Proteoglycan synthesis method

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology and can be used in various industries. The method involves the following. Initial material, e.g. cartilaginous tissue of fish, bird, shellfish or mammals is ground up. The ground initial material immersed in a 0.0025-0.05 n solution of an alkali metal salt. The obtained extract is centrifuged to separate the solid and oil phase. The obtained liquid phase is centrifuged to obtain a filtrate. The obtained filtrate is washed with distilled water which is 6 times more than the volume of the filtrate with subsequent separation and concentration of the obtained end product.

EFFECT: invention enables production of proteoglycan in unaltered undecomposed form in a short period of time.

1 dwg, 10 ex

 

The technical field

The present invention concerns a method of obtaining of proteoglycan, which can be used as a material for the production of medicines, medical supplies, cosmetics, food products, industrial goods and so on, which includes stages of its isolation from a biological sample containing proteoglycan, such as cartilage fish, shellfish, birds and mammals, and get him out of there.

Proteoglycan is the generic name related to glycoprotein with a very complex and diverse structure types and usually consisting of one crustal protein with several - several tens covalently attached linear polysaccharide chains. The most common polysaccharide chain, which is part of proteoglycan from cartilage is chondroitin sulfate.

Chondroitin sulfate is a component of attracting attention in the industry in light of the high applicability as having a good moisturizing properties, biocompatibility or lubricating properties, and developed many ways to efficiently retrieve and preparation of chondroitin sulfate from natural sources.

In cartilage chondroitin sulfate itself is not presented. Rather, it is represented in complex form with the protein, i.e. in the form proteoglycans this selection of proteoglycan without any changes is often difficult due to the complicated structure of the glycoprotein complex. For this reason, commonly used allocation method only chondroitin sulfate after the complete destruction of the part of the measles-containing protein glycoprotein. The result of this allocation method is mucopolysaccharide, such as chondroitin sulfate and so on.

Meanwhile, there have also been attempts to obtain, prepare and use proper proteoglycan instead of chondroitin sulfate. Especially in the cartilage tissue of fish, birds and mammal contains proteoglycan with chondroitin sulfate as the main polysaccharide chain. Moreover, in light of the fact that the cartilage are normally discarded as waste, it has been proposed several methods of obtaining of proteoglycan from cartilage tissue, as well as effective use of industrial waste.

For example, it was reported on the methods of isolation of proteoglycan from the cartilage of the nose salmon using guanidine hydrochloride (Patent document 1) and acetic acid (Patent document 2). However, unfortunately, we cannot say that such traditional methods reach the level of commercial applicability, as the cost of extraction and purification is very high.

Patent document 1: Laid patent application of Japan No. 2001-172296

Patent document 2: JP-A No. 2002-69097

Description of the INVENTION

The problem to be solved in the present invention

p> The aim of the present invention is to develop a low-cost method of obtaining proteoglycan for oral ingestion of fish, shellfish, bird, or mammal, especially of their parts, which is the waste.

The methods of problem solving

The authors of the present invention discovered that by using the alkaline solution, which was considered unsuitable for the selection and preparation of a protein or protein complex, in certain conditions, proteoglycan, which is a glycoprotein complex can be successfully distinguished from cartilage tissue and other biological samples containing proteoglycan, and therefore made each of the inventions described below.

(1) a Method of obtaining of proteoglycan, which includes stages of immersion biological sample containing proteoglycan, in 0,0025-0.1 N. the alkali solution and obtaining the solution after immersion.

(2) the Method described above in (1), which additionally includes the extraction of proteoglycan from the resulting solution.

(3) the Method described above in (1) or (2)where the alkaline solution is a solution of salts of alkaline metal.

(4) the Method described above in any of (1)to(3), where the biological sample containing proteoglycan, is a cartilage, muscle fibers or the skin of the fish, shellfish, bird, or mammal.

(5) the Method described above in (4), where biologicheskii sample, containing proteoglycan, is a cartilage fish, bird or mammal.

The effect of the invention

Compared with conventional methods of extraction, according to the method according to the present invention proteoglycan can be easily extracted in an unmodified and undecayed form within a short period of time, thereby considerably reducing the cost of production of proteoglycan. Moreover, proteoglycan, very useful in industry, can be obtained from the parts of the fish, bird or mammal, which is the waste and mostly rejected, thus facilitating the effective use of industrial waste and the reduction of industrial wastes as such.

Moreover, the present invention does not need to be added inhibitor of proteolytic enzymes to inactivate proteolytic enzymes in biological tissues. Since this inhibitor is not always effective for each proteolytic enzyme, and many of the inhibitors themselves are harmful to humans, it is undesirable to use the inhibitor for the production of proteoglycan as raw material for food products. In this regard, since according to the present invention the inhibitor is not required, the above problems can be avoided.

Brief opisaniemopyta

A drawing. Chart showing the number of proteoglycan obtained using sodium hydroxide and acetic acid.

