CN111087432A - Separation and extraction method of N-acetylneuraminic acid - Google Patents
Separation and extraction method of N-acetylneuraminic acid Download PDFInfo
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Abstract
The invention discloses a method for separating and extracting N-acetylneuraminic acid, which comprises the following steps: (1) carrying out acidolysis on the solution containing the polysialic acid to obtain acidolysis solution; (2) removing solid impurities from the acidolysis solution obtained in the step (1) through a first membrane to obtain a first permeate; passing the first permeate through a second membrane to remove macromolecular impurities to obtain a second permeate; passing the second permeate through a third membrane to remove monovalent salt, and collecting trapped fluid; (3) adjusting the pH of the trapped fluid to the isoelectric point of N-acetylneuraminic acid, removing multivalent salt through electrodialysis, washing impurities and eluting the solution without multivalent salt through anion exchange resin, collecting effluent liquid, and concentrating to obtain concentrated solution; (4) and (4) adjusting the pH value of the concentrated solution obtained in the step (3) to 1-3, adding a reverse solvent, cooling, precipitating crystals, centrifuging, washing and drying to obtain the compound. The whole separation process is simple to operate, the purity of the crystal product is over 99 percent, and the yield is more than 90 percent.
Description
Technical Field
The invention belongs to the technical field of separation and purification, and particularly relates to a method for separating and extracting N-acetylneuraminic acid.
Background
Sialic acid is the major nutrient component of bird's nest, a derivative of a 9-carbon monosaccharide named from greek, which is a negatively charged ion that produces a smooth sensation in saliva. At present, the cognition is the transmission transmitter of ganglioside and is a component of brain, sialic acid can prevent germ invasion, and the infant has higher sialic acid content in the juvenile period, thereby having important significance on the body development of the infant.
The production method of sialic acid mainly comprises fermentation method, enzyme synthesis method, and natural product extraction method. In the prior art, a method for continuously separating sialic acid is disclosed, wherein ethanol is required to perform polysialic acid precipitation, initial purification is performed through cetylpyridine, acidolysis is performed, anion and cation exchange resin is performed, and crystallization is performed on eluent in a ternary system of ethanol, water and ethyl acetate after freeze-drying, so that the separation cost in the whole process is high and the yield is only about 50%. In other documents, the method is only concentrated, acidified and crystallized after membrane filtration, and experiments prove that the method contains too many impurities in solution, the solution viscosity is too high, crystallization is difficult to complete, the product purity is low, the pigment content of the product is high, and the overall yield is less than 70%.
Since the thallus of the escherichia coli is small, the viscosity of the fermentation liquor is high, the difficulty and the efficiency of solid-liquid separation by conventional methods such as centrifugation, membrane filtration and plate-and-frame filtration are high, and therefore a new method needs to be developed to improve the separation yield and efficiency and the purity of the obtained product.
Disclosure of Invention
The purpose of the invention is as follows: the invention aims to solve the technical problem of providing a method for separating and extracting N-acetylneuraminic acid aiming at the defects of the prior art.
In order to solve the technical problem, the invention discloses a method for separating and extracting N-acetylneuraminic acid, which comprises the following steps:
(1) acidolyzing the solution containing polysialic acid to obtain acidolysis solution, namely the solution containing monomer sialic acid (N-acetylneuraminic acid);
(2) removing solid impurities such as thalli and the like from the acidolysis solution obtained in the step (1) through a first membrane to obtain a first permeate; passing the first permeate through a second membrane to remove macromolecular impurities such as protein and the like to obtain a second permeate; passing the second permeate through a third membrane, concentrating to remove monovalent salt (divalent or higher salt, which cannot be permeated by nanofiltration membrane), and collecting the trapped fluid;
(3) adjusting the pH of the trapped fluid to the isoelectric point of N-acetylneuraminic acid (sialic acid), removing multivalent salt by electrodialysis, adsorbing and washing impurities in the solution without multivalent salt by anion exchange resin, eluting, collecting effluent, and concentrating to obtain concentrated solution;
(4) and (4) adjusting the pH value of the concentrated solution obtained in the step (3) to 1-3, cooling, adding a reverse solvent, separating out coarse crystals, centrifuging, washing and drying to obtain the compound.
In the step (1), the solution containing N-acetylneuraminic acid is a fermentation solution containing polysialic acid; wherein the fermentation solution is prepared by inoculating Escherichia coli into culture medium composed of corn steep liquor, vitamins, inorganic salts and yeast extract, and fermenting.
In the step (1), the acidolysis is performed for 4-8 hours at a pH of 1.5-3.5 (preferably 1.9-3.0) and at a temperature of 65-90 ℃ (preferably 70-85 ℃), so that a solid-phase separation process of thalli and sialic acid can be effectively avoided.
