CN101628937B - Purification technology of macromolecular collagen protein - Google Patents
Purification technology of macromolecular collagen protein Download PDFInfo
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- CN101628937B CN101628937B CN2009101844815A CN200910184481A CN101628937B CN 101628937 B CN101628937 B CN 101628937B CN 2009101844815 A CN2009101844815 A CN 2009101844815A CN 200910184481 A CN200910184481 A CN 200910184481A CN 101628937 B CN101628937 B CN 101628937B
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Abstract
The invention relates to a purification technology of macromolecular collagen protein. The purification technology comprises the following steps: adding an acid solution to dissolve a crude product of the macromolecular collagen protein or directly taking an acid extracting solution of the macromolecular collagen protein as a raw material; adding a NaOH solution to the dissolved solution or the acid extracting solution to adjust a pH value; standing and taking out a suspension layer of the collagen protein; dissolving the suspension layer of the collagen protein by an acetate solution for direct dialysis; and freezing and drying. The purification technology comprises the step of directly adding the NaOH solution to the acid solution of the collagen protein to adjust acidity so as to omit the addition of a great amount of Tris and NaCl, greatly decrease the extraction cost, omit the freezing and centrifuging process of the collagen protein after being salted out, simplify the technical process and reduce the energy consumption. The direct distilled water dialysis technology greatly shortens the dialysis cost and the dialysis time, decreases the cost by more than one time, not only lowers the operation difficulty of the technology, but also saves the production cost, and the molecular weight of the extracted collagen protein is not less than 0.1 million Daltons.
Description
Technical field
The present invention relates to a kind of process for refining of protein purification technology, particularly a kind of macromolecular collagen protein.
Background technology
Collagen protein is one type of starting material in industry widespread uses such as medical surgical, beauty treatment filling, skin care item.At present China collagen protein production master be that raw material passes through extraction process and obtains the macromolecular collagen protein bullion with the skins of pig, ox etc., tendon etc.The macromolecular collagen protein bullion need pass through refining could be in sector applications such as medical surgical, beauty treatment filling, skin care item.The treating process of tradition macromolecular collagen protein is: in the collagen protein crude extract, at first add Tris to 0.01-1.0mol/L; Add sodium-chlor again to 1.5-3.5mol/L; Saltout and place 2-48h at low temperature or 4 ℃ of refrigerators; The centrifugal 10-100min of back 1000-20000rpm refrigerated centrifuge gets the collagen protein deposition; Resolution of precipitate in the acetic acid of 0.1-10mol/L, is saltoutd 1-5 time again, with 0.001-0.1mol/L acetic acid dialysis 10-100h, use distill water dialysis 10-100h again, lyophilize obtains macromolecular collagen protein.
In the tradition process for refining, at first add Tris, add sodium-chlor again, slowly stirring and dissolving, needs consume a large amount of Tris and analytical pure sodium-chlor, and Tris costs an arm and a leg, and has increased refining cost greatly, has restricted the industrial leaching process of macromolecular collagen protein.
In the tradition process for refining, need the freezing centrifugation method to obtain the collagen protein deposition after saltouing, complex equipments, energy consumption is high.
In the tradition process for refining, dialysis procedure adopts two-step approach, uses dilute acid soln earlier, uses the method for zero(ppm) water again, and the process of macromolecular collagen protein dialysis is complicated, and the technological operation difficulty is high.
Summary of the invention
Technical problem to be solved by this invention is the deficiency to prior art, and the process for refining of the macromolecular collagen protein that a kind of technology is simple, workable, cost is low is provided.
Technical problem to be solved by this invention is to realize through following technical scheme.The present invention is a kind of process for refining of macromolecular collagen protein, it is characterized in that, its step is following:
(1) in the macromolecular collagen protein bullion, add 0 ℃-30 ℃ acidic solution and dissolve, lysate, the weightmeasurement ratio of macromolecular collagen protein bullion and acidic solution is 1: 5-50; Perhaps directly get 0 ℃-30 ℃ macromolecular collagen protein acidic extraction liquid and make raw material, 0 ℃-30 ℃ of addings, concentration are the NaOH solution of 0.01-2.0mol/L in lysate or acidic extraction liquid, and its pH value is adjusted to 5-12, get collagen solution; With collagen solution 0 ℃-30 ℃ leave standstill 2-48 hour after, take out the collagen protein suspended layer of collagen solution top cohesion;
(2) the collagen protein suspended layer is dissolved in 0 ℃-30 ℃ the acidic solution, the weightmeasurement ratio of collagen protein suspended layer and acidic solution is 1: 5-50, and (1) described method is regulated pH value, is left standstill then set by step, must the collagen protein suspended layer;
(3) get of the acetum dissolving of collagen protein suspended layer with 0.05-10mol/L; The weightmeasurement ratio of collagen protein suspended layer and acetum is 1: 5-50; Directly dialyse with zero(ppm) water then, with the collagen protein suspension-s lyophilize that obtains, must make with extra care macromolecular collagen protein after the dialysis;
Described acidic solution is acetic acid, Hydrocerol A or hydrochloric acid soln.
