CN111733092B - Method for producing polysialic acid by fermentation and extraction and refining method thereof - Google Patents
Method for producing polysialic acid by fermentation and extraction and refining method thereof Download PDFInfo
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- CN111733092B CN111733092B CN202010396209.XA CN202010396209A CN111733092B CN 111733092 B CN111733092 B CN 111733092B CN 202010396209 A CN202010396209 A CN 202010396209A CN 111733092 B CN111733092 B CN 111733092B
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- 238000000855 fermentation Methods 0.000 title claims abstract description 71
- 230000004151 fermentation Effects 0.000 title claims abstract description 71
- 239000002253 acid Substances 0.000 title claims abstract description 56
- 238000000034 method Methods 0.000 title claims abstract description 28
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 21
- 238000007670 refining Methods 0.000 title abstract description 7
- 238000000605 extraction Methods 0.000 title abstract description 6
- 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 12
- 239000001963 growth medium Substances 0.000 claims abstract description 9
- 230000001105 regulatory effect Effects 0.000 claims abstract description 8
- 239000003957 anion exchange resin Substances 0.000 claims abstract description 7
- 238000001816 cooling Methods 0.000 claims abstract description 7
- 238000003756 stirring Methods 0.000 claims abstract description 5
- 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 32
- 239000008103 glucose Substances 0.000 claims description 32
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 23
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 claims description 15
- 239000011734 sodium Substances 0.000 claims description 15
- 229910052708 sodium Inorganic materials 0.000 claims description 15
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims description 11
- 235000011114 ammonium hydroxide Nutrition 0.000 claims description 11
- 238000011218 seed culture Methods 0.000 claims description 10
- 229910052799 carbon Inorganic materials 0.000 claims description 8
- 235000019441 ethanol Nutrition 0.000 claims description 8
- 239000007788 liquid Substances 0.000 claims description 8
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 6
- 230000001133 acceleration Effects 0.000 claims description 6
- 238000004321 preservation Methods 0.000 claims description 6
- 238000005406 washing Methods 0.000 claims description 6
- 239000000047 product Substances 0.000 claims description 5
- 230000001276 controlling effect Effects 0.000 claims description 4
- 238000012258 culturing Methods 0.000 claims description 4
- 239000012528 membrane Substances 0.000 claims description 4
- 238000002156 mixing Methods 0.000 claims description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 3
- 239000007640 basal medium Substances 0.000 claims description 2
- 238000004821 distillation Methods 0.000 claims description 2
- 239000000706 filtrate Substances 0.000 claims description 2
- 238000001291 vacuum drying Methods 0.000 claims description 2
- 230000003698 anagen phase Effects 0.000 claims 1
- 239000006228 supernatant Substances 0.000 claims 1
- 238000000108 ultra-filtration Methods 0.000 claims 1
- 239000002994 raw material Substances 0.000 abstract description 10
- 235000013305 food Nutrition 0.000 abstract description 9
- SQVRNKJHWKZAKO-UHFFFAOYSA-N beta-N-Acetyl-D-neuraminic acid Natural products CC(=O)NC1C(O)CC(O)(C(O)=O)OC1C(O)C(O)CO SQVRNKJHWKZAKO-UHFFFAOYSA-N 0.000 abstract description 6
- SQVRNKJHWKZAKO-PFQGKNLYSA-N N-acetyl-beta-neuraminic acid Chemical compound CC(=O)N[C@@H]1[C@@H](O)C[C@@](O)(C(O)=O)O[C@H]1[C@H](O)[C@H](O)CO SQVRNKJHWKZAKO-PFQGKNLYSA-N 0.000 abstract description 5
- 238000005265 energy consumption Methods 0.000 abstract description 4
- 239000003814 drug Substances 0.000 abstract description 3
- 239000000178 monomer Substances 0.000 abstract description 3
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- 229940079593 drug Drugs 0.000 abstract description 2
- 239000002158 endotoxin Substances 0.000 abstract description 2
- 230000007062 hydrolysis Effects 0.000 abstract description 2
- 238000006460 hydrolysis reaction Methods 0.000 abstract description 2
- SQVRNKJHWKZAKO-OQPLDHBCSA-N sialic acid Chemical compound CC(=O)N[C@@H]1[C@@H](O)C[C@@](O)(C(O)=O)OC1[C@H](O)[C@H](O)CO SQVRNKJHWKZAKO-OQPLDHBCSA-N 0.000 abstract description 2
- 239000000243 solution Substances 0.000 description 21
- 238000004128 high performance liquid chromatography Methods 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 4
