CN101280027A - Extracting method of chondroitin sulfate - Google Patents
Extracting method of chondroitin sulfate Download PDFInfo
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- CN101280027A CN101280027A CNA2007101132402A CN200710113240A CN101280027A CN 101280027 A CN101280027 A CN 101280027A CN A2007101132402 A CNA2007101132402 A CN A2007101132402A CN 200710113240 A CN200710113240 A CN 200710113240A CN 101280027 A CN101280027 A CN 101280027A
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- chondroitin sulfate
- extracting method
- enzymolysis
- ultrafiltration
- concentrated solution
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Abstract
The invention discloses an extraction method of chondroitin sulfate, belonging to the biological and chemical product field. The method adopts single enzymolysis technology to replace the traditional alkaline hydrolysis process and makes use of ultra-filtration membrane filtration to substitute for repeated alcohol precipitation so as to simplify the production process, ensure the integrity of chondroitin sulfate and increase the yield and purity, with the yield more than 37% and the purity more than 99.5%.
Description
[technical field]
The present invention relates to a kind of biochemical product, particularly directly from animal cartilage, extract the production technique of chondroitin sulfate.
[background technology]
The acidic mucopolysaccharide class material that extracts in the automatic thing cartilage of chondroitin sulfate prime system, the cartilage that is present in animal more, the larynx bone, bone in the nose, in the soft tissues such as tracheae, form by the mucopolysaccharide molecular chain, belong to glycosaminoglycan, chemical constitution is that D-glucuronic acid and acetylaminohexose (glucose and semi-lactosi) pass through 1, the superpolymer that 3-β-glycosidic bond links to each other has lipid and the lipoprotein removed in the body inner blood, remove the cholesterol of heart, prevent and treat atherosclerosis, promote cellular metabolism, anticoagulation, anti-inflammatory, accelerating wound healing and multiple pharmacological effect such as antitumor.Clinical treatment hyperlipidaemia, arteriosclerosis, nervous headache, neurodynia, the arthrodynia of being mainly used in, also can be used for the assisting therapy of, keratitis impaired and keratohelcosis because of dysacousis, chronic nephritis, liver function that Streptomycin sulphate causes, and the manufacturing that applies to the consolidant etc. of makeup, trauma wounds.
At present the chondroitin sulfate extracting method that generally adopts mainly contain that salt is separated, alkaline hydrolysis, enzymolysis process, its separation and purification has ethanol precipitation, ion exchange chromatography, quaternary ammonium salt companion method, absorption method and Mierocrystalline cellulose partition method etc.State Intellectual Property Office is disclosed on November 10th, 2004, number of patent application is No. 200310115383.9, name is called the application for a patent for invention of " double-enzyme method production chondroitin sulfate " and has adopted alkaline prozyme and two step of pancreatin enzymolysis process and ultrafiltration membrance filter, yield is between 16 ~ 22%, glucosamine content is 24 ~ 30%, but 16 ~ 17 hours consuming time of this two step of technology enzymolysis, and in twice enzymolysis process, also to regulate a pH value with acid, and must in 70% ethanol, can enter secondary enzymolysis by thick clean crystalline substance, the technical process complexity, acid and alcoholic acid consumption are big; And the ultra-filtration membrane aperture that this technology is chosen is 6 * 10
3Molecular weight, impurity albumen filters insufficient, causes purity not high.Though have the bibliographical information bear two beautiful (separation and purification of chondroitin sulfate peptide and evaluation. food scientific research technical college of Southern Yangtze University, 2006,67 ~ 70) etc. 27 (4): extract the chondroitin sulfate peptide with papoid, its purity reaches 99.09%, but because the purification process of its chondroitin sulfate peptide is the separation and purification of DEAE-Sepharose fastflow ion exchange chromatography, technology cost height is not suitable for industrialization production.And Gao Hua (chondroitin sulfate new process of production research, University Of Qingdao's journal, 55 ~ 58) etc. 2003,18 (4): adopt concentrated base-complex enzyme zymohydrolysis method, yield can reach 32.69%, but the dilute alkaline soln amount of using in the diluted alkaline enzymolysis process is too big, heavy greater than 6 times of cartilages, and ethanol sedimentation repeatedly, the ethanol consumption is big, as concentrating stoste, very power consumption.In sum, the problem that present production technique exists has: production stage is loaded down with trivial details, with alkali, acid, amount of alcohol is big, power consumption is high, the chondroitin sulfate damage is big, poor activity, and yield, purity are lower etc.
[summary of the invention]
The objective of the invention is to overcome the shortcoming of above prior art, a kind of extracting method of new chondroitin sulfate is provided, utilize present method can simplify technology significantly, save reagent, shorten the production time, reduce power consumption, improve product activity, yield, purity.
