CN103468771A - Method for extracting collagens from bovine achilles tendon - Google Patents
Method for extracting collagens from bovine achilles tendon Download PDFInfo
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- CN103468771A CN103468771A CN2013102004799A CN201310200479A CN103468771A CN 103468771 A CN103468771 A CN 103468771A CN 2013102004799 A CN2013102004799 A CN 2013102004799A CN 201310200479 A CN201310200479 A CN 201310200479A CN 103468771 A CN103468771 A CN 103468771A
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Abstract
The invention discloses a method for extracting collagens from bovine achilles tendon. The method comprises the following steps: refrigerating the bovine achilles tendon, cutting into blocks, refrigerating, crushing, filling crushed bovine achilles tendon into a temperature-controllable chromatography column, continuously injecting an extract liquid into the temperature-controllable chromatography column, controlling the temperature of circulating water in a jacket of the chromatography column consistent to the temperature of the extract liquid, and collecting the extract liquid from an outlet of the chromatography column; carrying out enzymatic hydrolysis, carrying out chromatographic separation in an affinity chromatography mode, wherein a ligand of an affinity chromatography media is a polypeptide; and leaching out uncombined proteins, and eluting to obtain proteins which can be combined with the chromatography media, that is, highly pure collagens. The method is simple to operate, and the prepared collagens have the advantages of high purity, low random degradation and the like.
Description
Technical field
The invention belongs to technical field of biological material, particularly, the present invention relates to a kind of method of extracting collagen from the ox heel string.
Technical background
Collagen accounts for 33% of total protein in mammalian body as the main component of extracellular matrix, almost be present in there is material impact in a organized way and to form, the structure and function of cell and tissue.It is slender rod shaped that tropocollagen molecule is, and is about 300nm, diameter 1.5nm, the about 300kDa of molecular weight.Tropocollagen molecule is comprised of 3 polypeptide chains, and every peptide chain is 1000 amino-acid residues approximately, and tropocollagen molecule has triple-helix structure, and every the polypeptide chain that forms collagen exists with the form of left hand rotation, and three polypeptide chains become collagen with the form winding-shaped of right-handed helix.According to the difference that forms the collagen polypeptide chain amino acid sequence, collagen can be divided into broad variety, has found 27 kinds of dissimilar collagens at present in mammalian body, and wherein the content of type i collagen is the highest.There is some difference for the performances such as the physical strength of different collagen and thermostability, and also there is some difference for the interaction between other biomacromolecule, cell or material interface, prepare different biomaterials collagen-type and ratio etc. is existed to certain requirement.
Collagen has good biological characteristics, comprise biocompatibility, poor antigen, biodegradable, Bioabsorbable and good water-absorbent etc., collagen is very extensive in the application of the fields such as organizational project, regenerative medicine, medicine equipment and medicine controlled releasing as a kind of desirable biomaterial, as collagen is the critical material for preparing the artificial organs such as styptic sponge, microbial film, operating sutures, cell culturing bracket, tissue filling material and artificial skin, artificial cornea, artificial liver.Collagen has good adhesiving effect to cell, therefore in organizational project and regenerative medicine field, collagen or a kind of desirable critical material that improves material surface performance and raising Biocompatibility, as aspect tissue engineering material, use three-dimensional rack prepared by collagen to can be used for inoculating the chondrocyte and for cartilaginous tissue reparation and regeneration, as aspect the tissue regeneration material, collagen for the acellular matrix of the bladder wall, transplant and regeneration effect good.In recent years, the medicine equipment of printing based on 3D and the development of organizational project technology of preparing are rapid, cause the demand of collagen is grown with each passing day.
