BG65654B1 - Method of obtaining microfibrillar water-soluble collagen for the food and pharmaceutical industries and cosmetics - Google Patents

Abstract

The invention relates to a method of obtaining microfibrillar water-soluble collagen, according to which the raw material (defatted and dehaired pork hides of young animals, processed to the "steepingö stage) is pre-subjected to vacuum-sublimation drying and subsequent (before the enzyme treatment) grinding and devilling. The enzyme treatment itself is performed with a combined enzyme preparationwith proteolytic, lipolytic and amylolytic activity. For the purpose of increasing the storage life of the resulting water-soluble collagen and for its use at any time, it is freeze-dried and, if need be, rehydrated to the concentration desired by the user. The product obtained y this method is intended for use in the food and pharmaceutical industries and in cosmetics.

Description

Technical field

The method of the present invention allows to increase the yield of purified microfibrillar collagen with improved water solubility. The finished product obtained by this method can be used in the food and pharmaceutical industry or for use in cosmetic compositions for hair and skin care. With further processing, it can serve as a starting material for the production of hemostatic products for medical purposes.

BACKGROUND OF THE INVENTION

Collagen is a major protein in mammals. It has a unique structure, the basic unit of which is the tropocollagen molecule. Most of its structure is slightly different in different mammalian species, which is the reason for the poor allergenicity and immunogenicity of collagen products. Purified collagen, with wholly or partially preserved native structure, is used in various fields of medicine (haemostatic products, dressings for implants and implants), cosmetics (nourishing and regenerating compositions) and the food industry (such as thickener stabilizer, hydrating agent, nutritional supplement, etc. .). In industrial conditions, purified collagen in various forms is obtained from hides, tendons, cartilage and other by-products of a number of farm animals by different methods.

The use of collagen films as a natural coating for filling sausages, ham or sausages is known (EP 1270672, US 5,736,180, US 5,885, 634). Collagen products (fibers and gels) are used as food additives (US 4,295,894) or included in pies, puddings, jelly candies, ice creams, mayonnaise sauces, snacks and more. (US 3,691,281).

JP 2001245614 describes a process for the preparation of dietetic food based on collagen jelly with added flavoring and correcting ingredients. Preparation of collagen and trace element-based nutritional supplement (GN1312019) is also suggested.

Collagen and collagen hydrolysis are applied in various cosmetic compositions: creams emulsion systems type B / M and M / B, lotions, hairspray, shampoos, bath foams and more. (US 4,131,650, US 3,691,281). Collagen products have some valuable properties - they are non-toxic, do not cause allergies, have a positive effect on the skin and its elasticity by improving its functional state and having a positive effect on protein and amino acid metabolism (Georgiev et al., 1990).

It is known to use collagen products as dressings for wounds with absorbent and epithelial-stimulating action (US 4,148,664; US 4,294,241; Chilkin et al. 1976) or for local haemostasis in capillary and parenchymal haemorrhages (US 4,320,201; US 4,3,231; US 4,3,231; No. 4,412,947).

Methods for the preparation of soluble collagen (from veal, porcine and sheep skin and from veal, porcine and bovine tendons) are known by enzymatic treatment with proteolytic enzymes and subsequent acid or alkaline treatment (WO 8704078, US 4,294,241, US 4,148,537, US 4,148,537, US 4,148,537, US 4,148,537, US 4,148,537, US US 4,837,258). The analytical yield can be increased by precisely mechanically shaking the enzyme-pretreated collagen (Hilkin et al., 1976). Many methods have been described for the preparation of acidic water-soluble collagen by continuous alkaline and acid treatment of the raw material and subsequent enzyme treatment, purification2

65654 Ballasting and bringing, by adding or removing water, to the required concentration (US 4,320,201).

US 4,295,894 discloses a method for the preparation of soluble collagen fibrils, which can be used for texturing meat, such as moisture retaining agents and a nutritional supplement or for use in cosmetic preparations. In the present technology, raw animal skins are treated with an aqueous alkali hydroxide solution and dehydrating agents to remove hair and fat, stabilized with an aqueous alkaline sulfate solution, and the resulting product neutralized and dissolved in dilute acid. The pH of the solution is adjusted to the isoelectric point to precipitate the collagen fibers, which are subsequently dried and preserved. However, this method does not adequately purify the collagen product from fats, globular proteins and telopeptides.

