CN102671231A - Method for preparing marine-derived compound collagen dressing - Google Patents
Method for preparing marine-derived compound collagen dressing Download PDFInfo
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- CN102671231A CN102671231A CN201110427726XA CN201110427726A CN102671231A CN 102671231 A CN102671231 A CN 102671231A CN 201110427726X A CN201110427726X A CN 201110427726XA CN 201110427726 A CN201110427726 A CN 201110427726A CN 102671231 A CN102671231 A CN 102671231A
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- 150000001875 compounds Chemical class 0.000 title abstract 7
- 241000251468 Actinopterygii Species 0.000 claims abstract description 72
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- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 81
- 239000000243 solution Substances 0.000 claims description 76
- SQDAZGGFXASXDW-UHFFFAOYSA-N 5-bromo-2-(trifluoromethoxy)pyridine Chemical compound FC(F)(F)OC1=CC=C(Br)C=N1 SQDAZGGFXASXDW-UHFFFAOYSA-N 0.000 claims description 43
- 229920001287 Chondroitin sulfate Polymers 0.000 claims description 43
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- Materials For Medical Uses (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention discloses a method for preparing a marine-derived compound collagen dressing, comprising the following steps of: extracting collagen from marine fish skins by an enzymic method; extracting chondroitin sulfuric acid from marine fish bones; simultaneously dissolving the extracted collagen and chondroitin sulfuric acid with a flocculant, a growth factor, an anti-inflammatory disinfectant and a cross-linking agent in acetum and mixing evenly; injecting the obtained mixed solution in a die for cross-linking and then quickly transferring the mixed solution to a freezer dryer so as to be freeze-dried in vacuum, thereby obtaining a crude compound collagen dressing; packaging the obtained crude compound collagen dressing, sterilizing the package under 60 DEG C irradiation, thereby obtaining the finished product marine-derived compound collagen dressing. The invention provides a method for preparing the compound collagen dressing from the collagen and the chondroitin sulfuric acid extracted from marine fishes; therefore, the multipurpose utilization rate of the marine fishes can be increased; and the compound collagen dressing prepared by the method provided by the invention is low in cost, good in safety, high in toughness and adhesiveness, and capable of promoting healing of wounds.
Description
Technical field
The present invention relates to biomedical materials field, especially relate to the method for preparing of source, a kind of ocean composite collagen dressing.
Background technology
Skin is to resist extraneous bacterial invasion, safeguard that organismic internal environment stablizes, prevents the natural cover for defense of water evaporates, but in daily life and work, unexpected skin trauma happens occasionally; And traditional dressing such as plant fiber cloth anthemorrhagic speeds such as gauze, absorbent cotton are slow; And only be to play isolated effect, itself not antibiotic, biocidal property must cooperate medicine to stop blooding or sterilizes; And these dressing can not waterproof, and weather or environmental factors can make the wound supervention infect.
Collagen protein is a kind of natural hemorrhage, has good biological characteristics and function, can promote new cell formation and promote epithelial cell, fibroblastic growth; Can adhere to by induced platelet simultaneously, promote platelet to focus on, activate the activity of thrombin; And have excellent biological compatibility with tissue and cell, collagen dressing then can make wound clean, slows down the bacterial infection phenomenon; Reduce wound and suppurate, increase the formation of granulation tissue, make the wound of healing can not produce contraction; And can form the cell needed aminoacid of growing after the collagen dressing degraded, and for wound repair provides nutrient substance, accelerating wound healing; In addition, the loose structure of the collagen dressing of sponge-type can be induced repair cell in dressing, to soak into and bred, and is used for the wound surface covering for a long time.
Chinese patent publication number: CN1569244A; Open day on January 26th, 2005; Disclosing a kind of is the biological collagen hemostasis silk floss and the production technology thereof of raw material production with the biological collagen, and its production stage is following: it is for use 1) biological collagen to be worn into 200～800 purpose biological collagen dry powder; 2) biological collagen dry powder and bioresorbable hemostasis is continuous with 0.5～1.5: 9 weight ratio mixes, and is filled into punching press in the mould then; 3) hemostasis of the biological collagen after punch forming silk floss is placed 120～170 degrees centigrade of oven dry down, get finished product.
But the dressing mechanical performance of processing with collagen protein merely is relatively poor, and haemostatic effect is undesirable, and adhesion is relatively poor, is prone to come off.
Chondroitin sulfate is a kind of acid mucopolysaccharide, owing to have a large amount of negative charges in the molecule, therefore has effects such as the fat of accent, antiinflammatory, accelerating wound healing; Can also promote the growth of fiber in the substrate, strengthen permeability, improve blood circulation; Quicken metabolism, promote the absorption of penetrating fluid and the elimination of inflammation, combine with collagen protein; Can form crosslinked action and electrostatic interaction, between hydroxyl, amido, be cross-linked with each other, improve the toughness of collagen sponge dressing.
But; Collagen protein and chondroitin sulfate are mainly derived from connective tissue and the cartilage in the Lu Sheng slaughtered animals, but since the appearance of disease such as bovine spongiform encephalopathy, foot and mouth disease in recent years with spread, make the collagen protein and the chondroitin sulfate in terrestrial animal source receive very big restriction in application facet; China is a sea fishery big country; The stock of fish is abundant, contains abundant collagen protein and chondroitin sulfate in epidermis of marine fishes and the cartilage, has very big potentiality to be exploited and value.