In the process for refining of above-described macromolecular collagen protein:
1, can repeating step (2) 1-5 time, the collagen protein suspended layer;
2, in step (1): the temperature of the acidic solution that in the macromolecular collagen protein bullion, adds is preferably 0 ℃-10 ℃; The weightmeasurement ratio of macromolecular collagen protein bullion and acidic solution is preferably 1: 5-20; Perhaps directly get 0 ℃-10 ℃ macromolecular collagen protein acidic extraction liquid and make raw material;
3, in step (1): the temperature of the NaOH solution that in lysate or acidic extraction liquid, adds is preferably 0 ℃-10 ℃, concentration and is preferably 0.5-1.0mol/L, preferably its pH value is adjusted to 7-11.
4, in step (1): preferably collagen solution is placed 0 ℃-10 ℃ when leaving standstill, time of repose is preferably 10-36 hour;
5, in step (2): the acidic solution of dissolving collagen protein suspended layer is preferably 0 ℃-10 ℃, and the weightmeasurement ratio of collagen protein suspended layer and acidic solution is preferably 1: 5-20.
6, in step (3): the concentration of the acetum of dissolving collagen protein suspended layer is preferably 0.1-5mol/L, and the weightmeasurement ratio of collagen protein suspended layer and acetum is preferably 1: 5-20.
7, technology of the present invention is applicable to that disclosed any acid system in the prior art is extracted the macromolecular collagen protein bullion that makes to be made with extra care, and is applicable to that also disclosed any acid system in the prior art is extracted the macromolecular collagen protein acidic extraction liquid that makes to be made with extra care.
Compared with prior art, technology of the present invention has the following advantages:
1, process for refining of the present invention directly adds NaOH solution acid adjustment degree in the acid collagen protein solution, adds a large amount of Tris, NaCl thereby saved, and the extraction expense is reduced greatly;
2, save the frozen centrifugation process of the back collagen protein of saltouing in the process for refining of the present invention, simplified technological process, reduced energy consumption;
3, the dialysis process using straight run distillation water dialysis technology of process for refining of the present invention shortens dialysis expense and time greatly;
4, the cost of process for refining of the present invention reduces more than one times; Both reduced the operation easier of technology; Practiced thrift production cost again, extracted the collagen molecules amount that obtains and be not less than 100,000 dalton, the collagen protein that its purity and traditional process for refining make is as good as basically.
Description of drawings
Fig. 1 is the electrophorogram of the macromolecular collagen protein that adopts the inventive method and make, among the figure:
1: with Tris, the NaCl traditional method refining collagen protein that obtains of saltouing;
2: the collagen protein that arrives with the inventive method system.
Embodiment
Below further describe concrete technical scheme of the present invention,, and do not constitute restriction its right so that those skilled in the art understands the present invention further.
Embodiment 1.A kind of process for refining of macromolecular collagen protein, its step is following:
(1) in the macromolecular collagen protein bullion, add 0 ℃ acidic solution and dissolve, lysate, the weightmeasurement ratio of macromolecular collagen protein bullion and acidic solution is 1: 5; 0 ℃ of adding, concentration are the NaOH solution of 0.01mol/L in lysate, and its pH value is adjusted to 5, get collagen solution; With collagen solution 0 ℃ leave standstill 2 hours after, take out the collagen protein suspended layer of collagen solution top cohesion;
(2) the collagen protein suspended layer is dissolved in 0 ℃ the acidic solution, the weightmeasurement ratio of collagen protein suspended layer and acidic solution is 1: 5, and (1) described method is regulated pH value, left standstill then set by step, must the collagen protein suspended layer;
(3) get of the acetum dissolving of collagen protein suspended layer with 0.05mol/L; The weightmeasurement ratio of collagen protein suspended layer and acetum is 1: 5; Directly dialyse with zero(ppm) water then, with the collagen protein suspension-s lyophilize that obtains, must make with extra care macromolecular collagen protein after the dialysis;
Described acidic solution is an acetum.
