-concentrcteds to 570ml, wherein CDP-
Choline concentration is 249.1g/L, and 2.3L(4 times of material liquid volume is slowly added dropwise) dehydrated alcohol, at 25 DEG C, 120rpm stirring knot
Brilliant 12h, with 80% ethyl alcohol, 1:1 is eluted by volume after crystal filtering, obtains the born of the same parents that HPLC purity is 99.9%, content is 98.2%
Two phosphorus choline sodium crude product 130.0g.Crude total recovery is 78.7%.
3) it is 1.1L by 130.0g citicoline sodium crude product dissolution constant volume, 1.2g(1% is added) SILVER REAGENT active carbon 723,
It is filtered after stirring at normal temperature 30min, cleaner liquid crosses 0.22 μm of filter membrane, 60 DEG C of vacuum-concentrcteds to 600ml, wherein CDP-
Choline concentration is 206.6g/L, and 3.6L(6 times of material liquid volume is slowly added dropwise) dehydrated alcohol, at 20 DEG C, 120rpm stirring knot
Brilliant 12h, with 80% ethyl alcohol, 1:1 is eluted by volume after crystal filtering, and 45 DEG C of vacuum drying obtain citicoline sodium finished product
118.1g.HPLC purity is 99.9%, content 99.3%.Refining yield is 91.9%.
Total recovery is 67.7%.
Embodiment 6
CDP-choline fermentation liquid 7.8L is prepared according to the method described above, and wherein CDP-choline concentration is 22.7g/L, is amounted to
177.1g.Treatment process is as follows:
1) 7.8L citicoline fermentation liquid obtains clear liquid through microfiltration of ceramic membrane, clear liquid is collected through ultrafiltration through 80 DEG C of heating 60min
Permeate, permeate collect trapped fluid through nanofiltration, obtain final pretreatment fluid.Wherein: microfiltration membranes are ceramic material, retention point
Son amount is 500000 dalton, and operating pressure 0.3-0.5MPa, operating temperature is 60 DEG C；Ultrafiltration membrane, nanofiltration membrane are polyethers
Sulfone material；Ultrafiltration retaining molecular weight is 20000 dalton, and ultrafiltration operating pressure is 0.9-1.1MPa, operating temperature 35-40
℃；Nanofiltration retaining molecular weight is 500 dalton, and nanofiltration operating pressure is 1.2-1.5MPa, and operating temperature is 35-40 DEG C.In advance
Treatment fluid amounts to 5.7L, and wherein CDP-choline concentration is 29.2g/L, and pretreatment yield is 94.0%.
2) pretreatment fluid is adjusted into pH11, with strong-base anion-exchange resin 202 on the flow velocity of 2.5BV/h, efflux
It is adsorbed again with 202 chromatographic columns on the flow velocity of 2.5BV/h.When CDP-choline concentration is concentration in upper prop liquid in absorption efflux
5% when, resin has been saturated, stop continue upper prop, then with 2BV deionized water wash column, with 0.05mol/L sodium chloride solution
Elution, elution flow rate 1.0BV/h collect 95% or more high-purity component of HPLC purity, adjust pH6.0 after merging, obtain 10.2L,
Wherein CDP-choline concentration 14.5g/L, purifying yield are 88.9%.Through 60 DEG C of vacuum-concentrcteds to 510ml, wherein CDP-
Choline concentration is 290.0g/L, and 2 L(4 times of material liquid volumes are slowly added dropwise) dehydrated alcohol, at 25 DEG C, 120rpm stirring knot
Brilliant 14h, with 80% ethyl alcohol, 1:1 is eluted by volume after crystal filtering, obtains the born of the same parents that HPLC purity is 99.9%, content is 98.5%
Two phosphorus choline sodium crude product 135.3g.Crude total recovery is 80.1%.
3) it is 1.0L by 135.3g citicoline sodium crude product dissolution constant volume, 1.3g(1% is added) SILVER REAGENT active carbon 767,
It is filtered after stirring at normal temperature 30min, cleaner liquid crosses 0.22 μm of filter membrane, 60 DEG C of vacuum-concentrcteds to 600ml, wherein CDP-
Choline concentration is 222.1g/L, and 3.0L(5 times of material liquid volume is slowly added dropwise) dehydrated alcohol, at 20 DEG C, 120rpm stirring knot
Brilliant 12h, with 80% ethyl alcohol, 1:1 is eluted by volume after crystal filtering, and 45 DEG C of vacuum drying obtain citicoline sodium finished product
122.1g.HPLC purity is 99.9%, content 99.1%.Refining yield is 90.8%.
Total recovery is 68.4%.
