RU2288729C2 - Method for isolating collagen from bone tissue of stock-farm animals - Google Patents

Abstract

FIELD: chemical engineering.

SUBSTANCE: method involves pretreating bone tissue of stock-farm animals, comminuting it, precipitating the end product and drying it by lyophilizing. The bone tissue is degreased with alcohol-ether mixture. Demineralization 0.5 and HCl is carried out after grinding during 20 h. Non-resolvable matrix is subjected to proteolysis in HCl of pH=2.0 in pepsin presence at 37°C during 18 h. Then it is centrifuged at 40000g. The end product is precipitated from supernatant with ammonium sulfate to 25% saturation and centrifuged at 6000g. The precipitate is lyophilized against distilled water and chromatographically purified using CM-Sephadex at pH=4.8 and dried with lyophilization.

EFFECT: low cost collagen production; high purity cosmetic preparation production.

2 dwg

Description

The invention relates to chemical technology, in particular to pharmaceutical production, namely the production of drugs, the action of which is aimed at improving the condition of connective tissue and its regeneration, and is intended for use in cosmetology, in addition, it can be used in the field of medicine.

It is now known that the collagen fibers of the connective tissue matrix play a structural, supporting and stabilizing role with respect to proteoglycans and water. In addition, the collagen frame has great elasticity under the action of tensile and compressive forces, playing a major role in limiting the degree of hydration of the connective tissue matrix (Miller EJ Biochemical characteristics and biological significance of the genetically distinct collagens // Molec. Cell. Biochem. - 1976. - Vol.13. - N.2. - P.165-192).

A known method of producing collagen from - non-mineralized connective tissue - dermis, which is subjected to alkaline-salt treatment, neutralization with acetic acid, followed by freezing, freeze drying and grinding, without subsequent sterilization (RF Patent No. 2076718, publ. 10.04.1997).

However, the known method involves the release of collagen from non-mineralized connective tissue, in the case of using mineralized raw materials, the isolation of collagen according to this scheme is difficult.

The present invention is to develop a cost-effective time and reagents method for producing collagen from bone tissue of farm animals.

The problem is solved in that in the method for isolating collagen from the bone tissue of farm animals, including tissue pretreatment, grinding, deposition of the target product and freeze drying, the bone tissue is degreased with an alcohol-ether mixture, after grinding 0.5 n is demineralized. HCl for 20 hours, the insoluble matrix is subjected to proteolysis in the presence of pepsin in HCl with a pH of 2.0 for 18 hours at 37 ° C, then centrifuged at 40000g, the target product is precipitated from the supernatant with ammonium sulfate to 25% saturation, centrifuged at 6000g, the pellet is dialyzed against distilled water and purified chromatography on CM-sephadex at pH 4.8, freeze-dried.

The present invention is illustrated by a detailed description, an example of laboratory performance and illustrations, in which:

Figure 1 - illustrates a scheme for the allocation of collagen from mineralized connective tissue;

Figure 2 - illustrates the chromatographic profile of collagen bone tissue on a cation exchanger.

The method is as follows.

After pretreatment consisting in the removal of soft tissues, the mineralized connective tissue was crushed to pieces about 5 × 5 mm in size. Then the tissue was subjected to demineralization of 0.5 N. HCl for 20 h, after this procedure, demineralized tissue was freed from a bone mineral solution, non-collagen proteins and pepsin proteolysis was performed in the presence of an enzyme in 0.1 M HCl with pH 2. Pepsin was taken at the rate of 1 mg per 100 mg of tissue. After 18 hours of incubation at a temperature of 37 ° С on a magnetic stirrer, the tissue was completely destroyed with the formation of a colloidal solution, from which collagen was precipitated by the addition of ammonium sulfate to 25% saturation. A precipitate formed for 1 h, after which it was separated by centrifugation (10 min, 40,000 gx). To obtain highly purified collagen, chromatographic separation on CM-Sephadex is used. After that, collagen is freeze-dried.

Examples of laboratory performance.

The bones of cattle are cleaned of soft tissues, degreased with an alcohol-ether mixture of 1: 1, crushed to pieces 5 × 5 mm in size.

100 g of crushed fat-free bone tissue is placed in a round bottom flask, into which 200 ml of 0.5 N are poured. HCl and left for 20 hours at room temperature. The insoluble bone matrix obtained after removal of the supernatant was poured with 0.1 M HCl and 1 mg of pepsin was added. The reaction mass is placed on a magnetic stirrer and enzymatic hydrolysis of the bone tissue is carried out for 18 hours until the bones are completely dissolved and a colloidal solution is formed. The resulting precipitate was separated by centrifugation (40,000 g × 30 minutes)

After that, a saturated solution of ammonium sulfate was poured into a solution of proteolysate to a concentration of 25%. The collagen precipitate formed in 24 hours was separated by centrifugation (6.000 g × 60 minutes), suspended and dialyzed against distilled water for 2 days, then freeze-dried. The resulting collagen was dissolved in a 0.02 M solution of CH ₃ COONa (pH 4.8) in 1.0 M urea, centrifuged, and applied to a 2.5 × 20 cm column with CM-Sephadex (ion exchange capacity 4.5 + 0, 5 meg / g, granule size 40-120 microns). A linear NaCl gradient (0.00-1.5 M) in acetate buffer (pH 4.8) was used for elution. Chromatography was performed under denaturing conditions. The fractions were densitometric at a wavelength of 226 nm, the collagen fraction within one chromatographic peak was collected, dialyzed against water and freeze-dried.

The proposed method is easy to use, allows you to play it in a laboratory of a medical institution, for its implementation does not require the use of expensive reagents and it allows you to get a highly purified product from cheap available raw materials.

In addition, the preparation of highly purified collagen can be carried out in combination with the release of other biologically active substances (GAG - glycosaminoglycans, NKB - not collagen proteins) of bone tissue, thereby reducing the cost of the target product.

Claims (1)

A method of isolating collagen from the bone tissue of farm animals, including pre-treatment of the tissue, grinding, sedimentation of the target product and freeze drying, characterized in that the bone tissue is degreased with an alcohol-ether mixture, after grinding, demineralization of 0.5 n HCl for 20 hours, the insoluble matrix is subjected to proteolysis in the presence of pepsin in HCl with a pH of 2.0 for 18 h at 37 ° C, then centrifuged at 40,000 g, the target product is precipitated from the supernatant with ammonium sulfate to 25% saturation, centrifuges comfort at 6,000g, the precipitate is dialyzed against distilled water and purified by chromatography on CM-Sephadex at pH 4.8 and freeze-dried.

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