CN101200497A - Jellyfish derived kindIIcollagen and preparation technique thereof - Google Patents
Jellyfish derived kindIIcollagen and preparation technique thereof Download PDFInfo
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- CN101200497A CN101200497A CNA2007101727102A CN200710172710A CN101200497A CN 101200497 A CN101200497 A CN 101200497A CN A2007101727102 A CNA2007101727102 A CN A2007101727102A CN 200710172710 A CN200710172710 A CN 200710172710A CN 101200497 A CN101200497 A CN 101200497A
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Abstract
The present invention provides type II collagen from cyanea arctica and the preparation process, pertaining to the natural biological active material and the technical art of preparation. The present invention provides novel natural halobios type II collagen. Being operated according to the present invention, 0.5 percent wt. type II collagen with purity up to more than 85 percents can be obtained. The type II collagen is originally white powder with fishy smell, which contains 85 to 99 percents wt. protein and almost no sugar. The type II collagen is nontoxic and has the reactivity of II collagen such as oral medication, inducement, immunological tolerance, prevention and treatment of chronic infectious arthritis.
Description
Technical field
This patent relates to a kind of class II Collagen Type VI and preparation technology thereof of jellyfish original, belongs to natural bioactivity substance and preparing technical field thereof.
Background technology
Rosy clouds jellyfish (Cyanea nozakii) is commonly called as fiber crops and bites, and is a kind of large ocean planktonic organism, belong to Cnidaria (Phylum Cnidaria), Scyphozoa (Class Scyphomedusae), Semaeostomeae (Order Phylum cnidaria), rosy clouds jellyfish section (Family Cyanea).The coastal adularescent rosy clouds jellyfish of having found of China (Cyanea nozaki Kishinouye), a shape rosy clouds jellyfish (Cyanea capillata), brown rosy clouds jellyfish (Cyanea ferrugineaEschscholtz), with 4 kinds of purple rosy clouds jellyfish (Cyaneapurpurea Kishinouye), wherein with white rosy clouds jellyfish quantity at most, distribution range is the widest.The rosy clouds jellyfish is a bait with small-sized zooplankton usually, its sexual gland prosperity, and fecundity is strong, and the speed of growth is exceedingly fast, and the annual production of China rosy clouds jellyfish has at present constituted a kind of new natural marine organism resource up to ten million ton.
The polysaccharide of marine source, proteoglycan and collagen protein are crucial natural marine food resources, usually have significant biological activity, as sea cucumber polysaccharide, Sargassum polysaccharides etc. many research reports are arranged all, confirm that it has significant biological activity.
The U.S. the someone carried out a kind of with in the jellyfish, sand is bitten (Stomolophus meleagris) and is raw material, separation and Extraction prepares the research of class II Collagen Type VI, and patent applied for [1], but be that any report is not seen in the research that the raw material separation and Extraction prepares class II Collagen Type VI both at home and abroad as yet with the rosy clouds jellyfish.[1]United States Patent：6,894,029
Summary of the invention
This patent has been invented a kind of class II Collagen Type VI and preparation technology thereof of jellyfish original.Comparatively unique is, it has realized the purpose that the rosy clouds jellyfish is developed and utilized with a kind of novel natural marine organism resource-Xia jellyfish (Cyanea nozakii)-process as the preparation raw material of class II Collagen Type VI.Operate according to this patent, can obtain high purity class II Collagen Type VI product with high productive rate.This is vital for the comprehensive utilization of this natural marine organism resource of rosy clouds jellyfish.
The active class II of the rosy clouds jellyfish Collagen Type VI of this patent invention is confirmed through preliminary research, substantially nontoxic, have the oral inducing immune tolerance prevention that the II Collagen Type VI had and the activity of treatment rheumatoid arthritis, will be widely used in foodstuffs industry and medicine industry with healthy food material as novel biological active materials and medicine.
Specifically, at first, we find fresh, freezing and refrigeration or bite through the commercially available fiber crops that three alum are pickled processing can be through being used as the preparation raw material of class II Collagen Type VI after the desalting treatment.It is stable that wherein contained class II Collagen Type VI is handled the water logging bubble, under the soaking technology condition that we taked, neither can be degraded, be extracted in the water and run off because of being subjected to soaking, can not influence its dispersiveness in follow-up hydrolysis treatment because of being subjected to soak yet, promptly the commercially available fiber crops of pickling processing through freezing and refrigeration or three alum bite with the fresh rosy clouds jellyfish of just having fished for disembarkation and have equal workability, all can be used as the raw material of preparation high purity high biological activity class II Collagen Type VI.Established thus with the rosy clouds jellyfish is the basis of raw material processing and preparing high purity high biological activity class II Collagen Type VI.
