CN115215949B - Sodium hyaluronate and purification method thereof - Google Patents
Sodium hyaluronate and purification method thereof Download PDFInfo
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- CN115215949B CN115215949B CN202210644688.1A CN202210644688A CN115215949B CN 115215949 B CN115215949 B CN 115215949B CN 202210644688 A CN202210644688 A CN 202210644688A CN 115215949 B CN115215949 B CN 115215949B
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- 229920002385 Sodium hyaluronate Polymers 0.000 title claims abstract description 95
- 229940010747 sodium hyaluronate Drugs 0.000 title claims abstract description 95
- YWIVKILSMZOHHF-QJZPQSOGSA-N sodium;(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- Chemical compound [Na+].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 YWIVKILSMZOHHF-QJZPQSOGSA-N 0.000 title claims abstract description 95
- 238000000034 method Methods 0.000 title claims abstract description 44
- 238000000746 purification Methods 0.000 title abstract description 13
- 238000001914 filtration Methods 0.000 claims abstract description 79
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 claims abstract description 63
- 239000012535 impurity Substances 0.000 claims abstract description 38
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 36
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 36
- 108091005658 Basic proteases Proteins 0.000 claims abstract description 11
- 230000001105 regulatory effect Effects 0.000 claims abstract description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 79
- 239000000706 filtrate Substances 0.000 claims description 58
- 230000001376 precipitating effect Effects 0.000 claims description 43
- 239000002244 precipitate Substances 0.000 claims description 34
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 28
- 230000000536 complexating effect Effects 0.000 claims description 23
- 238000001035 drying Methods 0.000 claims description 23
- 230000008569 process Effects 0.000 claims description 21
- 239000012528 membrane Substances 0.000 claims description 15
- 239000011780 sodium chloride Substances 0.000 claims description 14
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 13
- 239000011087 paperboard Substances 0.000 claims description 12
- 238000001556 precipitation Methods 0.000 claims description 11
- 238000005374 membrane filtration Methods 0.000 claims description 3
- 238000000926 separation method Methods 0.000 claims description 3
- 239000011148 porous material Substances 0.000 claims description 2
- 239000002994 raw material Substances 0.000 claims description 2
- WOWHHFRSBJGXCM-UHFFFAOYSA-M cetyltrimethylammonium chloride Chemical compound [Cl-].CCCCCCCCCCCCCCCC[N+](C)(C)C WOWHHFRSBJGXCM-UHFFFAOYSA-M 0.000 claims 4
- 239000012065 filter cake Substances 0.000 claims 1
- 239000008098 formaldehyde solution Substances 0.000 abstract description 6
- 238000002360 preparation method Methods 0.000 abstract description 4
- 239000003814 drug Substances 0.000 abstract description 2
- 238000004925 denaturation Methods 0.000 abstract 1
- 230000036425 denaturation Effects 0.000 abstract 1
- 238000003756 stirring Methods 0.000 description 37
- 239000007788 liquid Substances 0.000 description 30
- 238000007599 discharging Methods 0.000 description 22
- 239000006228 supernatant Substances 0.000 description 21
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 21
- 239000000047 product Substances 0.000 description 20
- UKHVLWKBNNSRRR-UHFFFAOYSA-M quaternium-15 Chemical compound [Cl-].C1N(C2)CN3CN2C[N+]1(CC=CCl)C3 UKHVLWKBNNSRRR-UHFFFAOYSA-M 0.000 description 19
- 244000005700 microbiome Species 0.000 description 15
- 238000000855 fermentation Methods 0.000 description 13
- 230000004151 fermentation Effects 0.000 description 13
- 238000004090 dissolution Methods 0.000 description 12
- 239000008213 purified water Substances 0.000 description 11
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 10
- 239000011248 coating agent Substances 0.000 description 10
- 238000000576 coating method Methods 0.000 description 10
- 238000010494 dissociation reaction Methods 0.000 description 9
- 230000005593 dissociations Effects 0.000 description 9
- 238000004519 manufacturing process Methods 0.000 description 7
- 241000561734 Celosia cristata Species 0.000 description 6
- 210000001520 comb Anatomy 0.000 description 6
- 238000012869 ethanol precipitation Methods 0.000 description 6
- 229960000583 acetic acid Drugs 0.000 description 5
- 230000000052 comparative effect Effects 0.000 description 5
- 239000012362 glacial acetic acid Substances 0.000 description 5
- 238000010438 heat treatment Methods 0.000 description 5
- 230000007480 spreading Effects 0.000 description 5
- 238000003892 spreading Methods 0.000 description 5
- 102000012479 Serine Proteases Human genes 0.000 description 4
- 108010022999 Serine Proteases Proteins 0.000 description 4
- 238000009395 breeding Methods 0.000 description 3
- 230000001488 breeding effect Effects 0.000 description 3
- 230000015556 catabolic process Effects 0.000 description 3
- 238000006731 degradation reaction Methods 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 239000002158 endotoxin Substances 0.000 description 3
- 239000012467 final product Substances 0.000 description 3
- 238000007689 inspection Methods 0.000 description 3
- 239000000123 paper Substances 0.000 description 3
- 230000002411 adverse Effects 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 235000010633 broth Nutrition 0.000 description 2
