CN112375160A - Method for separating and purifying hyaluronic acid from microbial fermentation liquor - Google Patents
Method for separating and purifying hyaluronic acid from microbial fermentation liquor Download PDFInfo
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- CN112375160A CN112375160A CN202011310307.3A CN202011310307A CN112375160A CN 112375160 A CN112375160 A CN 112375160A CN 202011310307 A CN202011310307 A CN 202011310307A CN 112375160 A CN112375160 A CN 112375160A
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- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 title claims abstract description 73
- 229920002674 hyaluronan Polymers 0.000 title claims abstract description 73
- 229960003160 hyaluronic acid Drugs 0.000 title claims abstract description 73
- 238000000855 fermentation Methods 0.000 title claims abstract description 54
- 230000004151 fermentation Effects 0.000 title claims abstract description 54
- 238000000034 method Methods 0.000 title claims abstract description 40
- 230000000813 microbial effect Effects 0.000 title claims abstract description 27
- 239000012528 membrane Substances 0.000 claims abstract description 97
- 238000001471 micro-filtration Methods 0.000 claims abstract description 40
- 238000000108 ultra-filtration Methods 0.000 claims abstract description 28
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 claims abstract description 24
- 238000000926 separation method Methods 0.000 claims abstract description 21
- 239000012141 concentrate Substances 0.000 claims abstract description 19
- 239000007788 liquid Substances 0.000 claims abstract description 13
- 239000000725 suspension Substances 0.000 claims abstract description 11
- 150000003839 salts Chemical class 0.000 claims abstract description 10
- 239000003456 ion exchange resin Substances 0.000 claims abstract description 9
- 229920003303 ion-exchange polymer Polymers 0.000 claims abstract description 9
- 238000001728 nano-filtration Methods 0.000 claims abstract description 9
- 230000002378 acidificating effect Effects 0.000 claims abstract description 5
- 230000007935 neutral effect Effects 0.000 claims abstract description 4
- 239000003957 anion exchange resin Substances 0.000 claims description 18
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 18
- 239000003729 cation exchange resin Substances 0.000 claims description 17
- 235000002639 sodium chloride Nutrition 0.000 claims description 16
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 14
- 239000003480 eluent Substances 0.000 claims description 11
- 239000000919 ceramic Substances 0.000 claims description 10
- 239000012607 strong cation exchange resin Substances 0.000 claims description 10
- 239000006228 supernatant Substances 0.000 claims description 10
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 9
- 239000002253 acid Substances 0.000 claims description 9
- 239000012610 weak anion exchange resin Substances 0.000 claims description 9
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 8
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 8
- 239000011780 sodium chloride Substances 0.000 claims description 7
- 239000012609 strong anion exchange resin Substances 0.000 claims description 7
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims description 6
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 6
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims description 6
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 6
- 244000005700 microbiome Species 0.000 claims description 6
- 239000012608 weak cation exchange resin Substances 0.000 claims description 6
- JIAARYAFYJHUJI-UHFFFAOYSA-L zinc dichloride Chemical compound [Cl-].[Cl-].[Zn+2] JIAARYAFYJHUJI-UHFFFAOYSA-L 0.000 claims description 6
- 241000194048 Streptococcus equi Species 0.000 claims description 4
- 241000120569 Streptococcus equi subsp. zooepidemicus Species 0.000 claims description 4
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 4
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 4
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 3
- 241000186226 Corynebacterium glutamicum Species 0.000 claims description 3
- 241000588724 Escherichia coli Species 0.000 claims description 3
- 241000194035 Lactococcus lactis Species 0.000 claims description 3
- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 claims description 3
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 claims description 3
- UIIMBOGNXHQVGW-DEQYMQKBSA-M Sodium bicarbonate-14C Chemical compound [Na+].O[14C]([O-])=O UIIMBOGNXHQVGW-DEQYMQKBSA-M 0.000 claims description 3
- 235000014897 Streptococcus lactis Nutrition 0.000 claims description 3
- WHMDKBIGKVEYHS-IYEMJOQQSA-L Zinc gluconate Chemical compound [Zn+2].OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C([O-])=O.OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C([O-])=O WHMDKBIGKVEYHS-IYEMJOQQSA-L 0.000 claims description 3
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 3
- 239000001110 calcium chloride Substances 0.000 claims description 3
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 3
- 235000011148 calcium chloride Nutrition 0.000 claims description 3
- 229910001629 magnesium chloride Inorganic materials 0.000 claims description 3
- 235000011147 magnesium chloride Nutrition 0.000 claims description 3
- 229910017604 nitric acid Inorganic materials 0.000 claims description 3
