Method for extracting pure type II collagen from chicken cartilage

A technology for chicken cartilage and collagen, which is applied in the field of biomedical material preparation, can solve the problems of inability to remove type IX, type X, type XI collagen and type II collagen, the extraction method is complex, and the type of collagen is not single. The effect of large-scale production, good biocompatibility, and low production cost

Inactive Publication Date: 2015-12-09
SICHUAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] At present, domestic and foreign literatures report a variety of methods for extracting collagen, but due to the complexity of type II collagen extraction methods, most of the extracted products are mixtures or crude extracts.
In the article "Preparation of Chicken Articular Cartilage Type II Collagen" published in "Food Science" 2007, Volume 28, No. 4, Cao Hui et al., neither the ion exchange chromatography nor the water washing method used can remove Type IX, Type X, and Collagen. Type Ⅺ collagen, so the type of collagen prepared is not single; the Chinese invention patent "non-denatured type Ⅱ collagen production method" with the authorization number CN102154425B adopts salting out under neutral conditions, and different types of collagen are obtained after salting out. Washing with deionized water can purify collagen, but it cannot remove other types of collagen in type II collagen, so the obtained type II collagen also contains other types of collagen

Method used

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  • Method for extracting pure type II collagen from chicken cartilage
  • Method for extracting pure type II collagen from chicken cartilage
  • Method for extracting pure type II collagen from chicken cartilage

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] 1) Pretreatment of cartilage

[0047] Remove residual meat and other non-cartilage components from the surface of fresh cartilage, weigh 100g and wash it fully with normal saline, add 10 times of degreasing agent (chloroform:methanol=1:1) to soak, replace the degreasing solution every 12h, pour it out after 60h solution, washed 3 times with cold ultrapure water, then soaked the cartilage in cold ultrapure water overnight, took it out and mashed it to obtain cartilage slurry;

[0048] 2) Removal of foreign proteins:

[0049] Prepare 4mol / LNaCl-0.05mol / LTris-HCl mixed solution H1, the pH is 7.5, add the above-mentioned mixed solution H1 to the cartilage slurry obtained in step 1), the ratio of solid to liquid is 1:25, stir for 24h, and centrifuge , the speed of the centrifuge is 9000r / min, the time is 10min, the temperature is 4°C, the precipitate is left, and the precipitate is washed repeatedly with cold ultrapure water for 5 times to obtain the precipitate;

[0050] ...

Embodiment 2

[0059] 1) Pretreatment of cartilage

[0060] Remove residual meat and other non-cartilage components from the surface of fresh cartilage, weigh 160g, wash it fully with normal saline, add 30 times of degreasing solution (chloroform:methanol=1:3) to soak, replace the degreasing solution every 10h, and pour it out after 36h Solution, wash 5 times with cold deionized water, then soak the cartilage in cold deionized water overnight, take out and mash to obtain cartilage slurry;

[0061] 2) Removal of foreign proteins:

[0062] Prepare 4mol / LNaCl-0.05mol / LTris-HCl mixed solution H1, the pH is 7.5, add the above-mentioned mixed solution H1 to the cartilage slurry obtained in step 1), the ratio of solid to liquid is 1:30, stir for 12h, and centrifuge , the speed of the centrifuge is 6000r / min, the time is 25min, the temperature is 4°C, the precipitate is left, and the precipitate is washed repeatedly with cold deionized water 4 times to obtain the precipitate;

[0063] Prepare 4mol...

Embodiment 3

[0072] 1) Pretreatment of cartilage

[0073] Remove residual meat and other non-cartilage components from the surface of fresh cartilage, weigh 120g and wash it fully with normal saline, add 20 times of degreasing solution (chloroform:methanol=1:4) to soak, replace the degreasing solution every 15h, and pour it out after 24h solution, washed 4 times with cold deionized water, then soaked the cartilage in cold deionized water overnight, took it out and mashed it to obtain cartilage slurry;

[0074] 2) Removal of foreign proteins:

[0075]Prepare 4mol / LNaCl-0.05mol / LTris-HCl mixed solution H1, the pH is 7.5, add the above-mentioned mixed solution H1 to the cartilage slurry obtained in step 1), the material-liquid ratio is 1:15, stir for 36h, and centrifuge , the speed of the centrifuge is 8000r / min, the time is 15min, the temperature is 4°C, the precipitate is left, and the precipitate is washed repeatedly with cold deionized water for 3 times to obtain the precipitate;

[007...

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Abstract

The invention discloses a method for extracting pure type II collagen from chicken cartilage. The method is characterized by including, using the fresh chicken cartilage as raw material, removing other non-cartilage components, performing degreasing pretreatment, using mixed solution of 4mol/L NaCl and 0.05mol/L Tris-HCl to remove soluble impure protein, using mixed solution (pH 7.5) of 4mol/L guanidine hydrochloride and 0.05mol/L Tris-HCl to remove insoluble impure protein, using acetic acid solution containing 1-2% pepsin for solid matter enzymolysis, then centrifuging to take supernate, adding NaCl until final concentration is 3-4.2mol/L, 1.2-1.8mol/L and 0.8-1.0mol/L, successively salting out to remove other types of collagen, repeatedly adding NaCl until the final concentration is 0.8-1.0mol/L, salting out for 2-5 times, and dialyzing to obtain the pure type II collagen. The type II collagen produced by the method is single in type, high in bioactivity, stable in physical and chemical property. Meanwhile, the method is simple to operate and capable of benefiting large-scale production.

Description

technical field [0001] The invention relates to a method for extracting pure type II collagen from chicken cartilage, belonging to the field of biomedical material preparation. Background technique [0002] Type II collagen is the main component of cartilage and vitreous body. It can induce the occurrence, differentiation and migration of chondrocytes, and is beneficial to the regeneration of articular cartilage and maintains the morphological structure and tensile strength of articular cartilage. The unique structure and chemical composition of type II collagen make it widely used in the fields of biomedical materials, tissue engineering, packaging materials, cosmetics and health food, especially for the prevention and treatment of rheumatoid arthritis. effect. Long-term studies at home and abroad have shown that type II collagen is an important scaffold material in cartilage tissue engineering and plays a vital role in the repair and reconstruction of cartilage tissue (Ch...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P21/06C07K14/78
Inventor 李国英吴雷刘文涛
Owner SICHUAN UNIV
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