CN101089021B - Process of separating and extracting hyaluronic acid from microbial fermented liquid - Google Patents
Process of separating and extracting hyaluronic acid from microbial fermented liquid Download PDFInfo
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Abstract
The process of separating and extracting hyaluronic acid from microbial fermented liquid includes the following steps: 1. complexing fermented liquid containing hyaluronic acid with chloro hexadecyl pyridine or precipitating the fermented liquid with ethanol; 2.collecting the complex or alcohol precipitate, and dissociating or dissolving the complex or alcohol precipitate in water solution of sodium chloride; and 3. collecting the hyaluronic acid precipitate, dissolving in water, and drying to obtain target product. The present invention has simple process, hyaluronic acid yield of about 90 %,molecular weight loss of about 15 % and low cost, and is suitable for industrial production.
Description
Technical field
The present invention relates to hyaluronic preparation method, relate in particular to the method for separating and extracting hyaluronic acid from microbial fermentation solution.
Background technology
Hyaluronic acid (Hyaluronic Acid, HA) be by glucuronic acid and acetylglucosamine by β, 1-3 and β, the repeating unit that the 1-4 glycosidic link constituted and the linear polymeric mucopolysaccharide that forms, the disaccharide units is that 300-1100 is right, molecular weight ranges 10
5-10
7Da.
1934, Meyer and Palmer isolated a kind of macromolecular polysaccharide from bovine vitreous body, and they are this polysaccharide called after hyaluronic acid.HA is water-soluble, is insoluble to organic solvents such as alcohol, ketone, ether.Owing to its solution has superpower retentiveness, visco-elasticity and biological easily absorptivity, quilt is extensively owing to fields such as Film with Preventing Adhesion, sacroiliitis and makeup in ophthalmologic operation, the abdominal operation.
Hyaluronic production mainly contains animal tissues's extraction method and two kinds of production technique of fermentation method.Mainly be extraction method in early days, can be used for producing hyaluronic animal organ's tissue and mainly contain cockscomb, people's umbilical cord and animal eyeball.Owing to be subjected to the restriction that raw material sources are few and hyaluronic acid contents is low, it is higher that extraction method is produced the hyaluronic acid cost.Compare with extraction method; the fermentative Production scale is not limited by raw material sources; hyaluronic acid exists with unbound state in the fermented liquid; be easy to separation and purification and form large-scale industrial production; so production cost is far below extraction method; and the danger of polluting of the Causative virus of animal origin-free, and can obtain the HA of macromolecule, high yield.Therefore, extraction method is replaced by fermentation method just gradually at present.
Separation of known use microorganism and the hyaluronic method of purifying are as follows: the purification process that U.S. Patent No. 4784990 is described comprises: add ethanol in the nutrient solution of streptococcus zooepidemicus hyaluronic acid is separated from microorganism, make its precipitation with the chloro-hexadecane yl pyridines then.The HA yield is 60-70% only, need and once cross post through heat, twice filtration, secondary complexing, four alcohol precipitations and just can make high purity HA.The purification process that U.S. Patent No. 5316926 is described comprises: lauryl sulfate (SLS) tensio-active agent of adding 0.01% is to separate attached to the hyaluronic acid on the cell walls in fermented liquid, add cats product chloro-hexadecane yl pyridines then and precipitate, and make its precipitation with alcohol to form hyaluronic acid.This process also comprises three filtrations and four alcohol precipitations in addition, handle with SLS pod membrane before also needing.U.S. Patent No. 5411874 has been described and added the formalin solution sterilization in fermented liquid, adding sodium lauryl sulphate (SDS) comes off pod membrane, after once filtration, a ultrafiltration, a complexing and three alcohol precipitations and after the HA that obtains, higher 0.2% (albumen/HA (g/g)) that be about of albumen is cosmetics-stage HA.The hyaluronic method of U.S. Patent No. 4157296 disclosed a kind of purifying comprises: the nutrient solution of handling Streptococcus pyrogenes with trichoroacetic acid(TCA) makes its precipitation with organic solvent then to remove bacterial strain.This method passes through to regulate fermented liquid pH sterilization, but owing to also need a large amount of repeated use organic solvents to precipitate, causes cost high and very consuming time.But membrane technique purifying HA, but generally all be as a kind of auxiliary separation means, be