CN105648017A - Extraction method of mammal collagen - Google Patents
Extraction method of mammal collagen Download PDFInfo
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- CN105648017A CN105648017A CN201610226706.9A CN201610226706A CN105648017A CN 105648017 A CN105648017 A CN 105648017A CN 201610226706 A CN201610226706 A CN 201610226706A CN 105648017 A CN105648017 A CN 105648017A
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- collagen protein
- collagen
- mammal
- extracting method
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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]
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
Abstract
The invention discloses an extraction method of mammal collagen. The method comprises the following steps: cleaning a raw material, removing fat and fasciae, immersing in a sodium carbonate solution, rinsing with distilled water, and airing; adding proteolytic enzyme into the treated raw material, adding an acetic acid solution, slowly stirring, centrifugating, and taking out the precipitate to obtain a crude extract collagen solution; adding an H2O2 solution into the crude extract collagen solution, uniformly mixing, standing for 3-6 hours, regulating the pH value to 5-6 with a trisodium citrate solution, centrifugating, carrying out salting-out treatment, putting the precipitate into a dialysis bag, dialyzing with an acetic acid solution for 1-1.5 days, and dialyzing with distilled water for 3-4 days, thereby finally obtaining the collagen water solution. The method greatly enhances the collagen purity, and completely maintains the activity of collagen.
Description
Technical field
The present invention relates to the extracting method of a kind of collagen protein, particularly to the extracting method of a kind of mammal collagen protein.
Background technology
Collagen protein, is a kind of functional protein, and it has very strong biological activity and biological function, is therefore widely used in the industrial circles such as medicine, food, daily-use chemical industry, biosynthesis.
Collagen protein is the protein that animal body intensive amount is maximum, account for the 25%-35% of human body protein, be equivalent to the 6% of human body weight, it is distributed across each histoorgan of whole body, such as skeleton, cartilage, ligament, skin, cornea, various inner membrance and fascia etc., especially in the skin of human body and connective tissue, containing substantial amounts of collagen protein.
Generally, the manufacturing process of collagen protein is complex, is mainly used in medical treatment and biomedical material aspect, and such as artificial skin, hemorrhage, artificial blood vessel and skin protection cosmetics etc., therefore price is higher.
The water-disintegrable collagen protein of presently commercially available some is almost the same with protein hydrolysate, and not only purity is relatively low, and its original physiologically active is also destroyed, and this is all owing to existing extracting mode causes. This use scope that also strongly limit collagen protein and result of use.
Summary of the invention
It is an object of the invention to: proposing the extracting method of a kind of mammal collagen protein, it not only substantially increases the purity of collagen protein, additionally it is possible to the activity retaining collagen protein of 100%.
The technical solution adopted for the present invention to solve the technical problems is:
The extracting method of a kind of mammal collagen protein, comprises the steps:
A, Feedstock treating: cleaned up by raw material, after removing fat and fascia, put into and soak in sodium carbonate liquor 3-6 hour, after then rinsing 3-4 time with distilled water again, dry;
B, enzymolysis processing: adding quality proportioning in step a raw materials treated is the proteolytic enzyme of 0.2WT%-0.3WT%, and adding concentration is 0.2mol/L acetic acid solution, and is slowly stirred 3-5 days, is then centrifuged for taking out precipitation, prepares and slightly carry collagen solution;
C, collagen protein purification: slightly carry that to add mass fraction in collagen solution be the H of 0.5%-1.5% prepared2O2Solution, stand 3-6 hour after mix homogeneously, afterwards, then regulate pH value to 5-6 with citric acid three sodium solution, after centrifugal, carry out process of saltouing again, again precipitate is loaded in bag filter, dialyse 1-1.5 days with the acetic acid solution of 0.2mol/L, then dialyse 3-4 days with distilled water, and every day change dialysis solution 2-3 time, finally give collagen protein water liquid;
D, vital preservation process: add the compound enzyme of 90-110ppm in collagen protein water liquid, and then under-50 DEG C of vacuum ultra low temperature state, lyophilization processes, and prepare mammal collagen protein.
Further, described principle process in after grease removal fat and fascia, first after freezing, place in sodium carbonate liquor at the temperature of 2-3 DEG C.
Further, described sodium carbonate liquor is 0.05%-0.1%.
Further, the concentration of described acetic acid solution is 0.3mol/L, and pH value is 2-3.
Further, described enzymolysis processing is slowly stirred carries out when 8-10 DEG C.
Further, described in saltout process at pH value under the acid condition of 4-6, by the concentration of the sodium chloride that concentration is 0.3mol/L, the collagen solution after centrifugal is processed.
Further, in the process of described vital preservation, compound enzyme includes cellulase, trehalase, sprout short stalk enzyme and mannase, and this compound enzyme can effectively reduce collagen and run off, and 100% retains the function of collagen protein activity and natural activity.