Secondly, after various extractants commonly used such as hydrolysising condition such as temperature, pressure and sodium hydroxide, hydrochloric acid, Collagenase have been carried out the investigation and contrast of system, we find, under suitable enzymatic hydrolysis condition, the class II Collagen Type VI that is contained in the rosy clouds jellyfish is without denaturing treatment, just can be directly dissolved, be distributed in the acidic solution, obtain the molten acid collagen of enzymatic.
We find that in soaking hydrolysis process parameters such as solvent, soak time, extractant, the temperature that is adopted when the kind of extractant and extracting is of paramount importance for the extracting yield.Wherein, effectively extractant is a kind of in common alkali (as oxyhydroxide or carbonate, the ammoniacal liquor etc. of sodium, potassium, calcium, barium), common acid (organic acids such as mineral acid such as example hydrochloric acid, phosphoric acid, sulfuric acid or formic acid, acetic acid, oxysuccinic acid, lactic acid, citric acid) or the various proteolytic enzyme (as stomach en-, trypsinase, pancreas Chymotrypsin, aspartic protease, Sumizyme MP, neutral protease, papoid, bromeline, Collagenase, elastoser etc.) or some kinds.When selecting for use sodium hydroxide to be extractant, the pH value should be within the 7.0-14.0 scope, and when selecting for use hydrochloric acid to be extractant, the pH value should be within the 0.5-6.8 scope, and when selecting for use protease to be extractant, the pH value should be within the optimal pH scope of selected enzyme; When selecting for use soda acid to be extractant, extraction temperature should preferably in 0-60 ℃ scope, when selecting for use protease to be extractant, should carry out extracting under the optimum temperuture of protease in-30-200 ℃ scope.Other parameters are soaked desalination and need be carried out 0-15 time, and soak required water consumption at every turn and be the rosy clouds jellyfish raw material thrown in 1-5 times, soaking temperature is in-50-98 ℃ scope, and soak time is 0.5-12hr time
-1Saltout and to select (NH for use
4)
2SO
4Or NaCl is salting-out agent, and saturation ratio is 0.1～10molL
-1It is that the tap water or the deionized water of certain certain value carries out that dialysis can be selected pH regulator for use, and its pH value is within the 2-12 scope.The buffering salt that is used to regulate pH can be Tris-HCl, phosphoric acid salt, acetic acid salt, citric acid salt, and the concentration of damping fluid is 0.01～10molL
-1The drying mode of liquid can be lyophilize, also can be spraying drying.
Embodiment
Following example will specify working method of the present invention, but can not be as limitation of the invention.
Example one
Get the fresh rosy clouds jellyfish of 1000g freezing, thaw, clean, handled 10 minutes at 10000rpm with high-speed homogenizer under the room temperature, get homogenate.In homogenate, add the NaOH of the 0.1mol/L of 2 times of volumes, under 4 ℃ of conditions, stirred 24 hours, use high speed freezing centrifuge then, 4 ℃, the centrifugal 10min of 8000rpm, supernatant discarded, collecting precipitation.Repeating above-mentioned extraction to homogenate operates 3 times.With the precipitation after centrifugal, wash repeatedly to neutrality with distilled water, add the acetic acid of 0.5mol/L then, under 4 ℃ of conditions, stirred 48 hours, use refrigerated centrifuge then, 4 ℃, the centrifugal 20min of 8000rpm, supernatant discarded, collecting precipitation.Repeat said extracted operation 2 times.The centrifugal precipitation that obtains is added the acetic acid of the 0.5mol/L of 2 times of volumes, add the stomach en-of jellyfish weight 5 ‰, stirred at least 48 hours, use refrigerated centrifuge then, 4 ℃, the centrifugal 20min of 8000rpm discards precipitation, the collection supernatant.In resulting supernatant liquor, add the NaCl solid, reach 2mol/L to the concentration of NaCl, be stirred to NaCl and all dissolve, place down at 4 ℃ then and spend the night.Use refrigerated centrifuge, 4 ℃, the centrifugal 20min of 8000rpm, supernatant discarded will be deposited in 0.02mol/LNa
2HPO
4Dialysis is 48 hours in the damping fluid, uses AgNO
3Detect extracellular fluid dialysis, no Cl
-Detect.With the dialyzate that obtains, lyophilize had both got 5g rosy clouds jellyfish class II Collagen Type VI product.