Concrete extracting method of the present invention may further comprise the steps:
(1) bubble material: the animal cartilage piece is put into retort, add suitable quantity of water and soaked 3-5 hour;
(2) enzymolysis: be warming up to 55-62 ℃, adjust pH, in retort, add hydrolysising protease then, after enzymolysis 7-9 hour, pH regulator between the 6.0-6.5, is warming up to 85-95 ℃, sedimentation 2-4 hour at 8.0-8.5;
(3) filtration, ultrafiltration: add 1% diatomite after the enzymolysis solution cooling and filter, filtrate is squeezed into the ultrafiltration jar, by the ultra-filtration membrane ultrafiltration;
(4) precipitation: concentrated solution transfers to pH5.5-6.0, and is to be cooled to below 30 ℃, adds the long-pending 3-4 of enzymolysis liquid 90% ethanol doubly, staticly settles 7-9 hour.Throw out washing with alcohol 2-3 time promptly gets described chondroitin sulfate after dehydrating.
In the extracting method of above-mentioned chondroitin sulfate, the hydrolysising protease that adds in (2) step is an alkaline endo proteolytic enzyme, and its enzyme concn is 3%-5%;
At the ultra-filtration membrane described in (3) step is 2 * 10
4Molecular weight aperture specification hollow fiber ultrafiltration membrane;
Add 0.9%NaCl solution in (3) step, concentrated solution is squeezed into setting tank, and adding is 1%NaCl with the concentrated solution weight ratio, to keep certain ionic concn, strengthens chondroitin sulfate sodium solubleness, and stablizes its activity;
Adjust pH to 10.5-11.5 before (4) step precipitation, adding is the 20%H of 5-10% with the concentrated solution weight ratio
2O
2Solution left standstill under 55-60 ℃ 8 hours, its objective is decolouring, and making the finished product is white.
Compared with prior art, the present invention has following outstanding beneficial effect:
1, simplified the technological process of production, with the single enzyme glycopeptide covalent linkage that dissociates; Replace repeatedly alcohol precipitation, crystallization with ultrafiltration membrance filter;
2, save cost, above-mentioned this method reaches alcohol precipitation repeatedly than existing technology owing to avoided alkaline hydrolysis, thereby has saved alkali, acid, ethanol consumption, when reducing cost, alleviates the pollution to environment, has reduced the difficulty and the energy consumption of downstream purification;
3, owing to avoid easy hurtful alkaline hydrolysis operation in the traditional technology, described in addition alkaline endo proteolytic enzyme unicity is strong, and chondroitin sulfate performance group damage is little, structural integrity, and molecular weight can remain on 2 * 10
4More than, selecting the aperture specification in view of the above is 2 * 10
4The ultra-filtration membrane of molecular weight filters, and has guaranteed that chondroitin sulfate and impurity are proteic effectively to separate, remove, and the yield with cartilage is reached more than 37%, and purity reaches more than 99.5%;
4, described chondroitin sulfate performance group damage is little, adds NaCl among ultra-filtration process successively in addition, to keep certain ionic concn, has guaranteed that the chondroitin sulfate structure is with active.
[embodiment]
Embodiment 1:
(1) pure white clean cartilage piece is put into retort, and add suitable quantity of water immersion 4 hours;
(2) be warming up to 58 ℃, keep pH, in retort, add 4% alkaline endo proteolytic enzyme, enzymolysis 8 hours at 8.0-8.5; PH regulator between the 6.0-6.5, is warming up to 90 ℃, sedimentation 3 hours;
(3) add 1% diatomite after the enzymolysis solution cooling and filter, the filtrate after the press filtration is squeezed into the ultrafiltration jar, is 2 * 10 by the molecule aperture
4The ultra-filtration membrane ultrafiltration, add 0.9% NaCl in the ultra-filtration process, concentrated solution is squeezed into setting tank, add with the concentrated solution weight ratio be 1%NaCl;
(4) concentrated solution is adjusted pH to 10.5-11.5, and adding is the 20%H of 5-10% with the concentrated solution weight ratio
2O
2Solution left standstill under 55-60 ℃ 8 hours, transferred PH to 5.5-6.0 then, and is to be cooled to below 30 ℃, adds 90% ethanol that concentrates long-pending 3.6 times of liquid, staticly settled 8 hours.Throw out washing with alcohol 3 times promptly get described chondroitin sulfate after dehydrating.
