CN104017073A - Method for preparing collagen - Google Patents
Method for preparing collagen Download PDFInfo
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- CN104017073A CN104017073A CN201410274732.XA CN201410274732A CN104017073A CN 104017073 A CN104017073 A CN 104017073A CN 201410274732 A CN201410274732 A CN 201410274732A CN 104017073 A CN104017073 A CN 104017073A
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- collagen
- collagen protein
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
Abstract
The invention relates to a method for preparing collagen. The method comprises the following steps of (1) taking tails of animals, sterilizing tails of animals, dissection tendons by a surgery, and collecting tendons; (2) immersing tendons in urea-containing solution, breaking tendons in the solution, extracting the broken mixed solution of tendons and urea at 2-25 DEG C; (3) filtering the mixed solution to remove undissolved impurities to obtain the crude extraction solution, and carrying out gel separation on the extraction solution; (4) desalting the crude extraction solution and removing impurities to obtain a refined liquid; and (5) lyophilizing the refined liquid with lyophilizer to obtain the medical grade collagen. According to the invention, the extraction and purification times can be greatly reduced, and the yield can reach above 90%, the efficiencies of extraction and purification are greatly enhanced, while collagen products obtained by the method provided by the invention is less in impurities and high in purity, the degradation caused by the extraction process is avoided so as to ensure the integrity and biological activity of the collagen product.
Description
Technical field
The present invention relates to collagen protein purification technique field, especially a kind of preparation method of collagen protein.
Background technology
Collagen protein is the biomaterial being most widely used in the world, and International Year output is 20,000 tons of left and right at present.At medical field, collagen protein is widely used in wound control and transforms medical science as high-biocompatibility, high biological activity material.2011, the market study data of the internationally famous commercial affairs test and appraisal Frost & Sullivan of mechanism also show, collagen protein has become the biomedical material that has the market competitiveness most, for regenerative medicine, and will further develop, becoming one of organ alternative medicine two large materials, the ready level of its technology---Technology readiness level (TRL) scoring is 8 (the highest by 9).Medicine food management board of the U.S. (FDA) prediction, will occupy the biomedical material market that exceedes 20% to collagen protein in 2015, and annual sales amount exceedes 20,000,000,000 dollars.
Collagen protein is the very abundant albumen of content in Mammals, is also very important a kind of protein.The change of type, quality and the distribution of collagen protein directly affects the normal function of animal body, as causes abnormal paralysed trace formation, cornea and lens abnormality, reticular tissue heredopathia etc. in the diabetes, wound healing of arteriosclerosis, blood vessel and the kidney collagen basilar membrane of degenerative osteoarthritis, the vascular stroma of hyaline cartilage.And studies show that collagen protein has very important biology performance:
(1) hemostatic function of collagen.Natural collagen aggregate itself is exactly good hemostatic agent, and it plays a role by two aspects: promote platelet aggregation and blood plasma caking.The mechanism of action between collagen and thrombocyte is also completely unclear, but relevant its interactional many phenomenons are confirmed.Collagen makes the ability of platelet aggregation seem to derive from free amine group, especially lysine side-chain amino.Sealing collagen carboxyl, can not cause the obvious decline of blood coagulation ability, but has but weakened the effect that collagen impels blood plasma to lump.So the natural structure of collagen is the quaternary structure of prosperity especially enough, is the basis that collagen has cohesion ability.The anastalsis of collagen makes it can be used as coagulant material, and physical aspect can be powdery, sheet, spongy etc.
(2) reduced immunogenicity.The immunogenicity of soluble collagen is very low, and the immunogenicity of insoluble collagen is lower.Though collagen protein is macromolecular substance, its structure repeatability is large, and compared with other has immunogenic protein, the immunogenicity of collagen is very low, and people were even once thinking that collagen did not have antigenicity.Nearest research shows, collagen has the antigenic factor of 3 types: the 1st class is to be caused by the non-helical end peptide of collagen peptide chain; The 2nd class is to be caused by the conformation of collagen helix; The 3rd class is to be caused by the amino-acid sequence of a chain helical region.The 2nd class antigenic factor exists only in natural collagen molecule, and the 3rd class only appears in denatured collagen, and the 1st class antigenic factor all exists in natural and denatured collagen.
