CN105985430A - Low-temperature macromolecular collagen extracting method - Google Patents
Low-temperature macromolecular collagen extracting method Download PDFInfo
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- CN105985430A CN105985430A CN201610052925.XA CN201610052925A CN105985430A CN 105985430 A CN105985430 A CN 105985430A CN 201610052925 A CN201610052925 A CN 201610052925A CN 105985430 A CN105985430 A CN 105985430A
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- 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 or cold insoluble globulin [CIG]
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Abstract
The invention discloses a low-temperature macromolecular collagen extracting method. The method includes steps: (1) preparing pigskin for standby application; (2) removing impurity proteins; (3) degreasing; (4) swelling after degreasing; (5) freezing preliminarily, pulping, crushing, extracting and filtering to obtain extract liquor and filter residues; (6) adding sodium chloride to perform salting-out precipitation, and performing freeze drying to obtain crude products of macromolecular collagen; (7) dissolving the crude products of macromolecular collagen, adjusting pH value, standing at a low temperature, and taking a collagen suspension layer; (8) dissolving, adjusting pH value, standing at a low temperature, and taking a collagen suspension layer; (9) dissolving, dialyzing, and performing freeze drying to obtain refined products of macromolecular collagen. The low-temperature macromolecular collagen extracting method has advantages that extraction time is shortened, energy consumption in an extraction process is reduced, a technical process is simplified, technical operation difficulty is lowered, production cost is saved, and the macromolecular collagen obtained by extraction is high in quality.
Description
Technical field
The present invention relates to a kind of extraction technology of macromolecular collagen protein, specifically a kind of low temperature extracts the method for macromolecular collagen protein.
Background technology
Collagen is human body skin, bone, the most important structural constituent of connective tissue.Collagen is that a class is at wide variety of raw material of industry such as medical surgical, beauty treatment filling, skin care item, food, chemical industry.Organism macromolecule has very big potentiality as artificial skin or the dressing of wound.Macromolecular collagen protein is mainly used in surgical hemostasis aspect, can make blood clotting, have coagulation function, is used as wound hemostasis dressing, and being mainly based upon collagen can combine closely with wound, penetrates in the middle of cambium, and support when growing as cell.Because spongiform collagen can absorb celiolymph, brain can be separated and organize and without serious inflammation phenomenon on brain, so the meninx substituent that can act also as breakage is used.Current China collagen produces and mainly obtains through extracting, purifying for raw material with the skin of pig, ox etc., tendon etc., extraction time length and also at the bottom of yield, energy consumption height.
Content of the invention
It is an object of the invention to provide a kind of method that low temperature extracts macromolecular collagen protein, with the problem solving to propose in above-mentioned background technology.
For achieving the above object, the present invention provides following technical scheme:
A kind of low temperature extracts the method for macromolecular collagen protein, comprises the following steps:
(1) fresh pigskin is selected, remove the pig hair on pigskin and impurity, drain after being cleaned by running water, be cut into the fragment of 1 × 1cm, the fragment being cut into soaks 10-15min in 75% ethanol, drains standby after the deionized water soaking flushing of 4-5 ° of C of taking-up 3～5 times；
(2) removal of impurities albumen: the removal of foreign protein uses sodium hydroxide solution to soak, and the concentration of sodium hydroxide solution is
0.6mol/L, soak time 20-30 hour, soaking temperature 4-5 DEG C, it in immersion process, is stirred continuously, every 5 hours, change sodium hydroxide solution 1 time；
(3) degreasing: use sodium carbonate and deionized water 4 DEG C stirring 3-6h, draining, add 4 ° of C of deionized water and clean, removing fat；
(4) adding acetum to carry out swelling according to the ratio of 1: 30 after degreasing, the concentration of acetum is 0.6mol/L, and swelling temperature is 4 DEG C, and swelling time is 24-36 hour；
(5) tentatively freezing after swelling, solidification point is-25 DEG C, and freeze-off time is 4 hours；Freezing the appropriate concentration of rear raw material addition is
The acetum of 0.5mol/L, carries out making beating broken 0.5 minute at 4 DEG C, after making beating, granular size is within 2mm, and making beating and environment temperature are 4 DEG C；Then the acetum being added thereto to 0.5mol/L once extracts 36 hours, and raw material is 1: 20 with the w/v of acetum, extracts after finishing, filters with 40-50 mesh gauze, obtain extract and filter residue；
(6) sodium chloride to concentration is added to be 2.5mol/L in extract
Carrying out salt precipitation, freeze-drying prepares macromolecular collagen protein crude product；