Wherein, the pH regulator is any one or combination of several of phosphoric acid, sulfuric acid and hydrochloric acid.
In the step (2), the first membrane is a ceramic membrane; the aperture of the ceramic membrane is 50-500 nm; the second membrane is a polypropylene ultrafiltration membrane, and the molecular weight cut-off of the second membrane is 2000-10000 Da (preferably 4000-8000 Da); the third membrane is a polypropylene nanofiltration membrane, and the molecular weight cut-off of the third membrane is 100-500 Da (preferably 150-300 Da).
In the step (2), washing is carried out by water in the process of concentration washing until the conductivity of the permeate is less than 500 us/cm.
In the step (3), the substances for adjusting the pH are hydrochloric acid, sulfuric acid, phosphoric acid, formic acid, acetic acid and the like.
In the step (3), the pH value of the isoelectric point of the sialic acid is 2.5-2.8.
In the step (3), due to the fact that divalent ions such as magnesium ions and sulfate ions are added in the fermentation process, the ions cannot selectively penetrate through a nanofiltration membrane, next sialic acid cannot be adsorbed by ion exchange resin, and the separation effect cannot be achieved; the operation mode of electrodialysis is a constant current mode; the running current is 0.1-1A; the volume ratio of the concentration chamber to the desalination chamber is 1: 1; controlling the flow rate to be 40-100 mL/min; the specification model is GJBMED-90 x 210-5, the area of the effective membrane of a single membrane is as follows: 90 x 210mm2The membrane stack repeating unit 20, the membrane stack configuration C-A-C electrode material is a titanium fetter coated electrode plate, and the membrane material in electrodialysis is styrene; the conductivity of the depleting compartment (containing sialic acid) after removal of the solution of multivalent salt is less than 2 ms/cm.
In the step (3), the anion exchange resin is anion exchange resin AD-1, the framework of the anion exchange resin AD-1 is an ethylene-divinylbenzene copolymer, and the particle size range of the ethylene-divinylbenzene copolymer is 0.315-1.25 mm; water content: 40% -45%; effective exchange capacity: 1.35 mmol/g.
In the step (3), the impurity washing agent is water; the flow rate of impurity washing is 1-2 BV/h; the volume of the washing impurities is 2-8 BV; the eluent of the elution is acid solution or salt solution; the flow rate of elution is 1 BV/h; the volume of elution is 3-15 BV; and (5) eluting until the eluent does not contain N-acetylneuraminic acid, and finishing the elution.
Wherein, the acid solution is any one or a combination of more of hydrochloric acid, sulfuric acid, formic acid and acetic acid; the salt solution is any one or a combination of more of sodium chloride aqueous solution, potassium chloride aqueous solution and magnesium chloride aqueous solution; the concentrations of the acid solution and the salt solution are both 0.05-1 mol/L; the binding capacity of the anion groups of the upper acid and the salt to the anion exchange resin is greater than the binding capacity of sialic acid to the anion exchange resin.
In the step (3), after triple membrane treatment, thalli, macromolecular compounds, salt micromolecule compounds and other compounds are removed, and a saliva solution with the conductivity of less than 2ms/cm is obtained through electrodialysis.
In the step (3), when the concentration of the effluent is less than 150g/L, nanofiltration concentration, MVR or rotary evaporation concentration can be performed under a neutral condition to obtain a concentrated solution, wherein the concentration of N-acetylneuraminic acid in the concentrated solution is 150-500 g/L (preferably 250-400 g/L). The driving force of crystallization is concentration gradient, and only a certain initial concentration is ensured, the supersaturation degree can be generated in the process of continuous cooling, so that the growth of crystals can be driven, and the crystallization can be completed within 2 hours after the propanol, the acetone or the isopropanol is added.
In the step (4), the substance for adjusting pH is hydrochloric acid, sulfuric acid, phosphoric acid, formic acid, acetic acid, etc.
In the step (4), the temperature is reduced to 20-35 DEG C
In the step (4), the reverse solvent is any one or combination of acetone, propanol and isopropanol; the volume ratio of the reverse solvent to the concentrated solution after the pH adjustment is 0.8-2: 1.
preferably, the reaction solvent is added while the seed crystal is added, and the temperature is slowly reduced in a light stirring and gradient cooling mode, specifically, the temperature of the concentrated solution after the pH is adjusted is reduced to 20-35 ℃, the seed crystal and the reverse solvent are added, the mixture is continuously stirred for 0.5h at the rotating speed of 50-800 rpm to perform crystal form conversion, crystal nuclei are obtained, then the temperature is reduced to 0-10 ℃ (preferably 2-5 ℃), preferably 2-5 ℃ within 1.5h, and the crystal nuclei continuously grow into crystals.
