CN104031274A - Preparation method of aquatic fish skin collagen hydrogel - Google Patents
Preparation method of aquatic fish skin collagen hydrogel Download PDFInfo
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Abstract
The invention relates to the technical field of biomedical materials and in particular relates to a preparation method of aquatic fish skin collagen hydrogel. The preparation method comprises the following steps: fully dissolving the aquatic fish skin collagen in an acetic acid solution with concentration of 0.5mol/L under the condition of continuous stirring at 4 DEG C to prepare a collagen solution; centrifuging the collagen solution after dissolution, and collecting the supernatant for later use; adjusting the pH value of the collagen supernatant with a filtered sodium hydroxide solution or mixing a filtered phosphate buffer solution with the collagen supernatant in a volume ratio of 1:1, continuously stirring the mixed solution, and then adjusting the pH value of the mixed solution with the filtered sodium hydroxide solution; putting the collagen supernatant mixed solution after the pH value is adjusted in a die, and then standing for 1-2 hours in a water bath at 30-40 DEG C so that the collagen supernatant mixed solution self aggregates, solidifies and is formed; taking the formed hydrogel out of the die, and soaking the hydrogel in ultrapure water for 20-30 minutes, thus obtaining the aquatic fish skin collagen hydrogel. The preparation method has the beneficial effects that the preparation process is simple, the materials are cheap and accessible, and the product is widely applied.
Description
Technical field
The present invention relates to bio-medical material art field, particularly relate to a kind of preparation method of aquatic products collagen of fish skin hydrogel.
Background technology
Collagen protein is that animal body intensive amount is the abundantest, protein the most widely distributes.Collagen protein has the features such as unique triple-helix structure, low antigenicity, nontoxicity, good biocompatibility and biodegradability, has been widely used in the bio-medical fields such as tissue engineering bracket material, styptic sponge, medicine control slow-released system, gene delivery vector, cosmetic surgery.At present, the collagen protein of domestic use is mainly from skin and the heel string of pig and ox, but along with the generation of the diseases such as mad cow disease and foot and mouth disease, makes people produce query to their security; In addition, due to reasons such as religion and customs, the application of the collagen protein in pig source in certain areas is restricted.Therefore, in the urgent need to seeking the better collagen protein of quality and security source.
The annual fish scale of China, the fishery products wastes such as fish-skin approximately have more than 40 ten thousand tons, these fishery products wastes all contain abundant collagen protein, its amino acid forms and Lu Sheng Mammals collagen protein no significant difference, and there is not the communicable disease of infecting both domestic animals and human in it, have great development and utilization and be worth, but these wastes are used to produce feed except small portion, major part is but dropped.Therefore, develop the new way of utilizing of the fishery products wastes such as fish-skin, not only can improve the added value of processing of aquatic products, and can reduce environmental pollution, there is good economic and social benefit.
Summary of the invention
The object of the invention is to provide a kind of preparation method of aquatic products collagen of fish skin hydrogel.
For achieving the above object, the present invention adopts technical scheme to be:
A preparation method for aquatic products collagen of fish skin hydrogel,
1) under condition aquatic products collagen of fish skin constantly being stirred at 4 ℃, be fully dissolved in the acetum that concentration is 0.5mol/L, be configured to collagen solution;
2) by the centrifugal 30min under 4 ℃, the condition of 20000g of the collagen solution after dissolving, fully remove insoluble impurity, with the careful sucking-off supernatant liquor of liquid-transfering gun, stand-by;
3) with the 10-15mol/L sodium hydroxide solution after filtering, regulate the pH to 7.0-7.8 of above-mentioned collagen supernatant liquor;
Or, with the phosphate buffered saline buffer after filtering and above-mentioned collagen supernatant liquor by volume the ratio of 1:1 mixes, and be constantly uniformly mixed liquid, then with the pH to 7.0-7.8 of the 10-15mol/L sodium hydroxide solution adjusting mixed solution after filtration;
4) by regulating the collagen supernatant liquor mixed solution after pH to be placed in mould, then at the 30-40 ℃ of standing 1-2h of water-bath, make its abundant autohemagglutination coagulation forming;
5) hydrogel after moulding is taken out from mould, be placed in ultrapure water and soak 20-30min, obtain aquatic products collagen of fish skin hydrogel.