- 239000001103 potassium chloride Substances 0.000 claims description 3
- 235000011164 potassium chloride Nutrition 0.000 claims description 3
- 229910052938 sodium sulfate Inorganic materials 0.000 claims description 3
- 235000011152 sodium sulphate Nutrition 0.000 claims description 3
- 239000011592 zinc chloride Substances 0.000 claims description 3
- 235000005074 zinc chloride Nutrition 0.000 claims description 3
- 239000011670 zinc gluconate Substances 0.000 claims description 3
- 235000011478 zinc gluconate Nutrition 0.000 claims description 3
- 229960000306 zinc gluconate Drugs 0.000 claims description 3
- 238000005194 fractionation Methods 0.000 claims description 2
- 238000005374 membrane filtration Methods 0.000 claims description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 abstract description 21
- 239000003960 organic solvent Substances 0.000 abstract description 9
- 102000004169 proteins and genes Human genes 0.000 abstract description 7
- 108090000623 proteins and genes Proteins 0.000 abstract description 7
- 230000007613 environmental effect Effects 0.000 abstract description 3
- 230000008569 process Effects 0.000 abstract description 3
- 238000011084 recovery Methods 0.000 abstract description 3
- 238000005406 washing Methods 0.000 description 9
- 238000010828 elution Methods 0.000 description 6
- 238000001694 spray drying Methods 0.000 description 5
- 238000001914 filtration Methods 0.000 description 4
- 238000000746 purification Methods 0.000 description 4
- 239000012535 impurity Substances 0.000 description 3
- 238000011068 loading method Methods 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- 230000001105 regulatory effect Effects 0.000 description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 238000003912 environmental pollution Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 239000000049 pigment Substances 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000005086 pumping Methods 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 239000011347 resin Substances 0.000 description 2
- 229920005989 resin Polymers 0.000 description 2
- 241000283690 Bos taurus Species 0.000 description 1
- 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 description 1
- 229920002683 Glycosaminoglycan Polymers 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 229940023913 cation exchange resins Drugs 0.000 description 1
- 150000001768 cations Chemical class 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
- PXEDJBXQKAGXNJ-QTNFYWBSSA-L disodium L-glutamate Chemical compound [Na+].[Na+].[O-]C(=O)[C@@H](N)CCC([O-])=O PXEDJBXQKAGXNJ-QTNFYWBSSA-L 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000003792 electrolyte Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000012869 ethanol precipitation Methods 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 230000001050 lubricating effect Effects 0.000 description 1
- 229940099596 manganese sulfate Drugs 0.000 description 1
- 239000011702 manganese sulphate Substances 0.000 description 1
- 235000007079 manganese sulphate Nutrition 0.000 description 1
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 235000013923 monosodium glutamate Nutrition 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 239000008213 purified water Substances 0.000 description 1
- 238000007670 refining Methods 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 229940073490 sodium glutamate Drugs 0.000 description 1
- 238000000638 solvent extraction Methods 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000012137 tryptone Substances 0.000 description 1
- 238000001291 vacuum drying Methods 0.000 description 1
- 238000009777 vacuum freeze-drying Methods 0.000 description 1
- 210000004127 vitreous body Anatomy 0.000 description 1
- 230000029663 wound healing Effects 0.000 description 1
Classifications
-
- 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
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
- C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
- C08B37/0072—Hyaluronic acid, i.e. HA or hyaluronan; Derivatives thereof, e.g. crosslinked hyaluronic acid (hylan) or hyaluronates
-
- 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
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Materials Engineering (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicinal Chemistry (AREA)
- Polymers & Plastics (AREA)
- Organic Chemistry (AREA)
- Sustainable Development (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
Abstract
A method for separating and purifying hyaluronic acid from a microbial fermentation broth, comprising the following steps: (1) adjusting the pH value of the microbial fermentation liquor to be acidic or neutral, and adding salt to form a suspension; (2) after solid-liquid separation of the suspension, concentrating by using a microfiltration membrane, an ultrafiltration membrane and a nanofiltration membrane to obtain a hyaluronic acid concentrated solution; (3) passing the hyaluronic acid concentrate through an ion exchange resin. The method has simple process, effectively removes residues, foreign proteins, heteropolysaccharide and decoloration, and completely does not use organic solvents such as ethanol and the like, thereby solving the problems of safety, recovery, environmental protection and the like of using a large amount of organic solvents such as ethanol and the like in the hyaluronic acid industry.