Embodiment 7
High performance liquid chromatography (HPLC) detection, the parameter of HPLC are as follows: using Agilent SB C18 4.6*150mm 5um, stream
Dynamic is mutually methanol and 10mM PBS (pH4.0), and 0.01-4.00 minutes methanol ratios of mobile phase ratio are 2%, 4.00-5.00 minutes
Methanol ratio rises to 10%, 5.00-5.01 minutes methanol ratios by 2% and is down to 2%, 5.10-10.0 minutes methanol ratios by 10%
It is 2%, is examined using UV detector, surveys 272 nm of wavelength；The flow velocity of mobile phase is 0.8 mL/min, 5 μ of applied sample amount of fermentation liquid
L, 30 DEG C of column temperature.CDPC appearance time is 2.745 minutes, and map is as shown in Figure 2.
Described above is only several embodiments of invention, it is noted that for those skilled in the art
For, under the premise of not departing from inventive principle, several improvement can also be made, these improvement also should be regarded as protection of the invention
Range.
Claims (10)
1. a kind of method of the purifying citicoline sodium from microbial fermentation solution, it is characterised in that: the following steps are included:
(1) citicoline fermentation liquid is heated, and flocculation, separation of solid and liquid obtains clear liquid, and clear liquid is located in advance through ultrafiltration, nanofiltration continous way
Reason obtains pretreatment fluid；
(2) pretreatment fluid is pre-processed by pre-column, the purified column absorption of efflux, then is eluted through sodium chloride solution, is eluted
Liquid is concentrated under reduced pressure again after merging, alcohol precipitation crystallizes, and obtains citicoline sodium crude product；
(3) citicoline sodium crude product is dissolved, is filtered after active carbon decoloring, cross cleaner liquid through vacuum-concentrcted, alcohol precipitation
It is dried in vacuo after crystallization, obtains citicoline sodium finished product.
2. the method for purifying citicoline sodium in fermentation liquid according to claim 1, it is characterised in that: described
Microbial fermentation solution is escherichia coli fermented broth.
3. the method for purifying citicoline sodium in fermentation liquid according to claim 2, it is characterised in that: the born of the same parents
After the heating of two phosphorus choline escherichia coli fermented broths, flocculant flocculation is added, heating temperature is 70-100 DEG C, heating time 30-
60min。
4. the method for the purifying citicoline sodium according to claim 1 or 3 from microbial fermentation solution, feature
Be: the flocculant is acid-soluble chitosan or polyethyleneimine, dosage 0.05-1%.
5. the method for the purifying citicoline sodium according to claim 1 from microbial fermentation solution, feature exist
In: solid-liquid separation method is plate-frame filtering perhaps tubular type centrifuge separation or 100000-500000 dongle in the step (1)
The microfiltration of ceramic membrane to pause；It is separated by solid-liquid separation gained ultrafiltration membrane ultrafiltration of the clear liquid through 2000-20000 dalton, 100-500 dalton
Nanofiltration membrane nanofiltration.
6. the method for the purifying citicoline sodium according to claim 1 from microbial fermentation solution, feature exist
In: pretreatment fluid adjusts pH2-14 in the step (2)；The eluent adjusts pH5.5-7.5.
7. the method for the purifying citicoline sodium according to claim 1 from microbial fermentation solution, feature exist
In: it is 200-400g/L that cytidine-5'-diphosphate choline na concn is concentrated under reduced pressure into the step (2)；Crystallization condition is: feed liquid is added
4-6 times of volume of ethyl alcohol stirs 12-24h under conditions of 4-25 DEG C, 80-120rpm；It is pressed after crystal filtering with 60-90% ethyl alcohol
Volume ratio 1:1 elution.
8. the method for the purifying citicoline sodium according to claim 1 from microbial fermentation solution, feature exist
In: the pre-column is strong-base anion-exchange resin, and the purification column is strong-base anion-exchange resin.
9. the method for the purifying citicoline sodium according to claim 8 from escherichia coli fermented broth, feature
Be: the strong-base anion-exchange resin is one or more of 201*7,201*4,202；Upper column flow rate 1-5BV/h,
Elution flow rate 0.5-3BV/h；The concentration of sodium chloride solution is 0.05-0.5mol/L.
10. the method for the purifying citicoline sodium according to claim 1 from microbial fermentation solution, feature exist
In: in the step (3), citicoline sodium crude product is redissolved in deionized water to 30-200g/L, the decoloration of active carbon room temperature
By 0.22 μm of membrane filtration after 30min, it is concentrated under reduced pressure into 200-400g/L；Crystallization condition is: being added 4-8 times of material liquid volume
Ethyl alcohol stirs 12-24h under conditions of 4-25 DEG C, 80-120rpm；With 60-90% ethyl alcohol, 1:1 drenches by volume after crystal filtering
It washes；Vacuum drying temperature is 40-45 DEG C.
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