Phosphate buffered saline buffer after described filtration is that concentration is the phosphate buffered saline buffer of the sodium-chlor that is 260mM containing concentration of 40mM, and filter paper filtering is stand-by.
Described phosphate buffered saline buffer is Sodium phosphate dibasic-phosphate sodium dihydrogen buffer solution, pH7.4.
Described aquatic products collagen of fish skin is Java tilapia skin enzymatic dissolubility collagen protein;
1) Java tilapia skin that cleans and blot surface-moisture is carried out to degreasing 24h with 10% propyl carbinol to fish-skin under magnetic agitation, during every 8h change a butanol solution; Then clean stand-by;
2) under 4 ℃ of conditions, with the sodium hydroxide of 0.1mol/L to above-mentioned processing after fish-skin carry out removing impurities protein 24 h, during every 8h change a sodium hydroxide solution, then clean stand-by;
3) fish-skin after above-mentioned removing impurities albumen is pulverized, after pulverizing, fish-skin powder mixes with 0.5mol/L acetum, adds the stomach en-(750U/mg, Sigma) of raw material fish-skin quality 0.5%-1% after mixing again, then at 4 ℃, stirs and extracts 48h;
4) by the centrifugal collection supernatant liquor of fish-skin extracting solution after said extracted, and under the condition of ice-water bath and stirring, to slowly adding in supernatant liquor, to grind sodium-chlor to final concentration in small, broken bits be 0.9mol/L, then liquid is placed in to 4 ℃ of refrigerator overnight and saltouts;
5) the above-mentioned liquid of saltouing is centrifugal, collecting precipitation, then redissolves precipitation in the acetum of 0.5mol/L; Gained redissolution liquid is loaded in 3.5kDa dialysis tubing, dialysis 24h, the disodium phosphate soln of dialyzate 0.02mol/L (pH8.6);
6) above-mentioned gained dialyzate is again with the acetum dialysis 24h of 0.1mol/L, after dialysis again with the pure water 48h that dialyses; Collagen solution after dialysis is met cold fast in-80 ℃, then-60 ℃ of lyophilize 48h, obtain Java tilapia skin enzymatic dissolubility collagen protein (PSC).
Advantageous effect of the present invention:
1. the present invention is raw materials used is the tankage Java tilapia skin of aquatic products processing, raw material sources safety, and the present invention makes full use of the fish-skin waste material producing in the tilapia aquatic products course of processing, turns waste into wealth, and increases economic efficiency.
2. the present invention's extracting method used is enzymatic extraction method, so not only can make the more thorough of collagen of fish skin extraction, and in leaching process, enzyme can excise the end end peptide of collagen protein, can reduce so the immune resistance of human body to it.
3. the present invention can develop Mammals (pig and ox) with the aquatic collagen protein medical material of external source, and its safety coefficient is higher, output is larger, and more cheap.
4. the aquatic products collagen of fish skin hydrogel that the present invention develops, to rely on the character of collagen protein autohemagglutination to prepare completely under the condition of simulation physiology potential of hydrogen and ionic strength environment, do not add any linking agent, so its security, stability and consistency in vivo can be better.
5. the aquatic products collagen of fish skin hydrogel that the present invention obtains, its low temperature (4-10 ℃) is lower is clear, colorless liquid, and when water-bath reaches certain temperature (30-40 ℃), just autohemagglutination moulding becomes solid state fast, and physical strength is good, there is certain supporting function, and degradable; When temperature during higher than certain value (50 ℃) just can again become again as liquid, but again drop to 30-40 ℃ when temperature, liquid cannot be gathered into solid again, this is an irreversible process.The aquatic products collagen of fish skin hydrogel obtaining by the present invention is of many uses, and as filled after operation on spinal cord etc., prospect is considerable.