Description
Technical Field
The present invention relates to, but is not limited to, a method for separating and purifying hyaluronic acid, and particularly, but not limited to, a method for separating and purifying hyaluronic acid from a microbial fermentation broth.
Background
Hyaluronic Acid (HA), also known as hyaluronic acid, is an acidic mucopolysaccharide that was first isolated from bovine vitreous humor by Meyer et al, university of columbia, 1934. Hyaluronic acid exhibits various important physiological functions in the body with its unique molecular structure and physicochemical properties, such as lubricating joints, regulating permeability of blood vessel walls, regulating proteins, regulating diffusion and operation of aqueous electrolytes, promoting wound healing, and the like.
At present, hyaluronic acid has two production methods, one of which is prepared by extracting and refining natural raw materials, namely animal tissues, with organic solvents such as acetone, ethanol, chloroform and the like. The extraction rate of the method is only about 1 percent, so the method not only has serious environmental pollution, but also has higher production cost. The other is prepared by a biological fermentation method. The biological fermentation method is not limited by raw material resources, is widely applied at present, and replaces a method for separating and extracting hyaluronic acid from animal tissues. Hyaluronic acid obtained by a biological fermentation method is widely applied to the fields of medicines, cosmetics, health-care foods, foods and the like.
Because the hyaluronic acid has large molecular weight and large viscosity, and thallus residues, proteins, heteropolysaccharides, pigments and the like in fermentation liquor are not easy to remove, the method for separating and purifying the hyaluronic acid from the fermentation liquor at present basically adopts a method of ethanol precipitation and diatomite filtration for more than 3 times, and the method has the problems of environmental pollution caused by using a large amount of ethanol, ethanol residues in products, difficult ethanol recovery and the like, and does not meet the current industrial development requirements of green ecology and environmental protection.
Disclosure of Invention
The following is a summary of the subject matter described in detail herein. This summary is not intended to limit the scope of the present application.
The application provides an extraction and purification method of hyaluronic acid which does not use organic solvents such as ethanol and is suitable for industrial amplification, industrial amplification production is easier to carry out due to no organic solvents, and the dosage of fermentation liquor can be 200 tons. According to the method for purifying the hyaluronic acid by membrane classification, the dispersion state of the hyaluronic acid in the fermentation liquor is changed by pretreatment of the fermentation liquor, solid-liquid separation of thalli is realized by ceramic membrane or centrifugal separation, the hyaluronic acid with different molecular weights is subjected to classification concentration by ultrafiltration membranes with different apertures, impurity proteins, heteropolysaccharides and the like are removed by ion exchange resin, organic solvents such as ethanol and the like are not used, and the method has high industrial application value.
The application provides a method for separating and purifying hyaluronic acid from microbial fermentation liquor, which comprises the following steps:
(1) adjusting the pH value of the microbial fermentation liquor to be acidic or neutral, and adding salt to form a suspension;
(2) after solid-liquid separation of the suspension, concentrating by using a microfiltration membrane, an ultrafiltration membrane and a nanofiltration membrane to obtain a hyaluronic acid concentrated solution;
(3) passing the hyaluronic acid concentrate through an ion exchange resin. Alternatively, the method for separating and purifying hyaluronic acid consists of the above steps.
In one embodiment, the microorganism is selected from any one or more of streptococcus equi, streptococcus zooepidemicus, corynebacterium glutamicum, lactococcus lactis, and escherichia coli.
In one embodiment, the solid-liquid separation in step (2) is to put the suspension into a centrifuge for centrifugal separation to obtain a supernatant; filtering the supernatant by using a microfiltration membrane to obtain a concentrated solution;
in one embodiment, the microfiltration membrane is a ceramic membrane;
in one embodiment, the centrifuge is a tube centrifuge or a butterfly centrifuge, and the centrifugal speed of the centrifugal separation is 4000r/min to 30000 r/min;
in one embodiment, the conductivity of the concentrate after filtration with the microfiltration membrane is from 1mS/cm to 7 mS/cm.