Example two
Getting 1000g three alum pickles the commercially available fiber crops of processing and bites, behind the draining, be immersed under the room temperature in the deionized water that 2-5 doubly measures and soak, and changed one time water in every 2-3 hour, (chlorion detects and is negative) obtained 980g desalination rosy clouds jellyfish till the content of sodium-chlor was lower than 1.5ppm in soaking water.Handled 10 minutes at 10000rpm with high-speed homogenizer under the desalination rosy clouds jellyfish room temperature, get homogenate.In homogenate, add the NaOH of the 0.1mol/L of 2 times of volumes, under 4 ℃ of conditions, stirred 24 hours, use high speed freezing centrifuge then, 4 ℃, the centrifugal 30min of 8000rpm, supernatant discarded, collecting precipitation.Repeating above-mentioned extraction to homogenate operates 3 times.With the precipitation after centrifugal, wash repeatedly to neutrality with distilled water, add the acetic acid of 0.5mol/L then, under 4 ℃ of conditions, stirred 48 hours, use refrigerated centrifuge then, 4 ℃, the centrifugal 60min of 8000rpm, supernatant discarded, collecting precipitation.Repeat said extracted operation 2 times.The centrifugal precipitation that obtains is added the acetic acid of the 0.5mol/L of 2 times of volumes, add the stomach en-of jellyfish weight 5 ‰, stirred at least 48 hours, use refrigerated centrifuge then, 4 ℃, the centrifugal 30min of 8000rpm discards precipitation, the collection supernatant.In resulting supernatant liquor, add the NaCl solid, reach 2mol/L to the concentration of NaCl, be stirred to NaCl and all dissolve, place down at 4 ℃ then and spend the night.Use refrigerated centrifuge, 4 ℃, the centrifugal 20min of 8000rpm, supernatant discarded will be deposited in 0.02mol/L Na
2HPO
4Dialysis is 48 hours in the damping fluid, uses AgNO
3Detect extracellular fluid dialysis, no Cl
-Detect.With the dialyzate that obtains, lyophilize had both got 5g rosy clouds jellyfish class II Collagen Type VI product.
Example three
A class II Collagen Type VI liquid sample has been carried out the mensuration of protein content, the mensuration that the mensuration of total sugar content and amino acid are formed.Measurement result shows, contains 98% protein in this sample, does not contain sugar substantially.Amino acid contained composition sees Table 1.
Table 1 is formed for the amino acid of test agent
| Amino acid | Content (mg/g) | ％ |
| Aspartic acid L-glutamic acid Serine Histidine glycine Threonine | 7.07883 5.80446 3.52608 1.17288 3.91896 2.30857 | 11.66 9.56 5.8 1.93 6.46 3.8 |
| Arginine L-Ala tyrosine halfcystine Xie Ansuan methionine(Met) phenylalanine Isoleucine leucine Methionin oxyproline proline(Pro) | 3.22697 2.12322 3.28675 1.45343 2.64805 0.472442 2.88379 3.29703 3.41924 3.11556 8.07391 2.9009 60.71107 | 5.32 3.5 5.41 2.39 4.36 0.78 4.75 5.43 5.63 5.13 13.3 4.78 99.99 |
Example four
With healthy Kunming kind small white mouse (male and female half and half, in 4 ages in week, body weight 18-22g provides [approval number: SCXK (Soviet Union) 2002-0006] by laboratory animal field, the south of the River, curry favour mountain, Wuxi) be laboratory animal, resulting rosy clouds jellyfish class II Collagen Type VI is carried out acute toxicity test, and the gained result is as follows:
Table 2 is for the influence of test agent to the mouse body weight
| Fate/group | Blank | Test group |
| 1d 2d 3d 4d 5d 6d 7d | 22.806g 22.985g 24.300g 24.688g 25.146g 26.084g 26.180g | 22.667 23.075 23.635 23.766 24.566 25.066 25.966 |
Table 3 acute toxicity test result
| Group | Mouse quantity | Dosage | One all body weight gain | Rate of increase | Death toll |
| The blank test group | 10 10 | 0.5ml/ 7500mg/kg only | 3.374g 3.299g | 14.79％ 14.55％ | 0 0 |
By observing, the interior mouse activity of 1h all reduces after the administration, recovers normal activity behind the 4h gradually.Food-intake increases gradually in the test group 7d, and mouse appetite, spirit, hair color are all normal; Behind the 7d, take off neck and put to death, gross necropsy is not seen main organs pathology and intoxicating phenomenons such as the heart, liver, spleen, lung, kidney.Weigh every day in the observation period 7d, and the body weight of test group mouse constantly increases.Result of study shows, under the situation for the heavy dose of filling of test agent stomach, dead mouse do not occur, and the overt toxicity reaction is not arranged yet.Illustrate that its nontoxicity or toxicity are very little, its maximum safe dose is more than 7500mg/Kg.