used for thalline or small molecular weight and remove, still need organic solvent to separate.As U.S. Patent No. 4517295, No.4801539 etc., adopt MWCO10000,20000, small-molecule substance is removed in the diafiltration of 30000Da ultra-filtration membrane.But U.S. Patent No. 6489467 described a kind of need not be with an organic solvent, but the method that makes the HA proterties change and use ultrafiltration to purify by the pH to 2.5 that regulates HA, but filtering velocity is very slow very consuming time, be unfavorable for scale operation, and it is in 2.5 environment that HA should not be in pH for a long time, easily causes molecular weight degradation.Chinese patent 200610057913.2 and 200610090586.0 has also been described the pH to 3 that regulates HA, carries out ultrafiltration under the help in electric field and magnetic field, to strengthen filtering velocity, avoided with an organic solvent simultaneously, but filtering velocity still only is 25.8L/ (m
2H), be unfavorable for amplifying.Japanese Patent Laid Open Publication No.63-012293 has described a kind of by using big netted anionite-exchange resin (DianionHPA-25, HPA-75, IRA-900, IRA-904) to handle to contain hyaluronic solution to be equal to or less than the method for low-molecular-weight hyaluronic acid and the pyrogen of 1500000Da to remove molecular weight.Korean Patent No.10-2002-0048915 has described fermented liquid and has obtained high purity HA through activated carbon treatment, the processing of aromatics polymeric adsorbent, Ultrafiltration and ethanol sedimentation effect etc.
Yet no matter extraction method or fermentation method have all comprised ethanol sedimentation and selective precipitation repeatedly in process of production.Operational path is long, complicated, time-consuming, the cost height; Adopt more organic solvent, easily cause secondary pollution.Cause hyaluronic production cost still very high, seriously limited the hyaluronic acid range of application.Various alternative methods are carried out removal of impurities as polymeric adsorbent or the ion exchange resin that adopts some more expensive stable performances, or adopt method removal of impurities such as frozen centrifugation, pH regulation and control, ultrafiltration, products obtained therefrom purity height, but HA yield and loss of molecular weight rate are then how not enough to some extent because of operation steps, relate to complicated treating processes and treatment facility again, thereby strengthened production cost.
Summary of the invention
The object of the present invention is to provide a kind of hyaluronic method of from microbial fermentation solution, collecting, to overcome the above-mentioned defective that prior art exists.
Method of the present invention comprises the steps:
(1) to containing hyaluronic fermented liquid, carries out complexing or carry out alcohol precipitation by ethanol by the chloro-hexadecane yl pyridines;
(2) collect complex compound or pure hypostasis, add sodium chloride aqueous solution, make dissociating or dissolving of complex compound or pure hypostasis;
(3) hyaluronic acid of collecting in dissociate thing or the solute precipitates, and uses water dissolution, and drying obtains target product;
Said hyaluronic molecular weight is 1～3MDa.
Said fermented liquid can adopt Tian Yihong to be published in " fermentation research of high molecular weight hyaluronic acid " reported method on " Chinese Medicine engineering magazine " in 2004, is prepared;
Said hyaluronic molecular weight is 1～3MDa;
Said chloro-hexadecane yl pyridines (being called for short CPC) can adopt the commercially available prod, as the product of Chemical Reagent Co., Ltd., Sinopharm Group;
Preferably, method of the present invention comprises the steps:
(1) will contain hyaluronic fermented liquid thin up to hyaluronic content is 0.3～0.5g/ml, adds the chloro-hexadecane yl pyridines then; Stir complexing;
CPC and hyaluronic weight ratio are: HA: CPC=1: 1.5～1: 2.5;
(2) with the product centrifugation of step (1), collect solid complex;
(3) add concentration be the sodium chloride aqueous solution of 0.4～1M to 0.9～1.1 times of original fermented solution volume, complex compound dissociates, and obtains HA solution;
(4) add ethanol, dosage is: HA solution: ethanol=1: 2, and volume ratio stirs, and precipitation obtains fibrous HA throw out;
(5) add the fermentating liquid volume of 0.1～0.2M sodium-chlor, be stirred to resolution of precipitate to twice;
(6) in lysate, add perlite and gac, stir, filter, obtain filtrate;
Said perlite is selected from more than one in No. 2 perlites or No. 4 perlites, and add-on is 2～4g/L, and the add-on of gac is 1.5～2.5g/L;
(7) add ethanol in filtrate, dosage is a HA solution: ethanol=1: 1.5～2.5, and volume ratio obtains the HA precipitation after the stirring;
(8) water dissolution precipitation, spraying or vacuum-drying get medical grade HA dry powder.
