CN101851339A - Method for preparing donkey skin collagens - Google Patents
Method for preparing donkey skin collagens Download PDFInfo
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Abstract
The invention discloses a method for preparing donkey skin collagens. In the method, fresh donkey skin is used as a raw material, and modified donkey skin collagen products are modified by steps of unhairing, degreasing, crushing, alkali solution soaking, low-temperature enzymolysis, purification, modification and the like. The donkey skin collagen products prepared by the method is characterized by retaining a triple helical structure, possessing biological activity, being homologous and compatible with human organisms and skin, along with high biological degradability, low antigenicity and the like; and the donkey skin collagens can be diluted by water in any ratio and be used for preparing surgical implants and tissue engineering medical products and developing medicaments, medical preparations, healthcare products and the like.
Description
Technical field
The invention belongs to the preparing technical field of collagen, particularly a kind of preparation method of donkey hide collagen.
Background technology
Collagen is maximum, the widest class protein family that distributes of animal body intensive amount, extensively is present in from low to wait vertebrates to mammiferous all tissues; Have the incomparable biocompatibility of other synthetic materialss, biodegradable and biological activity.Now find the encoding gene of kind of collagen chain surplus in the of 30 at least, can form the tropocollagen molecule more than 16 kinds.According to they distribution and functional characteristicss in vivo, at present collagen is divided into interstitial collagen, basilar membrane collagen and pericellular collagen, between matter Collagen Type VI molecule account for the overwhelming majority of whole machine body collagen, comprise I type, II type, III Collagen Type VI molecule, it mainly is distributed in tissues such as skin, tendon; Basilar membrane collagen typically refers to type. and it mainly is distributed in basilar membrane; The common middle finger collagen type v of cell week collagen exists in reticular tissue in a large number.
In recent years, foster donkey slowly receives livestock industry personage's concern, because not only donkey meat itself has higher economic use value, donkey hide also has very high pharmaceutical use, have natural enrich blood, anastalsis, be the main raw material of making donkey-hide gelatin.
Donkey hide rawhide material is extremely complicated material, is made up of protein and nonprotein two big components basically, and wherein, protein accounts for 30%～35% of fresh hide.Newly peeling the rawhide pH value of coming is 6.8～7.8, rotten gradually with cladding in depositing process, and the pH value is variation thereupon also.The kinds of protein of forming rawhide is a lot, and cladding protein is divided into scleroproein (structural protein), non-fibrous protein (Nonstructural Protein) and enzyme etc.Scleroproein is made up of collagen, angle protein, elastin.Collagen accounts for 29% of rawhide total amount (removing subcutaneous layer), accounts for 88% of all protein total amount.
At present, the application of donkey hide mainly is as the important source material that boils donkey-hide gelatin.According to the record of one one of Chinese Pharmacopoeia version in 2005, the method for making of donkey-hide gelatin is as follows:
Donkey hide (dry skin or the fresh hide of equine species donkey EquusasinusL.) is soaked unhairing, and stripping and slicing is cleaned, gradation decocting, group's mistake, merging filtrate concentrates (can add an amount of yellow rice wine, rock sugar and soya-bean oil respectively) to thick paste shape, condensation, stripping and slicing is dried, promptly.
Above-mentioned simple cooking method, do not carry out selective extraction and refining at the characteristic of contained collagen composition in the donkey hide, make in the donkey-hide gelatin except that containing collagen, also contain multiple amino acids such as Methionin that partial hydrolysis produces, arginine, Histidine, and compositions such as calcium, sulphur; And the poorly water-soluble of donkey-hide gelatin can only molten be taken, can not be as uses such as makeup, and its use is subjected to bigger restriction.
Summary of the invention
Main purpose of the present invention is to only limit to boil donkey-hide gelatin at the application to donkey hide that exists in the above-mentioned prior art, and complicated component in the donkey-hide gelatin, poorly water-soluble, except that being subjected to problems such as bigger restriction as other application the oral pharmaceutical, a kind of preparation method of donkey hide collagen is provided, with the donkey hide is raw material, by specific extraction and process for purification, obtain the donkey hide collagen of good water solubility, retains biological activity not only, with people's human body, skin homology and consistency, and have characteristics such as biodegradability and low antigenicity, can be by water with wilful dilution proportion, can be used for preparing surgical implant and organizational project medical product, exploitation medicine, medicinal agents, and healthcare products etc.