- 239000011111 cardboard Substances 0.000 description 2
- 238000010668 complexation reaction Methods 0.000 description 2
- 239000000306 component Substances 0.000 description 2
- 238000012444 downstream purification process Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 150000004676 glycans Chemical class 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 229920001282 polysaccharide Polymers 0.000 description 2
- 239000005017 polysaccharide Substances 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 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 description 1
- 206010067484 Adverse reaction Diseases 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 241000194017 Streptococcus Species 0.000 description 1
- 230000006838 adverse reaction Effects 0.000 description 1
- 238000005352 clarification Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000011143 downstream manufacturing Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 229920002674 hyaluronan Polymers 0.000 description 1
- 229960003160 hyaluronic acid Drugs 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 239000012452 mother liquor Substances 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 150000003242 quaternary ammonium salts Chemical class 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000001291 vacuum drying Methods 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
Abstract
The invention relates to the technical field of medicine preparation, in particular to sodium hyaluronate and a purification method thereof, wherein the purification method comprises the step of removing impurity proteins in sodium hyaluronate solution; the impurity protein removal step at least comprises any one of the following steps: (1) Adding formaldehyde into the sodium hyaluronate solution to denature impurity proteins; (2) And (3) regulating the pH value of the solution to be alkaline, adding alkaline protease, and carrying out enzymolysis on impurity proteins in the sodium hyaluronate solution. The formaldehyde solution is used for denaturation, so that the formaldehyde solution is easy to filter and intercept in the later period, and meanwhile, alkaline protease is added to adjust the pH to 10-11, so that on one hand, the mixed protein is removed through enzymolysis, meanwhile, the viscosity of the sodium hyaluronate solution can be effectively reduced, the problem of long filtering time is greatly solved, and finally, the product quality is improved.
Description
Technical Field
The invention relates to the technical field of medicine preparation, in particular to sodium hyaluronate and a purification method thereof.
Background
The conventional medical sodium hyaluronate purification method at present mainly comprises the following steps: ethanol precipitation, dissolution, filtration, quaternary ammonium salt precipitation, precipitation dissociation, organic solvent ethanol precipitation and the like.
Firstly, the components in the fermentation liquor are complex, including streptococcus metabolites, undigested culture medium components and the like, so that great difficulty is brought to subsequent purification and separation.
In addition, the sodium hyaluronate production process involves a dissolution step, and since it is slow to dissolve at normal temperature (dissolution at 0.3% concentration, typically about 6 hours), and is easily degraded at a temperature higher than 60 ℃, the dissolution temperature is typically set at about 45 ℃ (dissolution at 0.3% concentration, typically about 4 hours). At this temperature, the dissolution rate of sodium hyaluronate is increased, but a suitable growth environment for the microorganisms is given.
Finally, sodium hyaluronate solution belongs to high-viscosity polysaccharide substances, and the property of high viscosity makes the impurity filtering process very slow in the production process. The sodium hyaluronate solution is usually subjected to plate frame primary rate, cardboard fine filtration and 0.45 μm membrane filtration in sequence in the filtration process. Wherein, in the normal plate frame primary filtration process, 1m of liquid adopts a plate frame with a filtration area of 1 square meter; whereas fine filtration is performed on a paper sheet having a filtration area of 0.2 square meter (paper sheet having a pore size of about 2 μm is used for paper sheet). Thus, according to the above process, the impurity filtration of a conventional 1 ton of 0.3% sodium hyaluronate solution typically takes 4-6 hours to complete.
Therefore, the growth of microorganisms becomes a key to the quality control of the filtration stage under suitable temperature conditions for a long period of time with high nutrition.
Too high a protein content is considered to be a major cause of adverse reactions of the preparation products, so that some technical means are currently available for removing impurity proteins in sodium hyaluronate.
For example, CTAC precipitation is a very effective method for removing impurities (see in particular the purification of hyaluronic acid under application No. CN 200810040864.0), although most of the impurity proteins cannot be specifically complexed to be discharged with the mother liquor, a considerable portion of the impurity proteins will be encapsulated by the CTAC-HA precipitate and cannot be removed effectively and carried to downstream processes.
