CN102391374B - Preparation method of active collagen with triple-helix structure - Google Patents
Preparation method of active collagen with triple-helix structure Download PDFInfo
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Abstract
The invention relates to a preparation method of collagen; fresh pigskin is used as a raw material; the raw material is treated by pretreatment, fat removal, and acid-enzyme treatment; ultrasonic treatment is employed to further improve extraction efficiency; and finally purification is performed to prepare a collagen product with high purity. The preparation method of collagen has high extraction efficiency, and the product purity is up to above 98%. The product is active collagen with a maintained triple-helix structure, has low immunogenicity and high biocompatibility, is biodegradable, and is applicable to medical purposes.
Description
Technical field
The present invention relates to a kind of preparation method with the active collagen of triple-helix structure, belong to protein engineering field, this product is the active collagen keeping triple-helix structure, has reduced immunogenicity, high-biocompatibility, biodegradable, is applicable to medical use.
Background technology
Collagen is that vertebrates in-vivo content is the abundantest, distribution one group of scleroprotein the most widely, molecular weight is about 300000, all can form supramolecular aggregates in extracellular, be present in a large number in bone, cartilage, tendon and skin, account for the 25%-33% of human body or other animal body total protein contents.The normal collagen containing several types in same tissue, often based on certain.NTx is dispersed throughout each several part of human body, is mainly in skin, tendon and ligament, has very strong tensile energy.II Collagen Type VI is mainly present in hyaline cartilage, vitreum, has stronger anti-pressure ability.III Collagen Type VI is distributed widely in the large tissue of extensibility, as loose connective tissue etc., has extensibility and complaisance.Wherein, NTx accounts for 90% of the total collagen quantity of organism, and therefore, the use of NTx in biomaterial is also the most extensive.
Collagen is identical in molecular level structure, and be made up of 3 a chain polypeptide, each collagen chain is all left hand helix configuration.Article 3, left hand helix chain is wound in right-handed helix structure again mutually, and namely superhelix is the triple helices structure of collagen protein uniqueness, makes its molecular structure highly stable.Article 3, chain is rich in glycine, proline(Pro) and L-Ala, and lack halfcystine and tryptophane, tyrosine content is also very low, but containing unique hydroxylation amino acid (oxyproline and hydroxylysine).Due to fibriilar directivity and the difference becoming beam diameter and density, there is textural difference in the collagen of different tissues, and have respective function and structure feature.In reticular tissue, collagen is except mechanical support, or the important substance basis of cell adhesion and movement, and therefore, collagen is considered to form occurrence factor important in fetal development and tissue regeneration, is used widely in organizational project.
Collagen can be called as, must be that proteinoid that its triple-helix structure does not change, also remain with complete biological activity.And collagen protein is the hydrolysate of collagen, the triple-helix structure of collagen thoroughly unclamps, and becomes 3 peptide chains freely, and is degraded into polydisperse peptide section, comprising little peptide.Therefore collagen protein is polypeptide mixture, relative molecular weight from several thousand to several ten thousand, and molecular weight distribution is very wide, does not have biological activity, can be dissolved in cold water, and can be utilized by proteolytic enzyme.
Disclose a kind of preparation method of collagen in CN1110284, with acid extraction, salt precipitates, and membrane filtration, last freeze-drying, its extraction efficiency and purity are all lower.It take cod skin as the method that collagen protein prepared by raw material that CN101250218 discloses a kind of, and its preparation method can destroy the triple-helix structure of collagen.Method disclosed in prior art often destroys the triple-helix structure of collagen in the extraction preparation process of collagen, or extraction efficiency is lower, and cannot realize can either the triple-helix structure of retentive activity collagen, improves extraction efficiency and purity again simultaneously.