Accompanying drawing explanation
The concentration that Fig. 1 provides for the embodiment of the present invention is hydrogel prepared by 1mg/mL collagen solution;
The concentration that Fig. 2 embodiment of the present invention provides is hydrogel prepared by 2mg/mL collagen solution;
The concentration that Fig. 3 embodiment of the present invention provides is hydrogel prepared by 3mg/mL collagen solution;
Under the physiology salinity that Fig. 4 embodiment of the present invention provides, concentration is hydrogel prepared by 1mg/mL collagen solution;
Under the physiology salinity that Fig. 5 embodiment of the present invention provides, concentration is hydrogel prepared by 2mg/mL collagen solution;
Under the physiology salinity that Fig. 6 embodiment of the present invention provides, concentration is hydrogel prepared by 3mg/mL collagen solution.
Embodiment
With specific embodiment, further illustrate essentiality content of the present invention below, but content of the present invention is not limited to this.
The preparation of embodiment 1 aquatic products collagen of fish skin hydrogel:
The extraction of 1.1 enzymatic dissolubility Java tilapia skin collagen proteins:
1.1.1 clean fish-skin: take out the Java tilapia skin of a certain amount of being stored in-20 ℃, with a large amount of tap water, soak and rinse, remove its hetero-organizations such as the flesh of fish, fish scale of fish-skin remained on surface, by ultrapure water soaking and washing 3 times, then with thieving paper, blot the moisture on fish-skin surface,
1.1.2 degreasing: accurately take the above-mentioned fish-skin of 10g, fish-skin is cut into 1 * 2cm size, in 4 ℃ of refrigerator-freezers, under the condition constantly stirring at magnetic stirring apparatus, the 10% n-butanol aqueous solution degreasing 8h with 200mL, repeat above-mentioned skimming processes 3 times, then with tap water, rinse 30min, then clean 3 times with ultrapure water;
1.1.3 removing impurities albumen: in 4 ℃ of refrigerator-freezers, above-mentioned fish-skin after degreasing is soaked in to removing impurities albumen 8h in the 0.1mol/L sodium hydroxide solution of 200mL, during with glass stick, stir in good time, repeat above-mentioned skimming processes 3 times, then with tap water, rinse to neutral, then clean 3 times with ultrapure water;
1.1.4 enzymolysis and extraction: with organizing pulverizer that above-mentioned fish-skin is pulverized, after pulverizing, in fish-skin powder, add the 0.5mol/L acetum of 500mL and 0.05g (raw material fish-skin quality 0.5%) stomach en-(750U/mg Sigma), in 4 ℃ of refrigerator-freezers, by magnetic stirrer, extract 48h, then in 4 ℃, centrifugal 30min under 10000r/min condition, collects supernatant liquor;
1.1.5 saltout and dialyse: accurately taking 5.2596g and grind sodium-chlor in small, broken bits, slowly join enzyme and put on (final concn is 0.9mol/L) in clear liquid, whole process is all carried out under ice-water bath and the continuous condition stirring of magnetic stirring apparatus, then being placed on 4 ℃ of refrigerator overnight saltouts, in 4 ℃, centrifugal 30min under 10000r/min condition, collecting precipitation, precipitation is redissolved in the acetum of 0.5mol/L, the liquid that redissolves is loaded in the dialysis tubing of 3.5kDa, 24h successively dialyses in 0.02mol/L Sodium phosphate dibasic (pH8.6) solution, the 48h that dialyses in 24h and pure water dialyses in 0.1mol/L acetum, whole dialysis procedure is all carried out in 4 ℃ of refrigerator-freezers,
1.1.6 lyophilize: the collagen protein that dialysis is completed, at-80 ℃ of fast precoolings, then at-60 ℃ lyophilize 48h, obtain Java tilapia skin enzymatic dissolubility collagen protein.