(3) biocompatibility.Collagen protein depends on and stent material as Growth of Cells, can induce the propagation such as epithelial cell, breaks up and divide a word with a hyphen at the end of a line.The distinctive triple helix structure of tropocollagen molecule and the fiber being cross-linked to form thereof or network form cell important composition composition, therefore no matter collagenic material is to be absorbed previous crops for forming neoblastic skeleton, still be absorbed assimilation and enter host, become a part for host tissue, all there is good interaction with pericellular matrix, show interactional Harmony, and become cell and organize a normal physiological function entirety part.
These biology performances of collagen protein make it at medical science, food, same with occupying very consequence in the field such as chemical, bio-medical material, are considered to be worth forever studying and having multiduty bioabsorbable polymer material.
In the market of collagen protein application aspect, international well-known medicine equipment enterprise, all in the research and development of carrying out collagen protein medical use, comprises wound wrapper material, special open sore part control creme, collagen protein foam and sponge etc.Taking collagen product as substrate material, add cell and even stem cells technology, develop the organ transplantation of new cytoactive and organ and repair product and become the new technological trend of industry.
In recent years, international large-lot producer (as French Luo Sailuo (RUSSELOT) group) takes up to develop the research and development of synthetic collagen protein and analogue thereof, but limited by synthetic technology, level is lower, output is limited, expensive (price of market synthesis of high purity collagen protein is up to 3000 dollars/milligram at present), only can be used for research application.
All the time, collagen protein mainly extracts from the histoorgan of animal, as the skin of pig ox, bone and muscle tendon etc.Collagen protein extracting method has classical acid solution, neutral salt extraction process and enzyme process.All methods are inserted extraction solution after all first mouse tail being cleaned and are dissolved.After centrifugal, crude extract can be used as product stock also further processing treatment carry out essence and carry.Neutral salt extraction process has significant technology inferior position, be mainly the low less than 5% of productive rate, and impurity protein content is high, has substantially been eliminated.
At present, the still acid hydrolyzation based on traditional of the modal collagen protein extraction process of China, and also main raw material is Cowhells tendon (tendons of beef, mutton or pork) and mouse tail muscle tendon.Due to technology limitation, acid hydrolyzation extraction efficiency can only calculate from mouse tail muscle tendon weight in wet base, and productive rate is no more than 60%.In order to improve percentage extraction, in a lot of situations, also with trypsinase, muscle tendon is carried out to pre-treatment.Although it is collagen protein that animal muscle tendon exceedes 90% component, its stripping process manpower consumption from animal body is large, and is difficult for processing clean, causes the after stain of product assorted, and particularly the pollution of animal blood and muscle tissue is difficult to remove.In addition, the proteolytic enzyme of a lot of animal tissuess still exists, in acidolysis process, meeting and acid react on collagen protein simultaneously, cause the irregular hydrolysis of collagen protein, greatly reduce the controllability of flow process with repeatable, directly have influence on the quality, particularly its special biological function of collagen protein.Therefore, need a kind of method of simple possible to extract highly purified collagen protein.
Summary of the invention
The technical problem to be solved in the present invention is: overcome in prior art the extraction yield of collagen protein low, the technical problem such as the bad and preparation time of quality is long, a kind of preparation method of collagen protein is provided, and the method preparation time is short, the collagen protein quality better that extraction yield is high, obtain.
The technical solution adopted for the present invention to solve the technical problems is: a kind of preparation method of collagen protein, has following steps:
1. the preparation of animal tail tendon
Get animal tail, and animal tail is carried out after sterilising treatment, peeling operation muscle tendon, and collect muscle tendon;
2. the extraction of collagen protein
Use muscle tendon submergence step being obtained in 1. containing urea soln, in solution, by the fragmentation of muscle tendon, broken tail tendon urea mixed solution is mixed to 10-30h at 2-25 DEG C DEG C;
3. the purifying of collagen protein
The mixing solutions that 2. step is obtained carries out filtration treatment, obtains crude extract after removing undissolved impurity, and crude extract is carried out to gel separation, chooses the solution of suitable volumes section collect according to uv-absorbing;
4. refining
The crude extract that 3. step is collected obtains refined liquid after removing salt and decon processing, and in refined liquid, saltiness is lower than 0.1%;
5. freeze-drying
Get the refined liquid Freeze Drying Equipment freeze-drying that 4. step obtains, can obtain medical grade collagen protein.