Embodiment 2.A kind of process for refining of macromolecular collagen protein, its step is following:
(1) in the macromolecular collagen protein bullion, add 30 ℃ acidic solution and dissolve, lysate, the weightmeasurement ratio of macromolecular collagen protein bullion and acidic solution is 1: 50; 30 ℃ of addings, concentration are the NaOH solution of 2.0mol/L in lysate, and its pH value is adjusted to 12, get collagen solution; With collagen solution 30 ℃ leave standstill 48 hours after, take out the collagen protein suspended layer of collagen solution top cohesion;
(2) the collagen protein suspended layer is dissolved in 30 ℃ the acidic solution, the weightmeasurement ratio of collagen protein suspended layer and acidic solution is 1: 50, and (1) described method is regulated pH value, left standstill then set by step, must the collagen protein suspended layer;
(3) get of the acetum dissolving of collagen protein suspended layer with 10mol/L; The weightmeasurement ratio of collagen protein suspended layer and acetum is 1: 50; Directly dialyse with zero(ppm) water then, with the collagen protein suspension-s lyophilize that obtains, must make with extra care macromolecular collagen protein after the dialysis;
Described acidic solution is a citric acid solution.
Embodiment 3.With reference to Fig. 1.A kind of process for refining of macromolecular collagen protein, its step is following:
(1) in the macromolecular collagen protein bullion, add 4 ℃ acidic solution and dissolve, lysate, the weightmeasurement ratio of macromolecular collagen protein bullion and acidic solution is 1: 20; Perhaps directly get 4 ℃ macromolecular collagen protein acidic extraction liquid and make raw material, 4 ℃ of addings, concentration are the NaOH solution of 0.5mol/L in lysate or acidic extraction liquid, and its pH value is adjusted to 7, get collagen solution; With collagen solution 4 ℃ leave standstill 24 hours after, take out the collagen protein suspended layer of collagen solution top cohesion;
(2) the collagen protein suspended layer is dissolved in 4 ℃ the acidic solution, the weightmeasurement ratio of collagen protein suspended layer and acidic solution is 1: 20, and (1) described method is regulated pH value, left standstill then set by step, must the collagen protein suspended layer;
(3) get of the acetum dissolving of collagen protein suspended layer with 0.05-10mol/L; The weightmeasurement ratio of collagen protein suspended layer and acetum is 1: 40; Directly dialyse with zero(ppm) water then, with the collagen protein suspension-s lyophilize that obtains, must make with extra care macromolecular collagen protein after the dialysis;
Described acidic solution is a hydrochloric acid soln.
Adopting the SDS-PAGE electrophoretic detection that aforesaid method is made macromolecular collagen protein detects; Detailed process reference: Laemmli; U.K. (1970) .Cleavage of structural proteinsduring assembly head of bacteriophage T4.Nature, 277,680-685; Simultaneously the refining collagen protein that obtains of saltouing with Tris, NaCl traditional method is detected, the result is referring to Fig. 1.
As can be seen from Figure 1, the two sees that from electrophorogram purity difference is not obvious.
Embodiment 4.A kind of process for refining of macromolecular collagen protein, its step is following:
(1) in the macromolecular collagen protein bullion, add 10 ℃ acidic solution and dissolve, lysate, the weightmeasurement ratio of macromolecular collagen protein bullion and acidic solution is 1: 10; Perhaps directly get 10 ℃ macromolecular collagen protein acidic extraction liquid and make raw material, 10 ℃ of addings, concentration are the NaOH solution of 1.0mol/L in lysate or acidic extraction liquid, and its pH value is adjusted to 9, get collagen solution; With collagen solution 10 ℃ leave standstill 12 hours after, take out the collagen protein suspended layer of collagen solution top cohesion;
(2) the collagen protein suspended layer is dissolved in 10 ℃ the acidic solution, the weightmeasurement ratio of collagen protein suspended layer and acidic solution is 1: 10, and (1) described method is regulated pH value, left standstill then set by step, must the collagen protein suspended layer;
(3) get of the acetum dissolving of collagen protein suspended layer with 2mol/L; The weightmeasurement ratio of collagen protein suspended layer and acetum is 1: 10; Directly dialyse with zero(ppm) water then, with the collagen protein suspension-s lyophilize that obtains, must make with extra care macromolecular collagen protein after the dialysis;
Described acidic solution is an acetum.
