CN105777938B - A method of removing keratan sulfate from chondroitin sulfate crude extract - Google Patents
A method of removing keratan sulfate from chondroitin sulfate crude extract Download PDFInfo
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- 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
- C08B37/0069—Chondroitin-4-sulfate, i.e. chondroitin sulfate A; Dermatan sulfate, i.e. chondroitin sulfate B or beta-heparin; Chondroitin-6-sulfate, i.e. chondroitin sulfate C; Derivatives thereof
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- 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
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Abstract
The invention belongs to the method for keratan sulfate is removed in biotechnology more particularly to a kind of crude extract from chondroitin sulfate.After sodium hydroxide solution reaction is added in the present invention in chondroitin sulfate crude extract solution, hydrochloric acid is added and adjusts pH value, filters to get filtrate, it is 45 48ms/cm that sodium chloride is added in filtrate and adjusts electrical conductivity of solution, then ethanol solution is added, control ethanol solution flow velocity is 10 15mL/（L·min）, so that chondroitin sulfate is precipitated, filters to obtain sediment, sediment dry chondroitin sulfate finished product after dehydration.Present invention process condition is easy to control, at low cost, easy to operate, requires laboratory apparatus low, thoroughly solves that keratan sulfate is removed from chondroitin sulfate is difficult, it is difficult to the problem of industrialized production.
Description
Technical field
The invention belongs to biotechnologies, more particularly to one kind removing keratan sulfate from chondroitin sulfate crude extract
Method.
Background technology
Chondroitin sulfate (CS) is with animal（Pig, ox, horse, sheep, chicken, shark etc.）Cartilage be raw material a kind of macromolecule
Acid mucopolysaccharide, the repetition disaccharide unit institute group that molecular structure is formed for β-D-Glucose aldehydic acid and N- acetylamino galactosamines
At polysaccharide chain.It is it is a kind of white or micro-yellow powder, water imbibition is strong, it is soluble easily in water form thick liquid, insoluble in alcohol,
The organic solvents such as acetone, ether and chloroform, its esters relatively stablize heat.
Keratan sulfate (KS) is one kind of glycosaminoglycan, such polysaccharide by different sulphation modifications N- acetylaminos Portugal
Grape sugar and D- galactolipin disaccharides repetitive units composition, KS be present in the form of proteoglycan horn film, intervertebral disk, cartilage and
In artery, the study found that KS is largely present in cartilage and cornea of the mammals such as the cartilaginous fishes such as shark and whale, ox etc.
In, KS is usually mixed with chondroitin sulfate (CS), but the two content is different, such as CS in ox cartilage in mammals extract
Content is very high, and KS contents are higher in the cartilages class glycosaminoglycan such as shark, to detect the structure and content of chondroitin sulfate, just
CS is detached, then degradable is disaccharides, then is detected to disaccharides.At present to chondroitin sulfate and keratosulfate
The separation of element is all made of ion-exchange chromatography, and since the molecular structure of different glycosaminoglycans is different, the entrained quantity of electric charge is not
Together, the retention behavior on ion exchange resin is different, therefore different salinity elutions can be used and detached.Patent CN20
Chondroitin sulfate and keratan sulfate mixture are dissolved in deionized water by 1310695187, through anion exchange post separation, with not
It is eluted with concentration sodium-chloride water solution, it is CS components to be eluted through 1.2moL/L sodium chloride solutions, through 1.5moL/L chlorinations
It is KS components that sodium solution, which elutes, and this method is troublesome in poeration, of high cost, in industrialization a large amount of chondroitin sulfates it is pure
Change not applicable.
Invention content
It is difficult to solve to remove keratan sulfate from chondroitin sulfate at present, it is difficult to the problem of industrialized production, this hair
It is bright to provide a kind of method that keratan sulfate is removed in the crude extract from chondroitin sulfate, there is the sulphur that removal efficiency is good, obtains
The characteristics of aching and limp ossein purity is high, easy to operate, suitable for industrialized production.
