CN111733092A - Fermentation process of producing polysialic acid and its extracting and refining process - Google Patents
Fermentation process of producing polysialic acid and its extracting and refining process Download PDFInfo
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- 230000004151 fermentation Effects 0.000 title claims abstract description 77
- 239000002253 acid Substances 0.000 title claims abstract description 70
- 238000000034 method Methods 0.000 title claims abstract description 27
- 238000007670 refining Methods 0.000 title claims abstract description 9
- 238000004519 manufacturing process Methods 0.000 claims abstract description 21
- MYMOFIZGZYHOMD-UHFFFAOYSA-N Dioxygen Chemical compound O=O MYMOFIZGZYHOMD-UHFFFAOYSA-N 0.000 claims abstract description 16
- 241000588724 Escherichia coli Species 0.000 claims abstract description 14
- 239000002994 raw material Substances 0.000 claims abstract description 11
- 238000001816 cooling Methods 0.000 claims abstract description 8
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- 238000003756 stirring Methods 0.000 claims abstract description 5
- 239000007640 basal medium Substances 0.000 claims abstract description 3
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 33
- 239000008103 glucose Substances 0.000 claims description 33
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- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
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- OVRNDRQMDRJTHS-FMDGEEDCSA-N N-acetyl-beta-D-glucosamine Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O OVRNDRQMDRJTHS-FMDGEEDCSA-N 0.000 description 2
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- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 2
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- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
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- 229930003231 vitamin Natural products 0.000 description 2
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- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 1
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- 101100001161 Escherichia coli aggR gene Proteins 0.000 description 1
- 101100500479 Hafnia alvei eaeA gene Proteins 0.000 description 1
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- 108010080698 Peptones Proteins 0.000 description 1
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- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 1
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- 238000000862 absorption spectrum Methods 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
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- 229940126575 aminoglycoside Drugs 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
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- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 1
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- 239000002699 waste material Substances 0.000 description 1
- 150000003952 β-lactams Chemical class 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
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Abstract
The invention discloses a fermentation method for producing polysialic acid and an extraction and refining method thereof, which comprises the following steps: inoculating Escherichia coli strain SA-8(CGMCC No.5585) in basal medium, transferring from seeds to final fermentation, introducing into logarithmic growth phase, introducing pure oxygen and air, fermenting for 10 hr, gradually lowering pH and temperature, and fermenting with Ca (OH) in late stage2Adjusting the pH value to be more than 8.0, stopping stirring and cooling within 18-19 hours, only continuously introducing common air for 1.8-2.2 hours, and finally fermenting to prepare polysialic acid, wherein the obtained polysialic acid can be purified by anion exchange resin. The method of the invention is simple, saves cost, reduces energy consumption, and the obtained polysialic acid has high purity,Does not contain endotoxin, can meet the production requirement of continuously hydrolyzing to monomer sialic acid (N-acetylneuraminic acid), and can be used as raw materials for producing food, cosmetics and medicines.
Description
Technical Field
The invention relates to the technical field of bioengineering, in particular to a fermentation method for producing polysialic acid and an extraction and refining method thereof.
Background
There have been many studies and attempts to produce polysialic acid (PSA) by microbial fermentation. Can be finished by utilizing natural strains of escherichia coli. The polysialic acid has multiple purposes such as drug sustained release agent, immunologic adjuvant, prodrug and the like. And further hydrolyzing to obtain monomer N-acetylneuraminic acid (Neu5Ac), which is important food and medicine raw material.
The fermentation method for producing polysialic acid is reported in the literature at present, and the patent CN 103361283 discloses a method for producing poly N-acetylneuraminic acid by glucose fermentation by adopting a bacterial strain CGMCC No. 5585. However, the yield of poly-N-acetylneuraminic acid can only reach 6g/L at most by using the method.