In addition, impurities are often removed by a method of ethanol precipitation for 2-3 times in the actual production process, so that the product quality is improved. Therefore, not only is a certain hidden trouble caused in the aspect of safety and the production period is longer, but also the production cost is greatly improved.
Disclosure of Invention
The invention provides a purification method of sodium hyaluronate to overcome the defects of high endotoxin level in a preparation product caused by excessive impurity protein content in sodium hyaluronate in the prior art, and simultaneously provides sodium hyaluronate purified by the purification method.
In order to achieve the aim of the invention, the invention is realized by the following technical scheme:
a method for purifying sodium hyaluronate,
comprises the steps of removing impurity proteins in sodium hyaluronate solution;
the impurity protein removal step at least comprises any one of the following steps:
(1) Adding formaldehyde into the sodium hyaluronate solution to denature impurity proteins;
(2) And (3) regulating the pH value of the solution to be alkaline, adding alkaline protease, and carrying out enzymolysis on impurity proteins in the sodium hyaluronate solution.
The sodium hyaluronate in the present invention may employ any one or a combination of both of the above (1) or (2) in the purification process.
In the step (1), formaldehyde is added into the sodium hyaluronate solution, and the added formaldehyde can enable proteins in the sodium hyaluronate to be denatured and solidified, so that the later filtration and interception are facilitated, meanwhile, after the formaldehyde is added, the formaldehyde in the solution can enter a downstream purification process (CTAC precipitation dissociation and the like) along with a impurity removal process, so that the problem of mass microorganism breeding during filtration is effectively solved (the microorganism is increased in the filtration process, and adverse effects are often produced on the later sterilization filtration, endotoxin control and the like of a final product), and the quality of the product is improved.
The special characteristics of the sodium hyaluronate are considered in the step (2), and the viscosity of the sodium hyaluronate is reduced along with the increase of the pH value, so that the viscosity of the sodium hyaluronate solution is reduced by increasing the pH value, and the problem of long later filtering time (long filtering time, influence on the growth of microorganisms and the degradation of the molecular weight of the sodium hyaluronate in the process) is greatly reduced. Meanwhile, under the high alkaline condition, the breeding condition of microorganisms can be destroyed, and the growth of the microorganisms can be effectively inhibited.
In addition, after adjusting the sodium hyaluronate solution to be alkaline, alkaline protease is also added into the solution, which can carry out enzymolysis on proteins, so that the problem that impurity proteins can be coated by CTAC-HA precipitate formed in the subsequent CTAC precipitation dissociation process, and the impurity proteins cannot be effectively removed can be prevented.
Therefore, the invention can thoroughly remove the impurity protein in the sodium hyaluronate by combining the two protein removal methods, thereby effectively destroying the growth conditions of bacteria and effectively improving the quality of products.
Preferably, the formaldehyde in the sodium hyaluronate solution is added to a volume concentration of 0.03-0.1%.
Experiments conducted by the inventor of the present application show that when the added concentration of formaldehyde is lower than 0.03%, impurity proteins in the sodium hyaluronate solution may not be removed, and when the added amount of formaldehyde is too high, although the formaldehyde has a good removal effect on the impurity proteins, formaldehyde may remain in the sodium hyaluronate in a subsequent drying process, which is not beneficial to the improvement of the quality of the sodium hyaluronate.
Preferably, the pH of the sodium hyaluronate solution is adjusted to 10.5±0.5.
In the enzymolysis process, the pH of the sodium hyaluronate solution is adjusted to 10.5+/-0.5, which is considered based on the following two points:
first, when the pH of the sodium hyaluronate solution is less than 10, the viscosity of sodium hyaluronate is limited to be reduced, which results in insufficient viscosity reduction of sodium hyaluronate, which is unfavorable for the filtration of sodium hyaluronate, and the filtration time required for the filtration of sodium hyaluronate solution is longer.
When the pH of the sodium hyaluronate solution is more than 11, the filtering speed is effectively increased, but the degradation speed of the sodium hyaluronate under the condition of high pH is also increased, so that the molecular weight of the sodium hyaluronate is greatly reduced, and meanwhile, the pH alkaline protease with too high pH is deactivated.
Preferably, the alkaline protease is a serine protease.
Further preferably, the alkaline protease is added in an amount of 0.01% of the mass of sodium hyaluronate, and the enzymolysis process is continued for at least 1h.