Gained HA dry powder 1.2g, glucuronic acid purity is about 44.5%, and the HA yield is 91%, and molecular weight is 2.07MDa, and the loss of molecular weight rate is 13%, albumen/HA (g/g)=0.04%.
Preferably, method of the present invention comprises the steps:
(1) will contain hyaluronic fermented liquid thin up to hyaluronic content is 0.3～0.5g/ml, adds the chloro-hexadecane yl pyridines then; Stir complexing;
CPC and hyaluronic weight ratio are: HA: CPC=1: 1.5～1: 2.5;
(2) with the product centrifugation of step (1), collect solid complex;
(3) add the original fermented solution volume of sodium chloride aqueous solution to 1.8～2.2 times of 0.4～1M, complex compound dissociates, and obtains HA solution;
(4) in the solution of step (3), add perlite and gac, stir, filter, collect filtrate;
Said perlite is selected from more than one in No. 2 perlites or No. 4 perlites, and add-on is 2～4g/L, and the add-on of gac is 1.5～2.5g/L;
(5) add aluminum oxide in filtrate, dosage is: HA: aluminum oxide=1: 4～1: 8 (g/g), and other adds 1～2g/L gac, stirs, and filters, and collects filtrate;
(6) add ethanol in filtrate, dosage is a HA solution: ethanol=1: 1.5～2.5, and volume ratio obtains the HA precipitation after the stirring;
7) water dissolution precipitation, spraying or vacuum-drying get medical grade HA dry powder.
Gained HA dry powder 2.18g, glucuronic acid purity is about 46.1%, and the HA yield is 91%, and molecular weight is 1.92MDa, and the loss of molecular weight rate is 10.1%, albumen/HA (g/g)=0.0%.
Preferably, method of the present invention comprises the steps:
(1) will contain hyaluronic fermented liquid and add ethanol, dosage is a HA solution: ethanol=1: 1.5～2.5, and volume ratio obtains fibrous HA precipitation after the stirring;
(2) add the original fermented solution volume of sodium chloride aqueous solution to 1.8～2.2 times of 0.1～0.2M, stir resolution of precipitate;
(3) in the solution of step (2), add perlite and gac, stir, filter, obtain filtrate;
Said perlite is selected from more than one in No. 2 perlites or No. 4 perlites, and add-on is 2～4g/L, and the add-on of gac is 1.5～2.5g/L, filters, and collects filtrate;
(4) add aluminum oxide in the filtrate of step (3), dosage is: HA: aluminum oxide=1: 4～1: 8 (g/g), and other adds 1～2g/L gac, stirs, and filters, and collects filtrate;
(5) add ethanol in filtrate, add ethanol in filtrate, dosage is a HA solution: ethanol=1: 1.5～2.5, and volume ratio obtains the HA precipitation after the stirring;
(6 water dissolution precipitations, spraying or vacuum-drying get medical grade HA dry powder.
Gained HA dry powder 2.2g, glucuronic acid purity is about 45.9%, and the HA yield is 92%, and molecular weight is 1.90MDa, and the loss of molecular weight rate is 11.1%, albumen/HA (g/g)=0.0%.