Much collagen is insoluble in water under native state, and there is certain difficulty in direct preparation of high-purity collagen, is the focus of collagen research in technical field of biological material in recent years.Adopt engineered method can prepare recombinant collagen (recombined collagen, number of patent application CN102351954.A; A kind of recombination human source collagen protein and preparation method thereof, application number 102443057.A), but this technology can only to prepare the molecular weight of collagen low and be difficult to prepare the collagen with triple-helix structure.Duan Rui etc. adopt (a kind of process for refining of macromolecular collagen protein, publication number 200910184481.5) carrying out collagen after employing acidic solution facture collection collagen suspended layer makes with extra care, the method operation steps is simple, but what the collagen preparation process adopted is precipitation process, in the collagen that the co-precipitation that non-specific adsorption causes often causes preparing, contain a certain amount of foreign protein.Preparation patent (the application number: 200980101280.2) of high-purity collagen protein of the application such as Huang Linghui, to after raw materials pretreatment, be ground, obtained the collagen crude product, and the method that further adopts Acid precipitation to have carried out collagen refining, the method of removing foreign protein still belongs to the precipitator method, and the collagen treating process based on the precipitator method is difficult to prepare high-purity collagen.Li Jie etc. be take animal cartilage as raw material, after pulverizing, adopt stomach en-to be degraded, employing is saltoutd and centrifugal method obtains non-denatured collagen (non-sex change II collagen type production method, application number CN102154425.A), this process need saltout processing and centrifugal process repeatedly, process is comparatively loaded down with trivial details.
Summary of the invention
The problems such as the collagen purity existed for traditional collagen preparation process is low, sepn process is loaded down with trivial details, the object of the invention is to provide a kind of method of extracting collagen from the ox heel string, and the bovine collagen purity of preparation is high and keep the natural structure of triple helical.A kind of method of extracting collagen from the ox heel string of the present invention, the method comprises the following steps:
(1) weigh: take fresh ox heel string, WATER-WASHING METHOD is removed appurtenant.
(2) freezing: under-40 ℃ of conditions, freezing preservation is 12～24 hours.
(3) stripping and slicing: freezing ox heel string is cut into to bulk (0.5cm * 0.5cm).
(4) pulverize: will be cut into block ox heel string and within freezing 6～12 hours under-40 ℃ of conditions, be placed on crusher for crushing.
(5) extract: the ox heel string after pulverizing is placed in the empty chromatography column of temperature controllable, the aperture of its bottom screen cloth is 100～300 orders, the deionized water that is 38-45 ℃ by temperature continuously is injected into chromatography column from the chromatography column top, and the circulating water temperature of controlling the chromatography column chuck is consistent with the temperature of injected water in chromatography column; The solution that collection is flowed out from the chromatography column bottom is the collagen crude extract.
(6) enzymolysis: add proteolytic enzyme in the collagen crude extract, the ratio control of albumen and enzyme, stirs 24～36 hours to 200:1 at a certain temperature at 50:1.
(7) refining: that the solution after enzymolysis is crossed to loading to affine chromatography column, the aglucon of chromatography media is a peptide species, its aminoacid sequence is ICTTNEGVMYRIGDEWDKEHDMGHMMRCTCVGNGRGEWTCVAYSELRD, and applied sample amount is 5%～16% of chromatography media volume.Use albumen or the polypeptide drip washing that the aqueous solution will not adsorb to go out, adopt the albumen that 10%～30% acetonitrile-aqueous solution will be adsorbed on chromatography media to elute, collect elutriant.
(8) freeze-drying: the refining collagen lyophilize by affinity chromatography obtains high-purity collagen.
Preferably, in described step (4) leaching process, for the device that extracts collagen, be the temperature controllable chromatography column.
Preferably, in described step (4) leaching process, the circulating water temperature of chromatography column chuck is identical with the temperature of injected water in chromatography column.
Preferably, in described step (7) treating process, the chromatography mode that the collagen treating process adopts is affinity chromatography.
Preferably, in described step (7) treating process, the aglucon of affinity chromatography medium is a peptide species, and the aminoacid sequence of this polypeptide can be ICTTNEGVMYRIGDEWDKEHDMGH MMRCTCVGNGRGE WTCVAYSELRD, the polypeptide that also contains this aminoacid sequence.
Compared with prior art, its advantage comprises in the present invention:
(1) by the preprocessing process of ox heel string, be mechanical valve, not with an organic solvent.