Preferably, the sodium hyaluronate solution is first subjected to a precipitation separation step prior to the impurity protein, which is specifically as follows:
directly precipitating sodium hyaluronate fermentation broth prepared by microorganism fermentation method or cockscomb extraction method with 95% ethanol, discharging upper ethanol, rinsing with 95% ethanol for 2-3 times, discharging supernatant, transferring precipitate to dissolution tank, and dissolving with purified water according to sodium hyaluronate content of 0.2-0.4%.
Further preferably, 95% ethanol which is 1.5 times of the volume of the fermentation liquor is adopted for ethanol precipitation in the process of ethanol precipitation, and the fermentation liquor is rinsed for 2-3 times by one half of the volume of the ethanol solution after the ethanol precipitation.
Preferably, the sodium hyaluronate solution is further subjected to a filtration step after the impurity proteins, which is specifically as follows:
firstly, a diatomite filter layer is paved in advance, a sodium hyaluronate solution with impurity proteins removed is filtered to obtain primary filtrate, the primary filtrate is filtered by a paperboard for the second time to obtain secondary primary filtrate, and finally the secondary primary filtrate is filtered by a filter membrane to obtain refined filtrate, and the refined filtrate is collected for standby.
Further preferably, the membrane during membrane filtration is a 0.45 μm membrane.
Preferably, after the step of removing the impurity protein from the sodium hyaluronate solution, the method further comprises the steps of:
filtering the sodium hyaluronate solution with the impurity proteins removed, adding CTAC into the filtrate for complexing, collecting CTAC precipitate, then dissociating with sodium chloride solution to obtain dissociated solution, precipitating the dissociated solution with ethanol, collecting precipitate, and drying to obtain sodium hyaluronate raw material.
Preferably, during the CTAC complexation, glacial acetic acid is used to adjust the pH of the sodium hyaluronate solution to 6.0-7.5.
Preferably, the CTAC is added in an amount of 2 to 3 times the mass of sodium hyaluronate.
Preferably, the time of the complex precipitation during CTAC complexation is at least 30min.
Preferably, the molar concentration of the sodium chloride solution used in the dissociation process is 0.1-0.5 mol/L, and the dissociation time is more than 4 hours.
Further preferably, the molar concentration of the sodium chloride solution used in the dissociation process is 0.4mol/L, and the dissociation time is 4 hours or more.
Preferably, the dissociation solution is clarified and filtered and then subjected to alcohol precipitation with 3 volumes of 95% ethanol.
Preferably, after the precipitate is collected by alcohol precipitation, the precipitate is dehydrated and dried at least 3 times, and the time for vacuum drying is 12 hours (inclusive).
Sodium hyaluronate prepared by the purification method,
the microorganism content in the sodium hyaluronate is less than 100/g;
the protein content in the sodium hyaluronate is less than 0.05%.
Therefore, the invention has the following beneficial effects:
(1) The formaldehyde solution is used for denaturing the formaldehyde solution, so that the formaldehyde solution is easy to filter and retain in the later stage;
(2) Meanwhile, alkaline protease is added to adjust the pH to 10-11, on one hand, the foreign proteins are removed through enzymolysis, on the other hand, due to the special character of sodium hyaluronate, the higher the pH is, the lower the viscosity is, the viscosity of sodium hyaluronate solution is reduced through raising the pH, and the problem of long later filtering time (long filtering time, influence on the growth of microorganisms and the degradation of the molecular weight of sodium hyaluronate in the process) is greatly shortened;
(3) The method of rapid filtration of the cardboard frame is adopted to remove the impurity protein, so that the impurity protein can be effectively removed, the subsequent filtration time can be greatly reduced, the pressure of a downstream purification process (CTAC precipitation dissociation and the like) is reduced, the production cost is reduced, the production time is shortened, the impurity of a finished product is obviously reduced, and the quality of the finished product is improved;
(4) In addition, due to the characteristic of high viscosity, the sodium hyaluronate has long filtration time, is used as polysaccharide substances which are extremely easy to breed a large amount of microorganisms in the time period, and under the conditions of low concentration formaldehyde and high alkalinity, on one hand, the environment for inhibiting the growth of the microorganisms is generated, meanwhile, the low viscosity greatly shortens the filtration time, and effectively solves the problem of the breeding of the microorganisms in the filtration period (the increase of the microorganisms in the filtration process, the adverse effects on the post-sterilization filtration, the endotoxin control of the final product and the like) in the whole filtration process, so that the product quality is improved.
Detailed Description
The invention is further described below in connection with specific embodiments. Those of ordinary skill in the art will be able to implement the invention based on these descriptions. In addition, the embodiments of the present invention referred to in the following description are typically only some, but not all, embodiments of the present invention. Therefore, all other embodiments, which can be made by one of ordinary skill in the art without undue burden, are intended to be within the scope of the present invention, based on the embodiments of the present invention.