Patent application CN201811124478 discloses a method for producing polysialic acid by a fermentation method, but the adopted strain is different, namely CCTCC number M2018103, and the fermentation scheme is obviously different from the fermentation scheme of the patent application CGMCC No. 5585. The CCTCC number M2018103 fermentation scheme adopts sorbitol, and the CGMCC No.5585 does not need sorbitol; the CCTCC No. M2018103 adopts acetylglucosamine, the CGMCC No.5585 is common glucose, the CCTCC No. M2018103 adopts peptone and the CGMCC No.5585 adopts simplest and cheapest corn steep liquor in the aspect of a nitrogen source, and the CCTCC No. M2018103 needs to be added with vitamins and the CGMCC No.5585 does not need to be added with any vitamin independently. The fermentation time is CCTCC No. M2018103 more than 40 hours, and the CGMCC No.5585 only needs no more than 20 hours; in addition, as food raw materials, the acetylglucosamine and CTAB adopted by CCTCC number M2018103 fermentation are not in the food additive name list GB2760-2014 specified by the state and do not belong to food raw materials or food processing aids allowed to be used, and the CGMCC No.5585 adopts glucose, corn steep liquor and ammonia water which are food raw materials or food processing aids allowed to be used by GB 2760-2014. The fermentation method disclosed in patent application CN201811125667 for producing polysialic acid is similar to that disclosed in patent application CN201811124478, and as mentioned above, is significantly different from the fermentation process of the present patent CGMCC No. 5585. Patent application CN201811219956 discloses a method for adding resin adsorbent in fermentation process, and this patent does not adopt this method, and does not use any substance having adsorption, coupling and complexing effects on polysialic acid in fermentation process. The strain adopts CGMCC No.5585 which is subjected to antibiotic resistance identification and escherichia coli virulence identification.
Disclosure of Invention
The invention mainly solves the technical problem of providing the polysialic acid produced by the fermentation method and the extraction and refining method thereof, the method is simple, the cost is saved, the energy consumption is reduced, and the obtained polysialic acid has high purity and does not contain endotoxin, and can be used as a raw material for producing food, cosmetics and medicines.
In order to solve the technical problems, the invention adopts a technical scheme that: a method for producing polysialic acid by fermentation is provided, which comprises the following steps:
(1) inoculating 2-2.5 volume percent of escherichia coli SA-8 into a basic culture medium of a seeding tank, and performing fermentation culture to obtain a seed culture solution;
(2) inoculating seed culture solution into a fermentation tank containing the basic culture medium, introducing common sterile air at the initial stage of fermentation, controlling pH at 6.6-6.8 and 36-38 deg.C with ammonia water, performing fermentation culture, and adding carbon source, preferably, glucose;
(3) fermenting for 6 hr, introducing into logarithmic phase, introducing pure oxygen and air, controlling temperature at 36-38 deg.C for 6-10 hr, and controlling pH at 6.6-6.8 with ammonia water; after 10 hours, reducing the temperature to 33-35 ℃, controlling the pH value to 6.2-6.5 by ammonia water, and continuing carbon source supplementation and fermentation;
(4) fermenting for 17 hours, and then adopting Ca (OH)2Adjusting the pH value of the solution to 8.0-8.5, and cooling to 18-22 ℃; then stopping stirring, continuously keeping normal air for 1.8-2.2 hours, ending the fermentation process until 19-21 hours, finally fermenting to obtain the polysialic acid, wherein the yield is more than 10 g/L.