Summary of the invention
The objective of the invention is to extract collagen protein and the inevitable viral hidden danger of chondroitin sulfate in order to stop in terrestrial animal, and it is relatively poor to overcome the simple dressing mechanical performance of processing with collagen protein, haemostatic effect is undesirable; And adhesion is relatively poor, and caducous deficiency provides a kind of collagen protein that from marine fishes, extracts and chondroitin sulfate usually to prepare the method for composite collagen dressing; The present invention not only can improve the comprehensive utilization ratio of marine fishes, and lower through the composite collagen dressing cost of the present invention's preparation, and safety is good; Toughness is high; Adhesion is strong, can promote the healing of wound, can be widely used in the treatment of skin trauma face.
To achieve these goals, the present invention adopts following technical scheme:
The method for preparing of a kind of ocean of the present invention source composite collagen dressing may further comprise the steps:
One. utilize enzyme process from the marine fishes epidermis, to extract collagen protein.Because collagen protein is mainly derived from the connective tissue in the Lu Sheng slaughtered animals; There is certain bio-safety hidden danger in the collagen protein in terrestrial animal source; And the collagen protein that from marine fishes, extracts can be avoided this potential safety hazard, and simultaneously, China is a fishery big country; The discarded fish skin that produces in the marine fishes processing is used for extracting collagen protein, has improved the utilization rate of marine fishes when reducing the pollution to environment again.The chemical method hydrolysis is broken off the protein peptide bond with acid or alkali, because reaction environment is more extreme, is unfavorable for active maintenance, and enzyme process is with respect to the chemical method hydrolysis, and safety is higher, under temperate condition, positions hydrolysis, and hydrolytic process is also than being easier to control.Described marine fishes epidermis comprises the epidermis of various marine fishes such as squid skin, cod skin.
Two. from the marine fishes fishbone, extract chondroitin sulfate.Chondroitin sulfate is mainly derived from the cartilage in the Lu Sheng slaughtered animals; There is bio-safety hidden danger equally; And the chondroitin sulfate that from the marine fishes fishbone, extracts can be avoided this potential safety hazard; From the marine fishes fishbone, extract chondroitin sulfate, also can improve the utilization rate of marine fishes when reducing the pollution to environment.Described marine fishes fishbone can be the fishbone of various marine fishes such as anglerfish, Anguillar japonica, shark.
Three. the collagen protein of step 1 extraction, chondroitin sulfate and flocculating agent, somatomedin, antiinflammatory disinfectant, the cross-linking agent that step 2 is extracted are dissolved in mix homogeneously in the acetum that concentration is 0.05-0.2 mol/L simultaneously; In the mixed liquor, the quality percentage composition of each component is: collagen protein is 2.0～3.0%, and chondroitin sulfate is 1.0～1.5%; Flocculating agent is 0.03～0.05%; Somatomedin is 0.001～0.005%, and the antiinflammatory disinfectant is 0.1～0.4%, and cross-linking agent is 0.3～0.8%; The gained mixed liquor is injected mould, and pre-cooling 5～8h after leaving standstill 12～18 h under 0～4 ℃ goes to rapidly at last and just obtains composite collagen dressing bullion in the freezer dryer behind vacuum lyophilization 24～36h; After the composite collagen dressing bullion packing that obtains, with obtaining composite collagen dressing finished product behind 60Co irradiation sterilization 30～60 min.Collagen protein is a kind of natural hemorrhage, has good biological characteristics and function, can promote new cell formation and promote epithelial cell, fibroblastic growth; Can adhere to by induced platelet simultaneously, promote platelet to focus on, activate the activity of thrombin; And have excellent biological compatibility with tissue and cell, collagen dressing then can make wound clean, slows down the bacterial infection phenomenon; Reduce wound and suppurate, increase the formation of granulation tissue, make the wound of healing can not produce contraction; And can form the cell needed aminoacid of growing after the collagen dressing degraded, for wound repair provides nutrient substance, to promote the healing of wound.Chondroitin sulfate is a kind of acid mucopolysaccharide; Owing to have a large amount of negative charges in the molecule, therefore have effects such as the fat of accent, antiinflammatory, accelerating wound healing, can also promote the growth of fiber in the substrate; Strengthen permeability; Improve blood circulation, quicken metabolism, promote the absorption of penetrating fluid and the elimination of inflammation.Flocculating agent can make the collagen protein in the solution form flco more easily; Make the composite collagen dressing after the preparation be the spongy of loose structure, the loose structure of spongiform composite collagen dressing has also improved and the skin adhesion when improving the mechanical strength of composite collagen dressing, and it can be covered on the wound surface for a long time; Can also induce repair cell in dressing, to soak into and breed; Somatomedin can promote the Regeneration and Repair of wound face skin histology for having the active cytokine of stimulating cellular growth, thereby strengthens the healing ability of wound; And the antiinflammatory disinfectant can be when the collagen combine dressing be attached on the wound face; Can slowly discharge, strengthen partial antibacterial ability, avoid the infection of wound face; Cross-linking agent can make other compositions and the collagen cross-linking in the solution form RF together, improves the mechanical strength of composite collagen dressing with this.Described mould can be processed different shapes according to the needs of clinical use, and lyophilization is to utilize the distillation principle, after the material to be dried quick freezing, and the drying means of under high vacuum condition, ice distillation wherein being removed for steam again.In the vacuum lyophilization process, because taking away heat, the distillation of ice make the whole process of lyophilizing remain the sharp freezing state, organizational structure and mode of appearance are preserved preferably; Dried collagen protein can keep original physics and chemical property, and physical arrangement changes minimum, thereby can keep the activity of collagen protein to greatest extent; And there is not surperficial sclerosis problem in material; Exsiccant collagen protein is cellular, can stablize storage for a long time, and is prone to again rehydration and recovers active.Radiosiotope 60Co can radiate gamma-rays during decay, and gamma-ray penetration power is very strong, and is easy and simple to handle; Speed is fast, raises seldom generally only about 5 ℃ through the article temperature of 60Co radiation sterilization; Radiation dose is suitable; Can not destroy the effective ingredient of somatomedin and antiinflammatory disinfectant, also can not produce injury, and the advantages such as propagation again of sterilization back long period control antibacterial are arranged the people.