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 4
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 238000010521 absorption reaction Methods 0.000 description 4
- -1 carboxybenzyl Chemical group 0.000 description 4
- 238000000746 purification Methods 0.000 description 4
- 239000000600 sorbitol Substances 0.000 description 4
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 230000003115 biocidal effect Effects 0.000 description 3
- 239000011575 calcium Substances 0.000 description 3
- 238000010828 elution Methods 0.000 description 3
- 238000002329 infrared spectrum Methods 0.000 description 3
- 239000011347 resin Substances 0.000 description 3
- 229920005989 resin Polymers 0.000 description 3
- 238000001179 sorption measurement Methods 0.000 description 3
- 230000001018 virulence Effects 0.000 description 3
- DUKURNFHYQXCJG-UHFFFAOYSA-N Lewis A pentasaccharide Natural products OC1C(O)C(O)C(C)OC1OC1C(OC2C(C(O)C(O)C(CO)O2)O)C(NC(C)=O)C(OC2C(C(OC3C(OC(O)C(O)C3O)CO)OC(CO)C2O)O)OC1CO DUKURNFHYQXCJG-UHFFFAOYSA-N 0.000 description 2
- 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
- 239000006057 Non-nutritive feed additive Substances 0.000 description 2
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 2
- 239000004098 Tetracycline Substances 0.000 description 2
- 241000209149 Zea Species 0.000 description 2
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 2
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 2
- 229910021529 ammonia Inorganic materials 0.000 description 2
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 2
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 2
- 235000011130 ammonium sulphate Nutrition 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 159000000007 calcium salts Chemical class 0.000 description 2
- 150000001721 carbon Chemical group 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 229960005091 chloramphenicol Drugs 0.000 description 2
- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 description 2
- 235000005822 corn Nutrition 0.000 description 2
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 2
- 238000003306 harvesting Methods 0.000 description 2
- 238000005342 ion exchange Methods 0.000 description 2
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 2
- 235000019341 magnesium sulphate Nutrition 0.000 description 2
- 238000009629 microbiological culture Methods 0.000 description 2
- 229950006780 n-acetylglucosamine Drugs 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 108010009004 proteose-peptone Proteins 0.000 description 2
- GHMLBKRAJCXXBS-UHFFFAOYSA-N resorcinol Chemical compound OC1=CC=CC(O)=C1 GHMLBKRAJCXXBS-UHFFFAOYSA-N 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 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
- 235000019364 tetracycline Nutrition 0.000 description 2
- 150000003522 tetracyclines Chemical class 0.000 description 2
- 239000011782 vitamin Substances 0.000 description 2
- 229940088594 vitamin Drugs 0.000 description 2
- 229930003231 vitamin Natural products 0.000 description 2
- 235000013343 vitamin Nutrition 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 1
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 1
- 101100001161 Escherichia coli aggR gene Proteins 0.000 description 1
- 101100500479 Hafnia alvei eaeA gene Proteins 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- FKNQFGJONOIPTF-UHFFFAOYSA-N Sodium cation Chemical compound [Na+] FKNQFGJONOIPTF-UHFFFAOYSA-N 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- 238000000862 absorption spectrum Methods 0.000 description 1
- 239000003463 adsorbent Substances 0.000 description 1
- 238000005273 aeration Methods 0.000 description 1
- 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
- 229960000723 ampicillin Drugs 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 238000005452 bending Methods 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 125000004432 carbon atom Chemical group C* 0.000 description 1
- 239000000919 ceramic Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000000536 complexating effect Effects 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000002416 diarrheagenic effect Effects 0.000 description 1
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 1
- 235000019797 dipotassium phosphate Nutrition 0.000 description 1
- 101150107911 eae gene Proteins 0.000 description 1
- 238000012869 ethanol precipitation Methods 0.000 description 1
- 235000013373 food additive Nutrition 0.000 description 1
- 239000002778 food additive Substances 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 230000001571 immunoadjuvant effect Effects 0.000 description 1
