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

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

 

The invention relates to chemical technology, in particular to pharmaceutical production, namely the production of drugs, deustche aimed at improving the state of the connective tissue and its regeneration, and is intended for use in cosmetics, it is also possible its use in medicine.

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

A method of obtaining collagen from demineralizing connective tissue of the dermis, which is subjected to alkali-salt treatment, neutralization with acetic acid, followed by freezing, freeze drying and grinding, without further sterilization (RF Patent No. 2076718, publ. 10.04.1997).

However, the known method involves the secretion of collagen from demineralizing connective tissue, in the case of the mineralized materials selection collagen and according to this scheme difficult.

adaca of the present invention is to develop a consumption of time and reagents method of producing collagen from bone tissue of farm animals.

The problem is solved in that in the method of extraction of collagen from bone tissue of farm animals, including pre-treatment tissue, grinding, precipitation of the desired product and its freeze-dried bone degreasing alcohol-ether mixture after grinding spend the demineralization of 0.5 N. HCl for 20 hours, nerastvorim 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, from the supernatant precipitated target product ammonium sulfate up to 25% saturation, centrifuged at 6000g, sediment cialiswhat against distilled water and clean chromatographies on CM-Sephadex at pH of 4.8, freeze-dried.

The present invention explaining a detailed description of the sample lab execution and illustrations, in which:

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

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

The method is as follows.

After pre-processing, which consists in removing soft tissue, mineralized connective tissue were crushed to pieces of a size about 5×5 mm Then the fabric was subjected to demineralization of 0.5 N. HCl for 20 h, after that the procedure demineralized tissue was liberated from a solution of bone mineral Nikolayevich proteins and spent basenowy proteolysis in the presence of enzyme in 0.1 M HCl to pH 2. Pepsin was taken from a rate of 1 mg per 100 mg tissue. For 18 h incubation at 37 ° °With magnetic stirrer fabric was completely destroyed with the formation of a colloidal solution from which laid siege to the collagen by adding ammonium sulfate up to 25%saturation. The precipitate was formed within 1 h, after which it was separated by centrifugation (10 min, 40.000 gx). To obtain highly purified collagen using a chromatographic separation on CM-Sephadex. After that, the collagen freeze-dried.

Examples of laboratory execution.

Cattle bone is cleaned from soft tissue, degrease the alcohol-ether mixture 1:1, is crushed to pieces size 5×5 mm

100 g of degreased crushed bone tissue is placed in a round bottom flask, into which pour 200 ml of 0.5 N. HCl and left for 20 h at room temperature. Obtained after removal nadeshiko nerastvorim bone matrix filled with 0.1 M HCl and make 1 mg of pepsin. The reaction mass is placed on the magnetic stirrer and carry out the enzymatic hydrolysis of bone tissue for 18 h until complete dissolution of bones and formation of a colloidal solution. The precipitate was separated by centrifugation (40.000 g × 30 minutes)

Then to the solution FR is olidata was added a saturated solution of ammonium sulfate to a concentration of 25%. The precipitate of collagen, formed for 24 hours, was separated by centrifugation (6.000 g × 60 minutes), suspended and were dialyzed against distilled water for 2 days, then freeze-dried. The obtained collagen was dissolved in 0.02 M solution of CH3COONa (pH of 4.8) in 1.0 M urea, centrifuged and applied to the column with the dimensions of 2.5×20 cm with CM-Sephadex (ion-exchange capacity of 4.5 to+0.5 meg/g, grain size 40-120 μm). For elution used a linear gradient of NaCl (0,00-1.5 M) in acetate buffer (pH 4.8). Chromatography was performed under denaturing conditions. Fractions were densitometrically at a wavelength of 226 nm, the fraction of collagen within a single chromatographic peak were collected, were dialyzed against water and freeze-dried.

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

In addition, obtaining vysokochistogo collagen may be conducted in conjunction with the secretion of other biologically active substances (GAG is a glycosaminoglycan, NCU - not collagenous proteins of bone, thereby reducing the cost of the target product.

Method of extraction of collagen from the bone weste the animals, involving pre-treatment tissue, grinding, precipitation of the desired product and its freeze drying, characterized in that the bone degreasing alcohol-ether mixture after grinding spend the demineralization of 0.5 n HCl for 20 h, nerastvorim 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 40000g, from the supernatant precipitated target product ammonium sulfate up to 25% saturation, centrifuged at 6,000g, sediment deleteroute against distilled water and purified by chromatography on CM-Sephadex at pH 4,8, freeze-dried.



 

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