(2) adopt the chromatography column of temperature controllable to carry out collagen extraction, the collagen extracted shifts out in time, has avoided thermal destruction, is conducive to keep the triple-helix structure of collagen.
(3) adopt the method for affinity chromatography to carry out the refining of collagen, purification efficiency is high.
(4) collagen that adopts the present invention to prepare has the advantages such as high, the random degradation rate of purity is low.
Specific embodiment
Below by specific embodiment, the present invention is specifically described; what be worth proposition is; the present embodiment is just for to a kind of implementation method of the present invention; can not represent the whole of patent of the present invention; more can not be interpreted as limiting the scope of the invention; under the precondition of the design that does not break away from essence of the present invention, can also make some adjustment or improvement, these all belong to protection scope of the present invention.
Embodiment 1:
(1) weigh: get fresh ox heel string 50g, residue is removed in washing.
(2) freezing: under-40 ℃ of conditions, freezing preservation is 12 hours.
(3) stripping and slicing: the bulk that the ox heel string after freezing is cut into to 0.5cm * 0.5cm.
(4) pulverize: will be cut into block ox heel string and within freezing 8 hours under-40 ℃ of conditions, be placed on the crusher for crushing pulp.
(5) extract: the ox heel string after pulverizing is placed in to (diameter 5cm, height 20cm) in the temperature controllable chromatography column, and 300 purpose nylon wires are established in the extraction vessel bottom.The deionized water that is 42 ℃ by temperature continuously is injected in warm chromatography, and the circulating water temperature of controlling the chromatography column chuck is 42 ℃; The solution that collection is flowed out from the chromatography column outlet.
(6) enzymolysis: add stomach en-in the collagen crude extract obtained to step (5), the ratio control of albumen and enzyme, at 100:1, stirs 24 hours under 37 ℃.
(7) refining: the enzymolysis solution pH that step (6) is obtained is adjusted to 7.0, loading is to affine chromatography column (diameter 5cm, height 60cm), the matrix of chromatography media is agarose, aglucon is polypeptide, its aminoacid sequence is ICTTNEGVMYRIGDEWDKEHDMGHMMRCTCVGNGRGEWTCVAYSELRD, and applied sample amount is 60ml.Use albumen or the polypeptide drip washing that deionized water will not adsorb to go out, adopt the 30% acetonitrile solution wash-out of 80ml, collect elutriant.
(8) freeze-drying: the elutriant that step (7) is obtained carries out lyophilize, obtains high-purity collagen.
Claims (5)
1. a method of extracting collagen from the ox heel string is characterized in that extracting method comprises the following steps:
(1) weigh: take fresh ox heel string, WATER-WASHING METHOD is removed appurtenant.
(2) freezing: under-40 ℃ of conditions, freezing preservation is 12～24 hours.
(3) stripping and slicing: freezing ox heel string is cut into to bulk (0.5cm * 0.5cm).
(4) pulverize: will be cut into block ox heel string freezing 6-12 hour under-40 ℃ of conditions and be placed on crusher for crushing.
(5) extract: the ox heel string after pulverizing is placed in the empty chromatography column of temperature controllable, the aperture of its bottom screen cloth is 100～300 orders, the deionized water that is 38-45 ℃ by temperature continuously is injected into chromatography column from the chromatography column top, and the circulating water temperature of controlling the chromatography column chuck is consistent with the temperature of injected water in chromatography column; The solution that collection is flowed out from the chromatography column bottom is the collagen crude extract.
(6) enzymolysis: add proteolytic enzyme in the collagen crude extract, the ratio control of albumen and enzyme, stirs 24～36 hours to 200:1 at a certain temperature at 50:1.
(7) refining: that the solution after enzymolysis is crossed to loading to affine chromatography column, the aglucon of chromatography media is a peptide species, its aminoacid sequence is ICTTNEGVMYRIGDEWDKEHDMGHMMRCTCVGNGRGEWTC VAYSELRD, and applied sample amount is 5%～16% of chromatography media volume.Use albumen or the polypeptide drip washing that the aqueous solution will not adsorb to go out, adopt the albumen that 10%～30% acetonitrile-aqueous solution will be adsorbed on chromatography media to elute, collect elutriant.