Example five
(complete male with the healthy SD rat, body weight 150 ± 20g, provide [credit number: SCXK (Zhejiang) 2003-0001] by laboratory animal field, the south of the River, curry favour mountain, Wuxi) be laboratory animal, resulting rosy clouds jellyfish class II Collagen Type VI is used for the treatment of the arthritic physiologically active test of rat adjuvant type, and the gained result is as follows:
1, by irritating stomach rosy clouds jellyfish class II Collagen Type VI after 14 days, find that the two hind leg joints of administration group rat obviously reduce than model group, after two weeks, effect weakens to some extent.The rosy clouds jellyfish class II Collagen Type VI that proves high, normal, basic dosage all has certain result of treatment to adjuvant type sacroiliitis.See table 4 for details.
Table 4 for test agent to the influence of rat adjuvant type arthritis knuckle swelling (
, n=3)
| Group | Dose(mg/kg) | Before irritating stomach | The filling stomach is intact | Irritated a week | Irritated for two weeks |
| The positive group of the blank group treatment of model group collagen | 5 10 20 sprinkle Buddhist nun ground pine 25 | 0.96±0.05 0.98±0.09 0.97±0.09 1.00±0.15 0.78±0.02§ 0.89±0.07 | 0.95±0.08 0.86±0.03 * 0.79±0.05 * 0.85±0.05 ** 0.83±0.05 * 0.86±0.02 | 1.01±0.08 0.88±0.04 ** 0.86±0.05 ** 0.84±0.05§ 0.85±0.05 ** 0.94±0.05 | 0.90±0.04 0.87±0.02 0.88±0.07 0.87±0.06 0.83±0.03 ** 0.95±0.04 |
Each is organized data and all compares § P＜0.001 with the model group of corresponding time,
*P＜0.01,
*P＜0.05
2, by irritating stomach rosy clouds jellyfish class II Collagen Type VI after 14 days, find that NO obviously reduces than model group in the administration group rat blood serum, and effect is continual and steady, proves that the rosy clouds jellyfish class II Collagen Type VI of oral high, normal, basic dosage can effectively suppress the rising of the intravital NO content of adjuvant type rats with arthritis.See table 5 for details.
Table 5 for test agent to the influence of rat adjuvant type sacroiliitis serum NO level (
, n=3)
| Group | Dose(mg/kg) | Before irritating stomach | The filling stomach is intact | Irritated a week | Irritated for two weeks |
| The positive group of the blank group treatment of model group collagen | 5 10 20 sprinkle Buddhist nun ground pine 25 | 11.65±1.38 11.65±3.77 10.57±3.51 9.21±1.01 5.96±1.01 * 10.98±0.40 | 13.82±0.66 5.96±1.67 * 6.77±2.33 * 8.94±1.14 * 5.15±1.53 * 10.56±5.69 | 16.26±1.5 5.96±3.13 * 5.96±1.67 * 7.86±0.38 * 4.34±1.67 * 8.94±0.81 | 19.24±2.33 7.58±1.01 ** 8.67±1.38 ** 6.77±1.01 ** 5.69±1.76 ** 13.01±1.62 |
Each is organized data and all compares § P＜0.001 with the model group of corresponding time,
*P＜0.01,
*P＜0.05
3, by irritating stomach rosy clouds jellyfish class II Collagen Type VI after 14 days, find that SOD improves than the obvious activity of model group in the administration group rat blood serum, and effect is continual and steady, proves that the rosy clouds jellyfish class II Collagen Type VI of oral high, normal, basic dosage can effectively improve the activity of SOD in serum of adjuvant type rats with arthritis.See table 6 for details.