Preferably, in order to improve the purity of the crystals and reduce the influence of impurities in the crystallization mother liquor, the crystals are washed by using 20-100% ethanol water solution at the low temperature of 0-8 ℃, and the crystals are collected by centrifugation or suction filtration.
In the step (4), the manner of collecting the crystals is to centrifugally collect the coarse crystals, the centrifugal speed is 4000-10000 rpm (preferably 3000-7000 rpm), and the centrifugal time is 10-30 min.
In the step (4), the drying is blast drying or vacuum drying, the drying temperature is 40-100 ℃, preferably 50-80 ℃, and the drying time is 2-8 hours.
Wherein, the vacuum degree of the vacuum drying is 0-200 mbar, preferably 10-100 mbar.
The purity of the extracted N-acetylneuraminic acid prepared and extracted by the method is more than or equal to 99 percent, and the yield is more than or equal to 90 percent.
The invention innovatively adopts fermentation liquor for direct acidolysis, adopts three sets of membrane systems for crude separation to remove thalli and macromolecular impurities, removes multivalent salts through electrodialysis, and carries out one-step fine chromatographic separation through AD-1 anion exchange resin synthesized in a laboratory, eluent is concentrated, added with anti-solvent, cooled and crystallized, and crystals are obtained after washing and drying. The whole separation yield is more than 90 percent, the purity is higher than 99 percent, the yield is less than 70 percent and the purity is 97 percent which are superior to the prior art, and the process of the invention is simple and easy to operate and the separation cost is low.
Has the advantages that: compared with the prior art, the invention has the following advantages:
(1) the sialic acid of the invention adopts a direct acidolysis method, thereby omitting the solid-liquid separation process of sialic acid fermentation liquor.
(2) The sialic acid yield of the sialic acid obtained by separation of the sialic acid separation of the invention is more than 90 percent, which is far higher than that of the prior sialic acid separation technology.
(3) The invention initiates the combination of electrodialysis and ion exchange and is applied to the separation of sialic acid, so that the adsorption capacity of anion exchange resin is improved by more than 10 times, the resin dosage is reduced by 10 times, and the acid-base dosage and the operation time are indirectly saved.
(4) By adding the anti-solvent, the crystallization process can be shortened to within 2h, bright white crystals containing two crystal waters are obtained, the one-time crystallization yield is more than 90%, the purity of the crystals is more than 99%, and the product quality is higher than that of the prior art (sialic acid produced by the prior art is in an amorphous form of light yellow powder, does not contain crystal water, and has the purity of about 97%).
Drawings
FIG. 1 shows a crystalline product of sialic acid.
Detailed Description
The invention will be better understood from the following examples. However, those skilled in the art will readily appreciate that the description of the embodiments is only for illustrating the present invention and should not be taken as limiting the invention as detailed in the claims.
Example 1:
1) 2000mL of fermentation broth (polyacid hydrolysate (8.1g/L)) containing polysialic acid is subjected to acidolysis for 3h at 65 ℃ by adjusting pH to 1.5 with phosphoric acid to obtain 2050mL of acidolysis solution; wherein the concentration of sialic acid in the acidolysis solution is 7.9 g/L; the acidolysis solution contains sialic acid as main components, such as sugar, nucleic acid, salt, pigment, amino acids, protein, and organic acid.
Wherein the fermentation liquor containing sialic acid is prepared by converting glucose, corn steep liquor, vitamins, inorganic salts, yeast extract and the like by using escherichia coli to form polysialic acid.
2) Passing the acidolysis solution obtained in the step 1) through a 50nm ceramic membrane, collecting a permeate, washing the retentate with tap water until the retentate does not contain sialic acid, and collecting the ceramic membrane permeate.
3) Passing the ceramic membrane permeate obtained in the step 2) through a 2000Da ultrafiltration membrane to remove macromolecular impurities such as protein, nucleic acid and the like, continuously washing the trapped fluid until no sialic acid is contained, and collecting the ultrafiltration membrane permeate.
4) Concentrating the ultrafiltration membrane permeate obtained in the step 3) by a membrane of 100Da for desalting, continuously washing with deionized water until the conductivity of the permeate is less than 500us/cm, and collecting the trapped fluid.