Further, the sterilising method of step in is 1. for adopting 70% alcohol wipe some times.
Further, the step 1. method of middle peeling operation muscle tendon is: the animal tail after sterilizing is put into phosphate buffered saline buffer and preserve, in phosphate buffered saline buffer, remove exterior skin with scalpel, then white muscle tendon in animal tail is peeled off to extraction, avoid taking out of bone slag and meat mincing, animal tail tendon is put into phosphate buffered saline buffer and clean somely all over until filtrate, without bloodstain, is filtered with funnel, collect muscle tendon.
Further, step 2. in by animal tail tendon with can adopt shaking table to mix containing urea soln while mixing, shaking speed 50-320rpm; Also can adopt magnetic stirring apparatus to mix, the rotating speed 100-1200rpm of magnetic stirring apparatus.
Further, described containing in urea soln containing the urea of 1-10mol/L and the inorganic salt of 0-1mol/L.
Further, the filtration treatment of step described in is 3.: get the mixing solutions supernatant liquid that 2. step obtain and cross 0.22-10 μ m cellulose acetate film, obtain crude extract, then use protein purification system or peristaltic pump to carry out gel separation to crude extract, choose the solution of UV280>0.01 partial volume section according to filtrate uv-absorbing and collect, separated volume: effectively column volume is 1:10-1:100.As preferably, separated volume: effectively column volume is 1:50.
The first embodiment, the process for purification of step described in is 4.: adopt dialysis method, dialysis tubing material is cellulose acetate, and aperture is 10K dalton, through several times dialysis desalting, saltiness, lower than after 0.1%, obtains refining collagen solution.
The second embodiment, the process for purification of step described in is 4.: adopt ultrafiltration process, filter membrane adopts tubular type pollution-resistant membrane, aperture is 10K dalton, through the doubly continuous filter wash of 5-20, obtains high purity solutions, saltiness, lower than after 0.1%, obtains refining collagen solution.
Concrete, the freeze-drying container that step adopts when freeze-drying in is 5. centrifuge tube or watch-glass.
As preferably, step 5. in time of freeze-drying be 48-72 hour.Freeze-drying time is general to be determined according to the amount of producing, and amount greatly freeze-drying time extends, and measures little freeze-drying time and shortens.
Adopted technique scheme, the present invention has following beneficial effect:
(1) preparation time is short: this law adopts Wyler's process to extract only needs 1 day time, after adopt the ultra-filtration technique can be by purifying time shorten by several hours.And traditional acid hydrolyzation and enzymolysis process extraction purifying time need 3 days, even more than one week.
(2) yield is high: general neutral salt extraction method yield is less than 5%, and sour formulation or zymohydrolysis extracting method yield can reach 60% left and right, and present method is pressed tail tendon dry weight and calculated, and yield can reach more than 90%.
(3) quality better: acid is carried or zymohydrolysis extracting method will inevitably make degraded and the sex change of collagen protein in leaching process, causes collagen protein to lose its natural structure and activity.And this law has been avoided the degraded of leaching process, ensure integrity and the biological activity of collagen protein.
(4) treating processes is simple: adopt solvent urea soln to have very high bactericidal property, and can carry out inactivation of virus, significantly reduced bacterium radix, reduced endotoxin content.
(5) be widely used: the collagen protein the present invention relates to is applicable to biomedical research, be also applicable to doing medical device product, be particularly suitable for preparing cytoskeleton material, can also be applied to somatic dimensional culture; Also can be used as the substratum that cell attachment is cultivated.
Brief description of the drawings
Fig. 1 is polyacrylamide gel electrophoresis (sds-page) analysis chart of collagen protein of the present invention; The left side is marker, the collection of illustrative plates that middle different bands are different filtrates.