In one embodiment, the pH of the adjusted microbial fermentation broth is from 2 to 7, optionally the pH of the adjusted fermentation broth is from 3 to 5;
in one embodiment, the acid used to adjust the pH of the fermentation broth is selected from any one or more of hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, and acetic acid;
in one embodiment, the concentration of the acid is 0.1 to 6N, preferably the concentration of the acid is 0.5N.
In one embodiment, the salt is added in an amount such that the conductivity of the microbial broth is from 75mS/cm to 350 mS/cm;
in one embodiment, the salt is selected from any one or more of sodium chloride, potassium chloride, calcium chloride, zinc chloride, magnesium chloride, sodium bicarbonate, sodium sulfate, magnesium sulfate, and zinc gluconate.
In one embodiment, the microfiltration membrane is a microfiltration membrane with a molecular weight cut-off between 100000Da and 1000000 Da.
In one embodiment, the ultrafiltration membrane has a molecular weight cut-off of 5000Da to 100000 Da.
In one embodiment, the microfiltration membrane is arranged in a staged manner to intercept the hyaluronic acid with different molecular weights, and comprises a microfiltration membrane A with a molecular weight cut-off of above 1000000Da, a microfiltration membrane B with a molecular weight cut-off of 500000Da to 1000000Da, a microfiltration membrane C with a molecular weight cut-off of 200000Da to 500000Da, and a microfiltration membrane D with a molecular weight cut-off of 100000Da to 200000 Da;
in one embodiment, the ultrafiltration membrane is arranged in a grading way to intercept the hyaluronic acid with different molecular weights, and comprises an ultrafiltration membrane A with the molecular weight cut-off of 50000Da to 100000Da, an ultrafiltration membrane B with the molecular weight cut-off of 10000Da to 50000Da, and an ultrafiltration membrane C with the molecular weight cut-off of 5000Da to 20000 Da;
in one embodiment, the nanofiltration membrane has a molecular weight cut-off of between 300Da and 5000 Da;
in one embodiment, the concentrated solution after the microfiltration membrane or the ultrafiltration membrane is washed by pure water until the conductivity is 1mS/cm to 7 mS/cm.
In one embodiment, the ion exchange resin comprises an anion exchange resin and a cation exchange resin.
In one embodiment, the hyaluronic acid concentrate is passed through a cation exchange resin and an anion exchange resin in that order;
in one embodiment, the cation exchange resin is selected from any one or more of D001 macroporous cation exchange resin, D61 macroporous strong cation exchange resin, D62 macroporous strong cation exchange resin, D72 macroporous strong cation exchange resin, D113 weak cation exchange resin, D85 weak cation exchange resin;
in one embodiment, the anion exchange resin is selected from any one or more of D280 strong anion exchange resin, D301 macroporous weak anion exchange resin, D311 macroporous weak anion exchange resin, D201 macroporous strong anion exchange resin.
In one embodiment, the eluent from the cation exchange resin is pure water and the eluent from the anion exchange resin is water having a pH of 2 to 4.
In one embodiment, elution is performed at the fastest rate that can be tolerated by the equipment and ion exchange resin.
In one embodiment, the hyaluronic acid liquid obtained after elution is dried by headspace pulse spray drying, vacuum drying or freeze drying, and optionally headspace pulse spray drying to obtain hyaluronic acid powder; the headspace pulse spray drying method can select the air inlet temperature of 160-200 ℃, the air outlet temperature of 60-100 ℃, and 50 liters of purified water sprayed at intervals of 20 minutes to obtain the hyaluronic acid dry powder.
The method has simple process, effectively removes residues, foreign proteins, heteropolysaccharide and decoloration, and completely does not use organic solvents such as ethanol and the like, thereby solving the problems of safety, recovery, environmental protection and the like of using a large amount of organic solvents such as ethanol and the like in the hyaluronic acid industry. The product prepared by the purification method has the same effect as or better than the conventional organic solvent extraction method in the prior art.
Additional features and advantages of the application will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by the practice of the application. Other advantages of the present application may be realized and attained by the invention in its aspects as described in the specification.
Detailed Description
In order to make the objects, technical solutions and advantages of the present application more apparent, embodiments of the present application are described in detail below. It should be noted that the embodiments and features of the embodiments in the present application may be arbitrarily combined with each other without conflict.