5) Regulating the trapped liquid of the step 4) with formic acid to regulate pH to 2.7, passing through electrodialysis (specification: GJBMED-90 x 210-5, active membrane area of single membrane: 90 x 210mm2Stack repeat unit 20, stack configuration C-a-C electrode material: the titanium fetters electrode plate has the following operation modes: a constant current mode; operating current: 0.6A; the concentration chamber and the desalination chamber adopt a volume ratio of 1: 1; flow rate: 60mL/min), the sialic acid conductivity was less than 2ms/cm, and after passing through 200g of AD-1 anion exchange resin, the impurity was washed with deionized water at a flow rate of 1BV/h for 3 times the bed volume, and then eluted with 0.1mol/L sodium chloride solution (elution flow rate: 1BV/H, elution volume: 10BV, until the elution liquid is spotted without sialic acid), and the eluate is collected.
6) Concentrating the eluent obtained in the step 5) to obtain sialic acid with the concentration of 350g/L, adjusting the pH to 1 by using hydrochloric acid, cooling to 20 ℃, adding seed crystals and 0.8 times of acetone by volume, continuously stirring for 1h at 100rpm, slowly cooling to 1 ℃, and precipitating crystals;
7) and (3) carrying out suction filtration on the crystals obtained in the step 6), removing the mother liquor, adding 20 vt% ethanol water solution, washing twice, and carrying out suction filtration to obtain the crystals.
8) Putting the crystals obtained in the step 7) into a vacuum drying oven at 40 ℃, and drying for 24h under 10mbar, thereby obtaining 14.72g of sialic acid crystals.
The purity of the dried crystal was 99.2%, and the yield was 90.2%.
Comparative example 1: not passing through AD-1 anion exchange resin
1) Carrying out acidolysis on 2000mL of fermentation liquor (acidolysis solution (8.1g/L)) containing polysialic acid at 65 ℃ for 3h by adjusting the pH to 1.5 with phosphoric acid to obtain 2050mL of acidolysis solution; wherein the concentration of sialic acid in the acidolysis solution is 7.9 g/L;
2) passing the acidolysis solution obtained in the step 1) through a 50nm ceramic membrane, collecting a permeate, washing the retentate with tap water until the retentate does not contain sialic acid, and collecting the ceramic membrane permeate.
3) Passing the ceramic membrane permeate obtained in the step 2) through a 2000Da membrane to remove macromolecular impurities such as protein, nucleic acid and the like, continuously washing the trapped fluid until no sialic acid is contained, and collecting the ultrafiltration membrane permeate.
4) Concentrating the ultrafiltration membrane permeate obtained in the step 3) by a membrane of 100Da for desalting, continuously washing with deionized water until the conductivity of the permeate is less than 500us/cm, and collecting the trapped fluid.
5) Adjusting the pH of the retentate of step 4) to 2.7 with formic acid and passing the retentate through electrodialysis (specification: GJBMED-90 x 210-5, effective membrane area of single membrane: 90 x 210mm2Stack repeat unit 20, stack configuration C-a-C electrode material: the titanium fetters electrode plate has the following operation modes: a constant current mode; operating current: 0.6A; the concentration chamber and the desalination chamber adopt a volume ratio of 1: 1; flow rate: 60mL/min), sialic acid conductivity was less than 2ms/cm, not passed through AD-1 anion exchange resin.
6) Concentrating the eluent obtained in the step 5) to obtain sialic acid with the concentration of 350g/L, adjusting the pH to 1 by using hydrochloric acid, cooling to 20 ℃, adding seed crystals and 0.8 times of acetone by volume, continuously stirring for 1h at 100rpm, slowly cooling to 1 ℃, and precipitating crystals;
7) and (3) carrying out suction filtration on the crystals obtained in the step 6), removing the mother liquor, adding 20 vt% ethanol water solution, washing twice, and carrying out suction filtration to obtain 29.7g of crystals.
8) The crystals from step 7) were dried in a vacuum oven at 40 ℃ for 24h at 10 mbar.
The purity of the dried crystal was 49.2%, and the yield was 90.2%.
Comparative example 2: without electrodialysis
1) Carrying out acidolysis on 2000mL of fermentation liquor (8.1g/L of acidolysis solution) containing polysialic acid by using phosphoric acid to adjust the pH to 1.5 at 65 ℃ for 3h to obtain 2050mL of acidolysis solution; wherein the concentration of sialic acid in the acidolysis solution is 7.9 g/L.
2) Passing the acidolysis solution obtained in the step 1) through a 50nm ceramic membrane, collecting a permeate, washing the retentate with tap water until the retentate does not contain sialic acid, and collecting the ceramic membrane permeate.
3) Passing the ceramic membrane permeate obtained in step 2) through a 2000Da membrane to remove macromolecular impurities such as protein and nucleic acid, continuously washing the trapped fluid until no sialic acid is contained, and collecting the ultrafiltration membrane permeate
4) Concentrating the ultrafiltration membrane permeate obtained in the step 3) by a membrane of 100Da for desalting, continuously washing with deionized water until the conductivity of the permeate is less than 500us/cm, and collecting the trapped fluid.