Table 6 for test agent to the influence of rat adjuvant type sacroiliitis SOD in serum (
, n=3)
| Group | Dose(mg/kg) | Before irritating stomach | The filling stomach is intact | Irritated a week | Irritated for two weeks |
| The positive group of the blank group treatment of model group collagen | 5 10 20 sprinkle Buddhist nun ground pine 25 | 117.10±3.41 126.59±5.02 128.53±7.20 123.57±4.01 134.57±13.21 113.33±20.70 | 108.48±3.46 130.04±2.56 * 135.32±5.95 * 124.76±7.49 138.99±10.70 * 114.14±10.83 | 118.29±2.91 149.66±6.34 * 129.93±0.81 * 150.31±10.32 * 133.27±2.97 * 118.83±12.94 | 121.95±0.40 129.18±2.35 * 134.14±3.51 * 138.45±4.42 * 143.52±3.59 * 151.82±5.17 |
Each is organized data and all compares § P＜0.001 with the model group of corresponding time,
*P＜0.01,
*P＜0.05
4, by irritating stomach rosy clouds jellyfish class II Collagen Type VI after 14 days, find that MDA content obviously reduces than model group in the administration group rat blood serum, prove that the rosy clouds jellyfish class II Collagen Type VI of oral high, normal, basic dosage can effectively reduce the Content of MDA of adjuvant type rats with arthritis.See table 7 for details.
The arthritic physiologically active test-results of treatment rat adjuvant type shows, by irritating stomach rosy clouds jellyfish class II Collagen Type VI after 14 days, find that the two hind leg joints of administration group rat obviously reduce than model group, NO obviously reduces than model group in the rat blood serum, SOD is than obvious active raising of model group in the serum, and effect is continual and steady, and MDA content obviously reduces than model group in the serum.
This test-results proves that rosy clouds jellyfish class II Collagen Type VI has certain result of treatment to adjuvant type sacroiliitis.
Table 7 for test agent to the influence of rat adjuvant type sacroiliitis serum MDA (
, n=3)
| Group | Dose(mg/kg) | Before irritating stomach | The filling stomach is intact | Irritated a week | Irritated for two weeks |
| The positive group of the blank group treatment of model group collagen | 5 10 20 sprinkle Buddhist nun ground pine 25 | 7.63±0.45 6.57±1.57 6.92±1.51 6.48±1.22 3.22±0.76 * 7.87±0.46 | 6.70±0.33 3.17±1.35 * 3.44±1.10 * 2.91±1.31 * 2.69±1.02 * 7.87±0.42 | 6.44±0.59 3.88±0.16 * 3.70±0.28 * 4.50±0.49 ** 4.06±0.22 * 7.87±2.58 | 6.53±0.41 5.42±0.60 * 5.34±0.33 ** 4.72±41 * 3.83±0.29 ** 4.37±0.26 |
Each is organized data and all compares § P＜0.001 with the model group of corresponding time,
*P＜0.01,
*P＜0.05
Claims (10)
1. class II Collagen Type VI that separation and Extraction obtains in the rosy clouds jellyfish.
The rosy clouds jellyfish of being declared, being commonly called as fiber crops bites, mean the fresh product of fishing for of rosy clouds jellyfish section marine organisms white rosy clouds jellyfishes (Cyanea nozaki Kishinouye), shape rosy clouds jellyfish (Cyanea capillata), brown rosy clouds jellyfish (Cyanea ferruginea Eschscholtz) or purple rosy clouds jellyfish (Cyaneapurpurea Kishinouye), freezing and refrigeration product or the curing food of pickling through three alum, can can be head and the palpus of rosy clouds jellyfish for use as the position of raw material, also can be canopy or the rosy clouds jellyfish integral body of rosy clouds jellyfish.
The class II Collagen Type VI of being declared is for extracting a kind of product that obtains in the rosy clouds jellyfish.It is a kind of white powder, and extra large fishy smell is arranged, and wherein contains the protein of 85～99%wt., contains sugar hardly.Such II Collagen Type VI is nontoxic, has the oral inducing immune tolerance prevention that the II Collagen Type VI had and the activity of treatment rheumatoid arthritis.