The embodiment of the application provides a method for separating and purifying hyaluronic acid from a microbial fermentation broth, which comprises the following steps:
(1) adjusting the pH value of the microbial fermentation liquor to be acidic or neutral, and adding salt to form a suspension;
(2) after solid-liquid separation of the suspension, concentrating by using a microfiltration membrane, an ultrafiltration membrane and a nanofiltration membrane to obtain a hyaluronic acid concentrated solution;
(3) passing the hyaluronic acid concentrate through an ion exchange resin. Alternatively, the method for separating and purifying hyaluronic acid consists of the above steps.
In the present embodiment, the microorganism is selected from any one or more of streptococcus equi, streptococcus zooepidemicus, corynebacterium glutamicum, lactococcus lactis and escherichia coli.
In the embodiment of the application, the solid-liquid separation in the step (2) is to put the suspension into a centrifuge for centrifugal separation to obtain a supernatant; filtering the supernatant by using a microfiltration membrane to obtain a concentrated solution;
in the embodiments of the present application, the microfiltration membrane is a ceramic membrane;
in the embodiment of the application, the centrifugal machine is a tubular centrifugal machine or a butterfly centrifugal machine, and the centrifugal speed of the centrifugal separation is 4000r/min to 30000 r/min;
in the embodiment of the application, the conductivity of the concentrated solution after the microfiltration membrane filtration is 1mS/cm to 7 mS/cm.
In the embodiment of the present application, the pH of the adjusted microbial fermentation broth is 2 to 7, and optionally, the pH of the adjusted fermentation broth is 3 to 5;
in the embodiment, the acid used for adjusting the pH value of the fermentation liquor is selected from any one or more of hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid and acetic acid;
in the examples of the present application, the concentration of the acid is 0.1 to 6N, preferably, the concentration of the acid is 0.5N.
In the examples herein, the salt is added in an amount such that the conductivity of the microbial broth is from 75 to 350 mS/cm;
in embodiments of the present application, the salt is selected from any one or more of sodium chloride, potassium chloride, calcium chloride, zinc chloride, magnesium chloride, sodium bicarbonate, sodium sulfate, magnesium sulfate, and zinc gluconate.
In the embodiment of the application, the microfiltration membrane is a microfiltration membrane with the molecular weight cut-off of 100000Da to 1000000 Da.
In the examples of the present application, the ultrafiltration membrane is an ultrafiltration membrane with a molecular weight cut-off of 5000Da to 50000 Da.
In the present embodiment, the membrane fractionation arrangement intercepts the hyaluronic acid of different molecular weights, the microfiltration membrane comprises a microfiltration membrane a with a molecular weight cut-off above 1000000Da, a microfiltration membrane B with a molecular weight cut-off between 500000Da and 1000000Da, a microfiltration membrane C with a molecular weight cut-off between 200000Da and 500000Da, a microfiltration membrane D with a molecular weight cut-off between 100000Da and 200000 Da;
the ultrafiltration membrane comprises an ultrafiltration membrane A with the cut-off molecular weight of more than 50000Da to 100000Da, an ultrafiltration membrane B with the cut-off molecular weight of 10000Da to 50000Da and an ultrafiltration membrane C with the cut-off molecular weight of 5000Da to 20000 Da; or a combination of microfiltration membranes, ultrafiltration membranes and nanofiltration membranes with different molecular weight cut-off can be arranged.
In the embodiment of the application, the molecular weight cut-off of the nanofiltration membrane is between 300Da and 5000 Da;
in the embodiment of the application, the concentrated solution filtered by the microfiltration membrane or the ultrafiltration membrane is washed by pure water until the conductivity is 1mS/cm to 7 mS/cm.
In embodiments of the present application, the ion exchange resins include anion exchange resins and cation exchange resins.
In the embodiment of the application, the hyaluronic acid concentrated solution passes through cation exchange resin and anion exchange resin in sequence;
in the present embodiment, the cation exchange resin is selected from any one or more of D001 macroporous cation exchange resin, D61 macroporous strong cation exchange resin, D62 macroporous strong cation exchange resin, D72 macroporous strong cation exchange resin, D113 weak cation exchange resin, D85 weak cation exchange resin;
in the embodiment of the application, the anion exchange resin is selected from any one or more of D280 strong anion exchange resin, D301 macroporous weak anion exchange resin, D311 macroporous weak anion exchange resin and D201 macroporous strong anion exchange resin.
In the examples of the present application, the eluent of the cation exchange resin is pure water, and the eluent of the anion exchange resin is water with a pH value of 2-4.