The best way of carrying out the invention

The present invention relates to a method of extraction and obtaining of proteoglycan-containing proteins, which are usually unstable when heated and in alkali, based on the idea of using alkaline solution, although it certainly goes against conventional wisdom.

Proteoglycan is a complex compound of sugar and protein. However, because the connection polysaccharide chain and crustal protein has a weak binding force, they tend to be easily separating from each other. For this reason, selection or purification of proteoglycan are extremely heavy by themselves, and require a more careful approach compared to collagen, which consists only of protein, or chondroitin sulfate, which consists only of carbohydrate. Thus, conventional methods are not suitable for mass production of proteoglycan because of the complex or manual action, and so forth.

In addition to the above, proteins are generally unstable when heated, the action of acid, and particularly alkali, thus, they are easily denaturiruet and destructible. There is a method of destruction of proteins with active use of alkali, based on what waistbag proteins. However, it was not known that proteoglycan, which is a glycoprotein complex can be extracted with alkali, because its protein parts are protected from destruction.

The method according to the present invention can be applied to a biological sample containing proteoglycan, such as cartilage, muscle fibers or the skin of the fish, shellfish, bird, or mammal. Mostly, however, from the above it is applicable to the cartilage tissue. The cartilage used in the present invention may be cartilaginous tissue of fish, bird or mammal, especially parts that are wastes. Cartilage described in this invention, refers to the actual cartilage tissue, or other tissues containing the area surrounding the cartilage tissue, such as bone, muscle fibers, leather and so on.

According to the present invention mainly uses the cartilage of the nose salmon average weight in the head of a salmon is about 6%, and commonly called "hiz". When salmon are caught in the sea near Hokkaido (most of them are salmon-related Oncorhynchus keta) and butchered, their heads are generally considered to be waste. Although some of separated warheads are used for production of fish powder, most of them are thrown away as the industry is slowly waste. Thus, the "hiz" can be conveniently and stably obtained from such waste at a low price.

According to the present invention in addition to the above "hiz" can be used cartilage from fish such as manta and shark, and so on, cartilage poultry, such as chicken, and cartilage mammals, such as cartilage or bronchi cow and whale cartilage, and so on. It is well known that proteoglycan also found in the epidermis of molluscs such as squid or octopus, the skin of these mollusks can also be used according to the present invention. Especially for epidermis octopus, it was reported that found chondroitin-protein complex, which is almost free from sulfates, and this chondroitin up to 70% or more of all of the mucopolysaccharides found in the epidermis octopus (Suyama et al., "Use of a squid", published in November 1980, p.93, Kouseisha). Thus, in the present invention the skin of the octopus is a convenient example of a biological sample containing proteoglycan. Most of the above biological samples containing proteoglycan are industrial waste, as a result can be easily obtained. These materials preferably are cut up as smaller size to increase their surface area for the extraction of more proteoglycan is and before immersion in an alkaline solution as described below.

With regard to the alkaline solution of the present invention, accordingly, may be used an aqueous solution such as an aqueous solution of an alkali metal or its salts and an aqueous solution of alkaline earth metal or its salts. However, in view of the efficiency of extraction of proteoglycan and convenience of processing and so forth are preferably used an aqueous solution of an alkali metal, namely sodium hydroxide (NaOH), sodium bicarbonate, calcium carbonate and potassium hydroxide. Most preferably using sodium hydroxide.

The concentration of the alkaline solution is 0,0025-0.1 N., preferably 0.01 to 0.05 N. When used 0,0025 of 0.01 N. the alkaline solution, it is preferable to carry out the immersion for at least 9 hours. Further, when used from 0.01 to 0.05 N. the alkaline solution, it is preferable to carry out the immersion for about 9 hours. Moreover, when used as 0.05 to 0.1 N. the alkaline solution, it is preferable to carry out the immersion 9 hours or less. Moreover, when used from 0.01 to 0.1 N. the alkaline solution, it is preferable to carry out the immersion for about 2 hours, so that could be ingibirovany degradation crustal protein of proteoglycan. In accordance with such conditions processing proteoglycan higher molecular weight can be obtained and prepared.

Immersion cartilage Velodrom solution is carried out at a temperature between 0°C and room temperature, preferably between 0°C and 10°C, and most preferably between 0°C and 4°C. In particular, when the temperature of the immersion set between 0°C and 4°C, proteoglycan degraded only slightly, so it can be selected as polymer glycoprotein complex.

The immersion may be carried out with 2-15-fold excess of the alkaline solution by weight, preferably 4-12-fold, most preferably 6-13-fold by weight compared with the weight of cartilage. Preferably, the immersion is carried out under stirring with a mixer or agitator.

Extraction of proteoglycan from cartilage tissue can be monitored by detecting and counting the number of uronic acid in accordance with the known method, such as method Galambos (John et el., ANALYTICAL BIOCHEMISTRY, 1967, vol 19, str-132). However, other known methods can also be used to identify and track uronic acid.