5) Adjusting the pH of the trapped fluid obtained in the step 4) to 2.7 by using formic acid, directly passing through 200g of AD-1 anion exchange resin without electrodialysis, washing the trapped fluid with deionized water at the flow rate of 1BV/h by 3 times of the bed volume, and then eluting the trapped fluid by using 0.1mol/L sodium chloride solution (elution flow rate: 1BV/H, elution volume: 10BV, until the elution liquid is spotted without sialic acid), and the eluate is collected.
6) Concentrating the eluent obtained in the step 5) to obtain sialic acid with the concentration of 350g/L, adjusting the pH to 1 by using hydrochloric acid, cooling to 20 ℃, adding seed crystals and 0.8 times of acetone by volume, continuously stirring for 1h at 100rpm, slowly cooling to 1 ℃, and precipitating crystals;
7) and (3) carrying out suction filtration on the crystals obtained in the step 6), removing the mother liquor, adding 20 vt% ethanol water solution, washing twice, and carrying out suction filtration to obtain 6.08g of crystals.
8) The crystals from step 7) were dried in a vacuum oven at 40 ℃ for 24h at 10 mbar.
The purity of the dried crystal was 99.2%, and the yield was 37.2%.
Comparative example 3
1) Firstly, centrifuging 2000mL of fermentation liquor containing polysialic acid (polyacid hydrolysate (8.1g/L)) to remove solid content, then adjusting the pH to 1.5 by using phosphoric acid, and carrying out acidolysis for 3h at 65 ℃ to obtain 2050mL of acidolysis solution; wherein the concentration of sialic acid in the acidolysis solution is 6.5 g/L;
2) passing the acidolysis solution obtained in the step 1) through a 50nm ceramic membrane, collecting a permeate, washing the retentate with tap water until the retentate does not contain sialic acid, and collecting a first permeate and a first washing solution.
3) Passing the first permeate and the first washing solution obtained in the step 2) through a 2000Da membrane to remove macromolecular impurities such as protein and nucleic acid, continuously washing the trapped fluid until no sialic acid is contained, and collecting the permeate of the ultrafiltration membrane
4) Concentrating the second permeate and the second washing liquid obtained in the step 3) by a 100Da membrane for desalting, continuously washing by deionized water until the conductivity of the permeate is less than 500us/cm, and collecting trapped fluid.
5) Adjusting the pH of the retentate of step 4) to 2.7 with formic acid and passing the retentate through electrodialysis (specification:GJBMED-90 x 210-5, effective membrane area of single membrane: 90 x 210mm2Stack repeat unit 20, stack configuration C-a-C electrode material: the titanium fetters electrode plate has the following operation modes: a constant current mode; operating current: 0.6A; the concentration chamber and the desalination chamber adopt a volume ratio of 1: 1; flow rate: 60mL/min), the sialic acid conductivity was less than 2ms/cm, and after passing through 200g of AD-1 anion exchange resin, the impurity was washed with deionized water at a flow rate of 1BV/h for 3 times the bed volume, and then eluted with 0.1mol/L sodium chloride solution (elution flow rate: 1BV/H, elution volume: 10BV, until the elution liquid is spotted without sialic acid), and the eluate is collected.
6) Concentrating the eluent obtained in the step 5) to obtain sialic acid with the concentration of 350g/L, adjusting the pH to 1 by using hydrochloric acid, cooling to 20 ℃, adding seed crystals and 0.8 times of acetone by volume, continuously stirring for 1h at 100rpm, slowly cooling to 1 ℃, and precipitating crystals;
7) and (3) carrying out suction filtration on the crystals obtained in the step 6), removing the mother liquor, adding 20 vt% ethanol water solution, washing twice, and carrying out suction filtration to obtain 11.37g of crystals.
8) The crystals from step 7) were dried in a vacuum oven at 40 ℃ for 24h at 10 mbar.
The purity of the dried crystal was 99.2%, and the yield was 69.6%.
Comparative example 4
1) Fermentation broth containing polysialic acid (polyacid hydrolyzate (8.1g/L)
2) 2000mL of acid hydrolysis solution is obtained by adjusting the pH to 1.5 with phosphoric acid and carrying out acid hydrolysis for 3h at 65 ℃; wherein the concentration of sialic acid in the acidolysis solution is 7.9 g/L.
Wherein the fermentation liquor containing sialic acid is prepared by converting glucose, corn steep liquor, vitamins, inorganic salts, yeast extract and the like by using escherichia coli to form polysialic acid.