As preferably; Flocculating agent described in the step 3 is a heparin sulfate; Described somatomedin is one or more in fibroblast growth factor, hEGF, thrombopoietin or the angiogenin; Described antiinflammatory disinfectant is benzalkonium bromide or benzalkonium chloride, and described cross-linking agent is a glutaraldehyde.Heparin sulfate can with collagen cross-linking; Change the timbering material space structure; Make its one-tenth be cross-linked into the spongy of loose structure, the loose structure of spongiform collagen dressing has also improved and the skin adhesion when improving the mechanical strength of composite collagen dressing, and it can be covered on the wound surface for a long time; The loose structure of spongiform collagen dressing also can be induced repair cell in dressing, to soak into and bred, and more helps tissue repair and wound healing.Somatomedin be in fibroblast growth factor, hEGF, thrombopoietin or the angiogenin one or more, all can promote skin tissue regeneration, thereby accelerate the healing rate of wound face that described antiinflammatory disinfectant is benzalkonium bromide or benzalkonium chloride; It is little that benzalkonium bromide or benzalkonium chloride have toxicity, do not have accumulation property toxicity, nonirritant, not damage product; And stable in properties, fast light, heat-resisting, non-volatility; But long-term storage, easy to use, low cost and other advantages, and soluble in water, can combine rapidly with protein; Can change antibacterial endochylema membrane permeability, the thalline material is exosmosed, hinder its metabolism and a bactericidal action.Described cross-linking agent is a glutaraldehyde, the glutaraldehyde quick permeation, and crosslinked ability is strong, to proteinic good fixing effect.
As preferably, the precooling temperature described in the step 3 is-30～-50 ℃.In the pre-cooling stage, need strict control precooling temperature, if precooling temperature is low inadequately, then medicine possibly not freeze fully, the foaming of when evacuation distils, can expanding causes the variation of physical arrangement; If precooling temperature is too low, can increase unnecessary energy expenditure again.-30～-50 ℃ is a preferred precooling temperature scope.
As preferably, the cryodesiccated cooling scope described in the step 3 is-25 ℃～-60 ℃, and cooling rate is 3～4 ℃/min.Cryodesiccated rate of temperature fall can influence the size in the inner aperture of composite collagen dressing, and cooling rate is too fast, can make the aperture too small; Not only can make the degree of drying of lower floor's collagen protein, also can cause the permeability of collagen combine dressing relatively poor, and rate of temperature fall be excessive; Can make the aperture excessive again, cause relatively poor, the suitable rate of temperature fall of the mechanical property of collagen combine dressing; Can make the internal holes gauge structure in the collagen combine dressing of formation even, specific surface area is big.
As preferably, utilize enzyme process from the marine fishes epidermis, to extract collagen protein to may further comprise the steps described in the step 1:
(1) smash to pieces after the marine fishes epidermis is cleaned for use.Smash to pieces and can make the marine fishes epidermis can homodisperse in solution.
(2) in the marine fishes epidermis that step (1) obtains, add NaOH solution soaking 24-30h, whenever change NaOH solution 1 time, use distilled water to be washed till pH at last and be neutrality at a distance from 5～6 h.Add NaOH solution in order to remove foreign protein.
(3) use distilled water to be washed till pH behind the adding butanol solution stirring 16-24h and be neutrality.Butanol solution is used for defat, and the n-butyl alcohol lipotropy is strong, and the ability of particularly dissolving phospholipid is strong, and n-butyl alcohol has hydrophilic concurrently, in solubility range, can not cause the degeneration inactivation of enzyme, and in addition, the pH of butyl alcohol extraction method and the temperature range of choice are wider.
(4) by the addition adding distilled water of every gram marine fishes epidermis with 10～15ml; Re-adjustment pH to 6.5～7.5 backs are by the addition adding papain of every gram marine fishes epidermis with 0.01～0.02g; Under 45～60 ℃, be incubated and stir 3～5h; Re-adjustment enzymolysis pH value of solution to 7.5～9.0 backs are incubated and stir 3～5h by the addition adding trypsin of every gram marine fishes epidermis with 0.01～0.02g under 45～60 ℃, be heated to 90～100 ℃ of enzyme denaturing 5～10min at last.The hydrolysis effect of trypsin and papain is better relatively, and the collagen protein extraction ratio is higher, with papain and trypsin priority enzymolysis marine fishes epidermis, can make the abundant hydrolysis of marine fishes epidermis, to improve the extraction ratio of collagen protein.PH is 6.5～7.5; It is the optimum condition scope that papain carries out enzymolysis that temperature is 45～60 ℃, and pH is 7.5～9.0, and it is the optimum condition scope that trypsin carries out enzymolysis that temperature is 45～60 ℃; The extraction ratio of collagen protein within the specific limits can be along with the increase of the weight ratio of enzyme and marine fishes epidermis and is improved; But after to a certain degree, the collagen protein yield reduces in the fish skin, therefore needs the optimal addn scope of an enzyme; Every gram marine fishes epidermis adds 0.01～0.02g papain and 0.01～0.02g trypsin is a preferable range of the present invention, can obtain higher extraction ratio.
(5) with the enzymolysis solution that obtains 0～4 ℃ centrifugal, collect the upper strata stillness of night and carry out salt precipitation.In the time of 0～4 ℃, the character of collagen protein is the most stable.Salt precipitation is common protein separating method, and neutral salt such as adding solid sodium chloride, potassium chloride precipitate collagen protein fully and get final product, and salt precipitation can make various collagen protein fractional precipitations, and is not only simple to operate, and cost is also lower.