Example 1
The composition of the microbial fermentation broth used in example 1 was as follows: strain: streptococcus equi zooepidemicus (Streptococcus equi subsp. zoopeptimicus) SG1928, the strain preservation number is CGMCC No.20992, the strain is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, 15% of glucose, 4% of tryptone, 4% of yeast powder, 0.3% of dipotassium hydrogen phosphate, 0.7% of potassium dihydrogen phosphate, 0.2% of magnesium sulfate, 0.025% of manganese sulfate and 0.05% of sodium glutamate are added into a microorganism fermentation broth, the fermentation condition of the microorganism fermentation broth is that the fermentation temperature is 30-35 ℃, the pH value is 6.0-8.0, and DO 40-100%, so as to obtain the hyaluronic acid fermentation broth.
1) Taking 500L of hyaluronic acid fermentation liquor, adjusting the pH value to 3.0 by using hydrochloric acid with the concentration of 0.5N, adding 0.5 wt.% of sodium chloride, and fully and uniformly stirring to ensure that the conductivity of the hyaluronic acid fermentation liquor is 75 mS/cm;
2) performing solid-liquid separation by using a butterfly centrifuge with the rotating speed of 6000r/min to obtain supernatant; dialyzing the supernatant by using a ceramic membrane, concentrating and washing by water, and specifically operating as follows:
concentrating the supernatant with ceramic membrane A (trade name T19/60/400) from Xiamen Sanda Membrane science and technology Limited until the conductivity reaches 2mS/cm, and collecting concentrated solution A;
concentrating the dialysate of the microfiltration membrane A with ceramic membrane B of grade T19/60/400, available from Xiamen, Sanda, Limited Membrane science, and washing with water until the conductivity reaches 2mS/cm, and collecting the concentrated solution B for use;
concentrating the dialysate of the microfiltration membrane B by using a ceramic membrane C purchased from Xiamen Sanda Membrane science Limited T19/60/400, washing with water until the conductivity reaches 2mS/cm, and collecting the concentrated solution C for later use;
concentrating the dialysate of the microfiltration membrane C by using a ceramic membrane D which is purchased from Xiamen Sanda Membrane science Limited T19/60/400 brand, washing with water until the conductivity reaches 2mS/cm, and collecting a concentrated solution D for later use;
3) concentrating and washing the dialysate of the ceramic membrane D by using an 8040 mark ultrafiltration membrane A purchased from GE company until the conductivity reaches 2mS/cm, and collecting a concentrated solution 1 for later use;
4) concentrating and washing the dialysate obtained in the step 3) by using an 8040 mark ultrafiltration membrane B purchased from GE company until the conductivity reaches 2mS/cm, and collecting a concentrated solution 2 for later use;
5) concentrating and washing the dialysate obtained in the step 4) by using an 8040 mark ultrafiltration membrane C purchased from GE company until the conductivity reaches 2mS/cm, and collecting a concentrated solution 3 for later use;
6) concentrating and washing the dialysate obtained in the step 5) by using a 8040-grade nanofiltration membrane purchased from GE company until the conductivity reaches 2mS/cm, and collecting a concentrated solution 4 for later use;
7) collecting concentrate A, concentrate B, concentrate C, concentrate D, concentrate 1, concentrate 2, concentrate 3 and concentrateAnd (4) respectively loading the concentrated solution through a D001 macroporous cation exchange resin (chromatographic column:
volume of resin: 700L; eluent: pure water; elution flow rate: 600L/h; elution time: 2 h; sample loading operation: pumping the concentrated solution 1-5 into chromatographic column with a sample pump for separation), eluting with pure water, and collecting eluate containing hyaluronic acid;
8) the hyaluronic acid liquid (eluent) obtained in step 7) is loaded on a D280 strong anion exchange resin (chromatographic column:
volume of resin: 700L; eluent: pure water having a pH of 2.5; elution flow rate: 600L/h; elution time: 6 h; sample loading operation: taking cation eluent, pumping into a chromatographic column by using a sample pump for separation), directly flowing heteropolysaccharide and pigment out of the column without hanging the column, eluting by using pure water with the pH value of 2.5, washing hyaluronic acid, and collecting for later use;
9) and 8) spray drying the hyaluronic acid liquid obtained in the step 8 (spray drying negative pressure 100-.
Example 2
The difference from example 1 is that the volume of the fermentation broth is 1000L and the pH is adjusted to 5.0.
Example 3
The difference from example 1 is that the fermentation broth is adjusted to pH6.0 and the anion exchange resin is D301 macroporous weak anion exchange resin.