2) Passing the acidolysis solution obtained in the step 1) through a 50nm ceramic membrane, collecting a permeate, washing the retentate with tap water until the retentate does not contain sialic acid, and collecting the ceramic membrane permeate.
3) Passing the ceramic membrane permeate obtained in the step 2) through a 2000Da ultrafiltration membrane to remove macromolecular impurities such as protein, nucleic acid and the like, continuously washing the trapped fluid until no sialic acid is contained, and collecting the ultrafiltration membrane permeate.
4) Concentrating the ultrafiltration membrane permeate obtained in the step 3) by a membrane of 100Da for desalting, continuously washing with deionized water until the conductivity of the permeate is less than 500us/cm, and collecting the trapped fluid.
5) Adjusting the pH of the trapped fluid obtained in the step 4) to 2.7 by using formic acid, and performing electrodialysis (specification type: GJBMED-90 x 210-5, effective membrane area of single membrane: 90 x 210mm2Stack repeat unit 20, stack configuration C-a-C electrode material: the titanium fetters electrode plate has the following operation modes: a constant current mode; operating current: 0.6A; the concentration chamber and the desalination chamber adopt a volume ratio of 1: 1; flow rate: 60mL/min), sialic acid conductivity was less than 2ms/cm, and after passing through 2000g ad-1 anion exchange resin, 3 times the bed volume was washed with deionized water at a flow rate of 1BV/h, followed by elution with 0.1mol/L sodium chloride solution (elution flow rate: 1BV/H, elution volume: 10BV, until the elution liquid is spotted without sialic acid), and the eluate is collected.
6) Concentrating the eluent obtained in the step 5) to obtain sialic acid with the concentration of 350g/L, adjusting the pH to 1 by using hydrochloric acid, cooling to 20 ℃, adding seed crystals and 0.8 times of acetone by volume, continuously stirring for 1h at 100rpm, slowly cooling to 1 ℃, and precipitating crystals;
7) and (3) carrying out suction filtration on the crystals obtained in the step 6), removing the mother liquor, adding 20 vt% ethanol water solution, washing twice, and carrying out suction filtration to obtain the crystals.
8) Putting the crystals obtained in the step 7) into a vacuum drying oven at 40 ℃, and drying for 24h under 10mbar, thereby obtaining 14.72g of sialic acid crystals.
The purity of the dried crystal was 99.2%, and the yield was 90.2%.
Example 2
1) Carrying out acidolysis on 2000mL of fermentation liquor (polysialic acid hydrolysate 8.1g/L) containing polysialic acid at 80 ℃ for 5h by adjusting the pH to 2.5 with phosphoric acid to obtain 2040mL of acidolysis solution; wherein the concentration of sialic acid in the acidolysis solution is 7.94 g/L.
2) Passing the acidolysis solution obtained in the step 1) through a 50nm ceramic membrane, collecting a permeate, washing the retentate with tap water until the retentate does not contain sialic acid, and collecting the ceramic membrane permeate.
3) Passing the ceramic membrane permeate obtained in the step 2) through a 5000Da membrane to remove macromolecular impurities such as protein, nucleic acid and the like, continuously washing the trapped fluid until no sialic acid is contained, and collecting the ultrafiltration membrane permeate.
4) Concentrating the ultrafiltration membrane permeate obtained in the step 3) by a membrane of 100Da for desalting, continuously washing with deionized water until the conductivity of the permeate is less than 500us/cm, and collecting the trapped fluid.
5) Adjusting the pH of the retentate of step 4) to 2.6 with sulfuric acid and passing the retentate through electrodialysis (specification: GJBMED-90 x 210-5, effective membrane area of single membrane: 90 x 210mm2Stack repeat unit 20, stack configuration C-a-C electrode material: the titanium fetters electrode plate has the following operation modes: a constant current mode; operating current: 1A; the concentration chamber and the desalination chamber adopt a volume ratio of 1: 1; flow rate: 100mL/min), sialic acid conductivity was less than 2ms/cm, and after passing through 200g of ad-1 anion exchange resin, the bed volume was washed with deionized water at a flow rate of 1.5BV/h for 6 times the bed volume, followed by elution with 0.2mol/L formic acid solution (elution flow rate: 1 BV/H; the elution concentration was: 0.2mol/L, elution volume: 5BV, no sialic acid was spotted on the elution liquid) and the eluate was collected.
6) Concentrating the eluent obtained in the step 5) to obtain sialic acid with the concentration of 400g/L, adjusting the pH value to 1 by formic acid, cooling to 20 ℃, adding seed crystals and 0.8 times of acetone by volume, continuously stirring for 1h at 300rpm, slowly cooling to 4 ℃, and precipitating crystals.