The chondroitin sulfate of aforesaid method production is analyzed, and the result is as follows:
Proterties white or almost white powder art
Weight loss on drying 7.1%
Muriate＜0.50%
Mineral sulfates is (with SO
4Meter)＜0.24%
Heavy metal (in Pb)＜0.002%
Protein limit (in dry product) 0.18%
Purity (in dry product) 99.8%
Yield (for cartilage) 37.6%
Other embodiment referential datas (being followed successively by by embodiment 1 reaction sequence), see the following form:
| Soak time | Alkaline endo proteolytic enzyme | Temperature | Enzymolysis time | Heating temperature | Settling time | The ethanol volume | Sedimentation time |
| 3h | 3％ | 55℃ | 7h | 85℃ | 2h | 3 times | 7h |
| 5h | 5％ | 60℃ | 9h | 95℃ | 4h | 4 times | 9h |
Claims (7)
1, a kind of extracting method of chondroitin sulfate is characterized in that this method may further comprise the steps:
(1) bubble material: the animal cartilage piece is put into retort, add suitable quantity of water and soaked 3-5 hour;
(2) enzymolysis: be warming up to 55-62 ℃, adjust pH, in retort, add hydrolysising protease then, after enzymolysis 7-9 hour, pH regulator between the 6.0-6.5, is warming up to 85-95 ℃, get enzymolysis solution after sedimentation 2-4 hour at 8.0-8.5;
(3) filtration, ultrafiltration: add 1% diatomite after the enzymolysis solution cooling and filter, filtrate is squeezed into the ultrafiltration jar, by obtaining concentrated solution after the ultra-filtration membrane ultrafiltration;
(4) precipitation: it is 5.5-6.0 that concentrated solution transfers to pH, to be cooled to below 30 ℃, adds the long-pending 3-4 of enzymolysis liquid 90% ethanol doubly, staticly settles 7-9 hour.Throw out washing with alcohol 2-3 time gets final product after dehydrating.
2, the extracting method of chondroitin sulfate according to claim 1 is characterized in that the hydrolysising protease that adds is an alkaline endo proteolytic enzyme in (2) step.
3, the extracting method of chondroitin sulfate according to claim 2 is characterized in that described alkaline endo proteolytic enzyme enzyme concn is 3%-5%.
4, the extracting method of chondroitin sulfate according to claim 1 is characterized in that in the ultra-filtration membrane aperture described in (3) step be 2 * 10
4Molecular weight specification hollow fiber ultrafiltration membrane.
5, the extracting method of chondroitin sulfate according to claim 1 is characterized in that filtrate adds 0.9%NaCl solution before squeezing into the ultrafiltration jar in (3) step, and after concentrated solution was squeezed into setting tank, adding and concentrated solution weight ratio were 1% NaCl again.
6, the extracting method of chondroitin sulfate according to claim 1 is characterized in that the processing of decolouring before (4) step precipitation.
7, the extracting method of chondroitin sulfate according to claim 6 is characterized in that described decolouring processing mode for adjusting pH to 10.5-11.5, and adding is the 20%H of 5-10% with the concentrated solution weight ratio
2O
2Solution left standstill under 55-60 ℃ 8 hours.
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| CN2007101132402A CN101280027B (en) | 2007-10-18 | 2007-10-18 | Extracting method of chondroitin sulfate |
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| CN2007101132402A CN101280027B (en) | 2007-10-18 | 2007-10-18 | Extracting method of chondroitin sulfate |
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Cited By (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102125199A (en) * | 2010-11-24 | 2011-07-20 | 广东海洋大学 | Meretrix polysaccharide with function of assisting blood fat reduction |
| CN102153672A (en) * | 2010-12-28 | 2011-08-17 | 湖北远成药业有限公司 | Method for extracting chondroitin sulfate |
| CN102850466A (en) * | 2011-06-28 | 2013-01-02 | 西藏金稞集团有限责任公司 | Production process for chondroitin sulfate from yak bone |
| CN103497261A (en) * | 2013-10-21 | 2014-01-08 | 河北三鑫实业集团有限公司 | Chondroitin sulfate production process |
| CN103601815A (en) * | 2013-10-21 | 2014-02-26 | 青岛九龙生物医药有限公司 | Extraction of sodium chondroitin sulfate through combination of alkaline hydrolysis-enzymolysis method and flocculation precipitation method |
| CN103641935A (en) * | 2013-11-29 | 2014-03-19 | 济南天天香有限公司 | Extraction method of chondroitin sulfate |