In alkaline solution, obtained after the termination of immersion, after extraction of proteoglycan contains many residues. As such, they are preferably removed by filtration, centrifugation, or other means. The extract containing proteoglycan, can be used as a product itself. However, it is preferable to select and clear the appropriate method to purity, red is Dima for different uses.

The present invention does not necessarily require a special method of purification of proteoglycan. But it is mentioned centrifugation as the preferred method. Way centrifugation of small solid particles can be conveniently separated as a precipitate, and the oil phase of the raw material can be conveniently separated as floating on the surface of the mixture.

In addition, obtained after centrifugation, the liquid phase containing proteoglycan, can be further filtered using filter paper or device for the separation ultrafiltrate membrane with an appropriate molecular weight cast and the like. For the molecular weight exclusion amount can be used range from about 50000 daltons to 1,000,000 daltons. For this method, when using a filter with a drop of molecular weight of 500,000 daltons or more, even collagen can be removed from the liquid phase to the purity of proteoglycan could be increased. In addition, the penetration through the membrane can be facilitated by reducing the viscosity of the liquid phase by adding water to the liquid phase, containing proteoglycan. Moreover, by repeating this stage can be also removed the smell of fish, which is formed slightly in the process.

In addition, adding the thus obtained concentrate is to the ethanol, saturated sodium chloride, may be selected proteoglycan in the gel state. This gel proteoglycan can be turned into a solid using vacuum lyophilizate. Otherwise it can be dried using a spray dryer, which gives a solid in powder form.

Later in this document, the present invention is described in more detail with reference to the following examples. However, the present invention is not limited to them.

EXAMPLES

Example 1

Nasal cartilage extracted from the head of salmon Oncorhynchus keta, which was frozen and stored at -40°C, was crushed using an electric grinder chopped into small pieces. 200,00 g such crushed pieces were used as the starting material. A 5-liter extraction vessel poured 2397,60 g pre-cooled to 0°C distilled water, and then there was added to 2.40 g of solid sodium hydroxide, in order to get in the end 2400,00 g of an aqueous solution of sodium hydroxide (0,025 BC). The above starting material was added to this extraction vessel and held immersion for 9 hours while stirring the contents in the mixer.

After immersion, the content was transferred to another vessel, over which was placed a filter stainless steel (1 mm), that is s extract, containing proteoglycan could be obtained.

The extract was subjected to centrifugation in a centrifuge (IWAKI CFS-400 type) for 20 minutes at 3500 rpm As a result, solid particles and oil phase were separated, and the liquid phase containing proteoglycan, was obtained.

Next, the liquid phase was filtered through a filter paper (produced by Advantec), followed by adding distilled water, 6 times the volume of the filtrate. Then, using membrane PREP/SCALE TFF (cast molecular weight of 100,000 daltons, produced by Millipore, Japan), separation and concentration were carried out at the same time.

Sample collection concentrate and used to determine the percentage of dry matter in solution. A sample of the concentrate was dried in an oven (YAMATO DX401) at 105°C for 16 hours for complete evaporation. Thin weighing the remaining dry residue was carried out using digital scales (GF-400, produced by A&D Corp.). As a result, it was found that of 200,00 g of the starting material was obtained only 6.64 g of dry matter, which corresponds 3,32% of the starting material in the conversion.

In addition, the number of amino acids were determined by analyses concentrate on the automatic amino acid analyzer (L-8500 Amino Acid Analyzer, manufactured by Hitachi Ltd.) to determine the amount of collagen, helping the of amagosa in concentrate. Moreover, the subsequent method Galambos was determined amount of uronic acid in order to count the number of proteoglycan.

Moreover, using a high-speed liquid chromatography (column TSK-GEL G4000PWXL, produced by Shimadzu Corporation) was determined molecular weight of proteoglycan.

As a result of these analyses it was found that the dry residue contains 25,0% protein, 21.5 per cent of the ash components, 52,9% carbohydrates and 0.6% of lipids. Based on the description of the Patent document 1 mass fraction of the crustal protein proteoglycan is around 7.0%. Because the amount of carbohydrates obtained in this experience, there was some 52.9%, it is assumed that proteoglycan of the present invention has a purity of about 57%. In addition, it was shown that the molecular weight of proteoglycan is about 2200000 Dalton.

As for the workflow described for the above example, the concentration of sodium hydroxide was changed to 0.0025 N., of 0.025 or 0.05 N. N. and 0,666 N. acetic acid was used instead of sodium hydroxide for immersion and extraction within 24 hours. As a result, the observed change in the allocated amount of proteoglycan (i.e. the amount of uronic acids) depending on the period of time shown on the drawing.

Example 2

Nasal cartilage extracted from the head of salmon Oncorhynchus keta, which was amorous and stored at -40°C, was powdered using an electric grinder chopped into small pieces and held immersion in acetone, and nasal cartilage was dehydrated and degreased. 24,00 g processed nasal cartilage, after drying in air or under reduced pressure, was used as the starting material. A 5-liter extraction vessel poured 2997,75 g pre-cooled to 5°With distilled water and then there was added 2.25 g of solid sodium hydroxide, in order to get in the end 3000,00 g of an aqueous solution of sodium hydroxide (0,02 BC). The above starting material (24,00 g) was added to this extraction vessel and held immersion for 9 hours while stirring the contents in the mixer.