(7) adding the acetic acid of 0 DEG C-4 DEG C to dissolve in prepared macromolecular collagen protein crude product, obtaining lysate, macromolecular collagen protein crude product is 1: 40 with the w/v of acid solution；Addition 0 DEG C-4 DEG C in lysate or acidic extraction liquid, concentration are the sodium hydroxide solution of 2.0mol/L, its pH value is adjusted to 6-11, obtains collagen solution；After collagen solution is stood 24 hours at 0 DEG C-4 DEG C, take out the collagen suspension layer of collagen solution top cohesion；
(8) collagen suspension layer is dissolved in the acetic acid of 0 DEG C-4 DEG C, collagen suspension layer is 1: 20 with the w/v of acid solution, addition 0 DEG C-4 DEG C in lysate or acidic extraction liquid, concentration are the sodium hydroxide solution of 2.0mol/L, its pH value is adjusted to 6-11, obtains collagen solution；After collagen solution is stood 24 hours at 0 DEG C-4 DEG C, take out the collagen suspension layer of collagen solution top cohesion；
(9) acetum of the collagen suspension layer 0.05-10mol/L taking step (8) dissolves, collagen suspension layer is 1: 10 with the w/v of acetum, then directly dialysed by distilled water, the collagen suspension liquid freeze-drying that will obtain after dialysis, obtains macromolecular collagen protein fine work.
As the further scheme of the present invention: described pigskin is fresh black pig pigskin.
As the present invention further scheme: the stirring in step (3) uses mixer stirring.
As the present invention further scheme: repeat step (8) 1-3 time, obtain collagen suspension layer.
Compared with prior art, the invention has the beneficial effects as follows: reduce the time extracted, reduce the energy consumption of extraction process, simplify technical process, both reduced the operation easier of technique, saved again production cost, extract the macromolecular collagen protein quality obtaining good.
Detailed description of the invention
Below in conjunction with the embodiment of the present invention, the technical scheme in the embodiment of the present invention is clearly and completely described, it is clear that described embodiment is only a part of embodiment of the present invention, rather than whole embodiments.Based on the embodiment in the present invention, every other embodiment that those of ordinary skill in the art are obtained under the premise of not making creative work, broadly fall into the scope of protection of the invention.
Embodiment 1
In the embodiment of the present invention, a kind of low temperature extracts the method for macromolecular collagen protein, comprises the following steps:
(1) selecting fresh black pig pigskin, removing the pig hair on pigskin and impurity, drain, be cut into the fragment of 1 × 1cm after being cleaned by running water, the fragment being cut into soaks 10min in 75% ethanol, and taking-up is standby with draining after the deionized water soaking flushing 3 times of 4 ° of C；
(2) removal of impurities albumen: the removal of foreign protein uses sodium hydroxide solution to soak, and the concentration of sodium hydroxide solution is
0.6mol/L, soak time 20 hours, soaking temperature 4-5 DEG C, it in immersion process, is stirred continuously, every 5 hours, change sodium hydroxide solution 1 time；
(3) degreasing: use sodium carbonate and deionized water 4 DEG C to stir 3h, draining with mixer, adds 4 ° of C of deionized water and cleans, removing fat；
(4) adding acetum to carry out swelling according to the ratio of 1: 30 after degreasing, the concentration of acetum is 0.6mol/L, and swelling temperature is 4 DEG C, and swelling time is 24 hours；
(5) tentatively freezing after swelling, solidification point is-25 DEG C, and freeze-off time is 4 hours；Freezing the appropriate concentration of rear raw material addition is
The acetum of 0.5mol/L, carries out making beating broken 0.5 minute at 4 DEG C, after making beating, granular size is within 2mm, and making beating and environment temperature are 4 DEG C；Then the acetum being added thereto to 0.5mol/L once extracts 36 hours, and raw material is 1: 20 with the w/v of acetum, extracts after finishing, filters with 40 mesh gauzes, obtain extract and filter residue；
(6) sodium chloride to concentration is added to be 2.5mol/L in extract
Carrying out salt precipitation, freeze-drying prepares macromolecular collagen protein crude product；
(7) adding the acetic acid of 0 DEG C to dissolve in prepared macromolecular collagen protein crude product, obtaining lysate, macromolecular collagen protein crude product is 1: 40 with the w/v of acid solution；Addition 0 DEG C in lysate or acidic extraction liquid, concentration are the sodium hydroxide solution of 2.0mol/L, its pH value is adjusted to 6, obtains collagen solution；After collagen solution is stood 24 hours at 0 DEG C, take out the collagen suspension layer of collagen solution top cohesion；
(8) collagen suspension layer is dissolved in the acetic acid of 0 DEG C, collagen suspension layer is 1: 20 with the w/v of acid solution, addition 0 DEG C in lysate or acidic extraction liquid, concentration are the sodium hydroxide solution of 2.0mol/L, its pH value is adjusted to 6, obtains collagen solution；After collagen solution is stood 24 hours at 0 DEG C, take out the collagen suspension layer of collagen solution top cohesion；
(9) acetum of the collagen suspension layer 0.05mol/L taking step (8) dissolves, collagen suspension layer is 1: 10 with the w/v of acetum, then directly dialysed by distilled water, the collagen suspension liquid freeze-drying that will obtain after dialysis, obtains macromolecular collagen protein fine work.