(6) bag filter of packing into after precipitate is redissolved with 0.5～0.7mol/L acetum; With 0.1～0.2 mol/L acetum as extracellular fluid dialysis dialysis 24～36 h; Per 4～5h changes extracellular fluid dialysis; The reuse distilled water is as extracellular fluid dialysis dialysis 24～36 h, and per 4～5h changes extracellular fluid dialysis, at last with promptly getting collagen protein after the dialysis solution lyophilization.Dialysis improves the purity of collagen protein to remove the heteroion in the collagen protein, and lyophilization can prevent that collagen protein is rotten, can keep the natural structure and the characteristic of collagen protein again.
As preferably, the NaOH solution concentration described in the step (2) is 0.1～0.5mol/L, and adds by the addition of every gram marine fishes epidermis with 10～15ml.The excessive concentration of NaOH solution can make albumen generation degeneration, therefore needs the addition and the concentration of control NaOH solution.The NaOH solution concentration is 0.1～0.5mol/L, and it is a preferable range of the present invention that every gram marine fishes epidermis adds the described NaOH solution of 10～15ml.
As preferably, the percentage by volume of n-butyl alcohol is 10～15% in the butanol solution described in the step (3), and adds by the addition of every gram marine fishes epidermis with 15～20ml.The butanol solution excessive concentration also can make protein generation degeneration.The percentage by volume of n-butyl alcohol is 10～15% in the butanol solution, and it is a preferable range of the present invention that every gram marine fishes epidermis adds the described butanol solution body of 15～20ml.
As preferably, method for preparing according to claim 1, the chondroitin sulfate that from the marine fishes fishbone, extracts described in the step 1 may further comprise the steps:
A. the residual meat on the marine fishes fishbone being picked clean back cleans up in 40～50 ℃ of hot water.The hot water injection makes residual meat separate fully with cartilage.
B. add NaOH solution and NaCl solution in the marine fishes fishbone after handling, again the marine fishes fishbone is pulverized to form and pulverize liquid, gained is pulverized liquid and behind 50～60 ℃ of following stirring and leaching 6～8h, is filtered with 150～200 order filter clothes.Earlier cartilage is carried out alkali and carry, chondroitin sulfate tentatively is dissolved in the alkali liquor, add NaCl, stablize the activity of chondroitin sulfate simultaneously, filter through filter cloth again chondroitin sulfate is carried out crude separation to increase the dissolubility of chondroitin sulfate with alkali liquor.
C. the filtering residue that step b is obtained extracts once according to step b again, twice filtrating of combining step b and step c gained.Extract once with abundant extraction chondroitin sulfate again.
D. step c gained enzymolysis solution pH is transferred to 6.5～7.5, by the addition adding papain of every gram marine fishes fishbone, at 45～55 ℃ of following insulated and stirred 2～3 h post-heating to 90～100 ℃ enzyme denaturing 5～10min with 0.005～0.01g.Add papain and carry out enzymolysis protein in removing solution.
E. in the enzymolysis solution of steps d, constantly add water and stir that to add dehydrated alcohol alcoholic acid percentage by volume in solution be 60～65%; 0～4 ℃ leave standstill 18～24 h after centrifugalize; Precipitate is with absolute ethanol washing twice, and vacuum lyophilization gets chondroitin sulfate.Earlier carry out precipitate with ethanol with ethanol, make the chondroitin sulfate deposition, reuse ethanol dewaters to the chondroitin sulfate that precipitates, and carries out lyophilization after the dehydration and just obtains chondroitin sulfate.
As preferably; NaOH solution concentration described in the step a is 0.5～1.0 mol/L; And by of the addition adding of every gram marine fishes fishbone with 10～20ml; The mass percent of NaCl is 10～15% in the described NaCl solution, and adds by the addition of every gram marine fishes fishbone with 200～300ml.
The present invention is earlier through the collagen protein and the chondroitin sulfate of extraction respectively from the epidermises of marine fishes and cartilage; Collagen protein and chondroitin sulfate that reuse extracts usually prepare composite collagen dressing, and not only cost is lower, has also improved the comprehensive utilization ratio of marine fishes; The composite collagen dressing biological safety that the most important prepares gained is good; Can avoid viral hidden danger, and the toughness of the composite collagen dressing for preparing through the present invention is high, adhesion is strong; The healing of wound can be promoted, the treatment of skin trauma face can be widely used in.
The specific embodiment
With the specific embodiment the present invention is done further description below.
Embodiment 1
One. get smash to pieces after the 1.2kg cod skin is cleaned for use; Adding 12L concentration by every gram cod skin with the addition of 10ml in the cod skin after smashing to pieces is the NaOH solution of 0.1mol/L, soaks 30h, every at a distance from 1 NaOH solution of 6 h replacing, uses distilled water to be washed till pH at last and is neutrality; Adding the 24L percentage by volume by every gram cod skin with the addition of 20ml is 10% butanol solution, stirs to use distilled water to be washed till pH behind the 20h to be neutrality; By the addition adding 12L distilled water of every gram cod skin with 10ml; It is that 6.5 backs are by the addition adding 12g papain of every gram cod skin with 0.01g that reuse hydrochloric acid is regulated pH; Be incubated and stir 5h down at 60 ℃; Reuse ammonia is pressed the addition adding 12g trypsin of every gram cod skin with 0.01g after regulating enzymolysis pH value of solution to 8.5, and insulation and stirring 3h are heated to 100 ℃ of enzyme denaturing 10min at last under 60 ℃; With the enzymolysis solution that obtains 0 ℃ centrifugal, collect the upper strata stillness of night, add solid sodium chloride deposit-free in clear liquid and separate out; The bag filter of packing into after precipitate redissolved with the 0.5mol/L acetum; With 0.1 mol/L acetum as extracellular fluid dialysis 24 h that dialyse; Every 5h changes extracellular fluid dialysis; The reuse distilled water is as extracellular fluid dialysis 28 h that dialyse, and every 4h changes extracellular fluid dialysis, at last with obtaining collagen protein after the dialysis solution lyophilization.