The technical solution that the present invention proposes in order to solve the above problem：
A method of removing keratan sulfate from chondroitin sulfate crude extract, it is characterised in that use following steps：
（1）Chondroitin sulfate crude extract is weighed, adds deionized water to prepare the crude extract solution of mass fraction 10-15%, uses hydrogen
Sodium hydroxide solution tune PH is 12-14, is stirred at room temperature 2-3 hours, is then 8-9 with hydrochloric acid tune PH, is warming up to 45-50 DEG C, heat preservation
It 3-4 hours, is down to room temperature and continues with hydrochloric acid tune PH to be 6-7, filter to get filtrate；
（2）It is 45-48ms/cm that sodium chloride is added into filtrate and adjusts conductivity；
（3）It is 95-100% ethanol solutions, coutroi velocity 10- that alcoholic strength is added into the solution for regulate conductivity
15mL/（L·min）, it is stirred continuously, until alcoholic strength is 50-55%, staticly settles 2-4 hours, filter to obtain sediment；
（4）Dry chondroitin sulfate finished product after sediment is dehydrated.
Step（1）Described in chondroitin sulfate crude extract in content of chondroitin sulfate be 90-92%, keratan sulfate contains
Amount is 2-4%, protein content 1-3%.Step（1）Described in addition concentration of sodium hydroxide solution be 2moL/L.
Step（3）Described in flow velocity be 13mL/（L·min）.
Step（4）Described in finished product in content of chondroitin sulfate be 97-99%, the content of obtained keratan sulfate is
0.02-0.06%, protein content 0.5-0.8%.
Chondroitin sulfate and keratan sulfate are the similar proteoglycans of two kinds of structures, are typically to be mixed, sometimes
Only there are one protein portions for the two, therefore the separation of the two is extremely difficult, and sodium hydroxide solution is added in the solution, pass through
Controlling different temperature and PH can be such that sugar-peptide bond of proteoglycan is thoroughly broken, and not only reduce protein content in finished product
And chondroitin sulfate and keratan sulfate can be made to be easier to detach.Chondroitin sulfate belongs to amphoteric compound, molecule knot
Not only contained the amino that can be dissociated in structure containing the carboxyl that can be dissociated, the addition of sodium chloride increases the conductivity of solution, reduces
The dielectric constant of solution, when conductivity reaches certain numerical value in solution, the hydration shell of chondroitin sulfate molecular surface is broken
Bad, the attraction enhancing between polarity counter ion group, intermolecular mutual aggregation is added ethanol solution and adjusts lower alcohol
Degree will make its precipitation.And there was only amino in keratan sulfate molecular structure, without carboxyl, intermolecular aggregation ability is poor, needs
The sodium chloride solution of larger concentration is wanted, can just it be made mutually to assemble, needs the ethyl alcohol of larger alcoholic strength that can just make its precipitation.
Advantageous effect
(1) sodium hydroxide solution is added in chondroitin sulfate crude extract solution in the present invention, destroys polysaccharide chain and albumen
Covalent bond between matter makes sugar-peptide bond fracture of proteoglycan, can not only remove protein in crude extract, but also make chondroitin sulfate
Element and keratan sulfate are easier to detach.
（2）Conductivity is adjusted by the way that sodium chloride is added, makes the intermolecular mutual aggregation of chondroitin sulfate, by controlling to sulfuric acid
The rate of ethanol solution is added in chondroitin crude extract solution, chondroitin sulfate is made to be precipitated completely compared under alcoholic strength low, and sulphur
Tamarind quality then remains in supernatant, has not only saved the usage amount of ethanol solution but also can have been removed from chondroitin sulfate
Keratan sulfate obtains the chondroitin sulfate finished product of high-purity.
（3）Present invention process condition is easy to control, at low cost, easy to operate, low to laboratory apparatus requirement, is thoroughly solved
The problem of keratan sulfate difficulty is removed in industrial production from chondroitin sulfate.
Specific implementation mode
Embodiment and comparative example is set forth below, and the present invention will be described, but the invention is not limited in these embodiments.