Embodiment 2
In the embodiment of the present invention, a kind of low temperature extracts the method for macromolecular collagen protein, comprises the following steps:
(1) selecting fresh black pig pigskin, removing the pig hair on pigskin and impurity, drain, be cut into the fragment of 1 × 1cm after being cleaned by running water, the fragment being cut into soaks 12min in 75% ethanol, and taking-up is standby with draining after the deionized water soaking flushing 4 times of 4 ° of C；
(2) removal of impurities albumen: the removal of foreign protein uses sodium hydroxide solution to soak, and the concentration of sodium hydroxide solution is
0.6mol/L, soak time 25 hours, soaking temperature 4 DEG C, it in immersion process, is stirred continuously, every 5 hours, change sodium hydroxide solution 1 time；
(3) degreasing: use sodium carbonate and deionized water 4 DEG C to stir 4h, draining with mixer, adds 4 ° of C of deionized water and cleans, removing fat；
(4) adding acetum to carry out swelling according to the ratio of 1: 30 after degreasing, the concentration of acetum is 0.6mol/L, and swelling temperature is 4 DEG C, and swelling time is 30 hours；
(5) tentatively freezing after swelling, solidification point is-25 DEG C, and freeze-off time is 4 hours；Freezing the appropriate concentration of rear raw material addition is
The acetum of 0.5mol/L, carries out making beating broken 0.5 minute at 4 DEG C, after making beating, granular size is within 2mm, and making beating and environment temperature are 4 DEG C；Then the acetum being added thereto to 0.5mol/L once extracts 36 hours, and raw material is 1: 20 with the w/v of acetum, extracts after finishing, filters with 45 mesh gauzes, obtain extract and filter residue；
(6) sodium chloride to concentration is added to be 2.5mol/L in extract
Carrying out salt precipitation, freeze-drying prepares macromolecular collagen protein crude product；
(7) adding the acetic acid of 2 DEG C to dissolve in prepared macromolecular collagen protein crude product, obtaining lysate, macromolecular collagen protein crude product is 1: 40 with the w/v of acid solution；Addition 2 DEG C in lysate or acidic extraction liquid, concentration are the sodium hydroxide solution of 2.0mol/L, its pH value is adjusted to 8, obtains collagen solution；After collagen solution is stood 24 hours at 2 DEG C, take out the collagen suspension layer of collagen solution top cohesion；
(8) collagen suspension layer is dissolved in the acetic acid of 2 DEG C, collagen suspension layer is 1: 20 with the w/v of acid solution, addition 2 DEG C in lysate or acidic extraction liquid, concentration are the sodium hydroxide solution of 2.0mol/L, its pH value is adjusted to 8, obtains collagen solution；After collagen solution is stood 24 hours at 2 DEG C, take out the collagen suspension layer of collagen solution top cohesion；It is repeated 2 times；
(9) acetum of the collagen suspension layer 5mol/L taking step (8) dissolves, collagen suspension layer is 1: 10 with the w/v of acetum, then directly dialysed by distilled water, the collagen suspension liquid freeze-drying that will obtain after dialysis, obtain macromolecular collagen protein fine work.