In order to realize the foregoing invention purpose, the technical solution used in the present invention is as follows:
A kind of preparation method of donkey hide collagen comprises following key step:
(1) depilation, degreasing:
With check, the qualified bright donkey hide of quarantine is raw material, after the depilation of employing ordinary method, the degreasing, washes with water to neutrality again, and freezing preservation is standby below 0 ℃;
Because collagen is easy sex change inactivation under normal temperature and hot conditions, thereby need freezing preservation.
(2) onal, dipping by lye:
Getting the standby donkey hide in above-mentioned steps (1) processing back thaws, onal becomes 20-40 purpose particle, adding pH is that 7.5-9.5(can be preferably 7.5-9.0) sodium hydroxide solution, its add-on is according to the donkey hide granular mass: the sodium hydroxide solution volume=1g:(8-12) ml calculates, and soaks softening 48-96h; With deionized water rinsing to pH be 6.0-7.0, obtain neutral donkey hide particle;
The contriver finds by a large amount of tests, because the epidermal structure of donkey hide is tight, be not easy to soften, need earlier it to be broken into the small-particle of certain fineness, just help follow-up softening processing, the contriver confirms by a large amount of tests, when particle is coarser than 20 orders, because particle is too thick, immersion treatment surpasses 96h and also is difficult to whole softening behind the adding alkali lye; In theory, thin more helping more of the particle of onal softens, but because the physicochemical property of bright donkey hide, is difficult to again it is broken into surpass the thinner particle of 40 purposes, therefore, take all factors into consideration, with the 20-40 order for well;
Further, put into the alkali lye of appropriate pH value and carry out immersion treatment being broken to 20-40 purpose donkey hide particle, can soften colloid, help improving the efficient of subsequent step enzymolysis, but the pH value the low or too high bating effect that all is unfavorable for, a large amount of tests confirm, are that the sodium hydroxide solution of 7.5-9.5 is better with pH; And the selection of soak time is then closely related with pH value of donkey hide particulate fineness and alkali lye etc., interacts, and is that 20-40 order, pH are in the 7.5-9.5 scope at the donkey hide particle, and soak time can obtain the ideal bating effect usually in 48-96h;
Onal, dipping by lye by this step specified conditions are handled, and can effectively soften the colloid in the donkey hide, effectively extract donkey hide collagen for steps such as follow-up enzymolysis necessary assurance is provided.
(3) low temperature enzymolysis:
Get the neutral donkey hide particle after above-mentioned steps (2) is handled, adding pH is that 4.3-6.5(can be preferably 4.5-6.0) hydrochloric acid soln, its add-on is according to neutral donkey hide granular mass: the hydrochloric acid soln volume=1g:(45-50) ml calculates, adding 8-10UI(UI again is the enzyme activity international unit, refer under given conditions, transform 1 micromole's substrate, the perhaps required enzyme amount of the relevant group of 1 micromole in the substrate in 1 minute; In pepsic vitality test, be meant: at 37 ℃ ± 0.5 ℃, the required stomach en-amount of per minute energy catalytic hydrolysis hemoglobin production 1 μ mol tyrosine) stomach en-, low temperature (below 10 ℃) is placed 3-4d, stir and leave standstill at interval and handle, that stirs total time is about 1:(1.5-2.5 with the total time ratio that leaves standstill), obtain containing the first liquid of donkey hide collagen;
This step fully is dissolved in the solution collagen in the donkey hide by low temperature enzymolysis processing in hydrochloric acid soln, under the stomach en-effect, helps subsequent step and effectively extracts donkey hide collagen.
(4) extraction, purifying:
The first liquid that above-mentioned steps (3) obtains is centrifugal, obtain supernatant liquor, add NaCl, fully stir, make donkey hide collagen from solution, separate out, precipitate, left standstill 8-16 hour, get precipitation, use deionized water wash 1-2 time, obtain solubility in acid donkey hide collagen precipitation;
This step extracts the collagen that is dissolved in behind the enzymolysis in the solution, and further by the processing of saltouing, makes donkey hide collagen separate out precipitation from solution by centrifugal treating, and other impurity then is retained in the solution and is removed, thereby reaches the purpose of purifying donkey hide collagen;
But what this step obtained is acid soluble collagen, directly unsuitable and skin contact, and the contriver finds by a large amount of experimental studies, by specific modified method, can make it to become water miscible collagen, thereby satisfy the needs of multiple application.