1.2 Java tilapia skin enzymatic dissolubility collagen protein autohemagglutinations are prepared hydrogel:
1.2.1 different concns collagen protein autohemagglutination is prepared hydrogel:
Accurately take 10, 20, 30 and the collagen protein of 40mg join respectively in 10mL0.5mol/L acetum, in 4 ℃ of refrigerator-freezers, under magnetic stirrer condition, fully dissolve 48h, then in 4 ℃, centrifugal 30min under 20000g condition, with liquid-transfering gun, take out supernatant liquor, obtain concentration and be respectively 1, 2, 3 and the collagen solution of 4mg/mL, then use 10mol/L sodium hydroxide solution, regulate pH of mixed to 7.4, rapidly the collagen supernatant liquor that regulates pH is poured in mould, then be placed in 37 ℃ of water-bath autohemagglutination moulding 1h, then the hydrogel of the different concns of moulding is washed in ultrapure water, soak the hydrogel (referring to Fig. 1-3) that 30min obtains different concns.
1.2.2 simulate the impact that physiological saline concentration is prepared different collagen concentration autohemagglutination hydrogels:
Accurately take 20,40,60 and the collagen protein of 80mg be dissolved in respectively in 10mL0.5mol/L acetum, in 4 ℃ of refrigerator-freezers, under magnetic stirrer condition, fully dissolve 48h, then centrifugal 30min under 4 ℃, 20000g condition, with liquid-transfering gun, take out supernatant liquor, obtain concentration and be respectively 2,4,6 and the collagen solution of 8mg/mL, to slowly adding in each collagen solution the phosphate buffered saline buffer that 10mL40mM sodium chloride-containing 260mM pH is 7.4 (collagen solution and above-mentioned phosphate buffered saline buffer volume ratio are 1:1) to obtain the collagen liquid of different concns under physiology Variation of Salinity Condition, (final concentration is 1, 2, 3 and 4mg/mL, phosphoric acid salt final concentration is respectively 20mM, the final concentration of sodium-chlor is respectively 130mM), constantly stir during this time, then use 10mol/L sodium hydroxide solution, regulate pH of mixed to 7.4, rapidly the collagen mixed solution that regulates pH is poured in mould, then be placed in 37 ℃ of water-bath autohemagglutination moulding 1h, then the hydrogel of the different concns under the physiology Variation of Salinity Condition of moulding is washed in ultrapure water, soak the hydrogel (referring to Fig. 4-6) that 30min obtains different concns.
Embodiment 2 aquatic products fish-skin hydrogel anti-pressure abilities are measured:
The hydrogel that above-mentioned each condition is prepared is positioned on the sheet glass of level, the ergometer that is S with area applies a vertical pressure to hydrogel, slowly increase pressure, until hydrogel is cracked, record the instantaneous pressure F of hydrogel when cracked, the compressive resistance character of calculating collagen protein hydrogel product, formula is as follows:
Compressive resistance intensity (N/cm
2)=F/S
After measured, the above-mentioned different concns hydrogel that result demonstration the present invention makes or the hydrogel of the different concns under physiology salinity, its ultimate compression strength scope is all between 0.2-0.8, and anti-pressure ability character is good.
Claims (4)
1. a preparation method for aquatic products collagen of fish skin hydrogel, is characterized in that:
1) under condition aquatic products collagen of fish skin constantly being stirred at 4 ℃, be fully dissolved in the acetum that concentration is 0.5mol/L, be configured to collagen solution;
2) collagen solution after dissolving is centrifugal, collect supernatant liquor, stand-by;
3) with the 10-15mol/L sodium hydroxide solution after filtering, regulate the pH to 7.0-7.8 of above-mentioned collagen supernatant liquor;
Or, with the phosphate buffered saline buffer after filtering and above-mentioned collagen supernatant liquor by volume the ratio of 1:1 mixes, and be constantly uniformly mixed liquid, then with pH to 7.0-7.8 of the 10-15mol/L sodium hydroxide solution adjusting mixed solution after filtration;
4) by regulating the collagen supernatant liquor mixed solution after pH to be placed in mould, then at the 30-40 ℃ of standing 1-2h of water-bath, make its abundant autohemagglutination coagulation forming;
5) hydrogel after moulding is taken out from mould, be placed in ultrapure water and soak 20-30min, obtain aquatic products collagen of fish skin hydrogel.