After immersion, the content was transferred to another vessel, over which was placed a filter stainless steel (1 mm)that was removed nasal cartilage and could be obtained extract containing proteoglycan.

The extract was subjected to centrifugation in a centrifuge (Hitachi himacCF7D2 type) for 20 minutes at 3000 rpm/min As a result, solid particles and oil phase were separated, and the liquid phase containing proteoglycan, was obtained.

Next, the liquid phase was filtered through a filter paper (produced by Advantec), followed by adding distilled water, 6 times greater than the amount of Phil is a waste. Then, using BIOMAX 100K POLYETHERSULFONE (cast molecular weight of 100,000 daltons, produced by Millipore, Japan), separation and concentration were carried out at the same time.

Sample collection concentrate and used to determine the percentage of dry matter in solution. A sample of the concentrate was dried in a drying Cabinet (WMATA DX401) at 105°C for 16 hours for complete evaporation. Thin weighing the remaining dry residue was carried out using digital scales (GF-400, produced by A&D Corp.). As a result, it was found that of 24,00 g of the starting material was obtained 7.50 g of dry matter, which corresponds to 30,29% of the starting material in the conversion.

In addition, the number of amino acids were determined by analyses concentrate on the automatic amino acid analyzer (L-8500 Amino Acid Analyzer, produced by Hitachi Co. Ltd.) to determine the amount of collagen contained in concentrate. Moreover, according to the method Galambos was determined amount of uronic acid in order to calculate the amount of proteoglycan. Moreover, using a high-speed liquid chromatography (column TSK-GEL G4000PWXL, produced by Shimadzu Corporation) was determined molecular weight of proteoglycan.

As a result of these analyses it was found that the dry residue contains about 11.8% protein, 18.4% of the ash components, 67.8% of carbohydrates and 0,0% lipids. On on the basis of the description of the Patent document 1 mass fraction of the crustal protein proteoglycan around 7.0%. Thus, the estimated purity of proteoglycan of the present invention according to estimates amounted to 91.1% ((carbohydrates×0,07+lipids)/(carbs+protein+lipids)×100=for 91.1%). In addition, it was shown that the molecular weight of proteoglycan about 1200000 Dalton.

Example 3

Nasal cartilage extracted from the head of salmon Oncorhynchus keta, which was frozen and stored at -40°C, was crushed using an electric grinder chopped into small pieces, was conducted by immersion in acetone, and nasal cartilage was dehydrated and degreased. 17,90 g processed nasal cartilage, after drying in air or under reduced pressure, was used as the starting material. A 5-liter extraction vessel poured 2322,67 g pre-cooled to 5°With distilled water and then there was added 2,33 g of solid potassium hydroxide, to obtain the result 2325 g of an aqueous solution of potassium hydroxide (0,018 BC). The above starting material (17,90 g) was added to this extraction vessel and was conducted by immersion for 9 hours while stirring the contents in the mixer.

After immersion, the content was transferred to another vessel, over which was placed a filter stainless steel (1 mm)to the nasal cartilage was removed and the extract containing proteoglycan could be obtained.

Extract balodero to centrifugation in a centrifuge (Hitachi himacCF7D2 type) for 20 minutes at 3000 rpm/min As a result, solid particles and oil phase were separated, and the liquid phase containing proteoglycan, was obtained.

Next, the liquid phase was filtered through a filter paper (produced by Advantec), followed by adding distilled water, 6 times the volume of the filtrate. Then, using BIOMAX 100K POLYETHERSULFONE (cast molecular weight of 100,000 daltons, produced by Millipore, Japan), separation and concentration were carried out at the same time.

Sample collection concentrate and used to determine the percentage of dry matter in solution. A sample of the concentrate was dried in an oven (YAMATO DX401) at 105°C for 16 hours for complete evaporation. Thin weighing the remaining dry residue was carried out using digital scales (GF-400, produced by A&D Corp.). As a result, it was found that of 17,90 g of the starting material was obtained 5.29 g of dry matter, which corresponds to 29.61% of the starting material in the conversion.

In addition, the number of amino acids were determined by analyses concentrate on the automatic amino acid analyzer (L-8500 Amino Acid Analyzer, produced by Hitachi Co. Ltd.) to determine the amount of collagen contained in concentrate. In addition, according to the method Galambos was determined amount of uronic acid in order to count the number of proteoglycan. the more, using high-speed liquid chromatography (column TSK-GEL G4000PWXL, produced by Shimadzu Corporation) was determined molecular weight of proteoglycan.