Embodiment 1
Weighing chondroitin sulfate crude extract adds deionized water to prepare the solution that 10L mass fractions are 10%, the conductivity of solution
For 20ms/cm, content of chondroitin sulfate is 90% wherein in crude extract, and the content of keratan sulfate is 2%, and protein content is
1%,.It is 12 with 2moL/L sodium hydroxide solution tune PH, is stirred at room temperature 2 hours, is then 8 with hydrochloric acid tune PH, is warming up to 45 DEG C,
Heat preservation 3 hours, it is 6 to be down to room temperature hydrochloric acid conditioning solution PH, is filtered to get filtrate, and solid sodium chloride is added in filtered fluid and adjusts
The conductivity of solution is 45ms/cm, and 95% ethanol solution, control stream are extremely slowly added into the solution for regulate conductivity
Speed is 15mL/（L·min）, and be stirred continuously, until solution alcoholic strength is 50%, 2 hours are stood, sediment, sediment are filtered to obtain
Chondroitin sulfate finished product is dried to obtain after being dehydrated with absolute ethyl alcohol.
Embodiment 2
Weighing chondroitin sulfate crude extract adds deionized water to prepare the solution that 10L mass fractions are 15%, the conductivity of solution
For 22ms/cm, content of chondroitin sulfate is 92% wherein in crude extract, and the content of keratan sulfate is 4%, and protein content is
3%.It is 14 with 2moL/L sodium hydroxide solution tune PH, is stirred at room temperature 3 hours, is then 9 with hydrochloric acid tune PH, is warming up to 50 DEG C, protect
Temperature 4 hours, it is 7 to be down to room temperature hydrochloric acid conditioning solution PH, is filtered to get filtrate, and solid sodium chloride is added in filtrate and adjusts solution
Conductivity be 48ms/cm, ethanol solution is extremely slowly added into the solution for regulate conductivity, coutroi velocity is
10mL/（L·min）, and be stirred continuously, until solution alcoholic strength is 55%, 4 hours are stood, sediment, sediment nothing are filtered to obtain
Chondroitin sulfate finished product is dried to obtain after water-ethanol dehydration.
Embodiment 3
Weighing chondroitin sulfate crude extract adds deionized water to prepare the solution that 10L mass fractions are 15%, the conductivity of solution
For 21ms/cm, content of chondroitin sulfate is 90% wherein in crude extract, and the content of keratan sulfate is 2%, and protein content is
1%.It is 14 with 2mol/L sodium hydroxide solution tune PH, is stirred at room temperature 3 hours, is then 9 with hydrochloric acid tune PH, is warming up to 50 DEG C, protect
Temperature 4 hours, it is 6 to be down to room temperature hydrochloric acid conditioning solution PH, filtering, and the electricity that solid sodium chloride adjusts solution is added in filtered fluid
Conductance is 46ms/cm, and ethanol solution, coutroi velocity 13mL/ are extremely slowly added into the solution for regulate conductivity
（L·min）, and be stirred continuously, until solution alcoholic strength is 55%, 4 hours are stood, sediment, sediment absolute ethyl alcohol are filtered to obtain
Chondroitin sulfate finished product is dried to obtain after dehydration.
Comparative example
Weighing chondroitin sulfate crude extract adds deionized water to prepare the solution that 10L mass fractions are 15%, wherein in crude extract
Content of chondroitin sulfate is 92%, and the content of keratan sulfate is 4%, protein content 3%.Alcohol is rapidly joined into solution
Degree is 95% ethanol solution, and is stirred continuously, and when solution alcoholic strength is 55%, only minute quantity precipitation occurs in solution, continues
It adds and occurs a large amount of precipitations when alcoholic strength be 95% ethanol solution to alcoholic strength is 70%, staticly settle 4 hours, filter to obtain precipitation
Object, sediment dry to obtain chondroitin sulfate finished product after being dehydrated with absolute ethyl alcohol.
The measurement of various indexs, the following progress of evaluation in embodiment：
Content of chondroitin sulfate measures：It is detected according to liquid chromatography in GB/T 20365-2005；
Keratan sulfate assay：It is detected with high performance liquid chromatography；
The recovery rate (%) of chondroitin sulfate=（Sulphur in the quality of chondroitin sulfate/chondroitin sulfate crude extract in finished product
The quality of aching and limp ossein）×100；
Protein content determination：Folin- phenol reagent process.