in a preferred embodiment of the present invention, the Escherichia coli SA-8 described in step (1) has a preservation number of CGMCCNo.5585, has a high polysialic acid-producing ability, has a 10 cubic high-productivity fermentation level of more than 10g/L, and is sensitive to antibiotics, and is resistant to beta-lactams (benzylpenicillin, including ampicillin and carboxybenzyl), aminoglycosides (streptomycin, kanamycin, spectinomycin)And the like), tetracyclines (tetracyclines), chloramphenics (chloramphenicol), and the like are all very sensitive. Virulence gene sequencing shows that the strain does not contain important virulence genes carried by diarrheagenic escherichia coli such as stx1, stx2, eaeA, aggR, ipaH, It, stlb and the like; at present, the strain is subjected to patent preservation in China general microbiological culture Collection center, the preservation number is CGMCC No.5585, and the preservation unit is as follows: china general microbiological culture Collection center (CGMCC for short); address: xilu No. 1 Hospital, Beijing, Chaoyang, area; the preservation date is as follows: 12/13/2011, nomenclature of taxonomic nomenclature and Latin's name Escherichia coli(Escherichia coli)。
In a preferred embodiment of the present invention, the carbon source of the basal medium in step (1) is glucose, and the glucose content is 10-35 g/L.
In a preferred embodiment of the present invention, the carbon source fed in step (2) is glucose.
In a preferred embodiment of the invention, the glucose feeding is started after the seed culture solution is inoculated, the glucose concentration in the fermentation liquid is maintained to be 2-3 g/L at the feeding speed of 6-7 hours, the fermentation enters the logarithmic phase after 6 hours, and the glucose concentration in the fermentation liquid is maintained to be 5-8 g/L at the feeding speed of 8-12 hours; the glucose feeding speed is reduced after fermentation for 13-17 hours, so that the concentration of glucose in the fermentation liquor is 2-3 g/L, and the glucose is stopped after 17 hours.
In a preferred embodiment of the present invention, the concentration of the ammonia water in the step (2) is 20-33% by mass, and Ca (OH)2The mass percentage concentration of the solution is 0.5-0.6%.
In a preferred embodiment of the present invention, Ca (OH) is used in step (2)2The pH value of the solution is adjusted to 8.0-8.5.
In a preferred embodiment of the present invention, the pure oxygen used in step (3) is aerated with a mixture of pure oxygen and air, wherein the concentration of the pure oxygen is >96%, and the volume ratio is pure oxygen: air = 0.5-1: 1.
in order to solve the technical problem of polysialic acid extraction, the invention provides an embodiment of polysialic acid purification, which comprises the following steps:
(1) taking fermentation liquor of Escherichia coli CGMCC No.5585 as a raw material, wherein polysialic acid ammonia and calcium salt are formed in the fermentation process, and then changing the fermentation liquor into polysialic acid sodium by using weak-base anion exchange resin;
(2) ultrafiltering sodium polysialic acid with 70000Dalton ultrafiltration membrane, washing with pure water to adjust pH to below 7.2, discarding filtrate, and retaining the ultrafiltered concentrated solution;
(3) adding anhydrous ethanol with the volume being three times that of the concentrated solution of the polysialic acid sodium, standing, cooling, washing with ethanol with the mass percentage concentration being more than 70%, and then carrying out reduced pressure distillation or freeze vacuum drying to remove the ethanol, thus obtaining the polysialic acid finished product.
The invention has the beneficial effects that:
1. the fermentation yield of the polysialic acid can reach 10g/L, which is higher than the average level of the prior art, and the method is particularly suitable for industrial fermentation production of the polysialic acid;
2. the fermentation raw material of the invention uses cheap glucose to replace expensive sorbitol, thus further reducing the production cost;
3. the invention adopts the steps of introducing pure oxygen with a certain proportion, reducing the temperature midway and using Ca (OH) at the later stage of fermentation2The pH value is adjusted, so that the fermentation yield is greatly improved;
4. the purification method adopts ion exchange, firstly, sodium polysialic acid is changed into a product through weak-base anion exchange resin, then sodium chloride solution is eluted, and adsorption resin and ethanol elution are not adopted, so that the cost is saved, and the energy consumption is reduced.