Fig. 2 is that the Wester-Blot of collagen protein of the present invention analyzes collection of illustrative plates.C: positive control, adopts high purity collagen in contrast; Water: the aqueous solution of target collagen protein; Glue: other collagen proteins;
Analyze and Wester-Blot according to sds-page, product meets the feature of collagen protein, and has very high purity, inclusion-free protein band.
Embodiment
Embodiment 1
The preparation method of a kind of collagen protein of the present invention, has following steps:
1. the preparation of animal tail tendon
Get animal tail, desirable rat tail, mouse tail or kangaroo tail, and adopt ethanol for disinfection wiping some all over carrying out sterilising treatment to animal tail, animal tail after sterilizing is put into phosphate buffered saline buffer (PBS) and preserve, in phosphate buffered saline buffer, remove exterior skin with scalpel, then white muscle tendon in animal tail is peeled off to extraction, avoid taking out of bone slag and meat mincing, animal tail tendon is put into phosphate buffered saline buffer and clean somely all over until filtrate, without bloodstain, is filtered with funnel, collect muscle tendon;
2. the extraction of collagen protein
Use appropriate muscle tendon submergence step being obtained in 1. containing urea soln, in solution by the fragmentation of muscle tendon, broken tail tendon urea mixed solution is mixed at 2-25 DEG C, generally need 10-30 hour, by animal tail tendon with can adopt shaking table to mix containing urea soln while mixing, shaking speed 50-320rpm; Also can adopt magnetic stirring apparatus to mix, the rotating speed 100-1200rpm of magnetic stirring apparatus;
3. the purifying of collagen protein
The mixing solutions that 2. step is obtained carries out filtration treatment, get the mixing solutions supernatant liquid that 2. step obtain and cross 0.22-10 μ m cellulose acetate film, obtain crude extract, then use protein purification system (AKTA) or peristaltic pump to carry out gel separation to crude extract, choose the solution of UV280>0.01 partial volume section according to filtrate uv-absorbing and collect, separated volume: effectively column volume is 1:10-1:100.As preferably, separated volume: effectively column volume is 1:50;
Use the salts solution that damping fluid is 2M urea, flow velocity is 0.5-1mL/min, and effectively column volume is 50ml, and after 100ml damping fluid, the crude extract of loading 1ml through filtering, collects the solution of UV280>0.01 partial volume section.Then use 50ml buffer solution elution, continue loading.
4. refining
The crude extract that 3. step is collected obtains refined liquid after removing salt and decon processing, adopts dialysis method, and dialysis tubing material is cellulose acetate, aperture is 10K dalton, through several times dialysis desaltings, saltiness, lower than after 0.1%, obtains refining collagen solution;
5. freeze-drying
Get the refined liquid that 4. step obtain and put into the container such as centrifuge tube or watch-glass, with Freeze Drying Equipment freeze-drying 48-72 hour, can obtain medical grade collagen protein.
As the crucial solution that dissolves muscle tendon, described contains in urea soln containing the urea of 2mol/L and the inorganic salt of 0.5mol/L.
Embodiment 2
Embodiment 2 is for illustrating the preparation method of collagen protein provided by the present invention.
Adopt the method identical with embodiment 1 to prepare collagen product, different, the concentration containing urea in urea soln that step is used in is 2. 5mol/L, and the concentration of sodium-chlor is 0.1mol/L, acquisition collagen product 2.
Embodiment 3
Embodiment 3 is for illustrating the preparation method of collagen protein provided by the present invention.
Adopt the method identical with embodiment 1 to prepare collagen product, different, the concentration containing urea in urea soln that 2. step used is 1mol/L, and the concentration of sodium-chlor is 0.5mol/L, obtains collagen product 3.
Embodiment 4
Embodiment 4 is for illustrating the preparation method of collagen protein provided by the present invention.
Adopt the method identical with embodiment 1 to prepare collagen product, different, the concentration containing urea in urea soln that 2. step used is 8mol/L, obtains collagen product 4.