Embodiment 3
In the embodiment of the present invention, a kind of low temperature extracts the method for macromolecular collagen protein, comprises the following steps:
(1) selecting fresh black pig pigskin, removing the pig hair on pigskin and impurity, drain, be cut into the fragment of 1 × 1cm after being cleaned by running water, the fragment being cut into soaks 15min in 75% ethanol, and taking-up is standby with draining after the deionized water soaking flushing 5 times of 5 ° of C；
(2) removal of impurities albumen: the removal of foreign protein uses sodium hydroxide solution to soak, and the concentration of sodium hydroxide solution is
0.6mol/L, soak time 30 hours, soaking temperature 5 DEG C, it in immersion process, is stirred continuously, every 5 hours, change sodium hydroxide solution 1 time；
(3) degreasing: use sodium carbonate and deionized water 4 DEG C to stir 6h, draining with mixer, adds 4 ° of C of deionized water and cleans, removing fat；
(4) adding acetum to carry out swelling according to the ratio of 1: 30 after degreasing, the concentration of acetum is 0.6mol/L, and swelling temperature is 4 DEG C, and swelling time is 36 hours；
(5) tentatively freezing after swelling, solidification point is-25 DEG C, and freeze-off time is 4 hours；Freezing the appropriate concentration of rear raw material addition is
The acetum of 0.5mol/L, carries out making beating broken 0.5 minute at 4 DEG C, after making beating, granular size is within 2mm, and making beating and environment temperature are 4 DEG C；Then the acetum being added thereto to 0.5mol/L once extracts 36 hours, and raw material is 1: 20 with the w/v of acetum, extracts after finishing, filters with 50 mesh gauzes, obtain extract and filter residue；
(6) sodium chloride to concentration is added to be 2.5mol/L in extract
Carrying out salt precipitation, freeze-drying prepares macromolecular collagen protein crude product；
(7) adding the acetic acid of 4 DEG C to dissolve in prepared macromolecular collagen protein crude product, obtaining lysate, macromolecular collagen protein crude product is 1: 40 with the w/v of acid solution；Addition 4 DEG C in lysate or acidic extraction liquid, concentration are the sodium hydroxide solution of 2.0mol/L, its pH value is adjusted to 11, obtains collagen solution；After collagen solution is stood 24 hours at 4 DEG C, take out the collagen suspension layer of collagen solution top cohesion；
(8) collagen suspension layer is dissolved in the acetic acid of 4 DEG C, collagen suspension layer is 1: 20 with the w/v of acid solution, addition 4 DEG C in lysate or acidic extraction liquid, concentration are the sodium hydroxide solution of 2.0mol/L, its pH value is adjusted to 11, obtains collagen solution；After collagen solution is stood 24 hours at 4 DEG C, take out the collagen suspension layer of collagen solution top cohesion；It is repeated 3 times；
(9) acetum of the collagen suspension layer 10mol/L taking step (8) dissolves, collagen suspension layer is 1: 10 with the w/v of acetum, then directly dialysed by distilled water, the collagen suspension liquid freeze-drying that will obtain after dialysis, obtain macromolecular collagen protein fine work.
It is obvious to a person skilled in the art that the invention is not restricted to the details of above-mentioned one exemplary embodiment, and without departing from the spirit or essential characteristics of the present invention, the present invention can be realized in other specific forms.Therefore, no matter from the point of view of which point, embodiment all should be regarded as exemplary, and be nonrestrictive, the scope of the present invention is limited by claims rather than described above, it is intended that include all changes falling in the implication of equivalency and scope of claim in the present invention.
In addition, should be understood, although this specification is been described by according to embodiment, but not each embodiment only comprises an independent technical scheme, this narrating mode of specification is only for clarity sake, those skilled in the art should be using specification as an entirety, and the technical scheme in each embodiment also can form, through appropriately combined, other embodiments that it will be appreciated by those skilled in the art that.
