CN1259345C - Method of preparing transparent sodium protonate from transparent protonic acid fermentation liquid - Google Patents
Method of preparing transparent sodium protonate from transparent protonic acid fermentation liquid Download PDFInfo
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- CN1259345C CN1259345C CN 200410041680 CN200410041680A CN1259345C CN 1259345 C CN1259345 C CN 1259345C CN 200410041680 CN200410041680 CN 200410041680 CN 200410041680 A CN200410041680 A CN 200410041680A CN 1259345 C CN1259345 C CN 1259345C
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Abstract
The present invention relates to a method for preparing hyaluronic acid sodium from hyaluronic acid fermentation liquid, particularly to prepare hyaluronic acid sodium with a high molecular weight and a low protein content in the hyaluronic acid fermentation liquid. A thallus is removed by heating, protein is removed by adding proteinase, and small molecules of metal ions and colored substances are removed by using an ultrafiltration technology by the present invention so as to prepare the hyaluronic acid sodium from the fermentation liquid. The present invention has the specific technology that the hyaluronic acid sodium with the molecular weight of 1.8*10<6>, the protein content of 0.30% and the extraction yield of 98% is prepared by thallus removal, enzymolysis, ethanol precipitation, complexation, dissociation, ethanol precipitation, ultrafiltration, ethanol precipitation, wash, dehydration and dryness and finished product package from the hyaluronic acid fermentation liquid. The method of the present invention has the advantages of simple and convenient operation, high efficiency, low expenses, high purity of finished products, little environment pollution, easy industrialization, etc. The present invention is convenient for implementation in laboratories, and is also very easy to apply in industrial production.
Description
Technical field
The present invention relates to a kind of method that from hyaluronic acid fermentation liquid, prepares hyaluronate sodium, relate to the hyaluronate sodium that from hyaluronic acid fermentation liquid, prepares high molecular, lower protein content.
Background technology
(Hyaluronic acid HA), has another name called hyaluronic acid or glass acid to hyaluronic acid, is a kind of macromole mucopolysaccharide, extensively is present in the reticular tissue of organism.The physiological action of HA is different in different tissues, as mainly show as water retention in skin; In knuckle synovia, be mainly lubrication; Mainly be to regulate permeability in vessel wall.In addition, HA is as polyanion electrolyte, on the molecule with a large amount of negative charges, can regulate the concentration of negative ions on every side, the activity that suppresses plurality of enzymes, be widely used in ophthalmology viscosurgery, treatment joint disease, soft tissue repair and as pharmaceutical carrier etc., particularly in prevention with reduce after the surgical operation and obtained bigger progress in the tissue adhesion.
Adopt the Production by Microorganism Fermentation hyaluronic acid, the source of having widened HA has greatly promoted research and the application of HA.Microbial fermentation is produced HA and has not only been reduced the production cost of HA, and has higher quality and good quality.And the technological process of extracting HA from fermented liquid can directly affect output, quality and the production cost of HA.
It is the method for studying more deeply both at home and abroad that organic solvent is removed the thalline method, although the resulting HA of this method is very little by the degree of depolymerization.But the shortcoming that this method exists has: 1) even through repeatedly extracting repeatedly, still have a certain amount of thalline to exist among the finished product HA, thereby be difficult to improve extraction yield; 2) use a large amount of organic solvents, although the recyclable utilization of solvent, but still cause huge raw material consumption, increased extraction cost; 3) use of poisonous in a large number, explosive, volatile solvent has caused serious pollution to environment, has brought inconvenience to operation.
Extract the not only demanding extraction yield of HA and its molecule depolymerization does not take place, and demanding product purity.The protein that exists in the hyaluronic acid fermentation liquid is one of major impurity that influences the HA product purity.The protein that exists in the extracting solution can make it precipitation with common protein precipitant such as Tai-Ace S 150 etc., also can suitably transfer pH and interpolation trichoroacetic acid(TCA) etc. to make it the sex change precipitation.But these complex operations should not be applied in the suitability for industrialized production.If can adopt easy and simple to handle, inexpensive material to remove protein, can improve the quality of products and can reduce production costs, can be widely used in suitability for industrialized production.
At two valence metal ions such as the Ca that in the HA of ethanol sedimentation, also exist part
2+, Ba
2+, Zn
2+And coloring matter, directly affect application and the organoleptic quality of HA.Although remove metal ion effectively with the alcohol grading separation energy, this method operation is loaded down with trivial details and cost is higher, is difficult to adapt to suitability for industrialized production; Coloring matter is the another kind of impurity that is common among the HA, and removal method commonly used has oxidation style and reduction method, but these two kinds of methods can cause activity, desulfurization acidic group and the DeR of not terminal aldehyde group oxidation of HA, infringement HA.
