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Preparation method of pepsin-soluble high-purity superhelical-structured type-I collagen

A collagen and supercoil technology, applied in the field of preparation of high-purity type I collagen with low antigenicity, can solve the problems of cumbersome and complicated process, limited product purity, high production cost, etc., achieve simple and reasonable process flow, shorten purification time, The effect of shortening the production cycle

Active Publication Date: 2014-11-19
THIRD INST OF OCEANOGRAPHY STATE OCEANIC ADMINISTATION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantages of these preparation processes are: the process design did not pay attention to the influence of the two extraction methods of acid soluble and enzyme soluble on the antigenicity of collagen products, the process is cumbersome and complicated, the separation and purification cycle is long, the preparation cost is high, and the process is difficult to scale up and scale up Production; and the prepared enzyme-soluble collagen configuration is not clear, the triple helix structure is not clear, the value of the molecular weight is not exact, the purity of the product is limited - at most it can only reach the level of SDS-PAGE electrophoresis, and the yield of the product is not high. high

Method used

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  • Preparation method of pepsin-soluble high-purity superhelical-structured type-I collagen
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  • Preparation method of pepsin-soluble high-purity superhelical-structured type-I collagen

Examples

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Effect test

Embodiment 1

[0041] A preparation process of enzyme-soluble high-purity superhelical structure type I collagen, the steps are as follows:

[0042] (1) Raw material pretreatment

[0043] 2 kg of washed fish scales were pulverized first, then put into the reaction kettle, added an alkali solution 10 times the weight of the fish scales, stirred for 12 hours, washed with water, added an acid solution 10 times the weight of the fish scales, stirred for 12 hours, and then washed with water. Wherein the mass volume concentration of alkali solution is 3%, the mass volume concentration of acid solution is 5%, and pretreatment temperature is 25 ℃;

[0044] (2) Type Ⅰ collagen extraction

[0045] Add 0.1 mol / L of acetic acid, citric acid, oxalic acid or malic acid to the treated raw material as the extract solution, the extraction temperature is 4°C, the weight ratio of raw material to extractant is 1:10, and at the same time add 0.5% by weight of raw material The pepsin was stirred and extracted f...

Embodiment 2

[0051] A preparation process of enzyme-soluble high-purity superhelical structure type I collagen, the steps are as follows:

[0052] (1) Raw material pretreatment

[0053] Put 5 kg of washed fish scales into the reaction kettle, add an alkali solution 8 times the weight of the fish scales, stir for 10 hours, wash with water, add an acid solution 8 times the weight of the fish scales, stir for 10 hours, and then wash with water. The mass volume concentration is 2%, the mass volume concentration of the acid solution is 6%, and the pretreatment temperature is 20°C;

[0054] (2) Type Ⅰ collagen extraction

[0055] Add 0.25mol / L of acetic acid, citric acid, oxalic acid or malic acid to the treated raw material as the extracting solution, the extraction temperature is 4°C, the weight ratio of raw material to extractant is 1:10, and at the same time add 1% of raw material weight The pepsin was stirred and extracted for 12 hours. The extraction step could be repeated 3 times. The e...

Embodiment 3

[0061] A preparation process of enzyme-soluble high-purity superhelical structure type I collagen, the steps are as follows:

[0062] (1) Raw material pretreatment

[0063] Put 0.5 kg of washed fish scales into the reaction kettle, add an alkali solution 6 times the weight of the fish scales, stir for 4 hours, wash with water, add an acid solution 6 times the weight of the fish scales, stir for 12 hours, and then wash with water, the alkali solution The mass volume concentration of the acid solution is 1%, the mass volume concentration of the acid solution is 8%, and the pretreatment temperature is 15°C;

[0064] (2) Type Ⅰ collagen extraction

[0065] Add 1 mol / L of acetic acid, citric acid, oxalic acid or malic acid to the treated raw material as the extract solution, the extraction temperature is 8°C, the weight ratio of the raw material to the extractant is 1:10, and at the same time add 2% of the raw material weight The pepsin was stirred and extracted for 12 hours, and...

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Abstract

The invention discloses a preparation method of a pepsin-soluble high-purity superhelical-structured type-I collagen, i.e. a low-antigenicity high-purity superhelical-structured type-I collagen. The preparation method is characterized by comprising the following steps: carrying out alkali treatment on scales which mainly contain the type-I collagen and serves as a raw material to remove hybrid proteins and fats, then carrying out acid treatment to remove calcium salts of the scales, adding a weak acid solution as an extraction agent, simultaneously adding pepsin, carrying out low-temperature stirring and leaching, and after the leaching is completed, carrying out refrigerated centrifugation so as to obtain crude leach liquor of the pepsin-soluble type-I collagen; purifying the crude leach liquor of the pepsin-soluble type-I collagen by using a membrane separation technology, and freeze-drying, so that the pepsin-soluble type-I collagen with a purity of not less than 90% and kept at a triple helix structure is obtained. A collagen product prepared by using the method disclosed by the invention is low in antigenicity, clear in configuration, and integral in triple helix structure; HPLC detection shows that the product is a type-I collagen with a purity of greater than or equal to 90%, and MALDI-TOF-MS detection shows that a mass spectrogram of the product has no impure peak, and the molecular weight is 288 kDa.

Description

technical field [0001] The invention relates to a preparation process of enzyme-soluble high-purity type I collagen with a triple helix structure, and is also a preparation process of low-antigenic high-purity type I collagen, belonging to the technical field of biological extraction. Background technique [0002] Collagen is the most important and important type of protein in the support structure of animals. It is widely distributed in the skin, blood vessels, bones, cartilage and connective tissues of the human body. It has a special triple helix structure, also known as a superhelix structure, that is, three left-handed helical α chains are wound around each other through a right-handed helix. At the same time, the glycine content of collagen accounts for almost 1 / 3, and the content of proline and hydroxyproline is the highest among various proteins. Collagen also has hydroxylysine that does not exist in other proteins. Collagen's unique spatial structure and chemical c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P21/06C07K14/78C07K1/34
Inventor 陈思谨易瑞灶陈晖洪碧红谢全灵
Owner THIRD INST OF OCEANOGRAPHY STATE OCEANIC ADMINISTATION
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