Embodiment 5
Embodiment 5 is for illustrating the preparation method of collagen protein provided by the present invention.
Adopt the method identical with embodiment 1 to prepare collagen product, different, the concentration containing urea in urea soln that 2. step used is 10mol/L, obtains collagen product 5.
Embodiment 6
Embodiment 6 is for illustrating the preparation method of collagen protein provided by the present invention.
Adopt the method identical with embodiment 1 to prepare collagen product, different, the step 2. middle temperature condition dissolving is 4 DEG C, and dissolution time is 48h, obtains collagen product 6.
Embodiment 7
Embodiment 7 is for illustrating the preparation method of collagen protein provided by the present invention.
Adopt the method identical with embodiment 1 to prepare collagen product, different, in step, 3. filtering is to adopt 100 layers of sterile gauze to filter mixed solution to obtain coarse filtration liquid, obtains collagen product 7.
Embodiment 8
Embodiment 8 is for illustrating the preparation method of collagen protein provided by the present invention.
Adopt the method identical with embodiment 1 to prepare collagen product, different is, step refining employing 4. adopts ultrafiltration process, filter membrane adopts tubular type pollution-resistant membrane, and aperture is 10K dalton, through the doubly continuous filter wash of 5-20, obtain high purity solutions, saltiness, lower than after 0.1%, obtains refining collagen solution, obtains collagen product 8.
Comparative example 1
Adopt the method identical with embodiment 1 to prepare collagen product, different, the concentration of the urea soln using is 0.5mol/L, obtains collagen product 9.
Product yield and the collagen protein purity of the collagen product 1-9 that detection embodiment 1-8 and comparative example 1 make, the results are shown in table 1.
The yield of table 1 collagen protein and purity comparison
| Collagen product | Yield (% by weight) | Purity (% by weight) |
| 1 | 80 | 95 |
| 2 | 85 | 95 |
| 3 | 90 | 95 |
| 4 | 92 | 95 |
| 5 | 93 | 95 |
| 6 | 90 | 95 |
| 7 | 85 | 95 |
| 8 | 80 | 97 |
| 9 | 50 | 90 |
The result of the result of test case 1-8 and test case 9 is contrasted and can be found out, adopt the yield of product of the collagen protein that method provided by the present invention produces more than 80%, the purity of product is more than 95%.
By of the present invention to collagen protein and the performance of the collagen protein that obtains of additive method compare, in Table.
The Performance Ratio of table 2 collagen protein
| Index | Acid formulation | Enzyme formulation | This law |
| Extraction yield | 60％ | 75％ | 90％ |
| Purity (Sds-page method) | 90％ | 90％ | 95％ |
| Three-dimensional structure integrity | Imperfect | Imperfect | Complete structure |
| Intracellular toxin | <0.5EU/mg | <0.5EU/mg | <0.05EU/mg |
| Preparation time | 7-10 days | 5-7 days | 2-3 days |
Can find out from above embodiment and test case, produce collagen product according to method provided by the present invention and can greatly save step and the time of operation, yield and the purity of product are high, and have good biologic activity.
Taking above-mentioned foundation desirable embodiment of the present invention as enlightenment, by above-mentioned description, relevant staff can, not departing from the scope of this invention technological thought, carry out various change and amendment completely.The technical scope of this invention is not limited to the content on specification sheets, must determine its technical scope according to claim scope.
Claims (9)
1. a preparation method for collagen protein, is characterized in that having following steps:
1. the preparation of animal tail tendon
Get animal tail, and animal tail is carried out after sterilising treatment, peeling operation muscle tendon, and collect muscle tendon;
2. the extraction of collagen protein
Use muscle tendon submergence step being obtained in 1. containing urea soln, in solution by the fragmentation of muscle tendon, by broken tail tendon urea mixed solution 2-25 DEG C of extraction;
3. the purifying of collagen protein
The mixing solutions that 2. step is obtained carries out filtration treatment, obtains crude extract after removing undissolved impurity, and crude extract is carried out to gel separation, chooses the solution of suitable volumes section collect according to uv-absorbing;
4. refining
The crude extract that 3. step is collected obtains refined liquid after removing salt and decon processing, and in refined liquid, saltiness is lower than 0.1%;
5. freeze-drying
Get the refined liquid Freeze Drying Equipment freeze-drying that 4. step obtains, obtain medical grade collagen protein.