HA extracting method ubiquity in the past not high, the with low quality and shortcoming such as quality is unstable, environmental pollution is serious of cost height, extraction yield, is difficult to adapt to the needs of suitability for industrialized production and practical application.The process of extracting HA from fermented liquid is divided into just carries and makes with extra care two stages, just carries to removing the thalline in the fermented liquid and reclaiming and obtains primary HA, again through the refining HA that just carries gained of steps such as isolating protein, nucleic acid and heavy metal.Consider the continuous growth of domestic and international market, therefore, how on technical scale, to realize the extraction of high-quality HA, have important industrial application value high-quality HA demand.
Summary of the invention
The purpose of this invention is to provide a kind of hyaluronic acid fermentation liquid that utilizes and prepare hyaluronic method high-quality, high yield, the hyaluronic acid of preparation with the form of hyaluronate sodium as product.This method is easy and simple to handle, efficient, expense is cheap, finished product purity height, environmental pollution are little, be easy to industrialization.
Technical scheme of the present invention: the present invention adopts the fermented liquid heating, filters and remove thalline, add proteolytic enzyme and remove protein, use ultra-filtration technique to remove metal ion and micromolecular coloring matter, from fermented liquid, prepare hyaluronate sodium, the concrete operations operation for hyaluronic acid fermentation liquid through add heat abstraction thalline, enzymolysis, ethanol sedimentation, complexing, dissociate, to make molecular weight be 1.8 * 10 for ethanol sedimentation, ultrafiltration, ethanol sedimentation, washing dehydration drying, finished product packing
6, protein content is lower than 0.30%, the hyaluronate sodium of extract yield 98%.
The concrete operations step is as follows:
1, the removal of thalline: with one times of distilled water diluting of fermented liquid, centrifugal or filter press after the employing high-temperature steam is heated to 75～80 ℃ rapidly with fermented liquid discards thalline.
2, enzymolysis: the pH of supernatant liquor is transferred to 8.0, according to the volume of filtrate, take by weighing Sumizyme MP then, 37 ℃ of following enzymolysis 3 hours with mass volume ratio 0.05%.
3, ethanol sedimentation: the fermentation clear liquid pH behind enzymolysis is transferred to 7.0～7.2, add 95% ethanol that equates with the enzymolysis solution volume, fully stir, staticly settle HA.
4, resolution of precipitate: sedimentary HA uses and the isopyknic distilled water stirring and dissolving of initial fermented liquid.
5, complex-precipitation: (volume ratio is 10: 1=fermented liquid: CPC) gently to add 5% cetylpyridinium chloride (CPC) aqueous solution of 0.1 times of fermentating liquid volume in the above-mentioned abundant dissolved HA aqueous solution while stirring, thereby make the HA in the aqueous solution that complex-precipitation take place, leave standstill and make complex-precipitation sink abandoning supernatant.
6, dissociate: place the NaCl aqueous solution with the initial isopyknic 0.7～0.8mol/L of fermented liquid fully to stir to dissociate complex-precipitation and spend the night.
7, ethanol sedimentation: abundant dissociated solution is filtered, and elimination does not have thorough dissociated block, and dissociation solution is taken out throw out HA washing again with 95% ethanol sedimentation of 2 times of initial fermentating liquid volumes.
8, ultrafiltration: throw out HA is used and the isopyknic dissolved in distilled water of initial fermented liquid, is that 5000 polyethersulfone (PES) ultra-filtration membrane carries out ultrafiltration according to following condition: 0.2MPa, molecular weight cut-off, removes metal ion and micromolecular coloring matter.
9, ethanol sedimentation:, with 95% ethanol sedimentation of 2 times of initial fermentating liquid volumes, take out throw out HA-Na again with the sodium acetate of the adding of the filtrate after the ultrafiltration 1%.
10, washing dehydration drying: with HA-Na throw out absolute ethanol washing secondary, use final vacuum drying of washing with acetone again, promptly get the HA-Na finished product.
Beneficial effect of the present invention: the present invention adopts adds the heat abstraction thalline, add proteolytic enzyme removes protein, uses ultra-filtration technique to remove metal ion and micromolecular coloring matter, the operation sequence of preparation pharmaceutical grade hyaluronate sodium from fermented liquid, not only can in the laboratory, conveniently implement, and very easily in industrial production, use.The technology that employing adds thalline in the heat extraction fermented liquid can reduce the depolymerization of hyaluronan molecule, improves the extract yield of HA, gives hyaluronic acid good organoleptic quality simultaneously.Sumizyme MP is a macromole solution, by adding Sumizyme MP, carries out proteolysis widely in abstraction process, can well remove protein, and production cost is lower, is fit to suitability for industrialized production.Use is applicable to that the ultra-filtration technique of industrially scalable can effectively remove micromolecular metal ion and coloring matter, especially ultra-filtration technique and be fit to large-scale industrial production.