5. The high-purity polysialic acid sodium prepared by the purification and refining method has the HPLC purity of over 98 percent and the yield of 10 g/L.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments are briefly introduced below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without inventive efforts, wherein:
FIG. 1 is a UV absorption spectrum of polysialic acid of the present invention, sample: wavelength scanning range of PSA aqueous solution: 210-400 nm;
FIG. 2 is an infrared spectrum of polysialic acid of the present invention;
FIG. 3 is a high performance liquid chromatography of polysialic acid of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example one
The fermentation strain is Escherichia coli SA-8 obtained by mutagenesis screening in the industrial microorganism research laboratory of the institute of microbiology of Chinese academy of sciences, and has a patent collection number of CGMCC NO. 5585.
A method for producing polysialic acid by fermentation, comprising the steps of:
(1) inoculating 2% of Escherichia coli SA-8 by volume percentage in a basic culture medium of a seeding tank, and performing fermentation culture to obtain a seed culture solution, wherein the seeding tank adopts 1000L fermentation. The basic culture medium is glucose 25g/L, ammonium sulfate 5g/L, casein peptone 15g/L, dipotassium hydrogen phosphate 20g/L, magnesium sulfate 0.4g/L, after mixing uniformly, steam sterilization is conducted. After reaching 121 ℃, maintaining the tank pressure at 0.09MPa, and sterilizing for 30 minutes. The sterilization process requires continuous stirring at 200 rpm. After cooling, 9 liters of the cell culture solution was inoculated and cultured at 37 ℃ for 12 hours to obtain a seed culture solution.
(2) Inoculating the seed culture solution into a fermentation tank containing the basic culture medium, introducing air, controlling the pH to be 6.6-6.8 by ammonia water, carrying out fermentation culture at 37 ℃, adding a carbon source in parallel, fermenting the fermentation tank by adopting 10000 liters (10 m for thin film cultivation), and inoculating the seed culture solution, wherein the basic culture medium is 25g/L of glucose, 5g/L of ammonium sulfate, 15g/L of casein peptone, 20g/L of dipotassium phosphate and 0.4g/L of magnesium sulfate.
(3) After inoculating the seed culture solution, beginning to feed glucose, maintaining the concentration of the glucose in the fermentation liquid to be 2-3 g/L at the feeding speed of the first 6 hours, controlling the pH to be 6.6-6.8 by using ammonia water with the mass percent concentration of 25%, and after 6 hours, feeding pure oxygen, wherein the volume ratio of the pure oxygen to air is 0.75: 1, controlling the temperature to be 37 ℃ and the pH to be 6.6-6.8; after 10 hours, the pure oxygen to air volume ratio was 1: 1, controlling the pH value to be 6.3-6.5, and reducing the temperature from 37 ℃ to 34 ℃; maintaining the concentration of glucose in the fermentation liquid at a flow rate of 5-8 g/L from 6 hours to 12 hours of fermentation; starting to reduce the concentration of the glucose from 12 hours to ensure that the concentration of the glucose in the fermentation liquor is 2-3 g/L, and stopping adding the glucose after 17 hours; after fermenting for 17 hours, Ca (OH) with the mass percent concentration of 0.5 percent is adopted2Adjusting pH of the solution to 8.0-8.5, stopping stirring, cooling to 20 deg.C, stopping pure oxygen, introducing sterile air, maintaining ventilation for 2 hr, and ending the fermentation process after 20 hr.
(4) After the end of the fermentation, the PSA yield during the harvest period of the fermentation was determined, and the average PSA yield during the harvest period of the fermentation was 10g/L using the resorcinol method.
Example two
The fermentation broth obtained in example 1 was used. The polysialic acid is extracted and refined according to the following steps:
(1) and (3) bacterial liquid separation: removing thalli from a 200-nanometer ceramic membrane, and converting ammonium polysialic acid into sodium polysialic acid by using a clear solution through a weak-base anion exchange resin;
(2) removing small molecular impurities: ultrafiltering the polysialic acid sodium solution after ion exchange by using a pore size 70000Dalton membrane system, and concentrating macromolecules; and washing the concentrated sodium polysialic acid solution with pure water to pH below 7.2, discarding the filtered waste solution, and retaining the concentrated solution.