A method of producing microcrystalline collagen (US 3,691,281) is also known, in which raw or dried bovine skin is treated with a dilute acid solution and subsequently milled to a particle size under microns and dried under denaturing conditions. The microcrystalline collagen thus obtained is dispersed colloid when mixed with water and improves the stability of the suspended ingredients in liquids and gels. The technology proposed is based on the colloidal milling of acid-treated skins so that about 80-85% of the particles are submicron-sized, which, however, requires special equipment. The method does not clarify the issue of the physical stability of the resulting collagen suspensions and gels, especially during prolonged storage.

There are no known methods for producing acidic water-soluble collagen in which:

1. The raw material (skins or tendons), after being mechanically or chemically treated to remove the accompanying tissues, is subjected to vacuum freeze-drying. In this way it is achieved:

a) easier (at room temperature) and longer (2 and more years) storage of feedstock;

(b) grinding and milling of the raw material up to a size less than 1 mm, which is necessary for the full recovery of collagen;

c) subsequent enzyme treatment results in faster penetration of the enzyme solution into the lyophilized particles.

2. The enzymatic treatment of the feedstock is carried out with an enzyme complex containing at least three enzymes and having proteolytic, lipolytic and amylolytic activity, which helps to completely purify the starting material and to increase the yield of water-soluble collagen.

3. Skimmed and hairless pigskins of the "Refreshing" stage are used as feedstock only by young animals.

4. An additional vacuum-sublimation drying of the finished product is carried out, which significantly increases its storage time at room temperature and allows it to be used as intended after rehydration.

SUMMARY OF THE INVENTION

The essence of the invention is that by changing the basic production parameters of known technologies for the production of collagen, a method for the preparation of a biological product based on collagen has been devised, which has different fields of application in the food industry, pharmaceutical industry and cosmetics .

The starting material for collagen production is the use of skimmed and hairless skins of young animals (preferably pigs), treated to the stage of refreshment, well purified from accompanying tissue and other artifacts and washed with distilled water. They are cut into pieces of 250 to 500 cm ^{2 in size} and transferred to a low-temperature chamber at -30 to -50 ° C for at least 24 hours. After freezing, the skins are transferred to the drying chamber of a vacuum sublimation installation and subjected to lyophilization at a temperature of (-40 ° C) to + 25 ° C and a process duration of 24 to 32 h.

The residual moisture content of lyophilized skins should not be higher than 8%.

The raw material thus prepared is cut into 5-8 cm ² pieces and packed in water- and air-tight containers (for

65654 B1 preferably two or three-layer aluminum foil). The advantages of vacuum sublimation treatment of the raw material are the following: complete preservation of the native structure of collagen in the skin; possibility of storage for a long time at room temperature (more than 2 years); the ability to quickly use the raw material at any time; increased diffusion of the reaction mixtures during the enzymatic and acidic treatment of the feedstock, which aids / enhances the extraction of collagen from the skin and its dissolution in water, respectively; preventing additional bacterial contamination during the storage period.

A certain amount of lyophilized hides are ground on a knife mill with a sieve with a hole diameter of 0.5 to 1.5 mm. The raw material is subjected to enzymatic treatment. For this purpose it is transferred to a glass reactor with a jacket and stirrer, and a sequentially combined enzyme product with proteolytic, lipolytic and amylolytic activity (preferably pancreatin) is added in an amount of 0.5 to 1.0% w / w relative to the milled hides; 0.5-1.0% w / v nonionic surfactant (preferably polysorbate 80) and distilled water in a ratio of raw material from 1:20 to 1:40 (preferably 1:30). The stirrer was turned on for 1-2 minutes, after which the pH of the reaction mixture was adjusted to 7.0-7.5 by the addition of a 5% solution of sodium hydroxide or citric acid. Stirring was continued for 20-40 min at 300-500 rpm. Throughout the process, the temperature of the reaction mixture is maintained in the range of 30 to 32 ° C. The stirrer was switched off and the reaction mixture was allowed to rest for 1.5-2 hours, after which the inflow fluid was recovered in depth.

5-10 cm (about 5-61 cm) to remove secreted lipids, globular proteins, telopeptides, amino acids, mucopolysaccharides and other artifacts in the skin. Add distilled water in an adequate quantity to the decanted reaction mixture. The stirrer was again turned on and another 0.5% (relative to the original amount of feedstock) of the enzyme product was added. The procedure was repeated three times without the addition of an enzyme.