2. by the preparation method of aquatic products collagen of fish skin hydrogel claimed in claim 1, it is characterized in that: the phosphate buffered saline buffer after described filtration is that concentration is the phosphate buffered saline buffer of the sodium-chlor that is 260mM containing concentration of 40mM, filter paper filtering is stand-by.
3. by the preparation method of aquatic products collagen of fish skin hydrogel claimed in claim 2, it is characterized in that: described phosphate buffered saline buffer is Sodium phosphate dibasic-phosphate sodium dihydrogen buffer solution, pH7.4.
4. by the preparation method of aquatic products collagen of fish skin hydrogel claimed in claim 1, it is characterized in that: described aquatic products collagen of fish skin is Java tilapia skin enzymatic dissolubility collagen protein;
1) Java tilapia skin that cleans and blot surface-moisture is carried out to degreasing 24h with 10% propyl carbinol to fish-skin under magnetic agitation, during every 8h change a butanol solution; Then clean stand-by;
2) under 4 ℃ of conditions, with the sodium hydroxide of 0.1mol/L to above-mentioned processing after fish-skin carry out removing impurities protein 24 h, during every 8h change a sodium hydroxide solution, then clean stand-by;
3) fish-skin after above-mentioned removing impurities albumen is pulverized, after pulverizing fish-skin powder and 0.5mol/L acetum in mass ratio 1:50 mix, the stomach en-(750U/mg, Sigma) that adds again raw material fish-skin quality 0.5%-1% after mixing then stirs and extracts 48h at 4 ℃;
4) by the centrifugal collection supernatant liquor of fish-skin extracting solution after said extracted, and under the condition of ice-water bath and stirring, to slowly adding in supernatant liquor, to grind sodium-chlor to final concentration in small, broken bits be 0.9mol/L, then liquid is placed in to 4 ℃ of refrigerator overnight and saltouts;
5) the above-mentioned liquid of saltouing is centrifugal, collecting precipitation, then redissolves precipitation in the acetum of 0.5mol/L; Gained redissolution liquid is loaded in 3.5kDa dialysis tubing, dialysis 24h, the disodium phosphate soln of dialyzate 0.02mol/L (pH8.6);
6) above-mentioned gained dialyzate is again with the acetum dialysis 24h of 0.1mol/L, after dialysis again with the pure water 48h that dialyses; Collagen solution after dialysis is met cold fast in-80 ℃, then-60 ℃ of lyophilize 48h, obtain Java tilapia skin enzymatic dissolubility collagen protein (PSC).
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Cited By (14)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104436312A (en) * | 2014-11-12 | 2015-03-25 | 江苏德威兰医疗器械有限公司 | Artificial skin prepared by collagen and polymer material through ultrasonic treatment |
| CN104940113A (en) * | 2015-07-16 | 2015-09-30 | 广州赛莱拉干细胞科技股份有限公司 | 3D printed mask and manufacturing technology thereof |
| CN105213113A (en) * | 2015-10-30 | 2016-01-06 | 浙江优全护理用品科技有限公司 | A kind of energy-saving degradable diaper and production technology thereof |
| CN105815539A (en) * | 2016-03-18 | 2016-08-03 | 大连工业大学 | Preparation method of sea cucumber collagen fiber gel |
| CN105820355A (en) * | 2016-04-05 | 2016-08-03 | 李�杰 | Genipin crosslinked fish-derived collagen composite material and preparation method thereof |
| CN106831979A (en) * | 2015-11-30 | 2017-06-13 | 中国科学院深圳先进技术研究院 | Fish scale collagen hydrogel and preparation method and application |
| CN107137784A (en) * | 2017-05-08 | 2017-09-08 | 青岛大学 | Deep-sea fish collagen peptide hydrogel |