Embodiment 5.A kind of process for refining of macromolecular collagen protein, its step is following:
(1) in the macromolecular collagen protein bullion, add 8 ℃ acidic solution and dissolve, lysate, the weightmeasurement ratio of macromolecular collagen protein bullion and acidic solution is 1: 12; Perhaps directly get 8 ℃ macromolecular collagen protein acidic extraction liquid and make raw material, 8 ℃ of addings, concentration are the NaOH solution of 0.8mol/L in lysate or acidic extraction liquid, and its pH value is adjusted to 6, get collagen solution; With collagen solution 8 ℃ leave standstill 5 hours after, take out the collagen protein suspended layer of collagen solution top cohesion;
(2) the collagen protein suspended layer is dissolved in 8 ℃ the acidic solution, the weightmeasurement ratio of collagen protein suspended layer and acidic solution is 1: 30, and (1) described method is regulated pH value, left standstill then set by step, must the collagen protein suspended layer; Repeat above-mentioned steps again 1 time, get the collagen protein suspended layer;
(3) get of the acetum dissolving of collagen protein suspended layer with 8mol/L; The weightmeasurement ratio of collagen protein suspended layer and acetum is 1: 30; Directly dialyse with zero(ppm) water then, with the collagen protein suspension-s lyophilize that obtains, must make with extra care macromolecular collagen protein after the dialysis;
Described acidic solution is a citric acid solution.
Embodiment 6.A kind of process for refining of macromolecular collagen protein, its step is following:
(1) in the macromolecular collagen protein bullion, add 2 ℃ acidic solution and dissolve, lysate, the weightmeasurement ratio of macromolecular collagen protein bullion and acidic solution is 1: 18; Perhaps directly get 6 ℃ macromolecular collagen protein acidic extraction liquid and make raw material, 6 ℃ of addings, concentration are the NaOH solution of 1.5mol/L in lysate or acidic extraction liquid, and its pH value is adjusted to 11, get collagen solution; With collagen solution 6 ℃ leave standstill 15 hours after, take out the collagen protein suspended layer of collagen solution top cohesion;
(2) the collagen protein suspended layer is dissolved in 6 ℃ the acidic solution, the weightmeasurement ratio of collagen protein suspended layer and acidic solution is 1: 35, and (1) described method is regulated pH value, left standstill then set by step, must the collagen protein suspended layer; Repeat above-mentioned steps again 5 times, get the collagen protein suspended layer;
(3) get of the acetum dissolving of collagen protein suspended layer with 1.5mol/L; The weightmeasurement ratio of collagen protein suspended layer and acetum is 1: 16; Directly dialyse with zero(ppm) water then, with the collagen protein suspension-s lyophilize that obtains, must make with extra care macromolecular collagen protein after the dialysis;
Described acidic solution hydrochloric acid soln.
Embodiment 7.A kind of process for refining of macromolecular collagen protein, its step is following:
(1) in the macromolecular collagen protein bullion, add 5 ℃ acidic solution and dissolve, lysate, the weightmeasurement ratio of macromolecular collagen protein bullion and acidic solution is 1: 22; Perhaps directly get 5 ℃ macromolecular collagen protein acidic extraction liquid and make raw material, 5 ℃ of addings, concentration are the NaOH solution of 1.2mol/L in lysate or acidic extraction liquid, and its pH value is adjusted to 10, get collagen solution; With collagen solution 5 ℃ leave standstill 36 hours after, take out the collagen protein suspended layer of collagen solution top cohesion;
(2) the collagen protein suspended layer is dissolved in 5 ℃ the acidic solution, the weightmeasurement ratio of collagen protein suspended layer and acidic solution is 1: 45, and (1) described method is regulated pH value, left standstill then set by step, must the collagen protein suspended layer; Repeat above-mentioned steps again 3 times, get the collagen protein suspended layer;
(3) get of the acetum dissolving of collagen protein suspended layer with 3mol/L; The weightmeasurement ratio of collagen protein suspended layer and acetum is 1: 10; Directly dialyse with zero(ppm) water then, with the collagen protein suspension-s lyophilize that obtains, must make with extra care macromolecular collagen protein after the dialysis;
Described acidic solution is a citric acid solution.