Description of the invention
1: the fermentation broths selected in the following examples were all obtained in the same experimental batch
2: the thickness of the diatomite filter layers selected for clarification and filtration is 1cm
3: the examples are not limited to purification of sodium hyaluronate by microbial fermentation, but will also illustrate the effectiveness and versatility of the present invention in terms of cockscomb extraction.
Example 1
The invention comprises the following steps: taking 1L of fermentation liquor, adding 1.5L of 95% ethanol while stirring, precipitating for 30min, and discharging supernatant; adding ethanol 0.5L under stirring, standing for 15min, and discharging supernatant; repeating for one time, adding ethanol 0.5L under stirring, standing for 15min, and discharging supernatant; the precipitate was added with purified water to 3L (sodium hyaluronate content about 0.3 wt%), formaldehyde 1.0ml (volume concentration about 0.03%), heated to 45 ℃ in a water bath, stirred for 4h for dissolution, ph=10.5 adjusted, serine protease 0.3g added, and stirred for 1h. Spreading diatomite filter layer, filtering the solution to obtain primary filtrate, pre-coating paper board, filtering the primary filtrate again to obtain secondary primary filtrate, filtering the secondary primary filtrate with 0.45um filter membrane, and adjusting the pH=6.5 of the refined filtrate with glacial acetic acid. Detecting the conductivity range (1.2-1.4 MS/CM) of the refined filtrate, adding 16g of CTAC (cta), stirring after passing, complexing and precipitating for 30min, collecting the complexing and precipitating, dissociating the precipitate for 6h by using 2L of 0.4Mol/L sodium chloride solution, clarifying and filtering the dissociated liquid, precipitating the dissociated liquid with 6L of 95% ethanol, collecting the precipitate, dehydrating and drying for three times, and vacuumizing and drying for 12h at 40 ℃ to obtain the finished product.
Comparative example 1
The contrast process comprises the following steps: taking 1L of fermentation liquor, adding 2.5L of 95% ethanol while stirring, precipitating for 30min, and discharging supernatant; adding purified water to 3L (sodium hyaluronate content is about 0.3%), heating to 45 ℃ in a water bath, stirring for 4h to dissolve, pre-paving a diatomite filter layer, filtering the solution to obtain primary filtrate, pre-coating paper board, filtering the primary filtrate again to obtain secondary primary filtrate, filtering the secondary primary filtrate with a 0.45um filter membrane, detecting the conductivity range (1.2-1.4 MS/CM) of trapped liquid, adding 16g of CTAC (sodium hyaluronate) after passing the trapped liquid, stirring, complexing and precipitating for 30min, collecting complexing and precipitating, dissociating the precipitate with 2L of 0.4Mol/L sodium chloride solution for 6h, clarifying and filtering the dissociated liquid, precipitating with 6L of 95% ethanol, collecting the precipitate, dehydrating and drying three times, and vacuumizing and drying for 12h at 40 ℃ to obtain the finished product.
Example 2
The invention comprises the following steps: taking 1L of fermentation liquor, adding 1.5L of 95% ethanol while stirring, precipitating for 30min, and discharging supernatant; adding ethanol 0.5L under stirring, standing for 15min, and discharging supernatant; repeating for one time, adding ethanol 0.5L under stirring, standing for 15min, and discharging supernatant; the precipitate was added with purified water to 3L (sodium hyaluronate content about 0.3%), formaldehyde 3.0ml (0.1% by volume) and heated to 45℃in a water bath with stirring for 4h for dissolution. Spreading diatomite filter layer, filtering the solution to obtain primary filtrate, pre-coating paper board, filtering the primary filtrate again to obtain secondary primary filtrate, and filtering the secondary primary filtrate with 0.45um filter membrane. Detecting the conductivity range (1.2-1.4 MS/CM) of the refined filtrate, adding 16g of CTAC (cta), stirring after passing, complexing and precipitating for 30min, collecting the complexing and precipitating, dissociating the precipitate for 6h by using 2L of 0.4Mol/L sodium chloride solution, clarifying and filtering the dissociated liquid, precipitating the dissociated liquid with 6L of 95% ethanol, collecting the precipitate, dehydrating and drying for three times, and vacuumizing and drying for 12h at 40 ℃ to obtain the finished product.