- 239000000568 immunological adjuvant Substances 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 229960000318 kanamycin Drugs 0.000 description 1
- 229930027917 kanamycin Natural products 0.000 description 1
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 1
- 229930182823 kanamycin A Natural products 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 238000002703 mutagenesis Methods 0.000 description 1
- 231100000350 mutagenesis Toxicity 0.000 description 1
- 238000000655 nuclear magnetic resonance spectrum Methods 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 125000004430 oxygen atom Chemical group O* 0.000 description 1
- 235000019371 penicillin G benzathine Nutrition 0.000 description 1
- 229940056360 penicillin g Drugs 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 1
- 239000000651 prodrug Substances 0.000 description 1
- 229940002612 prodrug Drugs 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 229910001415 sodium ion Inorganic materials 0.000 description 1
- 229960000268 spectinomycin Drugs 0.000 description 1
- UNFWWIHTNXNPBV-WXKVUWSESA-N spectinomycin Chemical compound O([C@@H]1[C@@H](NC)[C@@H](O)[C@H]([C@@H]([C@H]1O1)O)NC)[C@]2(O)[C@H]1O[C@H](C)CC2=O UNFWWIHTNXNPBV-WXKVUWSESA-N 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 230000001502 supplementing effect Effects 0.000 description 1
- 229960002180 tetracycline Drugs 0.000 description 1
- 229930101283 tetracycline Natural products 0.000 description 1
- 229940040944 tetracyclines Drugs 0.000 description 1
- 238000009423 ventilation Methods 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
- 150000003952 β-lactams Chemical class 0.000 description 1
Classifications
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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
-
- 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
-
- 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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- Organic Chemistry (AREA)
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- Zoology (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
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- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
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- General Engineering & Computer Science (AREA)
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- Chemical Kinetics & Catalysis (AREA)
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- Virology (AREA)
- Tropical Medicine & Parasitology (AREA)
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Abstract
The invention discloses a method for producing polysialic acid by a fermentation method and an extraction and refining method thereof, which comprises the following steps: inoculating Escherichia coli strain SA-8 (CGMCC No. 5585) in basic culture medium, transferring from seed to final fermentation, introducing pure oxygen and air, fermenting for 10 hr, gradually regulating pH value and gradually lowering temperature, and adding Ca (OH) in late fermentation stage 2 And regulating the pH value to be above 8.0, stopping stirring for 18-19 hours, cooling, continuously introducing common air for 1.8-2.2 hours, and finally fermenting to prepare the polysialic acid, wherein the obtained polysialic acid can be purified by using anion exchange resin. The method is simple, saves cost, reduces energy consumption, ensures that the obtained polysialic acid has high purity and does not contain endotoxin, can meet the production requirement of continuous hydrolysis into monomer sialic acid (N-acetylneuraminic acid), and can be used as raw materials for producing foods, cosmetics and medicines.
Description
Technical Field
The invention relates to the technical field of bioengineering, in particular to a method for producing polysialic acid by a fermentation method and an extraction and refining method thereof.
Background
There have been many studies and attempts to produce polysialic acid (PSA) by microbial fermentation. The natural strain of the escherichia coli is utilized to finish the process. The polysialic acid has various purposes such as medicine slow release agent, immunoadjuvant, prodrug and the like. And further hydrolysis can give the monomer N-acetylneuraminic acid (Neu 5 Ac), which is an important food and pharmaceutical raw material.
The fermentation method for producing polysialic acid is reported in the literature, and patent CN 103361283 discloses a method for producing poly-N-acetylneuraminic acid by fermenting glucose by adopting a strain CGMCC No.5585. However, the yield of poly-N-acetylneuraminic acid can only reach 6g/L at maximum by using the method.