2. the preparation technology of class II Collagen Type VI as claimed in claim 1.This technology is made up of the following step:
1) is that solvent soaks the rosy clouds jellyfish with water, suitably removes the salinity that is contained in the raw material rosy clouds jellyfish.
2) the rosy clouds jellyfish that has removed salinity is carried out break process, obtain a white or a filemot homogenate.
3) extracting is carried out in resulting homogenate, the molten acid collagen of separation and Extraction enzymatic therefrom obtains the solution of the molten acid collagen of enzymatic.
4) to the processing of saltouing of resulting solution, obtain thick collagen throw out.
5) resulting thick collagen throw out is dialysed, obtain dialyzate.
6) resulting dialyzate is carried out drying, obtain white powder, be class II Collagen Type VI product.
3. the water system of declaring in the claim 2 refers to that pH regulator is the tap water or the deionized water of certain certain value, and its pH value is within the 2-12 scope.
4. the immersion of declaring in the claim 2 is carried out 0-15 time, the 1-5 that soaks required water consumption at every turn and be the rosy clouds jellyfish raw material of being thrown in doubly, soaking temperature is in-50-98 ℃ scope, soak time is 0.5-12hr time
-1
5. the fragmentation of declaring in the claim 2 can be used refiner, also can carry out with mortar, and broken temperature is in-50-98 ℃ scope, and the rotating speed of refiner is to be 1-100 minute the 1-20000rpm homogenate time.
6. the extracting of declaring in the claim 2 divides several stages to carry out, and each stage, used extraction solvent can be identical, also can be inequality.
7. the extracting of declaring in the claim 2, the extraction solvent that can Gong select for use are 0.01～10molL
-1Alkali (as oxyhydroxide or carbonate, the ammoniacal liquor etc. of sodium, potassium, calcium, barium), 0.01～10molL
-1Acid (organic acid such as mineral acid such as example hydrochloric acid, phosphoric acid, sulfuric acid or formic acid, acetic acid, oxysuccinic acid, lactic acid, citric acid) or various proteolytic enzyme (as stomach en-, trypsinase, pancreas Chymotrypsin, aspartic protease, Sumizyme MP, neutral protease, papoid, bromeline, Collagenase, elastoser etc.) in a kind of or some kinds.
8. that declares in the claim 2 saltouts, and the salt that can Gong select for use is (NH
4)
2SO
4Or NaCl, the saturation ratio of saltouing is 0.1～10molL
-1
9. the dialysis of declaring in the claim 2, the dialyzate that can Gong select for use are that pH regulator is the tap water or the deionized water of certain certain value, and its pH value is within the 2-12 scope.The buffering salt that is used to regulate pH can be Tris-HCl, phosphoric acid salt, acetic acid salt, citric acid salt, and the concentration of damping fluid is 0.01～10molL
-1
10. the drying of declaring in the claim 2 can be lyophilize, also can be spraying drying.
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| CNA2007101727102A CN101200497A (en) | 2007-12-21 | 2007-12-21 | Jellyfish derived kindIIcollagen and preparation technique thereof |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103060412A (en) * | 2013-01-15 | 2013-04-24 | 青岛贝尔特生物科技有限公司 | Preparation method of collagen for keeping skin elasticity |
| CN104379593A (en) * | 2012-08-23 | 2015-02-25 | 株式会社海月研究所 | Method for fractionally extracting mucin and collagen |
| CN111393521A (en) * | 2020-03-20 | 2020-07-10 | 张培华 | Extraction method of jellyfish collagen |
-
2007
- 2007-12-21 CN CNA2007101727102A patent/CN101200497A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104379593A (en) * | 2012-08-23 | 2015-02-25 | 株式会社海月研究所 | Method for fractionally extracting mucin and collagen |
| CN104379593B (en) * | 2012-08-23 | 2018-01-23 | 株式会社海月研究所 | The fractional extraction method of mucoprotein and collagen |
| CN103060412A (en) * | 2013-01-15 | 2013-04-24 | 青岛贝尔特生物科技有限公司 | Preparation method of collagen for keeping skin elasticity |
| CN111393521A (en) * | 2020-03-20 | 2020-07-10 | 张培华 | Extraction method of jellyfish collagen |
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