Two. get 1.2kg Anguillar japonica fishbone, the residual meat on the Anguillar japonica fishbone is picked clean back in 40 ℃ of hot water, clean up; Respectively adding NaOH solution that 12L concentration be 0.6 mol/L and 240L mass percent with the addition of 10ml and every gram Anguillar japonica fishbone with the addition of 200ml by every gram Anguillar japonica fishbone in the Anguillar japonica fishbone after processing is 10% NaCl solution; Again the Anguillar japonica fishbone is pulverized to form and pulverize liquid, gained is pulverized liquid and behind 50 ℃ of following stirring and leaching 8h, is filtered with 200 order filter clothes; Filtering residue on the filter cloth is extracted once according to last step, merge twice filtrating of gained; To filtrate and pH transferred to 7.5, by the addition adding 6g papain of every gram Anguillar japonica fishbone, at 45 ℃ of following insulated and stirred 2 h post-heating to 90 ℃ enzyme denaturing 10min with 0.005g with acetic acid; In enzymolysis solution, constantly add water and stir that to add dehydrated alcohol alcoholic acid percentage by volume in solution be 65%, 0 ℃ leave standstill 24 h after centrifugalize, precipitate is with absolute ethanol washing twice, vacuum lyophilization gets chondroitin sulfate.
Three. be dissolved in mix homogeneously in the acetum that concentration is 0.05mol/L simultaneously with the collagen protein of step 1 extraction, chondroitin sulfate and heparin sulfate, fibroblast growth factor, benzalkonium bromide, the glutaraldehyde that step 2 is extracted; In the mixed liquor, the quality percentage composition of each component is: collagen protein is 2.5%, and chondroitin sulfate is 1%; Heparin sulfate is 0.03%; Fibroblast growth factor is 0.001%, and benzalkonium bromide is 0.3%, and glutaraldehyde is 0.3%; The gained mixed liquor is injected mould, under 0 ℃, leave standstill 18 h, at-30 ℃ of pre-cooling 5h; Go in the freezer dryer rapidly at last; Cryodesiccated cooling scope-25 ℃～-60 ℃, cooling rate is 3 ℃/min, just obtains composite collagen dressing bullion behind the vacuum lyophilization 24h; After the composite collagen dressing bullion packing that obtains, with obtaining composite collagen dressing finished product behind 60Co irradiation sterilization 30 min.
Embodiment 2
One. get smash to pieces after the safe and comfortable fish skin of 1kg is cleaned for use; Adding 12L concentration by the safe and comfortable fish skin of every gram with the addition of 12ml in the safe and comfortable fish skin after smashing to pieces is the NaOH solution of 0.5mol/L, soaks 26h, whenever changes 1 NaOH solution at a distance from 5.5h, uses distilled water to be washed till pH at last and is neutrality; Adding the 15L percentage by volume by the safe and comfortable fish skin of every gram with the addition of 15ml is 15% butanol solution, stirs to use distilled water to be washed till pH behind the 18h to be neutrality; By the addition adding 12L distilled water of the safe and comfortable fish skin of every gram with 12ml; It is that 7.5 backs are by the addition adding 15g papain of the safe and comfortable fish skin of every gram with 0.015g that reuse hydrochloric acid is regulated pH; Be incubated and stir 4h down at 45 ℃; Reuse ammonia is pressed the addition adding 12g trypsin of the safe and comfortable fish skin of every gram with the 0.012g gram after regulating enzymolysis pH value of solution to 8, is incubated down and stirring 5h at 45 ℃, is heated to 100 ℃ of enzyme denaturing 5min at last; With the enzymolysis solution that obtains 2 ℃ centrifugal, collect the upper strata stillness of night, add solid sodium chloride deposit-free in clear liquid and separate out; The bag filter of packing into after precipitate redissolved with the 0.7mol/L acetum; With 0.2 mol/L acetum as extracellular fluid dialysis 36 h that dialyse; Every 4h changes extracellular fluid dialysis; The reuse distilled water is as extracellular fluid dialysis 26 h that dialyse, and every 4.5h changes extracellular fluid dialysis, at last with obtaining collagen protein after the dialysis solution lyophilization.
Two. get 1kg anglerfish fishbone, the residual meat on the anglerfish fishbone is picked clean back in 50 ℃ of hot water, clean up; Respectively adding NaOH solution that 20L concentration be 0.5 mol/L and 300L mass percent with the addition of 20ml and every gram anglerfish fishbone with the addition of 300ml by every gram anglerfish fishbone in the anglerfish fishbone after processing is 15% NaCl solution; Again the anglerfish fishbone is pulverized to form and pulverize liquid, gained is pulverized liquid and behind 60 ℃ of following stirring and leaching 7h, is filtered with 150 order filter clothes; Filtering residue on the filter cloth is extracted once according to last step, merge twice filtrating of gained; To filtrate and pH transferred to 6.5, by the addition adding 10g papain of every gram anglerfish fishbone, at 50 ℃ of following insulated and stirred 2.5 h post-heating to 100 ℃ enzyme denaturing 5min with 0.01g with acetic acid; In enzymolysis solution, constantly add water and stir that to add dehydrated alcohol alcoholic acid percentage by volume in solution be 62%, 2 ℃ leave standstill 22 h after centrifugalize, precipitate is with absolute ethanol washing twice, vacuum lyophilization gets chondroitin sulfate.