(5) modification:
The solubility in acid donkey hide collagen precipitation that above-mentioned steps (4) is obtained can be preferably 0.4-0.6M with 0.2-1.0M() the acetum dissolving, its consumption precipitates quality according to solubility in acid donkey hide collagen: the acetum volume=1g:(45-50) ml doubly measures calculating; Measure protein content, add acetone (C
3H
6O) as properties-correcting agent, its add-on is according to protein quality: the acetone volume=1g:(10-15) ml calculates, and under 4-8 ℃, is 10-12 with sodium hydrate regulator solution pH, and reaction 5-9h is 4.0-4.2 with HCl adjusting pH again, separates out precipitation, leaves standstill 8-16 hour; Getting precipitation, dry, is that the hydrochloric acid soln of 4.0-4.2 washs 1-3 time with pH, obtains the Colla Corii Asini original product after the modification, gets final product.
This step can effectively be removed the end peptide in the solubility in acid donkey hide collagen by under alkaline condition, as properties-correcting agent donkey hide collagen is carried out modification with acetone, becomes water miscible modification donkey hide collagen, is I type active collagen; Owing to only just removed the end peptide, proteic triple helix structure is not destroyed, so still can keep its original biological activity.Simultaneously, the precipitation after in this step modification being left standstill dries processing and also is very important, and can effectively remove the impurity such as non-fibrous protein that are dissolved in the acetum, to improve the purity of modification donkey hide collagen in the product.
The contriver is by a large amount of simultaneous tests, screen from numerous can be used as in theory protein modified dose the material, discovery is carried out modification with acetone to abovementioned steps (4) gained solubility in acid donkey hide collagen, can obtain the water-soluble collagen of ideal, and have some other properties-correcting agent the characteristics that can not have concurrently, as biological activity, consistency, low antigenicity, biodegradability etc. with people's human body, skin.
The various solution of using among the present invention when being not specifically noted, all are meant the aqueous solution.
Compared with prior art, the invention has the beneficial effects as follows:
The inventive method is a raw material with bright donkey hide, processing step by particular combinations and parameter condition are extracted with refinement treatment and are obtained solubility in acid collagen, adopt acetone under alkaline condition, solubility in acid collagen to be carried out modification again, obtain water miscible Colla Corii Asini original product, this water miscible donkey hide collagen has following characteristics:
(1) the triple helix structure of the collagen after the modification is not destroyed, still keeps its original biological activity;
(2) main component is an I type active collagen about the about 300KD of molecular weight in the product, and good water solubility can be diluted with arbitrary proportion by water;
(3) derive from vertebrate collagen and people's human body, skin have good homology and consistency, its molecular weight and structural similitude; Can dissolve in epidermis fast after being applied to skin;
(4) denaturation temperature of this collagen is about 36.5 ℃, after people's human body, skin absorption, in human body normal body temperature and body under the effect of collagenase, biodegradable is micromolecule polypeptide, single peptide, seed amino acid surplus can under the effect of other enzymes, continuing to resolve into 20 again, easily be absorbed, continue the performance trophism;
(5) by measuring, compare with the collagen that the end peptide is arranged before the modification, the collagen of peptide and the antigenic bonding force of collagen are poor for no reason after the modification, have low antigenicity.
Therefore, this kept biological activity and have good water solubility, with people's human body, skin homology and the active Colla Corii Asini original product of characteristics such as consistency is good, biodegradability and low antigenicity, can be used for preparing surgical implant and organizational project medical product, develop medicine, medicinal agents, reach healthcare products etc.
Description of drawings
Fig. 1 be step in the embodiment of the invention 1 (4) gained solubility in acid donkey hide collagen, step (5) gained water-soluble collagen respectively with the binding curve figure of collagen antibodies; Among Fig. 1, a is the solubility in acid donkey hide collagen of step (4) gained and the binding curve of collagen antibodies, and b is the water-soluble collagen of step (5) gained and the binding curve of collagen antibodies.
Fig. 2 is the temperature variant sex change graphic representation of the embodiment of the invention 1 gained collagen.
Embodiment
Below in conjunction with embodiment foregoing invention content of the present invention is described in further detail.
But this should be interpreted as that the scope of the above-mentioned theme of the present invention only limits to following embodiment.Not breaking away under the above-mentioned technological thought situation of the present invention, according to ordinary skill knowledge and customary means, make various replacements and change, all should comprise within the scope of the invention.