Summary of the invention
For above-mentioned situation, the object of this invention is to provide a kind of preparation method with the active collagen of triple-helix structure.The present invention uses sour enzyme to combine the preparation method processing and add gradual ultrasonication, can not only keep the triple-helix structure of collagen, improves the extraction efficiency of collagen significantly simultaneously.The present invention uses H
2o
2solution-treated acid adding dissolved salt is analysed purifying and is added the purity that dialysis purifying effectively raises collagen.
There is a preparation method for the active collagen of triple-helix structure, comprise the steps: that raw materials pretreatment, grease removal, sour enzyme combine process, ultrasonication, H
2o
2solution-treated, acid-soluble purifying of saltouing, dialysis purifying, drying.
Described raw material is be selected from the one in fresh porcine skin, ox-hide, ox heel string.
Described sour enzyme combines and is treated to: immerse in 0.6 ~ 0.7mol/L Glacial acetic acid and the pepsic mixed solution of 600 ~ 700mg/L through pretreated raw material, Keep agitation 25 ~ 30 hours.
Described ultrasonication preferably adopts gradual ultrasonication, first uses 100 ~ 200W supersound process 30 minutes, re-uses 200 ~ 300W supersound process 30 minutes, finally uses 300 ~ 400W supersound process 1 hour.
Described a kind of preparation method with the active collagen of triple-helix structure, concrete steps are as follows:
(1) get fresh porcine skin, oil layer and trichocutis are removed in cutting, remove impurity, cleaning, pigskin is broken into fine granularity, according to the ratio of solid-to-liquid ratio 1:30 ~ 1:40, adds the aqueous sodium hydroxide solution of 0.01 ~ 0.03mol/L, soak 1 ~ 2 hour in 6 ~ 8 DEG C, filter, for subsequent use;
(2) in above-mentioned pretreated pigskin, add the anhydrous diethyl ether that quality is pigskin quality 6 ~ 8 times or acetone, in 35 ~ 40 DEG C backflow 5-8 hour, afterwards with distilled water flushing to free from extraneous odour, for subsequent use;
(3) above-mentioned pigskin is immersed in 0.6 ~ 0.7mol/L Glacial acetic acid and the pepsic mixed solution of 600 ~ 700mg/L, Keep agitation 25 ~ 30 hours;
(4) gradual ultrasonication is adopted to said mixture, first use 100 ~ 200W supersound process 30 minutes, re-use 200 ~ 300W supersound process 30 minutes, finally use 300 ~ 400W supersound process 1 hour;
(5) add the H2O2 solution of 2 ~ 3%, mixing leaves standstill 2 ~ 4 hours, regulates pH to 5.0, centrifugal;
(6) get supernatant liquor, add NaCl, stir 20-30 hour, centrifugal, throw out adds 0.6-0.7moL/L Glacial acetic acid and dissolves, centrifugal, gets supernatant liquor, regulates pH to 7.5, adds NaCl, stirs 20-30 hour, centrifugal;
(7) precipitate dissolves step (6) obtained is in 0.6 ~ 0.7moL/L Glacial acetic acid, first with the Glacial acetic acid of 0.6 ~ 0.7moL/L for extracellular fluid dialysis is dialysed 2 times, each 4 hours, be extracellular fluid dialysis dialysis 5 ~ 7 times again with distilled water, each 4 hours, can't detect chlorion to outer liquid, obtain collagen liquid;
(8) by aforesaid liquid lyophilize, the collagen with triple-helix structure activity is obtained.
The described size with triple-helix structure active collagen is generally 20000 ~ 30000 dalton.
Acid enzyme combines process: sour enzyme of the present invention refers in conjunction with process and immerses in 0.6 ~ 0.7mol/L Glacial acetic acid and the pepsic mixed solution of 600 ~ 700mg/L through pretreated raw material, Keep agitation 25 ~ 30 hours.The method combines the feature that acid system and Enzymatic Extraction prepare collagen, prevents the destruction to active collagen triple-helix structure, effectively raises extraction efficiency.