| CN103665186A (en) * | 2013-10-21 | 2014-03-26 | 青岛九龙生物医药有限公司 | Method for increasing content of chondroitin sulfate sodium |
| CN103804518A (en) * | 2013-11-25 | 2014-05-21 | 青岛九龙生物医药有限公司 | Preparation method for improving chondroitin sulfate content |
| CN104130340A (en) * | 2014-08-13 | 2014-11-05 | 青岛万图明生物制品有限公司 | Method for preparing chondroitin sulfate from duck sternum |
| CN104140477A (en) * | 2014-08-13 | 2014-11-12 | 青岛万图明生物制品有限公司 | Preparation method for preparing chondroitin sulfate from chicken shanks |
| CN104177512A (en) * | 2014-08-26 | 2014-12-03 | 淮安麦德森制药有限公司 | Method for producing low-molecular chondroitin sulfate by catalytic process |
| CN104448037A (en) * | 2014-12-15 | 2015-03-25 | 美泰科技(青岛)有限公司 | Preparation process of potassium chondroitin sulfate salt |
| CN104448038A (en) * | 2014-12-15 | 2015-03-25 | 美泰科技(青岛)有限公司 | Preparation process of calcium chondroitin sulfate salt |
| CN107082829A (en) * | 2017-06-08 | 2017-08-22 | 福州大学 | A kind of preparation method of chondroitin sulfate and its application in vascular protection |
| CN107518307A (en) * | 2017-08-02 | 2017-12-29 | 安徽全康药业有限公司 | A kind of pregnant woman, the preparation method of wet nurse's type multivitamin several mineral materials piece |
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| CN1733809A (en) * | 2004-08-09 | 2006-02-15 | 上海日川生物科技有限公司 | Preparation method of low molecular weight chondroitin sulfate |
| CN1279063C (en) * | 2004-10-20 | 2006-10-11 | 天津科技大学 | Process for extracting chondroitin sulfate |
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- 2007-10-18 CN CN2007101132402A patent/CN101280027B/en not_active Expired - Fee Related
Cited By (19)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102125199A (en) * | 2010-11-24 | 2011-07-20 | 广东海洋大学 | Meretrix polysaccharide with function of assisting blood fat reduction |
| CN102153672A (en) * | 2010-12-28 | 2011-08-17 | 湖北远成药业有限公司 | Method for extracting chondroitin sulfate |
| CN102153672B (en) * | 2010-12-28 | 2012-11-21 | 湖北远成药业有限公司 | Method for extracting chondroitin sulfate |
| CN102850466A (en) * | 2011-06-28 | 2013-01-02 | 西藏金稞集团有限责任公司 | Production process for chondroitin sulfate from yak bone |
| CN102850466B (en) * | 2011-06-28 | 2016-08-17 | 西藏金稞集团有限责任公司 | A kind of method of Os Bovis grunniens chondroitin sulfate |
| CN103601815B (en) * | 2013-10-21 | 2016-04-13 | 青岛九龙生物医药有限公司 | Alkaline hydrolysis-enzymolysis process extracts Sodium chondroitin sulfate A in conjunction with flocculent precipitation |
| CN103497261A (en) * | 2013-10-21 | 2014-01-08 | 河北三鑫实业集团有限公司 | Chondroitin sulfate production process |
| CN103601815A (en) * | 2013-10-21 | 2014-02-26 | 青岛九龙生物医药有限公司 | Extraction of sodium chondroitin sulfate through combination of alkaline hydrolysis-enzymolysis method and flocculation precipitation method |
| CN103665186A (en) * | 2013-10-21 | 2014-03-26 | 青岛九龙生物医药有限公司 | Method for increasing content of chondroitin sulfate sodium |
| CN103804518A (en) * | 2013-11-25 | 2014-05-21 | 青岛九龙生物医药有限公司 | Preparation method for improving chondroitin sulfate content |
| CN103641935A (en) * | 2013-11-29 | 2014-03-19 | 济南天天香有限公司 | Extraction method of chondroitin sulfate |
| CN104140477A (en) * | 2014-08-13 | 2014-11-12 | 青岛万图明生物制品有限公司 | Preparation method for preparing chondroitin sulfate from chicken shanks |
| CN104130340A (en) * | 2014-08-13 | 2014-11-05 | 青岛万图明生物制品有限公司 | Method for preparing chondroitin sulfate from duck sternum |
| CN104177512A (en) * | 2014-08-26 | 2014-12-03 | 淮安麦德森制药有限公司 | Method for producing low-molecular chondroitin sulfate by catalytic process |
| CN104448037A (en) * | 2014-12-15 | 2015-03-25 | 美泰科技(青岛)有限公司 | Preparation process of potassium chondroitin sulfate salt |
| CN104448038A (en) * | 2014-12-15 | 2015-03-25 | 美泰科技(青岛)有限公司 | Preparation process of calcium chondroitin sulfate salt |
| CN104448038B (en) * | 2014-12-15 | 2017-01-11 | 美泰科技（青岛）股份有限公司 | Preparation process of calcium chondroitin sulfate salt |
| CN107082829A (en) * | 2017-06-08 | 2017-08-22 | 福州大学 | A kind of preparation method of chondroitin sulfate and its application in vascular protection |
| CN107518307A (en) * | 2017-08-02 | 2017-12-29 | 安徽全康药业有限公司 | A kind of pregnant woman, the preparation method of wet nurse's type multivitamin several mineral materials piece |
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