As a result of these analyses it was found that the dry residue contains 14,0% protein, 22.4% of ash components, 63.6% carbohydrates and 0,0% lipids. Based on the description of the Patent document 1 mass fraction of the crustal protein proteoglycan around 7.0%. Thus, the estimated purity of proteoglycan of the present invention according to estimates 87/7% ((carbohydrates×0,07+lipids)/(carbs+protein+lipids)×100=87,7%). Furthermore/ it was shown that the molecular weight of proteoglycan about 1200000 Dalton.

Example 4

Cartilage extracted from chicken keel, which was manually removed muscle tissue was crushed using an electric grinder chopped into small pieces, conducted by immersion in acetone, and cartilage from Kiel chicken was dehydrated and degreased. 44,40 g of the treated cartilage, after drying in air or under reduced pressure, was used as the starting material. A 5-liter extraction vessel poured 2997,75 g pre-cooled to 5°With distilled water and then there was added 2.25 g of solid sodium hydroxide, in order to get in the end 3000,00 g of an aqueous solution of sodium hydroxide (0,02 BC). The above starting material (44,40 g) was add the updates in this extraction vessel, and were immersion for 9 hours while stirring the contents in the mixer.

After immersion, the content was transferred to another vessel, over which was placed a filter stainless steel (1 mm)that was removed cartilage extract containing proteoglycan could be obtained.

The extract was subjected to centrifugation in a centrifuge (Hitachi himacCF7D2 type) for 20 minutes at 3000 rpm/min As a result, solid particles and oil phase were separated, and was obtained liquid phase containing proteoglycan.

Next, the liquid phase was filtered through a filter paper (produced by Advantec), followed by adding distilled water, 6 times the volume of the filtrate. Then, using BIOMAX 100K POLYETHERSULFONE (cast molecular weight of 100,000 daltons, produced by Millipore, Japan), separation and concentration were carried out at the same time.

The sample obtained concentrate was taken and used to determine the percentage of dry matter in solution. A sample of the concentrate was dried in an oven (YAMATO DX401) at 105°C for 16 hours for complete evaporation. Thin weighing the remaining dry residue was carried out using digital scales (GF-400, produced by A&D Corp.). As a result, it was found that of 44,40 g of the starting material was obtained 9,87 g dry matter, which is matched with the edge 22,23% of the starting material in the conversion.

In addition, the number of amino acids were determined by analyses concentrate on the automatic amino acid analyzer (L-8500 Amino Acid Analyzer, produced by Hitachi Co. Ltd.) to determine the amount of collagen contained in concentrate. In addition, according to the method Galambos was determined amount of uronic acid in order to count the number of proteoglycan. Moreover, using a high-speed liquid chromatography (column TSK-GEL G4000PWXL, produced by Shimadzu Corporation) was determined molecular weight of proteoglycan.

As a result of these analyses it was found that the dry residue contains 31,3% protein, 16.9% of ash components, 51.8% of carbohydrates and 0,0% lipids. Based on the description of the Patent document 1 mass fraction of the crustal protein proteoglycan around 7.0%. Thus, the estimated purity of proteoglycan of the present invention according to estimates accounted for 66.7% ((carbohydrates×0,07+lipids)/(carbs+protein+lipids)×100=66.7 per cent). In addition, it was shown that the molecular weight of proteoglycan has 920,000 about Dalton (16%) and about 460000 Dalton (84%).

Example 5

Cartilage, manually extracted from skate (Dipturus kwangtungensis)was crushed using an electric grinder chopped into small pieces, conducted by immersion in acetone, and the cartilage was dehydrated and degreased. 12.00 g of the treated cartilage, after drying in air or under reduced pressure and, was used as the starting material. A 5-liter extraction vessel poured 1678,74 g pre-cooled to 5°With distilled water and then there was added 1.26 g of solid sodium hydroxide, in order to get in the end 1680,00 g of an aqueous solution of sodium hydroxide (0,02 BC). The above starting material (12.00 g) was added to this extraction vessel and was conducted by immersion for 9 hours while stirring the contents in the mixer.

After immersion, the content was transferred to another vessel, over which was placed a filter stainless steel (1 mm)that was removed from the nasal cartilage and the extract containing proteoglycan could be separated.

The extract was subjected to centrifugation in a centrifuge (Hitachi himacCF7D2 type) for 20 minutes at 3000 rpm/min As a result, solid particles and oil phase were separated, and the liquid phase containing proteoglycan, was obtained.

Next, the liquid phase was filtered through a filter paper (produced by Advantec), followed by adding distilled water, 6 times the volume of the filtrate. Then, using BIOMAX 100K POLYETHERSULFONE (cast molecular weight of 100,000 daltons, produced by Millipore, Japan), separation and concentration were carried out at the same time.

The sample obtained concentrate was taken and used to determine d the Lee of dry matter in solution. A sample of the concentrate was dried in an oven (YAMATO DX401) at 105°C for 16 hours for complete evaporation. Thin weighing the remaining dry residue was carried out using digital scales (GF-400, produced by A&D Corp.). As a result, it was found that of 12.00 g of the starting material was obtained 2.15 g of dry matter, which corresponds to 17,92% of the starting material in the conversion.