Ultrasonication: sour enzyme of the present invention refers in conjunction with process and adopts gradual ultrasonication, first uses 100 ~ 200W supersound process 30 minutes, re-uses 200 ~ 300W supersound process 30 minutes, finally use 300 ~ 400W supersound process 1 hour.The present invention adopts ultrasonication combined acid Enzymatic Extraction to prepare collagen protein, through experimental studies have found that, adopts ultrasonication effectively can improve extraction efficiency.Adopt gradual ultrasonication compared with employing constant power supersound process, better can prevent the destruction of the triple-helix structure of collagen, and effectively reduce the production energy consumption in ultrasonication stage.
Beneficial effect of the present invention: the present invention uses sour enzyme to combine the preparation method processing and add gradual ultrasonication, decrease the destruction to collagen structure to the full extent, keep the triple-helix structure of active collagen, improve the extraction efficiency of collagen simultaneously significantly, reduce production cost.Meanwhile, gradual ultrasonication also reduces energy consumption to a certain extent in ultrasonication link.The present invention uses H
2o
2solution-treated acid adding dissolved salt is analysed purifying and is added the purity that dialysis purifying effectively raises collagen.The collagen that method of the present invention directly obtains has reduced immunogenicity and high-biocompatibility, by suitability for industrialized production, and is widely used in medical product, as: skin substitute products, bone surrogate, collagen protein dressing, biotechnology film etc.
Embodiment
Below in conjunction with embodiment, set forth the present invention further:
Embodiment 1
(1) get fresh ox-hide, oil layer and trichocutis are removed in cutting, remove impurity, and cleaning, is broken into fine granularity by ox-hide, according to the ratio of solid-to-liquid ratio 1:30, adds the aqueous sodium hydroxide solution of 0.03mol/L, soak 2 hours in 6 ~ 8 DEG C, filter, for subsequent use;
(2) in above-mentioned pretreated ox-hide, add the anhydrous diethyl ether that quality is ox-hide quality 8 times or acetone, in 35 DEG C backflow 8 hours, afterwards with distilled water flushing to free from extraneous odour, for subsequent use;
(3) above-mentioned ox-hide is immersed in 0.6mol/L Glacial acetic acid and the pepsic mixed solution of 700mg/L, Keep agitation 25 hours;
(4) gradual ultrasonication is adopted to said mixture, first use 100W supersound process 30 minutes, re-use 300W supersound process 30 minutes, finally use 400W supersound process 1 hour;
(5) H of 2% is added
2o
2solution, mixing leaves standstill 4 hours, regulates pH to 5.0, centrifugal;
(6) get supernatant liquor, add NaCl, stir 20 hours, centrifugal, throw out adds 0.7moL/L Glacial acetic acid and dissolves, centrifugal, gets supernatant liquor, regulates pH to 7.5, adds NaCl, stir 20 hours, centrifugal;
(7) precipitate dissolves step (6) obtained in 0.7moL/L Glacial acetic acid, first with the Glacial acetic acid of 0.7moL/L for extracellular fluid dialysis is dialysed 2 times, each 4 hours, be that extracellular fluid dialysis is dialysed 5 times again with distilled water, each 4 hours, can't detect chlorion to outer liquid, obtain collagen liquid;
(8) by aforesaid liquid lyophilize, the collagen with triple-helix structure activity is obtained.