The invention has the beneficial effects as follows: the extraction scheme of the present invention not only solves the problem that existing collagen protein purity is low, but also the complete triple-helix structure of tropocollagen molecule can be remain, accomplish the physiologically active retaining collagen protein of 100%, so that the result of use of the collagen protein extracted is better, the collagen protein extracted by this method possesses superpower repair ability, strengthening cutin repairs cuticular effect, reduce sebum film to run off, promote the synthesis of skin self moisturizing physiological compositions, powerful reparation power is had for sensitive flesh, red swelling of the skin, decortication, pox pox, dermatitis cicatrix even can use rubber originally to repair, and its superpower active component can promote skin regeneration, strengthen skin resistance, improve acne, small pox, the former environment that can form a micro-acid after being absorbed by the skin around small pox of rubber, this environment has the effect of antimicrobial antiphlogistic disease, help releive small pox flesh and dermatitis inflammation worsen, help dredging pore to reach to promote sebum normal secretions effect simultaneously, promote skin resistance to oxidation and antimicrbial power, and the early stage pigementation after acne healing can be improved, early stage shallow scars, defying age desalination microgroove bright color, life-time service can help skin to recover own metabolism function, it is suppressed that melanin generates and precipitation, bright color, supplements the collagen of loss continually for skin, makes cutin more healthy.
Detailed description of the invention
Below in conjunction with embodiment, the invention will be further described.
Embodiment 1:
The extracting method of a kind of mammal collagen protein, comprises the steps:
A, Feedstock treating: raw material is cleaned up, after removing fat and fascia, freezing processing at the temperature of 2 DEG C, place in 0.07% sodium carbonate liquor and soak 4 hours, after then rinsing 3 times with distilled water again, dry.
B, enzymolysis processing: adding quality proportioning in the raw material after drying is the proteolytic enzyme of 0.3WT%, add the acetic acid solution (PH is 2) that concentration is 0.2mol/L, and when 8 DEG C, be slowly stirred 4 days, it is then centrifuged for taking out precipitation, prepares and slightly carry collagen solution.
C, collagen protein purification: slightly carry that to add mass fraction in collagen solution be the H of 1.2% prepared2O2Solution, stand 5 hours after mix homogeneously, afterwards, then regulate pH value to 5 with citric acid three sodium solution, after centrifugal, at pH value under the acid condition of 5, put into the sodium chloride solution that concentration is 0.3mol/L and carry out precipitation process, then precipitate is loaded in bag filter, dialyse 1 day with the acetic acid solution of 0.2mol/L, dialyse 3 days with distilled water again, and change dialysis solution every day 2 times, finally give collagen protein water liquid.
D, vital preservation process: add the compound enzyme of 100ppm in collagen protein water liquid, described compound enzyme includes cellulase, trehalase, sprout short stalk enzyme and mannase, then under-50 DEG C of vacuum ultra low temperature state, lyophilization processes, and prepares mammal collagen protein.
Embodiment 2:
The extracting method of a kind of mammal collagen protein, comprises the steps:
A, Feedstock treating: raw material is cleaned up, after removing fat and fascia, freezing processing at the temperature of 3 DEG C, place in 0.1% sodium carbonate liquor and soak 6 hours, after then rinsing 4 times with distilled water again, dry.
B, enzymolysis processing: adding quality proportioning in the raw material after drying is the proteolytic enzyme of 0.26WT%, add the acetic acid solution (PH is 3) that concentration is 0.2mol/L, and when 10 DEG C, be slowly stirred 5 days, it is then centrifuged for taking out precipitation, prepares and slightly carry collagen solution.
C, collagen protein purification: slightly carry that to add mass fraction in collagen solution be the H of 1.5% prepared2O2Solution, stand 5 hours after mix homogeneously, afterwards, then regulate pH value to 6 with citric acid three sodium solution, after centrifugal, at pH value under the acid condition of 6, put into the sodium chloride solution that concentration is 0.3mol/L and carry out precipitation process, then precipitate is loaded in bag filter, dialyse 1.5 days with the acetic acid solution of 0.2mol/L, dialyse 4 days with distilled water again, and change dialysis solution every day 3 times, finally give collagen protein water liquid.
D, vital preservation process: add the compound enzyme of 100ppm in collagen protein water liquid, described compound enzyme includes cellulase, trehalase, sprout short stalk enzyme and mannase, then under-50 DEG C of vacuum ultra low temperature state, lyophilization processes, and prepares mammal collagen protein.
Comparative example:
Test group A: the mammal collagen protein that embodiment 1 produces
Experiment group B: mammal collagen protein produced by embodiment 2
Test group C: commercially available general mammal collagen protein
Analyze through Akta and show, the product purity of test group A and experiment group B respectively 98.3% and 98.8%.
From above analysis of experiments result: the mammal collagen protein that the present invention extracts possesses complete triple-helix structure, maintain complete biologically active structure, and commercially available general mammal collagen protein is Denatured protein, not possessing biological activity, molecular weight presents irregular distribution at random. But, collagen protein effect in Clinical practice is mainly soluble collagen, and makes the physiological function of collagen protein obtain sufficient embodiment owing to maintaining complete triple-helix structure.
The present invention extracts the collagen protein of gained can reach the rank of medical I type active collagen. The collagen constituting corium is exactly mainly NTx, and in human body, collagen about 90% is NTx.