In addition, the number of amino acids were determined by analyses concentrate on the automatic amino acid analyzer (L-8500 Amino Acid Analyzer, produced by Hitachi Co. Ltd.) to determine the amount of collagen contained in concentrate. Moreover, according to the method Galambos was determined amount of uronic acid in order to count the number of proteoglycan. Moreover, using a high-speed liquid chromatography (column TSK-GEL G4000PWXL, produced by Shimadzu Corporation) was determined molecular weight of proteoglycan.

As a result of these analyses it was found that the dry residue contains 43.5% of protein, 19.5% of the ash components, 37.0% of carbohydrates and 0,0% lipids. Based on the description of the Patent document 1 mass fraction of the crustal protein proteoglycan around 7.0%. Thus, the estimated purity of proteoglycan of the present invention according to estimates amounted to 49.2 percent (that is, (carbohydrates×0,07+lipids)/(outlawed+protein+lipids)×100=49,2%). In addition, it p is shown, the molecular weight of proteoglycan about 1700000 Dalton.

Example 6

Cartilage extracted manually from the sharks, was crushed using an electric grinder chopped into small pieces, conducted by immersion in acetone, and the cartilage was dehydrated and degreased. 12.00 g of the treated cartilage, after drying in air or under reduced pressure, was used as the starting material. A 5-liter extraction vessel poured 1678,74 g pre-cooled to 5°With distilled water and then there was added 1.26 g of solid sodium hydroxide, in order to get in the end 1680,00 g of an aqueous solution of sodium hydroxide (0,02 BC). The above starting material (12.00 g) was added to this extraction vessel and held immersion for 9 hours while stirring the contents in the mixer.

After immersion, the content was transferred to another vessel, over which was placed a filter stainless steel (1 mm)to the nasal cartilage was removed and the extract containing proteoglycan could be separated.

The extract was subjected to centrifugation in a centrifuge (Hitachi himacCF7D2 type) for 20 minutes at 3000 rpm/min As a result, solid particles and oil phase were separated, and the liquid phase containing proteoglycan, was obtained.

Next, the liquid phase was filtered through a filter paper (produced Advantc) followed by the addition of distilled water by volume, 6 times the volume of the filtrate. Then, using BIOMAX 100K POLYETHERSULFONE (cast molecular weight of 100,000 daltons, produced by Millipore, Japan), separation and concentration were carried out at the same time.

The sample obtained concentrate was taken and used to determine the percentage of dry matter in solution. A sample of the concentrate was dried in an oven (YAMATO DX401) at 105°C for 16 hours for complete evaporation. Thin weighing the remaining dry residue was carried out using digital scales (GF-400, produced by A&D Corp.). As a result, it was found that of 12.00 g of the starting material was obtained of 1.36 g of dry matter, which corresponds to 11,36% of the starting material in the conversion.

In addition, the number of amino acids were determined by analyses concentrate on the automatic amino acid analyzer (L-8500 Amino Acid Analyzer, produced by Hitachi Co. Ltd.) to determine the amount of collagen contained in concentrate. Moreover, according to the method Galambos was determined amount of uronic acid in order to count the number of proteoglycan. Moreover, using a high-speed liquid chromatography (column TSK-GEL G4000PWXL, produced by Shimadzu Corporation) was determined molecular weight of proteoglycan.

As a result of these analyses it was found that the dry residue contains 37.8 percent protein, compared to 27.4% of the ash component is s, 37.8% of carbohydrates and 0,0% lipids. Based on the description of the Patent document 1 mass fraction of the crustal protein proteoglycan around 7.0%. Thus, the estimated purity of proteoglycan of the present invention according to estimates accounted for 55.7% ((carbohydrates×0,07+lipids)/(carbs+protein+lipids)×100=55.7 per cent). In addition, it was shown that the molecular weight of proteoglycan about 1,500,000 daltons.

Example 7

The skin of the octopus was manually peeled conducted by immersion in acetone, and she was dehydrated and degreased. The treated skin after drying in air or under reduced pressure, was used as the starting material. After cutting the skin with scissors into small pieces and grind in a mortar and dried the skin of the octopus was cooked. A 10-liter extraction vessel poured 5036,20 g pre-cooled to 5°With distilled water and then there was added 3.80 g of solid sodium hydroxide, in order to get in the end 5040 g of an aqueous solution of sodium hydroxide (0,02 BC). The above dried skin (33,70 g) was added to this extraction vessel and held immersion for 9 hours while stirring the contents in the mixer.

After immersion, the content was transferred to another vessel, over which was placed a filter stainless steel (1 mm), so that the skin was removed and could be CTD is flax extract, containing proteoglycan.

The extract was subjected to centrifugation in a centrifuge (Hitachi himacCF7D2 type) for 20 minutes at 3000 rpm/min As a result, solid particles and oil phase were separated, and the liquid phase containing proteoglycan, was obtained.