Embodiment
Embodiment 1
The hyaluronic acid fermentation liquid that fermentation obtains, composition analysis result: thalline 4.0g/L; HA 4.83g/L; Protein 3.35%; Glucosamine 38.64%; Glucuronic acid 41.39%.Above-mentioned 1L fermented liquid is heated to 75 ℃～80 ℃ rapidly after with the 1L distilled water diluting, and with the centrifugal removal thalline of diluent after the heating, getting supernatant liquor, to regulate pH be 8.0.The Sumizyme MP that adds 1g then, enzyme digestion reaction 3h under 37 ℃ of constant temperature transfers pH7.0～7.2, adds the ethanol sedimentation HA of 2L 95%, the centrifugal supernatant liquor of removing, the dissolved in distilled water HA of adding 1L.Wait to precipitate the 5% cetylpyridinium chloride solution (CPC) that adds 0.1L after whole dissolvings and come complexing HA, staticly settle the centrifugal supernatant liquor that goes in back, throw out is put into the 24h that dissociates in the sodium chloride aqueous solution of 1L0.7～0.8mol/L.Dissociating adds the 2L95% ethanol sedimentation after finishing, centrifugal, abandoning supernatant, and the gained precipitation is HA.With the throw out HA 1L dissolved in distilled water of gained, be that 5000 polyethersulfone (PES) ultra-filtration membrane carries out ultrafiltration according to following condition: 0.2MPa, molecular weight cut-off, remove metal ion and micromolecular coloring matter.Filtrate after the ultrafiltration is added the 10g sodium acetate, with 95% ethanol sedimentation of 2L, take out throw out HA-Na again.Use absolute ethanol washing twice then, be refining HA-Na with the once back oven dry of washing with acetone again.This moment, the hyaluronate sodium molecular weight was 1.8 * 10
6, protein content is 0.30%, extract yield 98%, limiting viscosity is 43.7dl/g, the hyaluronate sodium flocculent substance that is silvery white in color has good organoleptic quality.
The comparative example 2
Use trichoroacetic acid(TCA) instead and remove thalline, other operations as removal protein are removed metal ion and coloring matter with embodiment 1.The result: the hyaluronate sodium molecular weight is 1.23 * 10
6, protein content is 0.45%, extract yield 90%, limiting viscosity is 34.1dl/g.
The comparative example 3
Use the Tai-Ace S 150 precipitating proteins instead or suitably transfer pH and add trichoroacetic acid(TCA) etc. and make protein denaturation precipitation, other operations as remove thalline are removed metal ion and coloring matter with embodiment 1.The result: the hyaluronate sodium molecular weight is 1.65 * 10
6, protein content is 0.76%, extract yield 92%, limiting viscosity is 40.5dl/g.
The comparative example 4
Adopt alcohol grading to separate and oxidation style removal small molecules metal ion and coloring matter, other operations are removed protein with embodiment 1 as removing thalline.The result: the hyaluronate sodium molecular weight is 1.67 * 10
6, protein content is 0.35%, extract yield 91%, limiting viscosity is 27.3dl/g.
Claims (2)
1 one kinds of methods that from hyaluronic acid fermentation liquid, prepare hyaluronate sodium, it is characterized in that adopting the fermented liquid heating, filter and remove thalline, add proteolytic enzyme and remove protein, use ultra-filtration technique to remove the method for metal ion and micromolecular coloring matter, concrete technology is for heating removes thalline, enzymolysis, ethanol sedimentation, complexing, dissociates, ethanol sedimentation, ultrafiltration, ethanol sedimentation, washing dehydration drying, finished product packing make the finished product hyaluronate sodium
Heating removes thalline: fermented liquid is removed thalline after filtration with behind one times of the distilled water diluting fermented liquid temperature being raised to 75 ℃～80 ℃;
Enzymolysis: the pH of supernatant liquor is transferred to 8.0, according to the volume of filtrate, take by weighing Sumizyme MP then, 37 ℃ of following enzymolysis 3 hours with mass volume ratio 0.05%;
Ethanol sedimentation: the fermentation clear liquid pH behind enzymolysis is transferred to 7.0～7.2, add 95% ethanol that equates with the enzymolysis solution volume, fully stir, staticly settle HA;
Resolution of precipitate: sedimentary HA uses and the isopyknic distilled water stirring and dissolving of initial fermented liquid;
Complex-precipitation: 5% the cetylpyridinium chloride aqueous solution that in the above-mentioned abundant dissolved HA aqueous solution, gently adds 0.1 times of fermentating liquid volume while stirring, thereby make the HA in the aqueous solution that complex-precipitation take place, leave standstill and make complex-precipitation sink abandoning supernatant;
Dissociate: place the NaCl aqueous solution with the initial isopyknic 0.7～0.8mol/L of fermented liquid fully to stir to dissociate complex-precipitation and spend the night;
Ethanol sedimentation: abundant dissociated solution is filtered, and dissociation solution is taken out throw out HA washing again with 95% ethanol sedimentation of 2 times of initial fermentating liquid volumes;
Ultrafiltration: throw out HA is used and the isopyknic dissolved in distilled water of initial fermented liquid, is that 5000 poly (ether-sulfone) ultrafiltration membrane carries out ultrafiltration according to following condition: 0.2MPa, molecular weight cut-off, removes metal ion and micromolecular coloring matter;
Ethanol sedimentation:, with 95% ethanol sedimentation of 2 times of initial fermentating liquid volumes, take out throw out HA-Na again with the sodium acetate of the adding of the filtrate after the ultrafiltration 1%;
Washing dehydration drying: with HA-Na throw out absolute ethanol washing secondary, use final vacuum drying of washing with acetone again, promptly get the HA-Na finished product;
Finished product packing: the HA-Na finished product that the organoleptic quality that obtains is good is packed.