Treatment with the combined enzyme product provides a more complete purification of collagen from the by-products. The enzyme complex simultaneously degrades the globular proteins and lipids and releases the telopeptides that bind end-to-end molecules of tropocollagen. Telopeptides are known to be primarily responsible for immunogenicity and allergenicity and should be removed when purifying collagen products (US 4,412,947; US 4,404,134). Treatment of insoluble collagen with amylase separates acidic glucoseaminoglycans (mucopolysaccharides) and leads to disorganization and disintegration of the structural elements of collagen, loosens or destroys intermolecular bonds and enables complete dissolution of collagen in a weak acid solution.

After completion of the enzyme treatment, the mixture is transferred to a metal reactor in which distilled water acidified with weak acid (acetic, tartaric, citric) has been added to pH 3.0-4.0 (preferably pH value 3.5). . The feedstock to water ratio is from 1: 120 to 1: 150. The mixture was homogenized and allowed to stand for 72-96 h at 1518 ° C. The resulting diluted colloidal solution of acidic collagen is centrifuged to remove undissolved particles and other impurities.

The acidic dilute solution of collagen is reconstructed by adding sodium hydroxide solution to the isoelectric point (pH 4.6-5.6), resulting in spontaneous fibrillation. The collagen dispersion was concentrated by centrifugation or filtration to the required concentration (1-2%).

The product obtained has the following parameters: pH value from 4.6 to 5.6; specific denaturation temperature from 38 to 41 ° C; a viscosity of 85 to 90 mPa.s (D = 64.6 s' ¹ ); dry matter content of 1 to 2%; total protein content from 0,800 to 0,950 g / g dry matter; lipid content below 0,030 g / g dry matter; an ash residue below 0,040 g / g dry matter; content of heavy metals below 1 microg / g product; hydroxyproline content of 110 to 140 mg / g dry matter; total number of viable micro-organisms below 1.10 ³ KOE / g product; total number of molds and yeasts below 1.10 ² KOE / g product; pathogenic microorganisms - not detected.

65654 Bl

Depending on the intended use of the product, it may be brought to pH 4,04,5 by the addition of citric acid, added a preservative authorized for use in food (preferably nipagin and nipazol) and packed in glass or plastic materials. banks. The acidic collagen gel thus obtained can be stored in a refrigeration chamber (0 to 4 ° C) for 3 months or subjected to vacuum freeze-drying. The shelf life of the lyophilized product is extended several times (3 years) and, if necessary, dissolved in water to the required concentration and used as intended.

The product of the present invention can also be used to produce porous collagen sponges intended for local hemostasis or as absorbent wound dressings. For this purpose, the collagen microfibrillar dispersion is cross-linked with a dilute solution of formaldehyde, glutaraldehyde or glyoxal (preferably glutaraldehyde) in an amount of 0.04 to 0.06% v / v, frozen and dried in a vacuum sublimation installation. The lyophilized product is sterilized with gamma rays and remains stable at room temperature for two years.

Examples of carrying out the invention

Example 1.

800 g of lyophilized pigskin from young animals are ground into a Frisch mill through a 1 mm diameter sieve. The milled hides are transferred to a stirred glass reactor. 20 g of Pancreatin (Merck), 8 g of Polysorbate 80 and 301 distilled water, warmed to 30-32 ° C, were added sequentially. The pH of the solution was adjusted to 7.0-7.5 by the addition of 40% w / v sodium hydroxide solution or 20% w / v citric acid solution dropwise. Hot water was supplied to the reactor jacket, providing a temperature of 30-32 ° C. Turn on the stirrer at 350 rpm for 30 minutes. The mixture was then left to rest for 90 minutes, decanted off the top layer at a depth of 5-10 cm and discarded. Distilled water is added in an amount adequate to the separated mixture. This procedure is performed three times.