2. the preparation method of collagen protein as claimed in claim 1, is characterized in that: the sterilising method of step in is 1. for adopting 70% alcohol wipe.
3. the preparation method of collagen protein as claimed in claim 1, it is characterized in that: the step 1. method of middle peeling operation muscle tendon is: the animal tail after sterilizing is put into phosphate buffered saline buffer and preserve, in phosphate buffered saline buffer, remove exterior skin with scalpel, then white muscle tendon in animal tail is peeled off to extraction, avoid taking out of bone slag and meat mincing, animal tail tendon is put into phosphate buffered saline buffer cleaning until filtrate, without bloodstain, with funnel filtration, is collected muscle tendon.
4. the preparation method of collagen protein as claimed in claim 1, is characterized in that: step 2. in by animal tail tendon with can adopt shaking table to mix containing urea soln while mixing, shaking speed 50-320rpm; Or adopt magnetic stirring apparatus to mix, the rotating speed 100-1200rpm of magnetic stirring apparatus.
5. the preparation method of the collagen protein as described in claim 1 or 4, is characterized in that: described contains in urea soln containing the urea of 1-10mol/L and the inorganic salt of 0-1mol/L.
6. the preparation method of collagen protein as claimed in claim 1, it is characterized in that: the filtration treatment of step described in is 3.: the supernatant liquid of getting the mixing solutions that 2. step obtain is crossed 0.22-10 μ m cellulose acetate film, obtain crude extract, then use protein purification system or peristaltic pump to carry out gel separation to crude extract, choose the solution of UV280>0.01 partial volume section according to filtrate uv-absorbing and collect, separated volume: effectively column volume is 1:10-1:100.
7. the preparation method of collagen protein as claimed in claim 1, it is characterized in that: the process for purification of step described in is 4.: adopt dialysis method, dialysis tubing material is cellulose acetate, aperture is 10K dalton, through several times dialysis desalting to saltiness lower than after 0.1%, obtain refining collagen solution.
8. the preparation method of collagen protein as claimed in claim 1, it is characterized in that: the process for purification of step described in is 4.: adopt ultrafiltration process, filter membrane adopts tubular type pollution-resistant membrane, aperture is 10K dalton, through the doubly continuous filter wash of 5-20, obtain high purity solutions to saltiness lower than after 0.1%, obtain refining collagen solution.
9. the preparation method of collagen protein as claimed in claim 1, is characterized in that: 5. the time of middle freeze-drying is 48-72h to step.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109810187A (en) * | 2017-11-20 | 2019-05-28 | 中国科学院大连化学物理研究所 | A kind of extracting method and application of animal tendon tendon collagen |
| WO2019169646A1 (en) * | 2018-03-07 | 2019-09-12 | 广州创尔生物技术股份有限公司 | Quality control method for biologically active collagen |
| CN106946987B (en) * | 2017-02-20 | 2020-02-28 | 福建农林大学 | High-concentration acid-soluble collagen solution and preparation method of water-soluble collagen solution |
| CN114452444A (en) * | 2022-01-27 | 2022-05-10 | 陕西巨子生物技术有限公司 | Preparation method of low endotoxin collagen |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106946987B (en) * | 2017-02-20 | 2020-02-28 | 福建农林大学 | High-concentration acid-soluble collagen solution and preparation method of water-soluble collagen solution |
| CN109810187A (en) * | 2017-11-20 | 2019-05-28 | 中国科学院大连化学物理研究所 | A kind of extracting method and application of animal tendon tendon collagen |
| WO2019169646A1 (en) * | 2018-03-07 | 2019-09-12 | 广州创尔生物技术股份有限公司 | Quality control method for biologically active collagen |
| CN114452444A (en) * | 2022-01-27 | 2022-05-10 | 陕西巨子生物技术有限公司 | Preparation method of low endotoxin collagen |
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Application publication date: 20140903 |