Comparative example 2
The contrast process comprises the following steps: taking 1L of fermentation liquor, adding 2.5L of 95% ethanol while stirring, precipitating for 30min, and discharging supernatant; adding purified water to 3L (sodium hyaluronate content is about 0.3%), heating to 45 ℃ in a water bath, stirring for 4h to dissolve, pre-paving a diatomite filter layer, filtering the solution to obtain primary filtrate, pre-coating paper board, filtering the primary filtrate again to obtain secondary primary filtrate, filtering the secondary primary filtrate with a 0.45um filter membrane, detecting the conductivity range (1.2-1.4 MS/CM) of trapped liquid, adding 16g of CTAC (sodium hyaluronate) after passing the trapped liquid, stirring, complexing and precipitating for 30min, collecting complexing and precipitating, dissociating the precipitate with 2L of 0.4Mol/L sodium chloride solution for 6h, clarifying and filtering the dissociated liquid, precipitating with 6L of 95% ethanol, collecting the precipitate, dehydrating and drying three times, and vacuumizing and drying for 12h at 40 ℃ to obtain the finished product.
Example 3
The invention comprises the following steps: taking 1L of fermentation liquor, adding 1.5L of 95% ethanol while stirring, precipitating for 30min, and discharging supernatant; adding ethanol 0.5L under stirring, standing for 15min, and discharging supernatant; repeating for one time, adding ethanol 0.5L under stirring, standing for 15min, and discharging supernatant; the precipitate was added with purified water to 3L (sodium hyaluronate content about 0.3%), heated to 45 ℃ in a water bath, stirred for 4h for dissolution, adjusted ph=10, added with serine protease 0.3g, and stirred for 1h. Spreading diatomite filter layer, filtering the solution to obtain primary filtrate, pre-coating paper board, filtering the primary filtrate again to obtain secondary primary filtrate, filtering the secondary primary filtrate with 0.45um filter membrane, and adjusting the pH=6 of the refined filtrate with glacial acetic acid. Detecting the conductivity range (1.2-1.4 MS/CM) of the refined filtrate, adding 16g of CTAC (cta), stirring after passing, complexing and precipitating for 30min, collecting the complexing and precipitating, dissociating the precipitate for 6h by using 2L of 0.4Mol/L sodium chloride solution, clarifying and filtering the dissociated liquid, precipitating the dissociated liquid with 6L of 95% ethanol, collecting the precipitate, dehydrating and drying for three times, and vacuumizing and drying for 12h at 40 ℃ to obtain the finished product.
Comparative example 3
The contrast process comprises the following steps: taking 1L of fermentation liquor, adding 2.5L of 95% ethanol while stirring, precipitating for 30min, and discharging supernatant; adding purified water to 3L (sodium hyaluronate content is about 0.3%), heating to 45 ℃ in a water bath, stirring for 4h to dissolve, pre-paving a diatomite filter layer, filtering the solution to obtain primary filtrate, pre-coating paper board, filtering the primary filtrate again to obtain secondary primary filtrate, filtering the secondary primary filtrate with a 0.45um filter membrane, detecting the conductivity range (1.2-1.4 MS/CM) of trapped liquid, adding 16g of CTAC (sodium hyaluronate) after passing the trapped liquid, stirring, complexing and precipitating for 30min, collecting complexing and precipitating, dissociating the precipitate with 2L of 0.4Mol/L sodium chloride solution for 6h, clarifying and filtering the dissociated liquid, precipitating with 6L of 95% ethanol, collecting the precipitate, dehydrating and drying three times, and vacuumizing and drying for 12h at 40 ℃ to obtain the finished product.
Example 4
The invention comprises the following steps: taking 1L of cockscomb enzymolysis liquid, adding 1.5L of 95% ethanol while stirring, precipitating for 30min, and discharging supernatant; adding ethanol 0.5L under stirring, standing for 15min, and discharging supernatant; repeating for one time, adding ethanol 0.5L under stirring, standing for 15min, and discharging supernatant; the precipitate was added with purified water to 3L (sodium hyaluronate content about 0.3%), formaldehyde 1.0ml (volume concentration about 0.03%), heated to 45 ℃ in a water bath, stirred for 4h for dissolution, ph=11, serine protease 0.3g, and stirred for 1h. Spreading diatomite filter layer, filtering the solution to obtain primary filtrate, pre-coating paper board, filtering the primary filtrate again to obtain secondary primary filtrate, filtering the secondary primary filtrate with 0.45um filter membrane, and adjusting the pH=7.5 of the refined filtrate with glacial acetic acid. Detecting the conductivity range (1.2-1.4 MS/CM) of the refined filtrate, adding 16g of CTAC (cta), stirring after passing, complexing and precipitating for 30min, collecting the complexing and precipitating, dissociating the precipitate for 6h by using 2L of 0.4Mol/L sodium chloride solution, clarifying and filtering the dissociated liquid, precipitating the dissociated liquid with 6L of 95% ethanol, collecting the precipitate, dehydrating and drying for three times, and vacuumizing and drying for 12h at 40 ℃ to obtain the finished product.