After the enzymatic treatment is complete, the mixture is transferred to a 1801 stirred metal reactor, which has previously measured 120-1301 distilled water and 120-140 g citric acid. Adjust the pH of the solution to 3,0-3,5 and add distilled water to the total volume of the solution 1601 if necessary. Turn on the stirrer at 500 rpm for 30 min, then turn off and allow the solution to stand 72 h at a temperature of 16-18 ° C, during which partial acid hydrolysis of the collagen takes place. The resulting diluted colloidal solution of acidic collagen was centrifuged on a sulfur flowing ultracentrifuge at 16 950 rpm for 20 min. Between the reactor and the centrifuge, the solution was passed through an adjustable flow water pump, which was adjusted so that 1601 of the solution would pass through it for 20-30 minutes. The centrifugation was transferred to a reactor with a capacity of 3001. Water was supplied to the reactor jacket to provide a mixture temperature of 18-20 ° C. The stirrer was turned on at 3 50 rpm and, with continuous stirring for 20 minutes, the pH was adjusted to 4.6-5.6 by the addition of small portions of 40% sodium hydroxide solution. After each addition of sodium hydroxide, pH is read. Upon reaching the isoelectric point, a spontaneous recovery (reconstruction) of the collagen fibrillar structure occurs. The collagen suspension thus obtained was allowed to stand for 2-3 h and then concentrated by centrifugation at 2500 rpm for 30 min. The centrifuge is discarded and the precipitated collagen suspension is analyzed for dry matter content. It is brought to the desired final concentration by the addition of distilled water. To the 10 kg of the mixture, 15 g of nipagine and 5 g of nigol are added as preservatives.

The analytical yield under these conditions is about 35-40 kg 1% collagen suspension.

Example 2.

Transfer 20 kg of reconstituted 1% microfibrillar collagen into a glass reactor and add from 20 to 50 g of citric acid to a solution pH of 4.0. The stirrer was turned on at 500 rpm and homogenized continued for 20-30 min until a homogeneous collagen gel was obtained. The resulting acidic water-soluble collagen is poured into trays and

65654 Bl undergoes lyophilization. Vacuum freeze drying was performed in a TG 16.50 lyophilizer. Cool the lyophilizer until the temperature in the chamber reaches (-40 ° C). Transfer the collagen solution trays to the lyophilizer chamber and allow to stand at this temperature for 2 h. Further, freeze-drying is carried out under automatic mode to a final product temperature of + 25 ° C for 24-26 h. The dry collagen product with a residual moisture content of less than 8% is packaged in air-tight and waterproof packaging and can be rehydrated and used as needed. It retains its properties unchanged at room temperature for 3 years.

Example 3.

The collagen suspension obtained according to Example 1, after reconstruction and before concentration, was adjusted to pH 5.5-6.0. Additional crosslinking and stabilization of the collagen microfibrils were performed by adding to the mixture 15 cm ^{3 a} 50% solution of glutaraldehyde, with continuous stirring for 15-20 min. The collagen suspension was allowed to stand for 2-3 h and then concentrated by centrifugation at 2500 rpm for 30 min to a final collagen concentration of 2%. Biologically active substances with haemostatic, antiseptic and epithelial stimulating action can be added to the collagen concentrate obtained, depending on the purpose of the final product (for local hemostasis in capillary and parenchymal haemorrhages, for the treatment of burn wounds, lacerations, injuries, injuries and others.). The collagen mixture thus prepared is poured into trays from the equipment of the vacuum sublimation installation at a layer thickness of 1 cm. The product was subjected to lyophilization under the conditions described in Example 2. The resulting collagen mushrooms are cut to the desired size, packaged and sterilized with gamma rays at a dose rate of 25 kGy.

Example 4.

The collagen suspension prepared under the technological conditions described in Example 1 is compressed by centrifugation to a concentration

4% and acidified with citric acid to a pH of 4.60. A homogeneous slightly opalescent gel is obtained which shows a plastic type of flow. At a temperature of 20.5 ° C, its viscosity is 9120.0 mPs (rotational viscometer Rheomat 108.7.61 s ¹ ). Another characteristic of this gel is mild thixotropy. The gel has no syneresis tendency and shows no significant changes in storage viscosity, which allows it to be used as a thickener and moisture retaining agent in the food industry.

Claims (4)

Claims 1. A method of producing microfibrillar water-soluble collagen using skimmed and non-skimmed pigskin from young animals, which includes grinding, flaking, enzymatic and acid treatment, centrifugation to separate insoluble particles, reconstruction and concentration, characterized in that they are defatted and hairless pigskins are previously subjected to vacuum freeze-drying. A method according to claim 1, characterized in that the enzymatic product of the raw material uses an enzyme product with combined action - with proteolytic, lipolytic and amylolytic activity. 3. The method according to claim 1, characterized in that the water-soluble microfibrillar collagen is acidified to pH 4,04,5 and subjected to vacuum freeze-drying. The method according to claims 1 and 2, characterized in that the water-soluble microfibrillar collagen after reconstruction is brought to pH 5.5-6.0 and further cross-linked with glutaraldehyde.