Example 4
The difference from example 1 is that the fermentation broth is adjusted to a conductivity of 100mS/cm with sodium chloride.
Example 5
The difference from example 1 is that the fermentation broth was adjusted to a conductivity of 90mS/cm with sodium chloride and the cation exchange resin was D72 macroporous strong cation exchange resin.
Example 6
The difference from example 5 is that the volume of the fermentation broth is 4000L, the pH of the eluate from the anion exchange resin is 2.0, and the anion exchange resin is D301 macroporous weak anion exchange resin.
Example 7
The difference from example 5 is that the anion exchange resin has an eluent pH of 3.0 and the anion exchange resin is D301 macroporous weak anion exchange resin.
Example 8
The difference from example 5 is that the volume of the fermentation broth was 2000L and the pH of the anion exchange resin eluate was 4.0.
Example 9
The difference from example 3 is that the fermentation broth was not adjusted in pH and maintained at a lower tank pH of 7.0.
Comparative example 1
The difference from the example 1 is that sodium chloride is not added during the pretreatment of the fermentation broth, and as a result, all hyaluronic acid, foreign proteins and other impurities are intercepted, and the next treatment cannot be carried out
The hyaluronic acid separation and purification results of each example are shown in table 1:
in the table: the hyaluronic acid average molecular weight (Da) is the average molecular weight of hyaluronic acid in the concentrated solution obtained after passing through the filter membrane;
the protein content (wt.%) is the weight ratio of protein in the concentrate obtained after passing through the filter membrane to the concentrate;
the hyaluronic acid content (wt.%) is the weight ratio of hyaluronic acid in the concentrate obtained after passing through the filter membrane to the concentrate;
the yield (%) is the weight ratio of the hyaluronic acid of a specific molecular weight fraction obtained after the treatment with the filter membrane to the hyaluronic acid of a corresponding molecular weight fraction in the dialysate of the previous membrane (or the supernatant obtained after the solid-liquid separation with the centrifuge).
Table 1: statistical table of hyaluronic acid separation and purification results
Table 2 shows the average molecular weight of the sample compared with the hyaluronic acid prepared by the applicant using the alcohol precipitation process, which is 80000Da, of the hyaluronic acid prepared in example 1 of the present application, and it can be seen from table 2 that the hyaluronic acid prepared by the preparation method provided by the present application is significantly better than the hyaluronic acid prepared by the conventional alcohol precipitation method in terms of purity, impurity content, and the like.
Table 2: hyaluronic acid sampling contrast
Although the embodiments disclosed in the present application are described above, the descriptions are only for the convenience of understanding the present application, and are not intended to limit the present application. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the disclosure as defined by the appended claims.
Claims (10)
1. A method for separating and purifying hyaluronic acid from a microbial fermentation broth, comprising the following steps:
(1) adjusting the pH value of the microbial fermentation liquor to be acidic or neutral, and adding salt to form a suspension;
(2) after solid-liquid separation of the suspension, concentrating by using a microfiltration membrane, an ultrafiltration membrane and a nanofiltration membrane to obtain a hyaluronic acid concentrated solution;
(3) passing the hyaluronic acid concentrate through an ion exchange resin.
2. The method for separating and purifying hyaluronic acid from a microbial fermentation broth of claim 1, wherein the microorganism is selected from any one or more of streptococcus equi, streptococcus zooepidemicus, corynebacterium glutamicum, lactococcus lactis and escherichia coli.
3. The method for separating and purifying hyaluronic acid from a microbial fermentation broth according to claim 1, wherein the solid-liquid separation in step (2) is that the suspension is put into a centrifuge for centrifugal separation to obtain a supernatant, and the supernatant is filtered by a microfiltration membrane to obtain a concentrated solution;
optionally, the microfiltration membrane is a ceramic membrane;
optionally, the centrifugal rotation speed of the centrifugal separation is 4000r/min to 30000 r/min;
optionally, the conductivity of the concentrate after microfiltration membrane filtration is 1mS/cm to 7 mS/cm.
4. The method for separating and purifying hyaluronic acid from a microbial fermentation broth of claim 1, wherein the adjusted pH of the microbial fermentation broth is 2 to 7, optionally the adjusted pH of the fermentation broth is 3 to 5;
optionally, the acid used to adjust the pH of the fermentation broth is selected from any one or more of hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, and acetic acid;
optionally, the concentration of the acid is 0.1-6N, preferably, the concentration of the acid is 0.5N.