Embodiment 1
The method preparation of present embodiment donkey hide collagen by comprising the steps:
(1) depilation, degreasing:
With check, the qualified bright donkey hide of quarantine is raw material, after depilation, the degreasing, washes with water to neutrality, and freezing preservation is standby below 0 ℃;
(2) onal, dipping by lye:
Get the standby donkey hide in above-mentioned steps (1) processing back and thaw, onal becomes 20-40 purpose particle, gets 500g donkey hide particle, and adding pH is 7.5 sodium hydroxide solution 6000ml, soaks softening 96h; Be about 6.0 with deionized water rinsing to pH, obtain neutral donkey hide particle;
(3) low temperature enzymolysis:
Get the neutral donkey hide particle 200g after above-mentioned steps (2) is handled, adding pH is 6.5 hydrochloric acid soln 9000ml, the stomach en-that adds 8UI again, low temperature below 10 ℃ is placed 3d, stir and leave standstill at interval and handle, it is 1:2.5 that the total time of stirring compared with the total time of leaving standstill, and obtains containing the first liquid of donkey hide collagen;
(4) extraction, purifying:
The first liquid that above-mentioned steps (3) obtains is centrifugal, obtain supernatant liquor, add NaCl, fully stir, make donkey hide collagen from solution, separate out, precipitate, left standstill 8 hours, get precipitation, use deionized water wash 1 time, obtain solubility in acid donkey hide collagen precipitation;
(5) modification:
Get the solubility in acid donkey hide collagen precipitation 10g that above-mentioned steps (4) obtains, acetum 400ml dissolving with 1.0M, measure protein content (being 6.9mg/ml), adding the about 27600ml of acetone as properties-correcting agent, at about 4 ℃, is 10.0 with sodium hydrate regulator solution pH, reaction 9h, regulating pH with HCl again is 4.0, separates out precipitation, leaves standstill 16 hours; Getting precipitation, dry, is that 4.0 hydrochloric acid soln washs 3 times with pH, obtains the Colla Corii Asini original product after the modification, gets final product.
The contriver entrusts Sichuan medicine equipment biomaterial and goods inspection center that the modification Colla Corii Asini original product that the foregoing description obtains is tested, survey report is presented at the corresponding colour band that α, β, γ are arranged in the electrophorogram, and molecular weight is respectively 100KD, 200KD and 300KD.
Further water-soluble, antigenicity, with tests such as the consistency demonstration of people's human body, skin: this product can be diluted with arbitrary proportion by water, good water solubility; Poor with the antigenic bonding force of collagen, in the binding curve figure (as shown in Figure 1) of collagen and collagen antibodies, be almost a straight line, have low antigenicity; Dissolve in epidermis rapidly after being applied to people's human body, skin, good with people's human body, skin-friendliness; 36.5 ℃ of left and right sides sex change degradeds and viscosity reduces (as shown in Figure 2).
Embodiment 2
The method preparation of present embodiment donkey hide collagen by comprising the steps:
(1) depilation, degreasing:
With check, the qualified bright donkey hide of quarantine is raw material, after depilation, the degreasing, washes with water to neutrality, and freezing preservation is standby below 0 ℃;
(2) onal, dipping by lye:
Get the standby donkey hide in above-mentioned steps (1) processing back and thaw, onal becomes 20-40 purpose particle, gets 500g donkey hide particle, and adding pH is 9.5 sodium hydroxide solution 4000ml, soaks softening 48h; Be about 7.0 with deionized water rinsing to pH, obtain neutral donkey hide particle;
(3) low temperature enzymolysis:
Get the neutral donkey hide particle 200g after above-mentioned steps (2) is handled, adding pH is 4.3 hydrochloric acid soln 10000ml, the stomach en-that adds 10UI again, low temperature below 10 ℃ is placed 4d, stir and leave standstill at interval and handle, it is 1:1.5 that the total time of stirring compared with the total time of leaving standstill, and obtains containing the first liquid of donkey hide collagen;
(4) extraction, purifying:
The first liquid that above-mentioned steps (3) obtains is centrifugal, obtain supernatant liquor, add NaCl, fully stir, make donkey hide collagen from solution, separate out, precipitate, left standstill 16 hours, get precipitation, use deionized water wash 2 times, obtain solubility in acid donkey hide collagen precipitation;
(5) modification:
Get the solubility in acid donkey hide collagen precipitation 10g that above-mentioned steps (4) obtains, acetum 500ml dissolving with 0.2M, measure protein content (being 4.1mg/ml), adding the about 30750ml of acetone as properties-correcting agent, at about 8 ℃, is 12.0 with sodium hydrate regulator solution pH, reaction 5h, regulating pH with HCl again is 4.2, separates out precipitation, leaves standstill 8 hours; Getting precipitation, dry, is that 4.2 hydrochloric acid soln washs 2 times with pH, obtains the Colla Corii Asini original product after the modification, gets final product.