Patent application CN201811124478 discloses a method for producing polysialic acid by a fermentation method, but different strains are adopted, the strain is CCTCC No. M2018103, and the fermentation scheme is obviously different from that of the patent application CGMCC No.5585. The CCTCC No. M2018103 fermentation scheme adopts sorbitol, while CGMCC No.5585 does not need sorbitol; 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, the CCTCC No. M2018103 needs to be added with vitamins, and the CGMCC No.5585 does not need to be added with any vitamins separately. The fermentation time CCTCC No. M2018103 is more than 40 hours, and the CGMCC No.5585 is only required to be not more than 20 hours; in addition, as food raw materials, acetylglucosamine and CTAB adopted by CCTCC No. M2018103 fermentation are not in the food additive list GB2760-2014 specified by the country, and are not food raw materials or food processing aids allowed to be used, while CGMCC No.5585 adopts glucose, corn steep liquor and ammonia water, which are both food raw materials or food processing aids allowed to be used by GB 2760-2014. The fermentation process disclosed in patent application CN201811125667 for the production of polysialic acid is similar to that disclosed in CN201811124478, and as described above, is significantly different from the fermentation process of this patent CGMCC No.5585. Patent application CN201811219956 discloses a method of adding resin adsorbent during fermentation, which is not adopted in the present patent, and any substances having adsorption, coupling and complexing functions on polysialic acid are not used during fermentation. The strain adopts CGMCC No.5585 which is identified by antibiotic resistance and escherichia coli virulence.
Disclosure of Invention
The invention mainly solves the technical problem of providing the method for producing the polysialic acid 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 no endotoxin, and can be used as a raw material for producing foods, cosmetics and medicines.
In order to solve the technical problems, the invention adopts a technical scheme that: there is provided a method for producing polysialic acid by fermentation, comprising the steps of:
(1) Inoculating escherichia coli SA-8 with the volume percentage of 2% -2.5% in a basic culture medium of a seed tank, and fermenting and culturing to obtain a seed culture solution;
(2) Inoculating the seed culture solution into a fermentation tank containing the basic culture medium, introducing normal sterile air at the initial stage of fermentation, fermenting and culturing at a pH of 6.6-6.8 and 36-38deg.C by adopting ammonia water, and adding carbon source, preferably glucose;
(3) Fermenting for 6 hr, mixing pure oxygen with air, ventilating, controlling temperature at 36-38deg.C for 6-10 hr, and controlling pH at 6.6-6.8 with ammonia water; after 10 hours, the temperature is reduced to 33-35 ℃, the pH value is controlled to 6.2-6.5 by ammonia water, and the fermentation is continued by supplementing carbon sources;
(4) After fermentation for 17 hours Ca (OH) was used 2 Regulating pH value of the solution to 8.0-8.5, and cooling to 18-22deg.C; and stopping stirring, continuously maintaining the normal air for 1.8-2.2 hours, ending the fermentation process for 19-21 hours, and finally fermenting to prepare the polysialic acid, wherein the yield is more than 10g/L.
In a preferred embodiment of the invention, the escherichia coli SA-8 in the step (1) has a preservation number of CGMCC No.5585, and the strain has the capacity of high-yield polysialic acid, and the maximum 10 cubic mass production fermentation level is more than 10g/L. Through antibiotic susceptibility identification, the antibiotic is very sensitive to beta lactams (benzyl penicillin, including ampicillin and carboxybenzyl), aminoglycosides (streptomycin, kanamycin, spectinomycin and the like), tetracyclines (tetracycline) and chloramphenicol (chloramphenicol). Sequencing of virulence genes shows that important virulence genes carried by diarrheagenic escherichia coli such as stx1, stx2, eaeA, aggR, ipaH, it, stlb and the like are not contained; at present, the strain has been subjected to patent preservation in China general microbiological culture collection center (CGMCC) No.5585, and the preservation number is: china general microbiological culture Collection center (CGMCC); address: beijingThe North Chen Xilu No. 1 hospital in the area of the city towards the sun; preservation date: 2011. the year 12 and 13, the classification name and Latin chemical name are Escherichia coli(Escherichia coli)。
In a preferred embodiment of the 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, glucose is added after seed culture solution is inoculated, the concentration of glucose in fermentation liquor is maintained to be 2-3 g/L in the first 6-7 hours, fermentation is carried out after 6 hours and enters a logarithmic phase, and the concentration of glucose in fermentation liquor is maintained to be 5-8 g/L in the last 8-12 hours; the glucose flow acceleration is reduced after 13-17 hours of fermentation, so that the concentration of glucose in the fermentation liquid is 2-3 g/L, and the glucose is stopped after 17 hours.