The central role mechanism of I type active collagen is as follows:
One, moisture retention
Triple-helix structure tropocollagen molecule, containing nature moisturizing factor and hydrophilic group, coordinates the polysaccharide component needed for three kinds of skin, it is possible to decrease transepidermal water loss rate (TEWL), moisturizing at the bottom of flesh;
Two, prosthetic
Skin exists in micro-damage situation, promotes epidermis cell normal growth, has the automatic repair function of uniqueness, guides epithelial cell to move into defective region, and plays support and lubrication when cell migration, improves epidermis cell microcirculation.
Three, film property
Complete triple-helix structure tropocollagen molecule forms protecting film at skin surface, improves its metabolism;Recover skin microenvironment balance, accelerate skin microcirculation; Simulation skin barrier function, recovers to improve skin self-resistance.
The ultimate principle of the present invention and principal character and advantages of the present invention have more than been shown and described. Skilled person will appreciate that of the industry; the present invention is not restricted to the described embodiments; described in above-described embodiment and description is that principles of the invention is described; without departing from the spirit and scope of the present invention; the present invention also has various changes and modifications, and these changes and improvements both fall within the claimed scope of the invention. Claimed scope is defined by appending claims and equivalent thereof.
Claims (7)
1. the extracting method of a mammal collagen protein, it is characterised in that comprise the steps:
A, Feedstock treating: cleaned up by raw material, after removing fat and fascia, put into and soak in sodium carbonate liquor 3-6 hour, after then rinsing 3-4 time with distilled water again, dry;
B, enzymolysis processing: adding quality proportioning in step a raw materials treated is the proteolytic enzyme of 0.2WT%-0.3WT%, and adding concentration is 0.2mol/L acetic acid solution, and is slowly stirred 3-5 days, is then centrifuged for taking out precipitation, prepares and slightly carry collagen solution;
C, collagen protein purification: slightly carry that to add mass fraction in collagen solution be the H of 0.5%-1.5% prepared2O2Solution, stand 3-6 hour after mix homogeneously, afterwards, then regulate pH value to 5-6 with citric acid three sodium solution, after centrifugal, carry out process of saltouing again, again precipitate is loaded in bag filter, dialyse 1-1.5 days with the acetic acid solution of 0.2mol/L, then dialyse 3-4 days with distilled water, and every day change dialysis solution 2-3 time, finally give collagen protein water liquid;
D, vital preservation process: add the compound enzyme of 90-110ppm in collagen protein water liquid, and then under-50 DEG C of vacuum ultra low temperature state, lyophilization processes, and prepare mammal collagen protein.
2. the extracting method of a kind of mammal collagen protein as claimed in claim 1, it is characterised in that described principle process in after grease removal fat and fascia, first after freezing, place in sodium carbonate liquor at the temperature of 2-3 DEG C.
3. the extracting method of a kind of mammal collagen protein as claimed in claim 2, it is characterised in that described sodium carbonate liquor is 0.05%-0.1%.
4. the extracting method of a kind of mammal collagen protein as claimed in claim 1, it is characterised in that the concentration of described acetic acid solution is 0.3mol/L, and pH value is 2-3.
5. the extracting method of a kind of mammal collagen protein as claimed in claim 1, it is characterised in that be slowly stirred in described enzymolysis processing and carry out when 8-10 DEG C.
6. the extracting method of a kind of mammal collagen protein as claimed in claim 1, it is characterized in that, described process of saltouing is at pH value under the acid condition of 4-6, by the concentration of the sodium chloride that concentration is 0.3mol/L, the collagen solution after being centrifuged is processed.
7. the extracting method of a kind of mammal collagen protein as claimed in claim 1, it is characterised in that in the process of described vital preservation, compound enzyme includes cellulase, trehalase, sprout short stalk enzyme and mannase.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610226706.9A CN105648017A (en) | 2016-04-13 | 2016-04-13 | Extraction method of mammal collagen |
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| CN201610226706.9A CN105648017A (en) | 2016-04-13 | 2016-04-13 | Extraction method of mammal collagen |
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| CN105648017A true CN105648017A (en) | 2016-06-08 |
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| CN201610226706.9A Pending CN105648017A (en) | 2016-04-13 | 2016-04-13 | Extraction method of mammal collagen |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106337074A (en) * | 2016-10-24 | 2017-01-18 | 广东工业大学 | Cirrhinus molitorella bone collagen extracting method |
| CN112022735A (en) * | 2020-09-08 | 2020-12-04 | 上海硅健生物材料有限公司 | Novel collagen mask and preparation method thereof |
-
2016
- 2016-04-13 CN CN201610226706.9A patent/CN105648017A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106337074A (en) * | 2016-10-24 | 2017-01-18 | 广东工业大学 | Cirrhinus molitorella bone collagen extracting method |
| CN112022735A (en) * | 2020-09-08 | 2020-12-04 | 上海硅健生物材料有限公司 | Novel collagen mask and preparation method thereof |
| CN112022735B (en) * | 2020-09-08 | 2023-10-10 | 上海硅健生物材料有限公司 | Preparation method of collagen facial mask |
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Application publication date: 20160608 |