Comparative example 4
The contrast process comprises the following steps: taking 1L of cockscomb enzymolysis liquid, adding 2.5L of 95% ethanol while stirring, precipitating for 30min, and discharging supernatant; adding purified water to 3L (sodium hyaluronate content is about 0.3%), heating to 45 ℃ in a water bath, stirring for 4h to dissolve, pre-paving a diatomite filter layer, filtering the solution to obtain primary filtrate, pre-coating paper board, filtering the primary filtrate again to obtain secondary primary filtrate, filtering the secondary primary filtrate with a 0.45um filter membrane, detecting the conductivity range (1.2-1.4 MS/CM) of trapped liquid, adding 16g of CTAC (sodium hyaluronate) after passing the trapped liquid, stirring, complexing and precipitating for 30min, collecting complexing and precipitating, dissociating the precipitate with 2L of 0.4Mol/L sodium chloride solution for 6h, clarifying and filtering the dissociated liquid, precipitating with 6L of 95% ethanol, collecting the precipitate, dehydrating and drying three times, and vacuumizing and drying for 12h at 40 ℃ to obtain the finished product.
Example 5
The invention comprises the following steps: taking 1L of cockscomb enzymolysis liquid, adding 1.5L of 95% ethanol while stirring, precipitating for 30min, and discharging supernatant; adding ethanol 0.5L under stirring, standing for 15min, and discharging supernatant; repeating for one time, adding ethanol 0.5L under stirring, standing for 15min, and discharging supernatant; the precipitate was added with purified water to 3L (sodium hyaluronate content about 0.3%), formaldehyde 1.0ml (volume concentration about 0.03%) and heated to 45℃in a water bath with stirring for 4h for dissolution. Spreading diatomite filter layer, filtering the solution to obtain primary filtrate, pre-coating paper board, filtering the primary filtrate again to obtain secondary primary filtrate, filtering the secondary primary filtrate with 0.45um filter membrane, and adjusting the pH=6.5 of the refined filtrate with glacial acetic acid. Detecting the conductivity range (1.2-1.4 MS/CM) of the refined filtrate, adding 16g of CTAC (cta), stirring after passing, complexing and precipitating for 30min, collecting the complexing and precipitating, dissociating the precipitate for 6h by using 2L of 0.4Mol/L sodium chloride solution, clarifying and filtering the dissociated liquid, precipitating the dissociated liquid with 6L of 95% ethanol, collecting the precipitate, dehydrating and drying for three times, and vacuumizing and drying for 12h at 40 ℃ to obtain the finished product.
Comparative example 5
The contrast process comprises the following steps: taking 1L of cockscomb enzymolysis liquid, adding 2.5L of 95% ethanol while stirring, precipitating for 30min, and discharging supernatant; adding purified water to 3L (sodium hyaluronate content is about 0.3%), heating to 45 ℃ in a water bath, stirring for 4h to dissolve, pre-paving a diatomite filter layer, filtering the solution to obtain primary filtrate, pre-coating paper board, filtering the primary filtrate again to obtain secondary primary filtrate, filtering the secondary primary filtrate with a 0.45um filter membrane, detecting the conductivity range (1.2-1.4 MS/CM) of trapped liquid, adding 16g of CTAC (sodium hyaluronate) after passing the trapped liquid, stirring, complexing and precipitating for 30min, collecting complexing and precipitating, dissociating the precipitate with 2L of 0.4Mol/L sodium chloride solution for 6h, clarifying and filtering the dissociated liquid, precipitating with 6L of 95% ethanol, collecting the precipitate, dehydrating and drying three times, and vacuumizing and drying for 12h at 40 ℃ to obtain the finished product.
The intermediate secondary primary filtrate obtained in the above examples was submitted for inspection, and the inspection results and process parameters were summarized in the following table:
TABLE 1
Finished products obtained in the above examples are inspected, and inspection results and process parameters are summarized in the following table:
TABLE 2
From the above table, it can be concluded that the use of formaldehyde or alkaline protease added to sodium hyaluronate can effectively reduce the content of impurity proteins in sodium hyaluronate, thereby effectively inhibiting the growth of microorganisms.
Meanwhile, the formaldehyde solution and the alkaline protease are combined for use, so that the process time is obviously shortened, the impurity protein of the product is further reduced, and particularly, the method has positive influence on the microbial control in the process and is greatly helpful for improving the quality of the final product. And the scheme cost is lower, and the operability is strong.