In a preferred embodiment of the present invention, the ammonia water in step (2) has a mass percentage concentration of 20% -33%, ca (OH) 2 The 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) 2 The pH value of the solution is regulated to 8.0-8.5.
In a preferred embodiment of the present invention, the concentration of pure oxygen in the step (3) is >96% when the pure oxygen is mixed with air for aeration, and the volume ratio is that of pure oxygen: air = 0.5-1:1.
in order to solve the technical problem of polysialic acid extraction, the invention provides an embodiment for purifying polysialic acid, 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, the polysialic acid ammonia and calcium salt are changed into polysialic acid sodium by using weak-alkaline anion exchange resin;
(2) Ultrafiltering sodium polysialide with 70000Dalton ultrafilter membrane, washing with pure water to adjust pH to below 7.2, discarding filtrate, and retaining ultrafiltered concentrated solution;
(3) Adding three times of volume of absolute ethyl alcohol into the concentrated solution of the polysialic acid sodium, standing for cooling, washing with the ethyl alcohol with the mass percent concentration of more than 70%, and then decompressing for distillation or freeze vacuum drying to remove the ethyl alcohol, thus obtaining the polysialic acid finished product.
The beneficial effects of the invention are as follows:
1. the fermentation yield of the polysialic acid can reach 10g/L, which is higher than the average level of the prior art, and is particularly suitable for industrialized fermentation production of polysialic acid;
2. the fermentation raw material of the invention uses cheap glucose to replace more expensive sorbitol, so that the production cost can be further reduced;
3. the invention adopts pure oxygen with a certain proportion to cool down in the middle, and Ca (OH) is used in the later period of fermentation 2 The pH is regulated, so that the fermentation yield is greatly improved;
4. the purification method adopts ion exchange, firstly changes the product into sodium polysialide through weak-base anion exchange resin, then elutes sodium chloride solution, and does not adopt adsorption resin and ethanol for elution, thereby saving the cost and reducing the energy consumption.
5. The purity of the high-purity polysialic acid sodium HPLC prepared by the purification and refining method is more than 98 percent, and the yield can reach 10g/L.
Drawings
For a clearer description of the technical solutions of the embodiments of the present invention, the drawings that are needed in the description of the embodiments will be briefly introduced below, it being obvious that the drawings in the description below are only some embodiments of the present invention, and that other drawings can be obtained according to these drawings without inventive effort for a person skilled in the art, wherein:
FIG. 1 is an ultraviolet absorbance spectrum of polysialic acid of the invention, sample: wavelength scan range of PSA aqueous solution: 210-400nm;
FIG. 2 is an infrared spectrum of polysialic acid of the present invention;
FIG. 3 is a high performance liquid spectrum of polysialic acid of the present invention.
Detailed Description
The following description of the technical solutions in the embodiments of the present invention will be clear and complete, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
The fermentation strain is Escherichia coli SA-8 subjected to mutagenesis screening by an industrial microbiology laboratory of China academy of sciences, and the patent preservation number is CGMCC NO.5585.
A method for producing polysialic acid by fermentation, comprising the steps of:
(1) Inoculating 2% of escherichia coli SA-8 in volume percentage in a basic culture medium of a seed tank, fermenting and culturing to obtain a seed culture solution, wherein 1000 liters of fermentation is adopted in the seed tank. The basic culture medium is prepared from 25g/L glucose, 5g/L ammonium sulfate, 15g/L casein peptone, 20g/L dipotassium hydrogen phosphate and 0.4g/L magnesium sulfate, and is sterilized by introducing steam after uniformly mixing. After reaching 121 ℃, the pot pressure was maintained at 0.09MPa and sterilized for 30 minutes. The sterilization process requires continuous stirring at 200 rpm. After cooling, 9 liters of the cell culture broth was inoculated, and the cell culture broth was cultured at 37℃for 12 hours to obtain a seed culture broth.
(2) Inoculating seed culture solution into fermentation tank containing the basic culture medium, introducing air, fermenting at 37 deg.C with ammonia water at pH of 6.6-6.8, adding carbon source, fermenting with 10000L (10 m 2) of fermentation tank, inoculating basic culture medium containing glucose 25g/L, ammonium sulfate 5g/L, casein peptone 15g/L, dipotassium phosphate 20g/L, magnesium sulfate 0.4g/L, and inoculating the seed culture solution.