The present invention adopts directly fermented liquid is carried out complexing or alcohol precipitation operation, can decolour, make the protein denaturation sterilization to be beneficial to the follow-up albumen that removes by filter, and can guarantee the HA yield again, and because of the contained HA concentration of fermented liquid height, HA almost can precipitate fully through alcohol precipitation.Therefore avoided employing acidification or high temperature to carry out sterilization, prevented that HA from being degraded; Also avoid these sterilant of adding formalin solution, SDS or SLS or tensio-active agent, prevented secondary pollution; Avoided the HA yield that first filter cake excessively causes to fermented liquid lower, because of filter operation need be diluted fermented liquid, the concentration of the contained HA of filtrate that obtains like this is just lower, adding ethanol this moment precipitates and can cause the HA yield lower, if filtrate redeposition behind thin film concentration has then increased running cost.
Adopt a complexing, once filter and during twice sedimentary method, fermented liquid is directly used the CPC complexing, CPC complexing HA key is to control well solution ion strength, therefore as long as being diluted enough multiples (10 times) back adding CPC, fermented liquid can generate complex compound, and the consumption of CPC is only relevant with the total mass of HA in the fermented liquid, so the fermented liquid dilution can't strengthen the CPC consumption.After the complexing complex compound is dissociated to the original fermented solution volume, use ethanol sedimentation this moment again, owing to be not the diluent of precipitation fermented liquid, guaranteed the HA high density, saved consumption of ethanol, and HA yield height, albumen clearance height.Then throw out is dissolved after-filtration, because the filtering consumption that depends on flocculating aids and sorbent material except that protein efficiency, can disposablely be removed when time in the absorption saturation range of foreign protein content at sorbent material, and CPC complexing or ethanol sedimentation remove protein efficiency only about 25%, therefore if filtration is arranged in the comparatively forward step, then the albumen clearance of final HA just can only reach about 95%, if will improve HA purity, need filter or carry out further purifications of operation such as adsorption column once more.Filtration is put behind and is carried out, and removes the protein efficiency height, and also because of sorbent materials such as the flocculating aids that adds and gac are easier to adsorb albumen after the sex change, and this moment, albumen obtained sufficient sex change already.Fermented liquid is carried out complexing earlier but not alcohol precipitation, and in order to prevent complexing behind the first alcohol precipitation, then the dissociation solution of complex compound is once filtered by only, and unnecessary CPC may not necessarily eliminate, and influences quality product.Long or the number of times of filter time adopts that once to filter be in order to enhance productivity, because too much can directly cause production efficiency low in the industrial production.Adopt the method for complexing, secondary filtration and a primary sedimentation, then mainly consider organic solvent use cost height, pollute greatly, substitute alcohol precipitation one time by increasing a filter operation, continuous filtration concentrates or other operations in the middle of can avoiding, and improves the HA yield.Adopt the method for secondary filtration and secondary sedimentation, consider mainly that then CPC is expensive, if can avoid using, can reduce production costs during scale operation, so adopt alcohol precipitation to substitute complexing.
Adopt simple equipment and simple FOUR EASY STEPS to make up in order, just make medical grade HA, and HA yield about 90%, loss of molecular weight rate about 15%, avoid complex apparatus to be unfavorable for amplifying or increasing production cost, need not the HA yield be reduced loaded down with trivial details step, the loss of molecular weight rate becomes and with an organic solvent increases cost greatly or in a large number, thereby is beneficial to the industrial amplification production hyaluronic acid.
Embodiment
Embodiment 1
1) get the 500ml fermented liquid, contain HA2g, molecular weight is 2.38MDa.Fermented liquid dilutes ten times with deionized water
2) add CPC, dosage is HA: CPC=1: 1.5 (g/g) after the stirring complexing, obtain complex compound behind the centrifugal 20min under 3000rpm.
3) sodium-chlor that adds 0.5M is stirred to and dissociates to the original fermented solution volume
4) add ethanol, dosage is a HA solution: ethanol=1: 2 (v/v) obtains fibrous HA precipitation after the stirring;
5) add the fermentating liquid volume of 0.1M sodium-chlor, be stirred to fully dissolving of precipitation to twice.