Claims (4)
1. the method that a low temperature extracts macromolecular collagen protein, it is characterised in that comprise the following steps:
(1) fresh pigskin is selected, remove the pig hair on pigskin and impurity, drain after being cleaned by running water, be cut into the fragment of 1 × 1cm, the fragment being cut into soaks 10-15min in 75% ethanol, drains standby after the deionized water soaking flushing of 4-5 ° of C of taking-up 3～5 times；
(2) removal of impurities albumen: the removal of foreign protein uses sodium hydroxide solution to soak, and the concentration of sodium hydroxide solution is
0.6mol/L, soak time 20-30 hour, soaking temperature 4-5 DEG C, it in immersion process, is stirred continuously, every 5 hours, change sodium hydroxide solution 1 time；
(3) degreasing: use sodium carbonate and deionized water 4 DEG C stirring 3-6h, draining, add 4 ° of C of deionized water and clean, removing fat；
(4) adding acetum to carry out swelling according to the ratio of 1: 30 after degreasing, the concentration of acetum is 0.6mol/L, and swelling temperature is 4 DEG C, and swelling time is 24-36 hour；
(5) tentatively freezing after swelling, solidification point is-25 DEG C, and freeze-off time is 4 hours；Freezing the acetum that rear raw material adds appropriate concentration to be 0.5mol/L, carrying out making beating broken 0.5 minute at 4 DEG C, after making beating, granular size is within 2mm, and making beating and environment temperature are 4 DEG C；Then the acetum being added thereto to 0.5mol/L once extracts 36 hours, and raw material is 1: 20 with the w/v of acetum, extracts after finishing, filters with 40-50 mesh gauze, obtain extract and filter residue；
(6) adding sodium chloride to concentration to be that 2.5mol/L carries out salt precipitation in extract, freeze-drying prepares macromolecular collagen protein crude product；
(7) adding the acetic acid of 0 DEG C-4 DEG C to dissolve in prepared macromolecular collagen protein crude product, obtaining lysate, macromolecular collagen protein crude product is 1: 40 with the w/v of acid solution；Addition 0 DEG C-4 DEG C in lysate or acidic extraction liquid, concentration are the sodium hydroxide solution of 2.0mol/L, its pH value is adjusted to 6-11, obtains collagen solution；After collagen solution is stood 24 hours at 0 DEG C-4 DEG C, take out the collagen suspension layer of collagen solution top cohesion；
(8) collagen suspension layer is dissolved in the acetic acid of 0 DEG C-4 DEG C, collagen suspension layer is 1: 20 with the w/v of acid solution, addition 0 DEG C-4 DEG C in lysate or acidic extraction liquid, concentration are the sodium hydroxide solution of 2.0mol/L, its pH value is adjusted to 6-11, obtains collagen solution；After collagen solution is stood 24 hours at 0 DEG C-4 DEG C, take out the collagen suspension layer of collagen solution top cohesion；
(9) acetum of the collagen suspension layer 0.05-10mol/L taking step (8) dissolves, collagen suspension layer is 1: 10 with the w/v of acetum, then directly dialysed by distilled water, the collagen suspension liquid freeze-drying that will obtain after dialysis, obtains macromolecular collagen protein fine work.
2. the method that low temperature according to claim 1 extracts macromolecular collagen protein, it is characterised in that described pigskin is fresh black pig pigskin.
3. the method that low temperature according to claim 1 extracts macromolecular collagen protein, it is characterised in that the stirring in step (3) uses mixer stirring.
4. the method that low temperature according to claim 1 extracts macromolecular collagen protein, it is characterised in that repeat step (8) 1-3 time, obtain collagen suspension layer.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107602692A (en) * | 2017-08-04 | 2018-01-19 | 青岛金典生化器材有限公司 | A kind of preparation method of collagen diaphragm material |
| CN112474552A (en) * | 2020-10-27 | 2021-03-12 | 广州创尔生物技术股份有限公司 | Cleaning method of beef tendon slices |
| CN114736945A (en) * | 2022-05-10 | 2022-07-12 | 成都奇璞生物科技有限公司 | Degreasing process and collagen extraction method |
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| CN101628937A (en) * | 2009-08-12 | 2010-01-20 | 江苏省海洋资源开发研究院 | Purification technology of macromolecular collagen protein |
| CN101628938A (en) * | 2009-08-12 | 2010-01-20 | 江苏省海洋资源开发研究院 | Extraction technology of macromolecular collagen protein |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107602692A (en) * | 2017-08-04 | 2018-01-19 | 青岛金典生化器材有限公司 | A kind of preparation method of collagen diaphragm material |
| CN112474552A (en) * | 2020-10-27 | 2021-03-12 | 广州创尔生物技术股份有限公司 | Cleaning method of beef tendon slices |
| CN114736945A (en) * | 2022-05-10 | 2022-07-12 | 成都奇璞生物科技有限公司 | Degreasing process and collagen extraction method |
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Application publication date: 20161005 |