2, a kind of method for preparing hyaluronate sodium from hyaluronic acid fermentation liquid according to claim 1 is characterized in that adopting high-temperature steam that the fermented liquid temperature is raised to 80 ℃ after fermented liquid dilutes one times removes thalline after filtration.
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Families Citing this family (18)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN100478360C (en) * | 2006-10-31 | 2009-04-15 | 山东福瑞达生物化工有限公司 | Process for producing hyaluronic acid or its salt by concentration |
| CN101633701B (en) * | 2008-07-23 | 2011-10-05 | 上海景峰制药有限公司 | Method for purifying hyaluronic acid |
| CN101550199B (en) * | 2009-05-09 | 2013-05-15 | 山东众山生物科技有限公司 | Method for preparing sodium hyaluronate from hyaluronic acid zymotic fluid |
| CN101935362B (en) * | 2009-06-30 | 2012-07-25 | 上海佰加壹医药有限公司 | Method for purifying hyaluronic acid by pre-laying filter aid |
| CN101704905B (en) * | 2009-09-28 | 2012-05-09 | 安徽丰原发酵技术工程研究有限公司 | Method for extracting hyaluronic acid |
| CN102020724B (en) * | 2010-12-23 | 2012-09-05 | 安徽丰原发酵技术工程研究有限公司 | Method for extracting sodium hyaluronate from fermentation liquor containing hyaluronic acid |
| CN105254779B (en) * | 2015-10-13 | 2017-11-21 | 青海大学 | A kind of method for extraction and purification of macromolecule hyaluronic acid |
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| CN105777937A (en) * | 2016-03-22 | 2016-07-20 | 山东众山生物科技有限公司 | Method for preparing sodium hyaluronate from hyaluronic acid fermentation liquor |
| CN105859911B (en) * | 2016-04-12 | 2018-09-14 | 广东东阳光药业有限公司 | A kind of method of separation purifying hyaluronic acid |
| CN105949351B (en) * | 2016-07-16 | 2019-07-23 | 山东阜丰发酵有限公司 | A kind of hyaluronic acid fermentation liquor extracting method |
| IT201600079633A1 (en) | 2016-07-28 | 2018-01-28 | Fidia Farm Spa | Preparation and purification process of the sodium salt of hyaluronic acid |
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| CN106883320B (en) * | 2017-04-28 | 2020-04-24 | 上海景峰制药有限公司 | Secondary dissociation preparation method of sodium hyaluronate for reducing protein content |
| CN109913518A (en) * | 2018-08-17 | 2019-06-21 | 上海昊海生物科技股份有限公司 | A kind of pipeline preparation method of Sodium Hyaluronate |
| CN110590972B (en) * | 2019-09-19 | 2021-06-25 | 华熙生物科技股份有限公司 | Drying method of hyaluronic acid |
| CN110734506A (en) * | 2019-11-19 | 2020-01-31 | 山东众山生物科技有限公司 | Preparation method of sodium hyaluronate |
| CN115215949B (en) * | 2022-06-09 | 2024-01-05 | 浙江驭远生物科技有限公司 | Sodium hyaluronate and purification method thereof |
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Assignee: Shanghai Bai Jiayi Pharmaceutical Co. Ltd. Assignor: Jiangnan University Contract fulfillment period: 2008.7.7 to 2013.7.7 contract change Contract record no.: 2008310000011 Denomination of invention: Method of preparing transparent sodium protonate from transparent protonic acid fermentation liquid Granted publication date: 20060614 License type: Exclusive license Record date: 2008.9.3 |
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Free format text: EXCLUSIVE LICENCE; TIME LIMIT OF IMPLEMENTING CONTACT: 2008.7.7 TO 2013.7.7 Name of requester: SHANGHAI BAIJIAYI PHARMACEUTICAL CO., LTD. Effective date: 20080903 |
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