7) And (3) carrying out suction filtration on the crystals obtained in the step 6), removing the mother liquor, adding 20 vt% ethanol water solution, washing twice, and carrying out suction filtration to obtain 14.72g of crystals.
8) The crystals from step 7) were dried in a vacuum oven at 40 ℃ for 24h at 10 mbar.
The purity of the dried crystal was 99.02%, and the yield was 90.07%.
Example 3
1) Carrying out acidolysis on 2000mL of fermentation liquor containing polysialic acid (polysialic acid hydrolysate 8.1g/L) at 85 ℃ for 3.5h by adjusting pH to 2.0 with phosphoric acid to obtain 2049mL of acidolysis solution; wherein the concentration of sialic acid in the acidolysis solution is 7.9 g/L.
2) Passing the acidolysis solution obtained in the step 1) through a 50nm ceramic membrane, collecting a permeate, washing the retentate with tap water until the retentate does not contain sialic acid, and collecting the ceramic membrane permeate.
3) Passing the ceramic membrane permeate obtained in the step 2) through a 4000Da membrane to remove macromolecular impurities such as protein and nucleic acid, continuously washing the trapped fluid until no sialic acid is contained, and collecting the ultrafiltration membrane permeate.
4) Concentrating the ultrafiltration membrane permeate obtained in the step 3) by a 200Da membrane for desalting, continuously washing with deionized water until the conductivity of the permeate is less than 500us/cm, and collecting the trapped fluid.
5) Adjusting the pH of the retentate of step 4) to 2.8 with phosphoric acid and performing electrodialysis (specification: GJBMED-90 x 210-5, effective membrane area of single membrane: 90 x 210mm2Stack repeat unit 20, stack configuration C-a-C electrode material: the titanium fetters electrode plate has the following operation modes: a constant current mode; operating current: 0.6A; the concentration chamber and the desalination chamber adopt a volume ratio of 1: 1; flow rate: 120mL/min), sialic acid conductivity was less than 2ms/cm, and after passing through 200g of ad-1 anion exchange resin, the bed volume was washed 5 times with deionized water at a flow rate of 1BV/h, and then eluted with 0.5mol/L acetic acid solution (elution flow rate: 1BV/H, elution volume: 7BV, no sialic acid was spotted on the elution liquid) and the eluate was collected.
6) Concentrating the eluent obtained in the step 5) to obtain the sialic acid with the concentration of 280g/L, adjusting the pH to 1 by using hydrochloric acid, cooling to 20 ℃, adding seed crystals and 0.8 times of acetone by volume, continuously stirring at 350rpm for 1h, slowly cooling to 1 ℃, and precipitating crystals.
7) And (3) carrying out suction filtration on the crystals obtained in the step 6), removing the mother liquor, adding 20 vt% ethanol water solution, washing twice, and carrying out suction filtration to obtain 14.80g of crystals.
8) The crystals from step 7) were dried in a vacuum oven at 40 ℃ for 24h at 10 mbar.
The purity of the dried crystal was 99.1%, and the yield was 90.6%.
Example 4
1) Carrying out acidolysis on 2000mL of fermentation liquor (polysialic acid hydrolysate 8.1g/L) containing polysialic acid at 90 ℃ for 3.5h by adjusting the pH to 3.0 with sulfuric acid to obtain 2052mL of acidolysis solution; wherein the concentration of sialic acid in the acidolysis solution is 7.89 g/L.
2) Passing the acidolysis solution obtained in the step 1) through a 50nm ceramic membrane, collecting a permeate, washing the retentate with tap water until the retentate does not contain sialic acid, and collecting the ceramic membrane permeate.
3) Passing the collected ceramic membrane permeate liquid obtained in the step 2) through a 10000Da membrane to remove macromolecular impurities such as protein, nucleic acid and the like, continuously washing the trapped liquid until no sialic acid is contained, and collecting the ultrafiltration membrane permeate liquid.
4) Concentrating the ultrafiltration membrane permeate obtained in the step 3) by a membrane of 100Da for desalting, continuously washing with deionized water until the conductivity of the permeate is less than 500us/cm, and collecting the trapped fluid.
5) Adjusting the pH of the trapped fluid obtained in the step 4) to 2.5 by hydrochloric acid, and performing electrodialysis (specification type: GJBMED-90 x 210-5, effective membrane area of single membrane: 90 x 210mm2Stack repeat unit 20, stack configuration C-a-C electrode material: the titanium fetters electrode plate has the following operation modes: a constant current mode; operating current: 0.1A; the concentration chamber and the desalination chamber adopt a volume ratio of 1: 1; flow rate: 40mL/min), sialic acid conductivity was less than 2ms/cm, and after passing through 200g of ad-1 anion exchange resin, 3 times the bed volume was washed with deionized water at a flow rate of 1BV/h, followed by elution with 0.5mol/L acetic acid solution (elution flow rate: 1BV/H, elution volume: 7BV, no sialic acid was spotted on the elution liquid) and the eluate was collected.