6) add No. 2 perlites of 3g/L in lysate, No. 4 perlites of 3g/L and the gac of 2g/L stirred after two hours, used cardboard filter, obtained filtrate.
7) add ethanol in filtrate, dosage is a HA solution: ethanol=1: 2 (v/v) obtains the HA precipitation after the stirring
8) use the pure water dissolution precipitation, spraying or vacuum-drying get medical grade HA dry powder.
Gained HA dry powder 1.2g, glucuronic acid purity is about 44.5%, and the HA yield is 91%, and molecular weight is 2.07MDa, and the loss of molecular weight rate is 13%, albumen/HA (g/g)=0.04%.
Embodiment 2
1) get the 500ml fermented liquid, contain HA2.4g, molecular weight is 2.14MDa.Fermented liquid needs earlier with ten times of deionized water dilutions
2) add CPC, dosage is HA: CPC=1: 1.5 (g/g) after the stirring complexing, obtain complex compound behind the centrifugal 20min under 3000rpm.
3) add the fermentating liquid volume of 0.5M sodium-chlor, be stirred to resolution of precipitate to twice;
4) add No. 2 perlites of 3g/L in lysate, No. 4 perlites of 3g/L and the gac of 2g/L stirred after two hours, used cardboard filter, filtrate at the beginning of obtaining.
5) just adding aluminum oxide in the filtrate, dosage is: HA: aluminum oxide=1: 5 (g/g), and other adds 1～2g/L gac, stirs after 2 hours, obtains filtrate with cardboard filter;
6) add ethanol in filtrate, dosage is a HA solution: ethanol=1: 2 (v/v) obtains the HA precipitation after the stirring
7) available pure water dissolution precipitation, spraying or vacuum-drying get medical grade HA dry powder.
Gained HA dry powder 2.18g, glucuronic acid purity is about 46.1%, and the HA yield is 91%, and molecular weight is 1.92MDa, and the loss of molecular weight rate is 10.1%, albumen/HA (g/g)=0.0%.
Embodiment 3
1) get the 500ml fermented liquid, contain HA2.4g, molecular weight is 2.14MDa.Fermented liquid does not dilute
2) add ethanol in the fermented liquid, dosage is a HA solution: ethanol=1: 2 (v/v) obtains fibrous HA precipitation after the stirring;
3) add the fermentating liquid volume of 0.1M sodium-chlor, be stirred to resolution of precipitate to twice;
4) add No. 2 perlites of 3g/L in lysate, No. 4 perlites of 3g/L and the gac of 2g/L stirred after two hours, used cardboard filter, filtrate at the beginning of obtaining.
5) just adding aluminum oxide in the filtrate, dosage is: HA: aluminum oxide=1: 5 (g/g), and other adds 1～2g/L gac, stirs after 2 hours, obtains filtrate with cardboard filter;
6) add ethanol in filtrate, dosage is a HA solution: ethanol=1: 2 (v/v) obtains the HA precipitation after the stirring
7) after this available pure water dissolution precipitation, spraying or vacuum-drying get medical grade HA dry powder.
Gained HA dry powder 2.2g, glucuronic acid purity is about 45.9%, and the HA yield is 92%, and molecular weight is 1.90MDa, and the loss of molecular weight rate is 11.1%, albumen/HA (g/g)=0.0%.
Claims (6)
1. the method for separating and extracting hyaluronic acid from microbial fermentation solution is characterized in that, comprises the steps:
(1) will contain hyaluronic fermented liquid thin up to hyaluronic content is 0.3～0.5g/ml, adds the chloro-hexadecane yl pyridines then; Stir complexing;
(2) with the product centrifugation of step (1), collect solid complex;
(3) add concentration be the sodium chloride aqueous solution of 0.4～1M to 0.9～1.1 times of original fermented solution volume, complex compound dissociates, and obtains hyaluronic acid solution;
(4) add ethanol, dosage is: hyaluronic acid solution: ethanol=1: 2, and volume ratio stirs, and precipitation obtains fibrous hyaluronic acid throw out;
(5) add the fermentating liquid volume of 0.1～0.2M sodium-chlor to 1.8～2.2 times, be stirred to resolution of precipitate;
(6) in lysate, add perlite and gac, stir, filter, obtain filtrate;
(7) add ethanol in filtrate, dosage is a hyaluronic acid solution: ethanol=1: 1.5～2.5, and volume ratio obtains the hyaluronic acid precipitation after the stirring;
(8) water dissolution precipitation, spraying or vacuum-drying get medical grade hyaluronic acid dry powder, and hyaluronic molecular weight is 1～3MD.