Next, the liquid phase was filtered through a filter paper (produced by Advantec), followed by adding distilled water six times the volume of the filtrate. Then, using BIOMAX 100K POLYETHERSULFONE (cast molecular weight of 100,000 daltons, produced by Millipore, Japan), separation and concentration were carried out at the same time.

The sample obtained concentrate was taken and used to determine the percentage of dry matter in solution. A sample of the concentrate was dried in an oven (Yamato DX401) at 105°C for 16 hours for complete evaporation. Thin weighing the remaining dry residue was carried out using digital scales (GF-400, produced by A&D Corp.). As a result, it was found that of 33,70 g of dried skin was obtained 16,10 g of dry matter, which corresponds to 47.7% of the starting material in the conversion.

In addition, the number of amino acids were determined by analyses concentrate on the automatic amino acid analyzer (L-8500 Amino Acid Analyzer, produced by Hitachi Co. Ltd.) to determine the amount of collagen contained in Konz is ntrate. Moreover, according to the method Galambos was determined amount of uronic acid in order to count the number of proteoglycan. Moreover, using a high-speed liquid chromatography (column TSK-GEL G4000PWXL, produced by Shimadzu Corporation) was determined molecular weight of proteoglycan.

As a result of these analyses it was found that the dry residue contains 91,7% protein, 1.9% of ash components, 6.4% carbohydrates and 0,0% lipids. Based on the description of the Patent document 1 mass fraction of the crustal protein proteoglycan around 7.0%. Thus, the estimated purity of proteoglycan of the present invention according to estimates amounted to 7.0% (ie (carbohydrate×0,07+lipids)/(carbs+protein+lipids)×100=7,0%). In addition, it was shown that the molecular weight of proteoglycan about 1700000 Dalton.

Examples of additional experiments

Experiment 1

Receipt of proteoglycan from the shell squid liver.

Shell squid liver was removed manually and subjected to immersion in acetone, and then was dehydrated and degreased. The treated membrane of the liver, after drying by air or drying under reduced pressure, was used as the starting material. After cutting the shell with scissors into small pieces, followed by grinding them in a mortar was obtained dried outer shell squid liver. The 3-liter extra is operating the vessel was added 1678,74 g of distilled water, pre-cooled to 5°C., and then dabwali 1.26 g of solid sodium hydroxide, in order to achieve 1680 g of an aqueous solution of sodium hydroxide (0,02 BC). The above dried shell squid liver (12 g) was added to this extraction vessel and held immersion for 24 hours at 5°C. while stirring the content in the mixer.

After immersion, the content was transferred to another vessel placed through the top of the vessel filter of stainless steel (1 mm), so that the upper shell can be removed, and the extract containing proteoglycan, could be selected.

The extract was subjected to centrifugation in a centrifuge (Hitachi type himacCF7D2) for 20 minutes at 3000 rpm/min As a result, solid particles and oil phase were separated, and was separated liquid phase containing proteoglycan.

The liquid phase was filtered through a filter paper (produced by Advantec), followed by adding distilled water, 6 times the volume of the filtrate. Then, using the BIOMAX membrane 100K POLYETHERSULFONE (cutoff molecular weight of 100,000 daltons, produced by Millipore, Japan), separation and concentration were carried out at the same time.

Sample collection concentrate and used to determine the percentage of solids in the liquid. A sample of the concentrate was dried in a drying SHK is fu (YAMATO DX401) at 105°C for 16 hours for complete evaporation. Accurate weighing the remaining solids was carried out using digital scales (GF-400, produced by A&D Corp.). As a result, it was found that of 12 g of dried membranes of the liver was obtained 5.31g dry matter, which corresponds to 44,25% in terms of source material.

In addition, the number of amino acids were determined by analyses concentrate on the automatic amino acid analyzer (L-8500 Amino Acid Analyzer, manufactured by Hitachi Ltd.) to determine the amount of collagen contained in concentrate. Moreover, the subsequent method Galambos was determined amount of uronic acid in order to calculate the amount of proteoglycan.

Moreover, using a high-speed liquid chromatography (column TSK-GEL G4000PWXL, produced by Shimadzu Corporation) was determined molecular weight of proteoglycan.

As a result of these analyses it was found that the dry residue contains 82.7% of protein, 2,43% ash components, 14,87% carbohydrates and 0,0% lipids. Based on the description of the Patent document 1 mass fraction of the crustal protein proteoglycan is around 7.0%. Thus, the estimated purity of proteoglycan present invention was calculated as the component of 16.3% (i.e., (carbohydrates×1,07+lipids)/(carbohydrates+proteins+lipids)×100=(14,87×1,07+0,0)/(82,7+14,87+0,0)×100=16,3%). In addition, it was found that the molecular weight proteoglycanes about 1450000 Dalton.

Experiment 2

Receipt of proteoglycan from the spine higfish(simenel, dumb, acne, monkey acne).