(3) After seed culture solution is inoculated, glucose starts to be fed in, the concentration of the glucose in fermentation liquid is maintained to be 2-3 g/L in the first 6 hours at the acceleration, the fermentation temperature is 37 ℃, the pH is controlled to be 6.6-6.8,6 hours by using ammonia water with the mass percent concentration of 25%, pure oxygen is introduced, and the volume ratio of the pure oxygen to air is 0.75:1, the temperature is still controlled at 37 ℃, and the pH is controlled at 6.6-6.8; after 10 hours, pure oxygenThe volume ratio of the air to the air is 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 broth at 5-8 g/l at a flow acceleration from 6 hours to 12 hours of fermentation; reducing the concentration of glucose from 12 hours to make the concentration of glucose in the fermentation liquor be 2-3 g/L, and stopping adding glucose after 17 hours; after 17 hours of fermentation, ca (OH) with the mass percentage concentration of 0.5% is adopted 2 The pH value of the solution is regulated to 8.0-8.5, stirring is stopped, the temperature is reduced to 20 ℃, pure oxygen is stopped at the moment, sterile air is used instead, ventilation is maintained for 2 hours, and the fermentation process is finished after 20 hours.
(4) After the fermentation was completed, the PSA yield in the fermentation harvest period was measured, and the PSA yield in the fermentation harvest period was measured by the resorcinol method and found to be 10g/L on average.
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 by a 200 nm ceramic membrane, and changing the ammonium polysialide salt into sodium polysialide by a weak alkaline anion exchange resin in clear liquid;
(2) Removing small molecule impurities: ultrafiltering the sodium polysialide solution subjected to ion exchange by using a membrane system with a pore diameter of 70000Dalton, and concentrating macromolecules; and washing the concentrated sodium polysialide solution with pure water to a pH below 7.2, discarding the filtered waste liquid, and retaining the concentrated solution.
(3) Ethanol precipitation PSA: adding three times of volume of absolute ethyl alcohol into the concentrated and washed polysialic acid sodium 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 then distilling under reduced pressure or lyophilizing to remove ethanol to obtain polysialic acid product.
Nuclear magnetic resonance spectrum of polysialic acid, 500Hz, D 2 Chemical shifts of O and carbon atoms are shown in Table 1.
TABLE 1
| The carbon atom position | C chemical shift (ppm) | The carbon atom position | 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 |
The separated polysialic acid is subjected to infrared spectrum analysis, and the infrared spectrum of the polysialic acid is 3358 cm -1 The position has wide and strong absorption, and the stretching vibration of the O-H bond of-OH and the N-H bond of-NH-is shown; at 1626 cm -1 There is a moderate absorption, shown by-COO-and-NHCOCH 3 C=o bond stretching and asymmetric stretching, and N-H bond bending; at 1384 and 1384 cm -1 There is moderate absorption showing C-O bond stretching vibration of-COO-and symmetrical stretching vibration of C=O; at 1080 cm -1 There is strong absorption, showing the stretching vibration of C-O-C (sugar ring). These groups are characteristic groups of an acetamido polysaccharide.
The HPLC diagram of polysialic acid is shown in fig. 3, and the HPLC method is as follows: the chromatographic column is ACE 5AQ C18:250mm×4.6mm; mobile phase 5Mm H 2 SO 4 (A) Methanol (B), gradient elution, elution time of 20min, flow rate of 1mL/min and sample injection amount of 10 mu L; column temperature: 35 ℃. As a result, referring to FIG. 3, the high-purity polysialic acid sodium prepared by the embodiment of the invention can be obtained by calculation of the graph, the HPLC purity is more than 98%, the specific optical rotation is +15 DEG to +17 DEG, the sodium ion content is lower than 8.5%, and the polysialic acid yield can reach more than 10g/L.