Embodiment 2
(1) get fresh ox heel string, oil layer and trichocutis are removed in cutting, remove impurity, and cleaning, is broken into fine granularity by ox heel string, according to the ratio of solid-to-liquid ratio 1:40, adds the aqueous sodium hydroxide solution of 0.01mol/L, soak 1 hour in 6 ~ 8 DEG C, filter, for subsequent use;
(2) in above-mentioned pretreated ox heel string, add the anhydrous diethyl ether that quality is ox heel string quality 6 times or acetone, in 40 DEG C backflow 5 hours, afterwards with distilled water flushing to free from extraneous odour, for subsequent use;
(3) above-mentioned ox heel string is immersed in 0.7mol/L Glacial acetic acid and the pepsic mixed solution of 600mg/L, Keep agitation 30 hours;
(4) gradual ultrasonication is adopted to said mixture, first use 100W supersound process 30 minutes, re-use 200W supersound process 30 minutes, finally use 300W supersound process 1 hour;
(5) H of 3% is added
2o
2solution, mixing leaves standstill 2 hours, regulates pH to 5.0, centrifugal;
(6) get supernatant liquor, add NaCl, stir 30 hours, centrifugal, throw out adds 0.6moL/L Glacial acetic acid and dissolves, centrifugal, gets supernatant liquor, regulates pH to 7.5, adds NaCl, stir 30 hours, centrifugal;
(7) precipitate dissolves step (6) obtained in 0.6moL/L Glacial acetic acid, first with the Glacial acetic acid of 0.6moL/L for extracellular fluid dialysis is dialysed 2 times, each 4 hours, be that extracellular fluid dialysis is dialysed 7 times again with distilled water, each 4 hours, can't detect chlorion to outer liquid, obtain collagen liquid;
(8) by aforesaid liquid lyophilize, the collagen with triple-helix structure activity is obtained.
Embodiment 3
(1) get fresh porcine skin, oil layer and trichocutis are removed in cutting, remove impurity, and cleaning, is broken into fine granularity by pigskin, according to the ratio of solid-to-liquid ratio 1:35, adds the aqueous sodium hydroxide solution of 0.02mol/L, soak 1.5 hours in 6 ~ 8 DEG C, filter, for subsequent use;
(2) in above-mentioned pretreated pigskin, add the anhydrous diethyl ether that quality is pigskin quality 7 times or acetone, in 37 DEG C backflow 6 hours, afterwards with distilled water flushing to free from extraneous odour, for subsequent use;
(3) above-mentioned pigskin is immersed in 0.65mol/L Glacial acetic acid and the pepsic mixed solution of 650mg/L, Keep agitation 28 hours;
(4) gradual ultrasonication is adopted to said mixture, first use 150W supersound process 30 minutes, re-use 250W supersound process 30 minutes, finally use 350W supersound process 1 hour;
(5) add the H2O2 solution of 2.5%, mixing leaves standstill 3 hours, regulates pH to 5.0, centrifugal;
(6) get supernatant liquor, add NaCl, stir 25 hours, centrifugal, throw out adds 0.65moL/L Glacial acetic acid and dissolves, centrifugal, gets supernatant liquor, regulates pH to 7.5, adds NaCl, stir 25 hours, centrifugal;
(7) precipitate dissolves step (6) obtained in 0.65moL/L Glacial acetic acid, first with the Glacial acetic acid of 0.65moL/L for extracellular fluid dialysis is dialysed 2 times, each 4 hours, be that extracellular fluid dialysis is dialysed 6 times again with distilled water, each 4 hours, can't detect chlorion to outer liquid, obtain collagen liquid;
(8) by aforesaid liquid lyophilize, the collagen with triple-helix structure activity is obtained.