(3) Ethanol precipitation of PSA: adding anhydrous ethanol with the volume being three times that of the concentrated and washed sodium polysialic acid solution, standing and cooling to below 8 ℃, maintaining for 1 hour, and centrifuging to collect polysialic acid precipitate; washing the precipitate with 70% ethanol for 2 times, and vacuum distilling or freeze vacuum drying to remove ethanol to obtain polysialic acid product.
NMR spectra of polysialic acid of the invention, 500Hz, D2O, chemical shift of carbon atom, as shown in table 1.
TABLE 1
Position of carbon atom | C chemical shift (ppm) | Position of carbon atom | C chemical shift (ppm) |
C-1 | 173.15 | C-7 | 69.01 |
C-2 | 100.92 | C-8 | 77.84 |
C-3 | 39.81 | C-9 | 61.18 |
C-4 | 68.29 | C-10 | 174.96 |
C-5 | 52.38 | C-11 | 22.46 |
C-6 | 73.15 |
Performing infrared spectrum analysis on the polysialic acid obtained by separation, wherein the infrared spectrum of the polysialic acid is 3358 cm-1Wide and strong absorption, showing stretching vibration of O-H bond of-OH and N-H bond of-NH-; at 1626 cm-1In the presence of moderate to strong absorption, are shown as-COO-and-NHCOCH3C = O key stretching vibration and asymmetric stretching vibration, and N — H key bending vibration; at 1384 cm-1Moderate absorption, C-O bond stretching vibration with-COO-and symmetric stretching vibration with C = O; at 1080 cm-1There is strong absorption, showing the stretching vibration of C-O-C (sugar ring). These groups are all characteristic groups of the acetamido polysaccharide.
the HPLC chart of polysialic acid is shown in FIG. 3, and the HPLC method comprises that the chromatographic column is ACE 5AQ C18:250Mm × 4.6Mm, and the mobile phase is 5Mm H2SO4(A) Methanol (B), gradient elution, elution time 20min, flow rate 1mL/min, sample size 10 μ L; column temperature: 35 ℃ is carried out. The results can be seen in FIG. 3, which shows that the high purity sodium polysialic acid prepared by the present invention can be calculated by the figure, the HPLC purity is more than 98%, the specific rotation is +15- +17 degrees, the sodium ion content is less than 8.5%, and the polysialic acid yield can reach more than 10 g/L.
Time of | A | B | |
0 | 75% | 25% | |
15 | 75% | 25% | |
15.01 | 100% | 0 |
The fermentation method for producing polysialic acid and the extraction and refining method have the beneficial effects that: the fermentation yield of the polysialic acid can reach 10g/L, which is higher than the average level of the prior art, and the method is particularly suitable for industrial fermentation production of the polysialic acid; the fermentation raw material of the invention uses cheap glucose to replace expensive sorbitol, thus further reducing the production cost; the invention adopts the steps of introducing pure oxygen with a certain proportion, reducing the temperature midway and using Ca (OH) at the later stage of fermentation2The pH value is adjusted, so that the fermentation yield is greatly improved; the purification method of the invention adopts weak base anion exchange resin to specifically adsorb the polysialic acid anion but not the micromolecular anion. After the product is changed into sodium polysialic acid through the weak-base anion exchange resin, the product is eluted by using a sodium chloride solution instead of using an adsorption resin and ethanol, so that the cost is saved, and the energy consumption is reduced; the high-purity polysialic acid sodium prepared by the purification and refining method has the HPLC purity of over 98 percent and the yield of 10 g/L. The monomeric sialic acid produced by hydrolyzing the polysialic acid obtained by the strain and the method meets the requirement of the national health commission on producing a new food raw material N-acetylnerveAmino acids "are the standard of admission.