Claims (5)
1. A method for purifying sodium hyaluronate is characterized in that,
the sodium hyaluronate solution is subjected to an alcohol precipitation separation step before impurity proteins are removed;
the impurity protein removal step sequentially comprises the following steps:
(1) Adding formaldehyde into the sodium hyaluronate solution to denature impurity proteins; the added volume concentration of formaldehyde in the sodium hyaluronate solution is 0.03-0.1%;
(2) Regulating the pH value of the solution to be 10.5+/-0.5, adding alkaline protease, and carrying out enzymolysis on impurity proteins in the sodium hyaluronate solution;
and filtering the sodium hyaluronate solution after removing the impurity protein, wherein the solution filtering step sequentially comprises diatomite filter cake filtering, paperboard filtering and membrane filtering.
2. A method for purifying sodium hyaluronate as set forth in claim 1, wherein,
after the step of removing the impurity protein in the sodium hyaluronate solution, the method further comprises the following steps:
adding CTAC into the filtrate for complexing, collecting CTAC precipitate, then dissociating with sodium chloride solution to obtain dissociated solution, precipitating the dissociated solution with ethanol, collecting precipitate, and drying to obtain sodium hyaluronate raw material.
3. A method for purifying sodium hyaluronate as set forth in claim 1, wherein,
the pore size of the filter membrane used in the membrane filtration was 0.45. Mu.m.
4. A method for purifying sodium hyaluronate as set forth in claim 2, wherein,
in the CTAC complexing process, the pH of the sodium hyaluronate solution is 6.0-7.5.
5. A method for purifying sodium hyaluronate as set forth in claim 4, wherein,
the addition amount of CTAC is 2-3 times of the mass of sodium hyaluronate.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5411874A (en) * | 1990-11-07 | 1995-05-02 | Fermentech Medical Limited | Production of hyaluronic acid |
| CN1597704A (en) * | 2004-08-10 | 2005-03-23 | 江南大学 | Method of preparing transparent sodium protonate from transparent protonic acid fermentation liquid |
| CN101512006A (en) * | 2006-07-06 | 2009-08-19 | 利莱恩斯生命科学有限公司 | Efficient process for purification of high molecular weight hyaluronic acid |
| CN106188338A (en) * | 2016-07-13 | 2016-12-07 | 浙江景嘉医疗科技有限公司 | A kind of reduce the method for foreign protein content in hyaluronic acid sodium raw materials |
| CN106349402A (en) * | 2016-09-30 | 2017-01-25 | 天津科技大学 | Method for removing protein in polysaccharide extraction liquid by dialdehyde cellulose |
| CN112940148A (en) * | 2021-04-06 | 2021-06-11 | 山东众山生物科技有限公司 | Hyaluronic acid purification method |
-
2022
- 2022-06-09 CN CN202210644688.1A patent/CN115215949B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5411874A (en) * | 1990-11-07 | 1995-05-02 | Fermentech Medical Limited | Production of hyaluronic acid |
| CN1597704A (en) * | 2004-08-10 | 2005-03-23 | 江南大学 | Method of preparing transparent sodium protonate from transparent protonic acid fermentation liquid |
| CN101512006A (en) * | 2006-07-06 | 2009-08-19 | 利莱恩斯生命科学有限公司 | Efficient process for purification of high molecular weight hyaluronic acid |
| CN106188338A (en) * | 2016-07-13 | 2016-12-07 | 浙江景嘉医疗科技有限公司 | A kind of reduce the method for foreign protein content in hyaluronic acid sodium raw materials |
| CN106349402A (en) * | 2016-09-30 | 2017-01-25 | 天津科技大学 | Method for removing protein in polysaccharide extraction liquid by dialdehyde cellulose |
| CN112940148A (en) * | 2021-04-06 | 2021-06-11 | 山东众山生物科技有限公司 | Hyaluronic acid purification method |
Non-Patent Citations (3)
| Title |
|---|
| "Effect of pH on the Behavior of Hyaluronic Acid in Dilute and Semidilute Aqueous Solutions";Atoosa Maleki等;《Macromol. Symp.》;第274卷;第131-140页 * |
| "Role of the pH on Hyaluronan Behavior in Aqueous Solution";Iuliana Gatej等;《Biomacromolecules》;第6卷;第61-67页 * |
| "Viscosity and Wettability of Hyaluronic Acid according to Antimicrobial Supplementation, Ionic Strength, and pH";Hong-Seop Kho等;《Journal of Oral Medicine and Pain》;第39卷(第3期);第90-95页 * |
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