Three. be dissolved in mix homogeneously in the acetum that concentration is 0.1 mol/L simultaneously with the collagen protein of step 1 extraction, chondroitin sulfate and heparin sulfate, fibroblast growth factor, angiogenin, benzalkonium bromide, the glutaraldehyde that step 2 is extracted; In the mixed liquor, the quality percentage composition of each component is: collagen protein is 3%, and chondroitin sulfate is 1.2%; Heparin sulfate is 0.04%; Fibroblast growth factor and thrombopoietin are 0.005% altogether, and benzalkonium bromide is 0.3%, and glutaraldehyde is 0.4%; The gained mixed liquor is injected mould, under 2 ℃, leave standstill 13 h, at-40 ℃ of pre-cooling 6h; Go in the freezer dryer rapidly at last; Cryodesiccated cooling scope is-25 ℃～-60 ℃, and cooling rate is 4 ℃/min, and vacuum lyophilization just obtains composite collagen dressing bullion after 36 hours; After the composite collagen dressing bullion packing that obtains, with obtaining composite collagen dressing finished product behind 60Co irradiation sterilization 40 min.
Embodiment 3
One. get smash to pieces after the 1.5kg cod skin is cleaned for use; Adding 18L concentration by every gram cod skin with the addition of 12ml in the cod skin after smashing to pieces is the NaOH solution of 0.2mol/L, soaks 24h, every at a distance from 1 NaOH solution of 6 h replacing, uses distilled water to be washed till pH at last and is neutrality; Adding the 27L percentage by volume by every gram cod skin with the addition of 18ml is 13% butanol solution, stirs to use distilled water to be washed till pH behind the 16h to be neutrality; By the addition adding 22.5L distilled water of every gram cod skin with 115ml; It is that 7 backs are by the addition adding 30g papain of every gram cod skin with 0.02g that reuse hydrochloric acid is regulated pH; Be incubated and stir 3h down at 50 ℃; Reuse ammonia is pressed the addition adding 30g trypsin of every gram cod skin with 0.02g after regulating enzymolysis pH value of solution to 7.5, and insulation and stirring 4h are heated to 95 ℃ of enzyme denaturing 8min at last under 50 ℃; With the enzymolysis solution that obtains 4 ℃ centrifugal, collect the upper strata stillness of night, add solid sodium chloride deposit-free in clear liquid and separate out; The bag filter of packing into after precipitate redissolved with the 0.6mol/L acetum; With 0.15 mol/L acetum as extracellular fluid dialysis 26 h that dialyse; Every 4.5h changes extracellular fluid dialysis; The reuse distilled water is as the extracellular fluid dialysis 24h that dialyses, and every 4h changes extracellular fluid dialysis, at last with obtaining collagen protein after the dialysis solution lyophilization.
Two. get 1kg morrhua fishbone, the residual meat on the morrhua fishbone is picked clean back in 45 ℃ of hot water, clean up; Respectively adding NaOH solution that 15L concentration be 1.0 mol/Ls and 300L mass percent with the addition of 15ml and every gram morrhua fishbone with the addition of 300ml by every gram morrhua fishbone in the morrhua fishbone after processing is 12% NaCl solution; Again the morrhua fishbone is pulverized to form and pulverize liquid, gained is pulverized liquid and behind 55 ℃ of following stirring and leaching 6h, is filtered with 180 order filter clothes; Filtering residue on the filter cloth is extracted once according to last step, merge twice filtrating of gained; To filtrate and pH transferred to 7, by the addition adding 6g papain of every gram morrhua fishbone, at 55 ℃ of following insulated and stirred 3 h post-heating to 95 ℃ enzyme denaturing 8min with 0.006g with acetic acid; In enzymolysis solution, constantly add water and stir that to add dehydrated alcohol alcoholic acid percentage by volume in solution be 65%, 4 ℃ leave standstill 24 h after centrifugalize, precipitate is with absolute ethanol washing twice, vacuum lyophilization gets chondroitin sulfate.
Three. be dissolved in mix homogeneously in the acetum that concentration is 0.15 mol/L simultaneously with the collagen protein of step 1 extraction, chondroitin sulfate and heparin sulfate, fibroblast growth factor, hEGF, thrombopoietin, angiogenin, benzalkonium chloride, the glutaraldehyde that step 2 is extracted; In the mixed liquor; The quality percentage composition of each component is: collagen protein is 2.0%; Chondroitin sulfate is 1.5%, and heparin sulfate is 0.05%, and fibroblast growth factor, hEGF, thrombopoietin, angiogenin are 0.003% altogether; Benzalkonium bromide is 0.4%, and glutaraldehyde is 0.6%; The gained mixed liquor is injected mould, under 4 ℃, leave standstill 15h, at-50 ℃ of pre-cooling 7h; Go in the freezer dryer rapidly at last; Cryodesiccated cooling scope is-25 ℃～-60 ℃, and cooling rate is 3.5 ℃/min, and vacuum lyophilization just obtains composite collagen dressing bullion after 28 hours; After the composite collagen dressing bullion packing that obtains, with obtaining composite collagen dressing finished product behind 60Co irradiation sterilization 50 min.