The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications of equivalent structures and equivalent processes, which are made by the present specification, or directly or indirectly applied to other related technical fields, are included in the scope of the present invention.
Claims (9)
1. A method for producing polysialic acid by fermentation, comprising the steps of:
(1) inoculating 2-2.5 volume percent of escherichia coli SA-8 into a basic culture medium of a seeding tank, and performing fermentation culture to obtain a seed culture solution;
(2) inoculating the seed culture solution into a fermentation tank containing the basic culture medium, introducing air, controlling the pH to be 6.6-6.8 by adopting ammonia water, performing fermentation culture at 36-38 ℃, and adding a carbon source;
(3) fermenting for 6 hr, introducing into logarithmic phase, introducing pure oxygen and air, controlling temperature at 36-38 deg.C for 6-10 hr, and controlling pH at 6.6-6.8 with ammonia water; after 10 hours, reducing the temperature to 33-35 ℃, controlling the pH value to 6.2-6.5 by ammonia water, and continuing carbon source supplementation and fermentation;
(4) fermenting for 17 hours, and then adopting Ca (OH)2Adjusting the pH value of the solution to 8.0-8.5, and cooling to 18-22 ℃; then stopping stirring, continuously keeping normal air for 1.8-2.2 hours, ending the fermentation process until 19-21 hours, finally fermenting to obtain the polysialic acid, wherein the yield is more than 10 g/L.
2. The method for producing polysialic acid by fermentation according to claim 1, wherein the preservation number of Escherichia coli SA-8 in step (1) is CGMCC No. 5585.
3. The method for producing polysialic acid by fermentation according to claim 1, wherein the carbon source of the basal medium in step (1) is glucose, and the glucose content is 10 to 35 g/l.
4. The method for producing polysialic acid by fermentation according to claim 1, wherein the carbon source fed to step (2) is glucose.
5. The method for producing polysialic acid by fermentation according to claim 4, wherein the method for feeding glucose comprises: after inoculating the seed culture solution, beginning to add glucose, maintaining the concentration of the glucose in the fermentation liquor at a rate of 2-3 g/L within 6-7 hours, and maintaining the concentration of the glucose in the fermentation liquor at a rate of 5-8 g/L within 8-12 hours; the glucose feeding speed is reduced after fermentation for 13-17 hours, so that the concentration of glucose in the fermentation liquor is 2-3 g/L, and the glucose is stopped after 17 hours.
6. The method for producing polysialic acid by fermentation according to claim 1, wherein the concentration of ammonia water in step (2) is 20% to 33% by mass, and Ca (OH)2The mass percentage concentration of the solution is 0.5-0.6%.
7. The method for producing polysialic acid by fermentation according to claim 1, wherein Ca (OH) is used in the step (2)2The pH value of the solution is adjusted to 8.0-8.5.
8. The method for producing polysialic acid by fermentation according to claim 1, wherein the pure oxygen used in step (3) is aerated with a mixture of pure oxygen and air at a concentration of >96%, and the volume ratio is pure oxygen: air = 0.5-1: 1.
9. a method for purifying and refining polysialic acid, which is characterized by comprising the following steps:
(1) using fermentation liquor of Escherichia coli CGMCC No.5585 as raw material, forming polysialic acid ammonia and calcium salt in the fermentation process, and then using weak base anion exchange resin to change into polysialic acid sodium;
(2) ultrafiltering sodium polysialic acid with 70000Dalton ultrafiltration membrane, washing with pure water to adjust pH to below 7.2, discarding filtrate, and retaining the ultrafiltered concentrated solution;
(3) adding anhydrous ethanol with the volume being three times that of the concentrated solution of the polysialic acid sodium, standing, cooling, washing with ethanol with the mass percentage concentration being more than 70%, and then carrying out reduced pressure distillation or freeze vacuum drying to remove the ethanol, thus obtaining the polysialic acid finished product.
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