Embodiment 3
The method preparation of present embodiment donkey hide collagen by comprising the steps:
(1) depilation, degreasing:
With check, the qualified bright donkey hide of quarantine is raw material, after depilation, the degreasing, washes with water to neutrality, and freezing preservation is standby below 0 ℃;
(2) onal, dipping by lye:
Get the standby donkey hide in above-mentioned steps (1) processing back and thaw, onal becomes 20-40 purpose particle, gets 500g donkey hide particle, and adding pH is 8.0 sodium hydroxide solution 5000ml, soaks softening 72h; Be about 6.5 with deionized water rinsing to pH, obtain neutral donkey hide particle;
(3) low temperature enzymolysis:
Get the neutral donkey hide particle 200g after above-mentioned steps (2) is handled, adding pH is 4.5 hydrochloric acid soln 9500ml, the stomach en-that adds 8UI again, low temperature below 10 ℃ is placed 3d, stir and leave standstill at interval and handle, it is 1:2 that the total time of stirring compared with the total time of leaving standstill, and obtains containing the first liquid of donkey hide collagen;
(4) extraction, purifying:
The first liquid that above-mentioned steps (3) obtains is centrifugal, obtain supernatant liquor, add NaCl, fully stir, make donkey hide collagen from solution, separate out, precipitate, left standstill 12 hours, get precipitation, use deionized water wash 2 times, obtain solubility in acid donkey hide collagen precipitation;
(5) modification:
Get the solubility in acid donkey hide collagen precipitation 10g that above-mentioned steps (4) obtains, acetum 460ml dissolving with 0.4M, measure protein content (being 7.5mg/ml), adding the about 41400ml of acetone as properties-correcting agent, at about 6 ℃, is 11.0 with sodium hydrate regulator solution pH, reaction 6h, regulating pH with HCl again is 4.2, separates out precipitation, leaves standstill 12 hours; Getting precipitation, dry, is that 4.2 hydrochloric acid soln washs 2 times with pH, obtains the Colla Corii Asini original product after the modification, gets final product.
The method preparation of present embodiment donkey hide collagen by comprising the steps:
(1) depilation, degreasing:
With check, the qualified bright donkey hide of quarantine is raw material, after depilation, the degreasing, washes with water to neutrality, and freezing preservation is standby below 0 ℃;
(2) onal, dipping by lye:
Get the standby donkey hide in above-mentioned steps (1) processing back and thaw, onal becomes 20-40 purpose particle, gets 500g donkey hide particle, and adding pH is 9.0 sodium hydroxide solution 5500ml, soaks softening 60h; Be about 6.8 with deionized water rinsing to pH, obtain neutral donkey hide particle;
(3) low temperature enzymolysis:
Get the neutral donkey hide particle 200g after above-mentioned steps (2) is handled, adding pH is 6.0 hydrochloric acid soln 9800ml, the stomach en-that adds 9UI again, low temperature below 10 ℃ is placed 4d, stir and leave standstill at interval and handle, it is 1:2 that the total time of stirring compared with the total time of leaving standstill, and obtains containing the first liquid of donkey hide collagen;
(4) extraction, purifying:
The first liquid that above-mentioned steps (3) obtains is centrifugal, obtain supernatant liquor, add NaCl, fully stir, make donkey hide collagen from solution, separate out, precipitate, left standstill 12 hours, get precipitation, use deionized water wash 1 time, obtain solubility in acid donkey hide collagen precipitation;
(5) modification:
Get the solubility in acid donkey hide collagen precipitation 10g that above-mentioned steps (4) obtains, acetum 480ml dissolving with 0.6M, measure protein content (being 5.2mg/ml), adding the about 25000ml of acetone as properties-correcting agent, at about 4 ℃, is 11.0 with sodium hydrate regulator solution pH, reaction 7h, regulating pH with HCl again is 4.1, separates out precipitation, leaves standstill 14 hours; Getting precipitation, dry, is that 4.1 hydrochloric acid soln washs 1 time with pH, obtains the Colla Corii Asini original product after the modification, gets final product.
