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Preparation method of low-endotoxin collagen

A collagen and endotoxin technology, applied in the field of low-endotoxin collagen preparation, can solve the problems of increased operation difficulty, endotoxin reduction, collagen denaturation, etc., and achieve the effect of simple and convenient operation, high removal efficiency and low cost

Active Publication Date: 2021-05-11
胶原蛋白(武汉)生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method is also not suitable for removing endotoxins in collagen, and the reasons are as follows: ① continuous two-step alkali treatment (vacuum high concentration (29.5g / L) sodium hydroxide hydrolysis treatment-ultrafiltration-low concentration (1.5g / L) ) Sodium hydroxide treatment) technical scheme increases the difficulty of operation, and high-concentration alkaline solution can easily lead to collagen denaturation, decomposition or inactivation; ②Researchers usually use alkaline hydrolysis to extract collagen peptides in animal tissues, while alkaline Treatment conditions will cause collagen to be hydrolyzed into collagen peptides. It can be seen that alkali treatment generally cannot be used in the extraction process of collagen; ③ direct alkali treatment of collagen enzymatic hydrolyzate and collagen products will affect the quality of collagen and stability, so that the yield of collagen is greatly reduced; ④ It can be seen from the records of this patent specification that the reduction efficiency of endotoxin in the technical solution is up to 80%. The endotoxin is reduced to below 0.5Eu / mL stipulated by the pharmaceutical industry standard

Method used

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  • Preparation method of low-endotoxin collagen
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  • Preparation method of low-endotoxin collagen

Examples

Experimental program
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Effect test

Embodiment 1

[0036] Embodiment 1 Preparation process 1 of low endotoxin collagen

[0037] Wash the yak tendons, remove foreign matter, and crush them into small pieces; degrease the crushed small pieces with 10% n-butanol, take the precipitate, and wash it with water until neutral; wash the precipitate obtained in the previous step with 0.5M hydrochloric acid Decalcification, decalcification is over, take the precipitate, wash it with water until neutral; soak the tissue precipitate obtained in the previous step in 0.1M sodium hydroxide solution, stir at 25°C for 4 hours, take the precipitate, wash it with water until neutral; Extract collagen with 0.5M acetic acid solution of 1g / L pepsin to obtain a crude collagen extract; adjust the pH to neutral to inactivate the enzyme; perform dialysis with an 8-14kDa dialysis bag, and freeze-dry after the dialysis to obtain Collagen with low endotoxin content.

Embodiment 2

[0038] Embodiment 2 The preparation process 2 of low endotoxin collagen

[0039]Wash the yak tendons, remove foreign matter, and crush them into small pieces; degrease the crushed small pieces with 10% n-butanol, take the precipitate, and wash it with water until neutral; wash the precipitate obtained in the previous step with 0.5M hydrochloric acid Decalcification, decalcification is over, take the precipitate, wash it with water until neutral; soak the tissue precipitate obtained in the previous step in 0.5M sodium hydroxide solution, stir at 10°C for 2 hours, take the precipitate, wash it with water until neutral; Extract collagen with 0.5M acetic acid solution of 1g / L pepsin to obtain a crude collagen extract; adjust the pH to neutral to inactivate the enzyme; perform dialysis with 8-14kDa dialysis bags, and freeze-dry after dialysis to obtain Collagen with low endotoxin content.

Embodiment 3

[0040] Embodiment 3 Preparation process 3 of low endotoxin collagen

[0041] Wash the yak tendons, remove foreign matter, and crush them into small pieces; degrease the crushed small pieces with 10% n-butanol, take the precipitate, and wash it with water until neutral; wash the precipitate obtained in the previous step with 0.5M hydrochloric acid Decalcification, decalcification is over, take the precipitate, wash it with water until neutral; soak the tissue precipitate obtained in the previous step in 0.01M sodium hydroxide solution, stir at 20°C for 10 hours, take the precipitate, wash it with water until neutral; Extract collagen with 0.5M acetic acid solution of 1g / L pepsin to obtain a crude collagen extract; adjust the pH to neutral to inactivate the enzyme; perform dialysis with 8-14kDa dialysis bags, and freeze-dry after dialysis to obtain Collagen with low endotoxin content.

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Abstract

The invention belongs to the field of collagen preparation, and particularly relates to a preparation method of low-endotoxin collagen. According to the method, a procedure of low-temperature aqueous alkali treatment is added in a procedure of raw material treatment for collagen, so that the collagen with low endotoxin is prepared while ensuring undamaged activity of the collagen. The preparation method is high in endotoxin removal efficiency, and the endotoxin content of the prepared collagen is remarkably lower than 0.5 Eu / mL specified by the pharmaceutical industry standard (YY 0954-2015). The method provided by the invention is simple and convenient in operation, low in cost, and capable of removing endotoxin without special devices, so as to obtain the low-endotoxin collagen.

Description

technical field [0001] The invention belongs to the field of collagen preparation, and in particular relates to a preparation method of low-endotoxin collagen. Background technique [0002] Collagen is the most abundant protein in the human body. As a natural biological resource, collagen has the advantages of unparalleled biological activity, biocompatibility, biodegradability and low immunogenicity, and is widely used in tissue engineering and drug delivery. However, in the process of natural collagen extraction and preparation, if the removal of endotoxin is not thorough, the quality of collagen materials will be reduced, and the application of collagen materials in the biomedical field will be limited. [0003] At present, researchers usually use acid-soluble enzymolysis to prepare collagen. The commonly used acid is glacial acetic acid, and the enzymes include pepsin and trypsin. In order to make the extracted collagen meet the standards for medical application, the e...

Claims

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Application Information

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IPC IPC(8): C07K14/78C12P21/06C07K1/14
CPCC07K14/78C12P21/06
Inventor 肖建喜么林妍
Owner 胶原蛋白(武汉)生物科技有限公司
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