5. The method for separating and purifying hyaluronic acid from a microbial fermentation broth of claim 1, wherein the salt is added in an amount such that the conductivity of the microbial fermentation broth is from 75mS/cm to 350 mS/cm;
optionally, the salt is selected from any one or more of sodium chloride, potassium chloride, calcium chloride, zinc chloride, magnesium chloride, sodium bicarbonate, sodium sulfate, magnesium sulfate, and zinc gluconate.
6. The method for separating and purifying hyaluronic acid from a microbial fermentation broth of any of claims 1-5, wherein the microfiltration membrane has a molecular weight cut-off of 100000Da to 1000000Da and the ultrafiltration membrane has a molecular weight cut-off of 5000Da to 100000 Da.
7. The method for separating and purifying hyaluronic acid from a microbial fermentation broth of claim 6, wherein the membrane fractionation setup intercepts the hyaluronic acid of different molecular weights;
the microfiltration membrane comprises a microfiltration membrane A with the molecular weight cutoff of more than 1000000Da, a microfiltration membrane B with the molecular weight cutoff of 500000Da to 1000000Da, a microfiltration membrane C with the molecular weight cutoff of 200000Da to 500000Da and a microfiltration membrane D with the molecular weight cutoff of 100000Da to 200000 Da;
the ultrafiltration membrane comprises an ultrafiltration membrane A with the molecular weight cutoff of 50000Da to 100000Da, an ultrafiltration membrane B with the molecular weight cutoff of 10000Da to 50000Da and an ultrafiltration membrane C with the molecular weight cutoff of 5000Da to 20000 Da;
optionally, the nanofiltration membrane has a molecular weight cut-off of 300Da to 5000 Da;
optionally, the concentrated solution filtered by the microfiltration membrane or the ultrafiltration membrane is washed by pure water until the conductivity is 1mS/cm to 7 mS/cm.
8. The method for separating and purifying hyaluronic acid from a microbial fermentation broth of any of claims 1-5, wherein the ion exchange resin comprises an anion exchange resin and a cation exchange resin.
9. The method for separating and purifying hyaluronic acid from a microbial fermentation broth of claim 8, wherein optionally, the hyaluronic acid concentrate is sequentially passed through a cation exchange resin and an anion exchange resin;
optionally, the cation exchange resin is selected from any one or more of D001 macroporous cation exchange resin, D61 macroporous strong cation exchange resin, D62 macroporous strong cation exchange resin, D72 macroporous strong cation exchange resin, D113 weak cation exchange resin, D85 weak cation exchange resin;
optionally, the anion exchange resin is selected from any one or more of D280 strong anion exchange resin, D301 macroporous weak anion exchange resin, D311 macroporous weak anion exchange resin, D201 macroporous strong anion exchange resin.
10. The method for separating and purifying hyaluronic acid from a microbial fermentation broth of claim 9, wherein the eluent of the cation exchange resin is pure water and the eluent of the anion exchange resin is water with a pH value of 2-4.
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| EP0694616A2 (en) * | 1994-07-26 | 1996-01-31 | POLI INDUSTRIA CHIMICA S.p.A. | Process for the preparation of hyaluronic acid by fermentation with streptococcus |
| CN101914169A (en) * | 2008-12-01 | 2010-12-15 | 思德生物科技股份有限公司 | Process for purifying hyaluronic acid |
| CN105985994A (en) * | 2015-03-11 | 2016-10-05 | 上海其胜生物制剂有限公司 | Method for preparing sodium hyaluronate by means of salt-free and organic solvent-free purification technology |
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| EP0694616A2 (en) * | 1994-07-26 | 1996-01-31 | POLI INDUSTRIA CHIMICA S.p.A. | Process for the preparation of hyaluronic acid by fermentation with streptococcus |
| CN101914169A (en) * | 2008-12-01 | 2010-12-15 | 思德生物科技股份有限公司 | Process for purifying hyaluronic acid |
| CN105985994A (en) * | 2015-03-11 | 2016-10-05 | 上海其胜生物制剂有限公司 | Method for preparing sodium hyaluronate by means of salt-free and organic solvent-free purification technology |
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| WO2023129082A1 (en) * | 2021-12-31 | 2023-07-06 | Turhan Irfan | Increasing the productivity of hyaluronic acid fermentation by online recovery from fermentation medium |
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