Claims (1)
1. there is a preparation method for the active collagen of triple-helix structure, it is characterized in that, comprise the steps: that raw materials pretreatment, grease removal, sour enzyme combine process, ultrasonication, H
2o
2solution-treated, acid-soluble purifying of saltouing, dialysis purifying, drying, concrete steps are as follows:
(1) get fresh porcine skin, oil layer and trichocutis are removed in cutting, remove impurity, cleaning, pigskin is broken into fine granularity, according to the ratio of solid-to-liquid ratio 1:30 ~ 1:40, adds the aqueous sodium hydroxide solution of 0.01 ~ 0.03mol/L, soak 1 ~ 2 hour in 6 ~ 8 DEG C, filter, for subsequent use;
(2) in above-mentioned pretreated pigskin, add the anhydrous diethyl ether that quality is pigskin quality 6 ~ 8 times or acetone, in 35 ~ 40 DEG C backflow 5-8 hour, afterwards with distilled water flushing to free from extraneous odour, for subsequent use;
(3) above-mentioned pigskin is immersed in 0.6 ~ 0.7mol/L Glacial acetic acid and the pepsic mixed solution of 600 ~ 700mg/L, Keep agitation 25 ~ 30 hours;
(4) gradual ultrasonication is adopted to said mixture, first use 100 ~ 200W supersound process 30 minutes, re-use 200 ~ 300W supersound process 30 minutes, finally use 300 ~ 400W supersound process 1 hour;
(5) H of 2 ~ 3% is added
2o
2solution, mixing leaves standstill 2 ~ 4 hours, regulates pH to 5.0, centrifugal;
(6) get supernatant liquor, add NaCl, stir 20-30 hour, centrifugal, throw out adds 0.6-0.7moL/L Glacial acetic acid and dissolves, centrifugal, gets supernatant liquor, regulates pH to 7.5, adds NaCl, stirs 20-30 hour, centrifugal;
(7) precipitate dissolves step (6) obtained is in 0.6 ~ 0.7moL/L Glacial acetic acid, first with the Glacial acetic acid of 0.6 ~ 0.7moL/L for extracellular fluid dialysis is dialysed 2 times, each 4 hours, be extracellular fluid dialysis dialysis 5 ~ 7 times again with distilled water, each 4 hours, can't detect chlorion to outer liquid, obtain collagen liquid;
(8) by aforesaid liquid lyophilize, the collagen with triple-helix structure activity is obtained.
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| CN103966294B (en) * | 2013-02-02 | 2017-06-20 | 深圳兰度生物材料有限公司 | Biological active collagen extracting method |
| CN106317909A (en) * | 2016-08-25 | 2017-01-11 | 董晓 | Preparing method of antibacterial medical grade thin film material |
| 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 |
| CN106591406B (en) * | 2016-11-23 | 2020-03-24 | 江南大学 | Method for preparing bone gelatin by using ultrasonic-assisted enzyme method |
| CN106866816A (en) * | 2017-04-14 | 2017-06-20 | 桂林融通科技有限公司 | The acid-enzyme binding-method of collagen is extracted from pigskin |
| CN107441549A (en) * | 2017-06-16 | 2017-12-08 | 无锡贝迪生物工程股份有限公司 | A kind of preparation method of collagen Heparan sulfate combine dressing |
| CN108192941A (en) * | 2018-03-07 | 2018-06-22 | 广州创尔生物技术股份有限公司 | A kind of method of quality control of biologically active collagen |
| CN110563834A (en) * | 2019-09-25 | 2019-12-13 | 成都奇璞生物科技有限公司 | Collagen extraction method |
| CN111388742A (en) * | 2020-04-26 | 2020-07-10 | 无锡贝迪生物工程股份有限公司 | Collagen dressing capable of releasing antibiotics in sustained and controlled manner and preparation method thereof |
| CN113667009B (en) * | 2021-07-13 | 2023-04-28 | 尚诚怡美(成都)生物科技有限公司 | Three-helix structure collagen and preparation method thereof |
| CN114634563A (en) * | 2022-02-16 | 2022-06-17 | 惠州华阳医疗器械有限公司 | Active collagen extracting solution, preparation method and system thereof, skin care product and preparation method thereof |
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Address after: 214000, Jiangsu, Wuxi province Mashan biological medicine industry park (business place, Wuxi, Binhu District, Mashan, Mei Liang Road, No. 132) Patentee after: Wuxi Betty biological engineering Limited by Share Ltd Address before: 214063, Mashan biological pharmaceutical industry park (Ziyun Road), Binhu District, Jiangsu, Wuxi Province Patentee before: Wuxi Biot Bio-technology Co., Ltd. |