Embodiment 4
One. get smash to pieces after the 1.5kg cod skin is cleaned for use; Adding 21L concentration by every gram cod skin with the addition of 14ml in the cod skin after smashing to pieces is the NaOH solution of 0.1mol/L, soaks 30h, every at a distance from 1 NaOH solution of 5 h replacing, uses distilled water to be washed till pH at last and is neutrality; Adding the 24L percentage by volume by every gram cod skin with the addition of 16ml is 15% butanol solution, stirs to use distilled water to be washed till pH behind the 24h to be neutrality; By the addition adding 15L distilled water of every gram cod skin with 10ml; It is that 6.5 backs are by the addition adding 15g papain of every gram cod skin with 0.01g that reuse hydrochloric acid is regulated pH; Be incubated and stir 3h down at 60 ℃; Reuse ammonia is pressed the addition adding 15 gram g protease of every gram cod skin with 0.01g after regulating enzymolysis pH value of solution to 9.0, is incubated down and stirring 5h at 50 ℃, is heated to 100 ℃ of enzyme denaturing 10min at last; With the enzymolysis solution that obtains 0 ℃ centrifugal, collect the upper strata stillness of night, add solid sodium chloride deposit-free in clear liquid and separate out; The bag filter of packing into after precipitate redissolved with the 0.5mol/L acetum; With 0.2 mol/L acetum as extracellular fluid dialysis 36 h that dialyse; Every 5h changes extracellular fluid dialysis; The reuse distilled water is as extracellular fluid dialysis 36 h that dialyse, and every 5h changes extracellular fluid dialysis, at last with obtaining collagen protein after the dialysis solution lyophilization.
Two. get 1kg anglerfish fishbone, the residual meat on the anglerfish fishbone is picked clean back in 40 ℃ of hot water, clean up; Respectively adding NaOH solution that 10L concentration be 0.7 mol/L and 250L mass percent with the addition of 10ml and every gram anglerfish fishbone with the addition of 250ml by every gram anglerfish fishbone in the anglerfish fishbone after processing is 10% NaCl solution; Again the anglerfish fishbone is pulverized to form and pulverize liquid, gained is pulverized liquid and behind 50 ℃ of following stirring and leaching 8h, is filtered with 200 order filter clothes; Filtering residue on the filter cloth is extracted once according to last step, merge twice filtrating of gained; To filtrate and pH transferred to 7.5, by the addition adding 10g papain of every gram anglerfish fishbone, at 50 ℃ of following insulated and stirred 3 h post-heating to 90 ℃ enzyme denaturing 5min with 0.01g with acetic acid; In enzymolysis solution, constantly add water and stir that to add dehydrated alcohol alcoholic acid percentage by volume in solution be 60%, 0 ℃ leave standstill 18 h after centrifugalize, precipitate is with absolute ethanol washing twice, vacuum lyophilization gets chondroitin sulfate.
Three. be dissolved in mix homogeneously in the acetum that concentration is 0.2 mol/L simultaneously with the collagen protein of step 1 extraction, chondroitin sulfate and heparin sulfate, hEGF, thrombopoietin, benzalkonium chloride, the glutaraldehyde that step 2 is extracted; In the mixed liquor, the quality percentage composition of each component is: collagen protein is 2%, and chondroitin sulfate is 1.3%; Heparin sulfate is 0.03%; HEGF and thrombopoietin are 0.005% altogether, and benzalkonium chloride is 0.1%, and glutaraldehyde is 0.8%; The gained mixed liquor is injected mould, under 3 ℃, leave standstill 12 h, at-35 ℃ of pre-cooling 8h; Go in the freezer dryer rapidly at last; Cryodesiccated cooling scope is-25 ℃～-60 ℃, and cooling rate is 3 ℃/min, and vacuum lyophilization just obtains composite collagen dressing bullion after 26 hours; After the composite collagen dressing bullion packing that obtains, with obtaining composite collagen dressing finished product behind 60Co irradiation sterilization 60 min.
The foregoing description all adopts the collagen protein and the chondroitin sulfate that from marine fishes, extract usually to prepare the dressing of collagen composite collagen; Not only can improve the comprehensive utilization ratio of marine fishes; And the composite collagen dressing cost of preparation is lower, and safety is good, but preventing virus hidden danger; Simultaneously in the preparation process, all add somatomedin and antiinflammatory disinfectant, can increase the healing of wound.
Claims (9)
1. the method for preparing of source, ocean composite collagen dressing is characterized in that, described method for preparing may further comprise the steps:
Utilize enzyme process from the marine fishes epidermis, to extract collagen protein;
From the marine fishes fishbone, extract chondroitin sulfate;
The collagen protein of step 1 extraction, chondroitin sulfate and flocculating agent, somatomedin, antiinflammatory disinfectant, the cross-linking agent that step 2 is extracted are dissolved in mix homogeneously in the acetum that concentration is 0.05-0.2 mol/L simultaneously; In the mixed liquor, the quality percentage composition of each component is: collagen protein is 2.0～3.0%, and chondroitin sulfate is 1.0～1.5%; Flocculating agent is 0.03～0.05%; Somatomedin is 0.001～0.005%, and the antiinflammatory disinfectant is 0.1～0.4%, and cross-linking agent is 0.3～0.8%; The gained mixed liquor is injected mould, and pre-cooling 5～8h after leaving standstill 12～18 h under 0～4 ℃ goes to rapidly at last and just obtains composite collagen dressing bullion in the freezer dryer behind vacuum lyophilization 24～36h; After the composite collagen dressing bullion packing that obtains, with obtaining composite collagen dressing finished product behind 60Co irradiation sterilization 30～60 min.
2. method for preparing according to claim 1; It is characterized in that; Flocculating agent described in the step 3 is a heparin sulfate; Described somatomedin is one or more in fibroblast growth factor, hEGF, thrombopoietin or the angiogenin, and described antiinflammatory disinfectant is benzalkonium bromide or benzalkonium chloride, and described cross-linking agent is a glutaraldehyde.
3. method for preparing according to claim 1 and 2 is characterized in that, the precooling temperature described in the step 3 is-30～-50 ℃.