Recovery rate in above-described embodiment and content are detected, data are as follows：
Claims (5)
1. a kind of method for removing keratan sulfate in crude extract from chondroitin sulfate, it is characterised in that use following steps：
（1）Chondroitin sulfate crude extract is weighed, adds deionized water to prepare the crude extract solution of mass fraction 10-15%, uses hydroxide
Sodium solution tune pH is 12-14, is stirred at room temperature 2-3 hours, is then 8-9 with hydrochloric acid tune pH, is warming up to 45-50 DEG C, keeps the temperature 3-4
Hour, it is down to room temperature and continues with hydrochloric acid tune pH to be 6-7, filter to get filtrate；
（2）It is 45-48ms/cm that sodium chloride is added into filtrate and adjusts conductivity；
（3）It is 95-100% ethanol solutions, coutroi velocity 10-15mL/ that alcoholic strength is added into the solution for regulate conductivity
（L·min）, it is stirred continuously, until alcoholic strength is 50-55%, staticly settles 2-4 hours, filter to obtain sediment；
（4）Dry chondroitin sulfate finished product after sediment is dehydrated.
2. the method as described in claim 1, it is characterised in that step（1）Described in chondroitin sulfate crude extract in sulfuric acid it is soft
Ossein content is 90-92%, and keratosulfate cellulose content is 2-4%, protein content 1-3%.
3. the method as described in claim 1, it is characterised in that step（1）Described in addition concentration of sodium hydroxide solution be
2moL/L。
4. the method as described in claim 1, it is characterised in that step（3）Described in flow velocity be 13mL/（L·min）.
5. the method as described in claim 1, it is characterised in that step（4）Described in finished product in content of chondroitin sulfate be
The content of 97-99%, obtained keratan sulfate are 0.02-0.06%, protein content 0.5-0.8%.
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| CN201610180646.1A CN105777938B (en) | 2016-03-28 | 2016-03-28 | A method of removing keratan sulfate from chondroitin sulfate crude extract |
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| CN201610180646.1A CN105777938B (en) | 2016-03-28 | 2016-03-28 | A method of removing keratan sulfate from chondroitin sulfate crude extract |
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| CN107151685B (en) * | 2017-06-23 | 2020-06-02 | 山东众山生物科技有限公司 | Method for producing chondroitin sulfate by fermentation method |
| CN112321750A (en) * | 2020-12-09 | 2021-02-05 | 美泰科技（青岛）股份有限公司 | Method for controlling content of chondroitin sulfate by adjusting conductivity of feed liquid |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101698686A (en) * | 2009-10-27 | 2010-04-28 | 赵静 | Method for purifying chondroitin sulfate |
| CN103819578A (en) * | 2013-11-22 | 2014-05-28 | 青岛九龙生物医药有限公司 | Method for improving yield of chondroitin sulfate by adding sodium hydroxide |
| WO2015140281A1 (en) * | 2014-03-21 | 2015-09-24 | Altergon S.A. | Chondroitin sulphate purification method |
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2016
- 2016-03-28 CN CN201610180646.1A patent/CN105777938B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101698686A (en) * | 2009-10-27 | 2010-04-28 | 赵静 | Method for purifying chondroitin sulfate |
| CN103819578A (en) * | 2013-11-22 | 2014-05-28 | 青岛九龙生物医药有限公司 | Method for improving yield of chondroitin sulfate by adding sodium hydroxide |
| WO2015140281A1 (en) * | 2014-03-21 | 2015-09-24 | Altergon S.A. | Chondroitin sulphate purification method |
Non-Patent Citations (1)
| Title |
|---|
| Selective removal of keratan sulfate in chondroitin sulfate samples by sequential precipitation with ethanol;Fabio Galeotti, et al.;《Analytical Biochemistry》;20131101;第448卷;第113-115页 * |
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Effective date of registration: 20190605 Address after: 276800 No. 777 Yuquan Second Road, Rizhao City, Shandong Province Patentee after: Rizhao Lanshan Biochemical Products Co., Ltd. Address before: 276807 No. 98 Lanshan Road, Rizhao City, Shandong Province Patentee before: Shandong Topscience Bio-Tech Co.,Ltd. |