Claims (7)
1. the process for refining of a macromolecular collagen protein is characterized in that, its step is following:
(1) extract the acidic solution that adds 0 ℃-30 ℃ in the macromolecular collagen protein bullion that makes to acid system and dissolve, get lysate, the weightmeasurement ratio of macromolecular collagen protein bullion and acidic solution is 1: 5-50; Perhaps directly getting 0 ℃-30 ℃ acid system extracts the macromolecular collagen protein acidic extraction liquid make and makes raw material; 0 ℃-30 ℃ of addings, concentration are the NaOH solution of 0.01-2.0mol/L in lysate or acidic extraction liquid; Its pH value is adjusted to 5-12, gets collagen solution; With collagen solution 0 ℃-30 ℃ leave standstill 2-48 hour after, take out the collagen protein suspended layer of collagen solution top cohesion;
(2) the collagen protein suspended layer is dissolved in 0 ℃-30 ℃ the acidic solution, the weightmeasurement ratio of collagen protein suspended layer and acidic solution is 1: 5-50, and (1) described method is regulated pH value, is left standstill then set by step, must the collagen protein suspended layer;
(3) get of the acetum dissolving of collagen protein suspended layer with 0.05-10mol/L; The weightmeasurement ratio of collagen protein suspended layer and acetum is 1: 5-50; Directly dialyse with zero(ppm) water then, with the collagen protein suspension-s lyophilize that obtains, must make with extra care macromolecular collagen protein after the dialysis;
Described acidic solution is acetic acid, Hydrocerol A or hydrochloric acid soln.
2. the process for refining of macromolecular collagen protein according to claim 1 is characterized in that, repeating step (2) 1-5 time gets the collagen protein suspended layer.
3. the process for refining of macromolecular collagen protein according to claim 1 and 2 is characterized in that, in step (1): the temperature of the acidic solution that in the macromolecular collagen protein bullion, adds is 0 ℃-10 ℃; The weightmeasurement ratio of macromolecular collagen protein bullion and acidic solution is 1: 5-20; Perhaps directly get 0 ℃-10 ℃ macromolecular collagen protein acidic extraction liquid and make raw material.
4. the process for refining of macromolecular collagen protein according to claim 1 and 2; It is characterized in that; In step (1): the temperature of the NaOH solution that in lysate or acidic extraction liquid, adds is that 0 ℃-10 ℃, concentration are 0.5-1.0mol/L, and its pH value is adjusted to 7-11.
5. the process for refining of macromolecular collagen protein according to claim 1 and 2 is characterized in that, in step (1): when leaving standstill collagen solution is placed 0 ℃-10 ℃, time of repose is 10-36 hour.
6. the process for refining of macromolecular collagen protein according to claim 1 and 2 is characterized in that, in step (2): the acidic solution of dissolving collagen protein suspended layer is 0 ℃-10 ℃, and the weightmeasurement ratio of collagen protein suspended layer and acidic solution is 1: 5-20.
7. the process for refining of macromolecular collagen protein according to claim 1 and 2; It is characterized in that; In step (3): the concentration of the acetum of dissolving collagen protein suspended layer is 0.1-5mol/L, and the weightmeasurement ratio of collagen protein suspended layer and acetum is 1: 5-20.
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| CN102621213A (en) * | 2012-01-13 | 2012-08-01 | 淮海工学院 | Evaluation method for degree of thermal denaturation of macromolecular collagen by using pepsin process |
| CN103352063B (en) * | 2013-06-14 | 2014-12-10 | 张仲文 | Method for extracting and concentrating type II collagen |
| CN105985430A (en) * | 2016-01-27 | 2016-10-05 | 成都市科乐生物技术有限公司 | Low-temperature macromolecular collagen extracting method |
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| CN101445821A (en) * | 2008-12-24 | 2009-06-03 | 东北林业大学 | Method for preparing deer byproduct collagen and functional collagen protein taken as medical and function foods |
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| CN101445821A (en) * | 2008-12-24 | 2009-06-03 | 东北林业大学 | Method for preparing deer byproduct collagen and functional collagen protein taken as medical and function foods |
Non-Patent Citations (2)
| Title |
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| 张俊杰 等.鲤鱼鱼皮胶原蛋白的提取及其性质研究.《氨基酸和生物资源》.2008,18-21. * |
| 段蕊 等.鲤鱼鱼皮和鱼骨酶溶性胶原蛋白的性质比较.《食品与发酵工艺》.2008,10-13. * |
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