(8) freeze-drying: the refining collagen lyophilize by affinity chromatography obtains high-purity collagen.
2. a kind of method of extracting collagen from the ox heel string according to claim 1, is characterized in that being the temperature controllable chromatography column for the device that extracts collagen in step (4) leaching process.
3. a kind of method of extracting collagen from the ox heel string according to claim 1, is characterized in that the circulating water temperature of chromatography column chuck in step (4) leaching process is identical with the temperature of injected water in chromatography column.
4. a kind of method of extracting collagen from the ox heel string according to claim 1, is characterized in that the chromatography mode that step (7) collagen treating process adopts is affinity chromatography.
5. a kind of method of extracting collagen from the ox heel string according to claim 1, the aglucon that it is characterized in that affinity chromatography medium in step (7) collagen treating process is a peptide species ICTTNEGVMYRIGDEWDKEHDM GHMMRCTCVGNGRGEWTCVAYSELRD, or the polypeptide that contains this sequence.
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| CN201310200479.9A CN103468771B (en) | 2013-05-27 | 2013-05-27 | Method for extracting collagens from bovine achilles tendon |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105601731A (en) * | 2014-09-03 | 2016-05-25 | 浙江百谷医疗科技有限公司 | Cow achilles tendon collagen purification method and sponge preparation thereof |
| CN106148466A (en) * | 2016-08-30 | 2016-11-23 | 夏佳文 | A kind of preparation method of yak collagen protein |
| CN106589113A (en) * | 2016-11-17 | 2017-04-26 | 北京华信佳音医疗科技发展有限责任公司 | Method for extracting collagen from bovine achilles tendons |
| CN107227330A (en) * | 2017-07-10 | 2017-10-03 | 山东省千佛山医院 | A kind of extracting method of ox heel string NTx |
| CN107913785A (en) * | 2016-10-11 | 2018-04-17 | 浙江崇山生物制品有限公司 | A kind of efficiently animal tendon breaking method |
| CN110467668A (en) * | 2019-09-10 | 2019-11-19 | 河北考力森生物科技有限公司 | A kind of extracting method of type III collagen |
| CN111363772A (en) * | 2020-04-08 | 2020-07-03 | 平凉市华科生物技术有限公司 | Method for preparing collagen peptide by hydrolyzing bovine bone and collagen peptide thereof |
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| CN101363040A (en) * | 2008-09-19 | 2009-02-11 | 无锡贝迪生物工程有限公司 | Method for preparing collagen protein |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105601731A (en) * | 2014-09-03 | 2016-05-25 | 浙江百谷医疗科技有限公司 | Cow achilles tendon collagen purification method and sponge preparation thereof |
| CN106148466A (en) * | 2016-08-30 | 2016-11-23 | 夏佳文 | A kind of preparation method of yak collagen protein |
| CN107913785A (en) * | 2016-10-11 | 2018-04-17 | 浙江崇山生物制品有限公司 | A kind of efficiently animal tendon breaking method |
| CN106589113A (en) * | 2016-11-17 | 2017-04-26 | 北京华信佳音医疗科技发展有限责任公司 | Method for extracting collagen from bovine achilles tendons |
| CN107227330A (en) * | 2017-07-10 | 2017-10-03 | 山东省千佛山医院 | A kind of extracting method of ox heel string NTx |
| CN110467668A (en) * | 2019-09-10 | 2019-11-19 | 河北考力森生物科技有限公司 | A kind of extracting method of type III collagen |
| CN111363772A (en) * | 2020-04-08 | 2020-07-03 | 平凉市华科生物技术有限公司 | Method for preparing collagen peptide by hydrolyzing bovine bone and collagen peptide thereof |
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| CN103468771B (en) | 2015-07-15 |
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