| Time | A | B |
| 0 | 75% | 25% |
| 15 | 75% | 25% |
| 15.01 | 100% | 0 |
The method for producing polysialic acid by the fermentation method has 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 is particularly suitable for industrialized fermentation production of polysialic acid; the fermentation raw material of the invention uses cheap glucose to replace more expensive sorbitol, so that the production cost can be further reduced; the invention adopts pure oxygen with a certain proportion to cool down in the middle, and Ca (OH) is used in the later period of fermentation 2 The pH is regulated, so that the fermentation yield is greatly improved; the purification method of the invention adopts weak alkaline anion exchange resin to specifically adsorb sialic acid anions but not small molecule anions. After the product is changed into sodium polysialide through the weak-alkaline anion exchange resin, the adsorption resin and ethanol are not used for eluting, but sodium chloride solution is used for eluting, so that the cost is saved, and the energy consumption is reduced; the purity of the high-purity polysialic acid sodium HPLC prepared by the purification and refining method is more than 98 percent, and the yield can reach 10g/L. The monomer sialic acid produced by hydrolyzing the polysialic acid obtained by the strain and the method of the invention meets the admission standard of the national health commission for producing new food raw material N-acetylneuraminic acid.
The foregoing description is only illustrative of the present invention and is not intended to limit the scope of the invention, and all equivalent structures or equivalent processes or direct or indirect application in other related arts are included in the scope of the present invention.
Claims (4)
1. A method for producing polysialic acid by a fermentation method, which comprises the following steps:
(1) Inoculating 2-2.5% of escherichia coli SA-8 with the preservation number of CGMCC No.5585 into a basic culture medium of a seed tank, and fermenting and culturing to obtain a seed culture solution;
(2) Inoculating the seed culture solution into a fermentation tank containing the basic culture medium, introducing air, adopting ammonia water to control the pH value to be 6.6-6.8, carrying out fermentation culture at 37 ℃, and feeding glucose in the first 6 hours, wherein the concentration of the glucose in the fermentation liquid is maintained to be 2-3 g/L by the flow acceleration;
(3) After fermenting for 6 hours, entering a logarithmic growth phase, mixing and ventilating pure oxygen and air, wherein the volume ratio of the pure oxygen to the air is 0.75:1, the concentration of the pure oxygen is more than 96%, the temperature is still controlled at 37 ℃ for 6-10 hours, and the pH is controlled at 6.6-6.8 by ammonia water; reducing the temperature to 34 ℃ after 10 hours, controlling the pH value to 6.3-6.5,6-12 hours by ammonia water, and continuing to ferment the fed-batch glucose, wherein the concentration of the glucose in the fermentation liquid is maintained to be 5-8 g/L by the flow acceleration, and mixing and aerating pure oxygen and air after 10 hours to 17 hours, wherein the volume ratio of the pure oxygen to the air is 1:1, and the concentration of the pure oxygen is more than 96%;
(4) After fermentation for 17 hours Ca (OH) was used 2 Regulating the pH value of the solution to 8.0-8.5, and cooling to 20 ℃; and stopping stirring, continuously maintaining common air for 1.8-2.2 hours, ending the fermentation process until 19-21 hours, and finally fermenting to obtain polysialic acid, wherein glucose is continuously fed in the fermentation liquor after 13-17 hours, and stopping feeding glucose after 17 hours after the glucose concentration in the fermentation liquor is maintained to be 2-3 g/L by the acceleration of the feeding.
2. The method for producing polysialic acid by fermentation according to claim 1, wherein the carbon source of the basal medium in the step (1) is glucose, and the glucose content is 10-35 g/l.
3. A method for producing polysialic acid by fermentation according to claim 1The method is characterized in that the mass percentage concentration of the ammonia water in the step (2) is 20-33 percent, and Ca (OH) in the step (4) 2 The mass percentage concentration of the solution is 0.5-0.6%.
4. A method according to any one of claims 1 to 3, further comprising the step of purifying the purified polysialic acid:
(1) Subjecting the supernatant obtained by separating the polysialic acid fermentation broth obtained by the method of any one of claims 1, 2 and 3 to weak-base anion exchange resin to obtain polysialic acid sodium;
(2) Ultrafiltering the obtained sodium polysialide with 70000Dalton ultrafiltration membrane, washing with pure water to adjust pH to below 7.2, discarding filtrate, and retaining ultrafiltered concentrated solution;
(3) Adding three times of volume of absolute ethyl alcohol into the concentrated solution of the polysialic acid sodium, standing for cooling, washing with the ethyl alcohol with the mass percent concentration of more than 70%, and then decompressing for distillation or freeze vacuum drying to remove the ethyl alcohol, thus obtaining the polysialic acid finished product.
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