| CN107936579A (en) * | 2017-12-19 | 2018-04-20 | 中国科学院烟台海岸带研究所 | A kind of preparation method of double-network hydrogel |
| CN108335910A (en) * | 2017-01-20 | 2018-07-27 | 中国科学院烟台海岸带研究所 | A kind of composite gel material and preparation method thereof, solar cell module and preparation method thereof |
| CN109141693A (en) * | 2018-06-25 | 2019-01-04 | 厦门大学 | A kind of pliable pressure sensor and preparation method thereof |
| CN109535447A (en) * | 2018-11-06 | 2019-03-29 | 福建农林大学 | A kind of thermal sensitivity collagen nanofiber/PNIPAM semi-intercrossing network formula hydrogel and preparation method thereof |
| CN112961374A (en) * | 2021-02-03 | 2021-06-15 | 南方医科大学 | Preparation and application of natural swim bladder-derived conductive hydrogel |
| CN114702692A (en) * | 2022-04-01 | 2022-07-05 | 中国科学院青岛生物能源与过程研究所 | Method for improving stability of protein hydrogel |
| CN116082717A (en) * | 2023-02-02 | 2023-05-09 | 领博生物科技(杭州)有限公司 | Hydrogel, vascular fiber skeleton, preparation method and application thereof |
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| CN104436312A (en) * | 2014-11-12 | 2015-03-25 | 江苏德威兰医疗器械有限公司 | Artificial skin prepared by collagen and polymer material through ultrasonic treatment |
| CN104436312B (en) * | 2014-11-12 | 2017-05-31 | 江苏德威兰医疗器械有限公司 | The artificial skin prepared by ultrasonically treated collagen and macromolecular material |
| CN104940113A (en) * | 2015-07-16 | 2015-09-30 | 广州赛莱拉干细胞科技股份有限公司 | 3D printed mask and manufacturing technology thereof |
| CN105213113A (en) * | 2015-10-30 | 2016-01-06 | 浙江优全护理用品科技有限公司 | A kind of energy-saving degradable diaper and production technology thereof |
| CN106831979A (en) * | 2015-11-30 | 2017-06-13 | 中国科学院深圳先进技术研究院 | Fish scale collagen hydrogel and preparation method and application |
| CN105815539A (en) * | 2016-03-18 | 2016-08-03 | 大连工业大学 | Preparation method of sea cucumber collagen fiber gel |
| CN105820355A (en) * | 2016-04-05 | 2016-08-03 | 李�杰 | Genipin crosslinked fish-derived collagen composite material and preparation method thereof |
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| CN108335910A (en) * | 2017-01-20 | 2018-07-27 | 中国科学院烟台海岸带研究所 | A kind of composite gel material and preparation method thereof, solar cell module and preparation method thereof |
| CN107137784A (en) * | 2017-05-08 | 2017-09-08 | 青岛大学 | Deep-sea fish collagen peptide hydrogel |
| CN107936579A (en) * | 2017-12-19 | 2018-04-20 | 中国科学院烟台海岸带研究所 | A kind of preparation method of double-network hydrogel |
| CN109141693A (en) * | 2018-06-25 | 2019-01-04 | 厦门大学 | A kind of pliable pressure sensor and preparation method thereof |
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| CN109535447B (en) * | 2018-11-06 | 2021-08-24 | 福建农林大学 | Thermosensitive collagen nanofiber/PNIPAM semi-interpenetrating network type hydrogel and preparation method thereof |
| CN112961374A (en) * | 2021-02-03 | 2021-06-15 | 南方医科大学 | Preparation and application of natural swim bladder-derived conductive hydrogel |
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| CN114702692B (en) * | 2022-04-01 | 2024-04-23 | 中国科学院青岛生物能源与过程研究所 | Method for improving stability of protein hydrogel |
| CN116082717A (en) * | 2023-02-02 | 2023-05-09 | 领博生物科技(杭州)有限公司 | Hydrogel, vascular fiber skeleton, preparation method and application thereof |
| CN116082717B (en) * | 2023-02-02 | 2024-06-18 | 领博生物科技(杭州)有限公司 | Hydrogel, vascular fiber skeleton, preparation method and application thereof |
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Application publication date: 20140910 |