6) Concentrating the eluent obtained in the step 5) to obtain sialic acid with the concentration of 250g/L, adjusting the pH to 1 by using hydrochloric acid, cooling to 20 ℃, adding seed crystals and 0.8 times of acetone by volume, continuously stirring at 350rpm for 1h, slowly cooling to 1 ℃, and precipitating crystals;
7) and (3) carrying out suction filtration on the crystals obtained in the step 6), removing the mother liquor, adding 20 vt% ethanol water solution, washing twice, and carrying out suction filtration to obtain 15.15g of crystals.
8) The crystals from step 7) were dried in a vacuum oven at 40 ℃ for 24h at 10 mbar.
The purity of the dried crystal was 99.1%, and the yield was 92.7%.
The present invention provides a method and a concept for separating and extracting N-acetylneuraminic acid, and a plurality of methods and ways for implementing the technical scheme, 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, many modifications and embellishments can be made without departing from the principle of the present invention, and these modifications and embellishments 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.
Claims (10)
1. A method for separating and extracting N-acetylneuraminic acid is characterized by comprising the following steps:
(1) carrying out acidolysis on the solution containing the polysialic acid to obtain acidolysis solution;
(2) removing solid impurities from the acidolysis solution obtained in the step (1) through a first membrane to obtain a first permeate; passing the first permeate through a second membrane to remove macromolecular impurities to obtain a second permeate; passing the second permeate through a third membrane to remove monovalent salt, and collecting trapped fluid;
(3) adjusting the pH of the trapped fluid to the isoelectric point of N-acetylneuraminic acid, removing multivalent salt through electrodialysis, washing impurities and eluting the solution without multivalent salt through anion exchange resin, collecting effluent liquid, and concentrating to obtain concentrated solution;
(4) and (4) adjusting the pH value of the concentrated solution obtained in the step (3) to 1-3, cooling, adding a reverse solvent, separating out crystals, centrifuging, washing and drying to obtain the compound.
2. The method as claimed in claim 1, wherein in the step (1), the acid hydrolysis is performed at a pH of 1.5-3.5 and at a temperature of 65-90 ℃ for 4-8 h.
3. The method according to claim 2, wherein the pH regulator is any one or combination of phosphoric acid, sulfuric acid and hydrochloric acid.
4. The method according to claim 1, wherein in step (2), the first membrane is a ceramic membrane; the aperture of the ceramic membrane is 50-500 nm; the second membrane is an ultrafiltration membrane, and the molecular weight cutoff of the second membrane is 2000-10000 Da; the third membrane is a nanofiltration membrane, and the molecular weight cut-off of the third membrane is 100-500 Da.
5. The method according to claim 1, wherein in step (3), the electrodialysis is operated in a constant current mode; the running current is 0.1-1A; the volume ratio of the concentration chamber to the desalination chamber is 1: 1; controlling the flow rate to be 40-100 mL/min; the conductivity of the desalting chamber after removing the solution of the multivalent salt is lower than 2 ms/cm.
6. The method according to claim 1, wherein in step (3), the anion exchange resin is anion exchange resin AD-1.
7. The method according to claim 1, wherein in the step (3), the impurity washing agent is water; the flow rate of impurity washing is 1-2 BV/h; the volume of the washing impurities is 2-8 BV; the eluent of the elution is acid solution or salt solution; the flow rate of elution is 1 BV/h; the volume of elution is 3-15 BV; and (5) eluting until the eluent does not contain N-acetylneuraminic acid, and finishing the elution.
8. The method according to claim 7, wherein the acid solution is any one or a combination of hydrochloric acid, sulfuric acid, formic acid and acetic acid; the salt solution is any one or a combination of more of sodium chloride aqueous solution, potassium chloride aqueous solution and magnesium chloride aqueous solution; the concentration of the acid solution and the concentration of the salt solution are both 0.05-1 mol/L.
9. The method according to claim 1, wherein in the step (3), the concentration of N-acetylneuraminic acid in the concentrated solution is 150-500 g/L.
10. The method according to claim 1, wherein in the step (4), the temperature is reduced to 20-35 ℃; the reverse solvent is any one or combination of acetone, propanol and isopropanol; the volume ratio of the reverse solvent to the concentrated solution after the pH adjustment is 0.8-2: 1.
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