4. according to claim 1 or 2 or 3 described method for preparinies, it is characterized in that the cryodesiccated cooling scope described in the step 3 is-25 ℃～-60 ℃, cooling rate is 3～4 ℃/min.
5. method for preparing according to claim 1 is characterized in that, utilizes enzyme process from the marine fishes epidermis, to extract collagen protein to may further comprise the steps described in the step 1:
Smash to pieces for use after the marine fishes epidermis cleaned;
In the marine fishes epidermis that step (1) obtains, add NaOH solution soaking 24-30h, whenever change NaOH solution 1 time, use distilled water to be washed till pH at last and be neutrality at a distance from 5～6 h;
Use distilled water to be washed till pH behind the adding butanol solution stirring 16-24h and be neutrality;
By the addition adding distilled water of every gram marine fishes epidermis with 10～15ml; Re-adjustment pH to 6.5～7.5 backs are by the addition adding papain of every gram marine fishes epidermis with 0.01～0.02g; Under 45～60 ℃, be incubated and stir 3～5h; Re-adjustment enzymolysis pH value of solution to 7.5～9.0 backs are incubated and stir 3～5h by the addition adding trypsin of every gram marine fishes epidermis with 0.01～0.02g under 45～60 ℃, be heated to 90～100 ℃ of enzyme denaturing 5～10min at last;
With the enzymolysis solution that obtains 0～4 ℃ centrifugal, collect the upper strata stillness of night and carry out salt precipitation;
The bag filter of packing into after precipitate redissolved with 0.5～0.7mol/L acetum; With 0.1～0.2 mol/L acetum as extracellular fluid dialysis dialysis 24～36 h; Per 4～5h changes extracellular fluid dialysis; The reuse distilled water is as extracellular fluid dialysis dialysis 24～36 h, and per 4～5h changes extracellular fluid dialysis, at last with promptly getting collagen protein after the dialysis solution lyophilization.
6. method for preparing according to claim 5 is characterized in that, the NaOH solution concentration described in the step (2) is 0.1～0.5mol/L, and adds by the addition of every gram marine fishes epidermis with 10～15ml.
7. described according to claim 5 or 6, it is characterized in that the percentage by volume of n-butyl alcohol is 10～15% in the butanol solution described in the step (3), and add by the addition of every gram marine fishes epidermis with 15～20ml.
8. method for preparing according to claim 1 is characterized in that, the chondroitin sulfate that from the marine fishes fishbone, extracts described in the step 1 may further comprise the steps:
Residual meat on the marine fishes fishbone is picked clean back to be cleaned up in 40～50 ℃ of hot water;
Add NaOH solution and NaCl solution in the marine fishes fishbone after the processing, again the marine fishes fishbone is pulverized to form and pulverize liquid, gained is pulverized liquid and behind 50～60 ℃ of following stirring and leaching 6～8h, is filtered with 150～200 order filter clothes;
The filtering residue that step b is obtained extracts once according to step b again, twice filtrating of combining step b and step c gained;
PH transfers to 6.5～7.5 with step c gained filtrating, by the addition adding papain of every gram marine fishes fishbone with 0.005～0.01g, at 45～55 ℃ of following insulated and stirred 2～3 h post-heating to 90～100 ℃ enzyme denaturing 5～10min;
In the enzymolysis solution of steps d, constantly add water and stir that to add dehydrated alcohol alcoholic acid percentage by volume in solution be 60～65%, 0～4 ℃ leave standstill 18～24 h after centrifugalize, precipitate is with absolute ethanol washing twice, vacuum lyophilization gets chondroitin sulfate.
9. method for preparing according to claim 8; It is characterized in that; NaOH solution concentration described in the step a is 0.5～1.0 mol/L; And by the addition adding of every gram marine fishes fishbone with 10～20ml, the mass percent of NaCl is 10～15% in the described NaCl solution, and adds by the addition of every gram marine fishes fishbone with 200～300ml.
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| CN109503733A (en) * | 2018-12-29 | 2019-03-22 | 福州宏东食品有限公司 | A kind of extracting method of shark chondroitine |
| CN112358018A (en) * | 2020-10-30 | 2021-02-12 | 浙江工商大学 | Protein flocculant and preparation method thereof |
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| CN103386144B (en) * | 2013-07-23 | 2014-10-01 | 海南光宇生物科技有限公司 | Preparation method of fish collagen combined chitosan biological dressing |
| CN106943620A (en) * | 2017-03-17 | 2017-07-14 | 广州赛莱拉干细胞科技股份有限公司 | A kind of gel dressing of medical use liquid containing extracellular matrix components and application |
| CN107899080A (en) * | 2017-11-30 | 2018-04-13 | 振德医疗用品股份有限公司 | A kind of dermis restoration stent preparation method |
| CN108939153A (en) * | 2018-08-17 | 2018-12-07 | 振德医疗用品股份有限公司 | A kind of collagen-based packing material and preparation method thereof and its application method |
| CN109503733A (en) * | 2018-12-29 | 2019-03-22 | 福州宏东食品有限公司 | A kind of extracting method of shark chondroitine |
| CN112358018A (en) * | 2020-10-30 | 2021-02-12 | 浙江工商大学 | Protein flocculant and preparation method thereof |
| CN112358018B (en) * | 2020-10-30 | 2022-05-27 | 浙江工商大学 | Protein flocculant and preparation method thereof |
| CN115154643A (en) * | 2022-06-27 | 2022-10-11 | 福建医科大学附属协和医院 | Preparation method and application of aloe polysaccharide-collagen composite dressing |
| CN115154643B (en) * | 2022-06-27 | 2024-03-12 | 福建医科大学附属协和医院 | Preparation method and application of aloe polysaccharide-collagen composite dressing |
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