The modification Colla Corii Asini original product that the contriver entrusts same unit that the various embodiments described above are obtained carry out electrophoresis test, and water-soluble, antigenicity, with the tests such as consistency of people's human body, skin, the result is all similar to the test result of the original product of embodiment 1 gained.
Claims (4)
1. the preparation method of a donkey hide collagen comprises the steps:
(1) depilation, degreasing:
With check, the qualified bright donkey hide of quarantine is raw material, after depilation, the degreasing, washes with water to neutrality, and freezing preservation is standby below 0 ℃;
(2) onal, dipping by lye:
Getting the standby donkey hide in above-mentioned steps (1) processing back thaws, onal becomes 20-40 purpose particle, adding pH is the sodium hydroxide solution of 7.5-9.5, and its add-on is according to the donkey hide granular mass: the sodium hydroxide solution volume=1g:(8-12) ml calculates, and soaks softening 48-96h; With deionized water rinsing to pH be 6.0-7.0, obtain neutral donkey hide particle;
(3) low temperature enzymolysis:
Get the neutral donkey hide particle after above-mentioned steps (2) is handled, adding pH is the hydrochloric acid soln of 4.3-6.5, its add-on is according to neutral donkey hide granular mass: the hydrochloric acid soln volume=1g:(45-50) ml calculates, the stomach en-that adds 8-10UI again, low temperature below 10 ℃ is placed 3-4d, stir and leave standstill at interval and handle, the total time of stirring and the total time of leaving standstill are than being 1:(1.5-2.5), obtain containing the first liquid of donkey hide collagen;
(4) extraction, purifying:
The first liquid that above-mentioned steps (3) obtains is centrifugal, obtain supernatant liquor, add NaCl, fully stir, make donkey hide collagen from solution, separate out, precipitate, left standstill 8-16 hour, get precipitation, use deionized water wash 1-2 time, obtain solubility in acid donkey hide collagen precipitation;
(5) modification:
Use the acetum of 0.2-1.0M to dissolve the solubility in acid donkey hide collagen precipitation that above-mentioned steps (4) obtains, its consumption precipitates quality according to solubility in acid donkey hide collagen: the acetum volume=1g:(45-50) ml doubly measures calculating; Measure protein content, add acetone as properties-correcting agent, its add-on is according to protein quality: the acetone volume=1g:(10-15) ml calculates; Under 4-8 ℃, be 10.0-12.0 with sodium hydrate regulator solution pH, reaction 5-9h, regulating pH with HCl again is 4.0-4.2, separates out precipitation, leaves standstill 8-16 hour; Getting precipitation, dry, is that the hydrochloric acid soln of 4.0-4.2 washs 1-3 time with pH, obtains the Colla Corii Asini original product after the modification, gets final product.
2. preparation method according to claim 1 is characterized in that:
The sodium hydroxide solution pH that adds in the described step (2) is 7.5-9.0.
3. preparation method according to claim 1 is characterized in that:
The hydrochloric acid soln pH that adds in the described step (3) is 4.5-6.0.
4. preparation method according to claim 1 is characterized in that:
In the described step (5), with the acetum dissolving acid dissolubility donkey hide collagen precipitation of 0.4-0.6M.
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| CN112608379A (en) * | 2020-12-03 | 2021-04-06 | 青岛大学附属医院 | Preparation method of donkey skin collagen |
| CN112625122A (en) * | 2020-12-30 | 2021-04-09 | 福建纽伯尔生物科技有限公司 | Collagen fiber extraction method |
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| CN107619845A (en) * | 2017-10-16 | 2018-01-23 | 吴义峰 | A kind of preparation method of collagen |
| CN108795909A (en) * | 2018-05-08 | 2018-11-13 | 北京开元鲁采餐饮管理有限公司 | A kind of method of purification of neutral proteinase |
| CN108795909B (en) * | 2018-05-08 | 2020-07-03 | 北京开元鲁采餐饮管理有限公司 | Preparation method of donkey skin for diet |
| CN112608379A (en) * | 2020-12-03 | 2021-04-06 | 青岛大学附属医院 | Preparation method of donkey skin collagen |
| CN112625122A (en) * | 2020-12-30 | 2021-04-09 | 福建纽伯尔生物科技有限公司 | Collagen fiber extraction method |
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| CN101851339B (en) | 2011-10-26 |
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