2. method according to claim 1 is characterized in that, chloro-hexadecane yl pyridines and hyaluronic weight ratio are: hyaluronic acid: chloro-hexadecane yl pyridines=1: 1.5～1: 2.5.
3. method according to claim 1 is characterized in that, said perlite is selected from more than one in No. 2 perlites or No. 4 perlites, and add-on is 2～4g/L, and the add-on of gac is 1.5～2.5g/L.
4. the method for separating and extracting hyaluronic acid from microbial fermentation solution is characterized in that, comprises the steps:
(1) will contain hyaluronic fermented liquid thin up to hyaluronic content is 0.3～0.5g/ml, adds the chloro-hexadecane yl pyridines then; Stir complexing;
(2) with the product centrifugation of step (1), collect solid complex;
(3) add the original fermented solution volume of sodium chloride aqueous solution to 1.8～2.2 times of 0.4～1M, complex compound dissociates, and obtains hyaluronic acid solution;
(4) in the solution of step (3), add perlite and gac, stir, filter, collect filtrate;
(5) add aluminum oxide in filtrate, dosage is: hyaluronic acid: aluminum oxide=1: 4～1: 8, and g/g, other adds 1～2g/L gac, stirs, and filters, and collects filtrate;
(6) add ethanol in filtrate, dosage is a hyaluronic acid solution: ethanol=1: 1.5～2.5, and volume ratio obtains the hyaluronic acid precipitation after the stirring;
(7) water dissolution precipitation, spraying or vacuum-drying get medical grade hyaluronic acid dry powder, and hyaluronic molecular weight is 1～3MD.
5. method according to claim 4 is characterized in that, chloro-hexadecane yl pyridines and hyaluronic weight ratio are: hyaluronic acid: chloro-hexadecane yl pyridines=1: 1.5～1: 2.5.
6. method according to claim 4 is characterized in that, said perlite is selected from more than one in No. 2 perlites or No. 4 perlites, and add-on is 2～4g/L, and the add-on of gac is 1.5～2.5g/L.
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| CN101935363B (en) * | 2009-06-30 | 2012-08-08 | 上海佰加壹医药有限公司 | Method for producing pharmaceutical grade hyaluronic acid |
| CN102516409A (en) * | 2011-11-29 | 2012-06-27 | 上海景峰制药有限公司 | Method for carrying out secondary complexing precipitation and purification on sodium hyaluronate |
| CN103304685B (en) * | 2013-06-28 | 2015-06-17 | 四川柯森油田化学有限公司 | Alcohol granulating method of hyaluronic acid fermentation liquor |
| CN106018391A (en) * | 2015-03-30 | 2016-10-12 | 广东东阳光药业有限公司 | Method for quantitative detection of content of hyaluronic acid in hyaluronic acid fermentation liquid |
| 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 |
| CN106243243A (en) * | 2016-07-29 | 2016-12-21 | 黄毅 | A kind of hyaluronic acid purifying technique |
| CN110721341A (en) * | 2018-07-17 | 2020-01-24 | 上海美白臻生物科技有限公司 | Preparation process of long-acting hyaluronic acid |
Citations (1)
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| US5411874A (en) * | 1990-11-07 | 1995-05-02 | Fermentech Medical Limited | Production of hyaluronic acid |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US5411874A (en) * | 1990-11-07 | 1995-05-02 | Fermentech Medical Limited | Production of hyaluronic acid |
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