Ridge eel (simangele) was subjected to immersion in acetone, and then was dehydrated and degreased. The processed range, after drying in air or under reduced pressure, was used as the starting material. After cutting the ridge with scissors into small pieces and grind in a mortar was obtained dried range of acne. The 3-liter extraction vessel was added 1678,74 g of distilled water, pre-cooled to 5°C. and then was added 1.26 g of solid sodium hydroxide, in order to achieve 1680 g of an aqueous solution of sodium hydroxide (0,02 BC). The above dried ridge (12 g) was added to this extraction vessel and was conducted by immersion for 9 hours at 5°C. while stirring the content in the mixer.

After immersion, the content was transferred to another vessel, over which was placed a filter stainless steel (1 mm)to remove the ridge, and select extract containing proteoglycan.

The extract was subjected to centrifugation in a centrifuge (Hitachi himacCF7D2 type) for 20 minutes at 3000 rpm/min As a result, solid particles and oil phase were separated, and the liquid phase containing proteoglycan, was selected.

The liquid phase was profilemanager filter paper (produced by Advantec), followed by adding distilled water by volume, six times the volume of the filtrate. Then, using BIOMAX 100K POLYETHERSULFONE (cutoff molecular weight of 100,000 daltons, produced by Millipore, Japan), separation and concentration were carried out at the same time.

The sample obtained concentrate was taken and used to determine the percentage of solids in the liquid. A sample of the concentrate was dried in an oven (YAMATO DX401) at 105°C for 16 hours for complete evaporation. Accurate weighing of the remaining dry residue was carried out using digital scales (GF-400, produced by A&D Corp.). As a result, it was found that of 12 g of dried ridge received a 3.83 g of dry matter, which corresponds to 31.9% in terms of source material.

In addition, the number of amino acids were determined by analyses concentrate on the automatic amino acid analyzer (L-8500 Amino Acid Analyzer, produced by Hitachi Co. Ltd.) to determine the amount of collagen contained in concentrate. Moreover, according to the method Galambos was determined amount of uronic acid in order to calculate the amount of proteoglycan. Moreover, using a high-speed liquid chromatography (column TSK-GEL G4000PWXL, produced by Shimadzu Corporation) was determined molecular weight of proteoglycan.

As a result of these analyses it was found that the dry residue contains 86,68% protein, 3,32% ash component is s, 10,0% carbohydrates and 0,0% lipids. Based on the description of the Patent document 1, the mass fraction of measles-containing protein proteoglycan is around 7.0%. Thus, the estimated purity of proteoglycan of the present invention according to estimates equaled 11.07% ((carbohydrates×1,07+lipids)/(carbohydrates+proteins+lipids)×100=(10,0×1,07+0,0)/(86,68+10,0+0,0)×100=11,07%). In addition, it was shown that the molecular weight of proteoglycan about 1450000 Dalton.

Experiment 3

Receipt of proteoglycan from cartilage whale minke whales (Balaenoptera acutorostrata).

Cartilage extracted from whale minke whales (Balaenoptera acutorostrata), which was frozen and stored at -40°C, was crushed using an electric grinder into small pieces, conducted by immersion in acetone, and the cartilage was dehydrated and degreased. The treated cartilage, after drying in air or under reduced pressure, was used as the starting material. After cutting the cartilage scissors into small pieces and grind in a mortar and dried cartilage China ball obtained. The 3-liter extraction vessel was added 1678,74 g pre-cooled to 0°C distilled water and then there was added 1.26 g of solid sodium hydroxide, in order to get in the end 1680,00 g of an aqueous solution of sodium hydroxide (0,02 BC). The above starting material (12, 00 g) was added to this extraction vessel, and about what odiles immersion for 24 hours while stirring the contents in the mixer.

After immersion, the content was transferred to another vessel, over which was placed a filter stainless steel (1 mm)that was removed cartilage, and the extract containing proteoglycan, could be selected.

The extract was subjected to centrifugation in a centrifuge (IWAKI CFS-400 type) for 20 minutes at 3500 rpm As a result, solid particles and oil phase were separated, and was separated liquid phase containing proteoglycan.

The liquid phase was filtered through a filter paper (produced by Advantec), followed by adding distilled water, 6 times the volume of the filtrate.

Then, using a PREP/SCALE TFF membrane (cutoff molecular weight of 100,000 daltons, produced by Millipore, Japan), separation and concentration were carried out at the same time.

The sample obtained concentrate was taken and used to determine the percentage of solids in the liquid. A sample of the concentrate was dried in an oven (YAMATO DX401) at 105°C for 16 hours for complete evaporation. Accurate weighing of the remaining dry residue was carried out using digital scales (GF-400, produced by A&D Corp.). As a result, it was found that of 12.00 g of the starting material was obtained 4.61 in g dry matter, which corresponds to 38,42% in terms of source material.

In addition, the number of amino the slot was determined by analyses concentrate on the automatic amino acid analyzer (L-8500 Amino Acid Analyzer, produced by Hitachi Co. Ltd.) to determine the amount of collagen contained in concentrate. Moreover, according to the method Galambos was determined amount of uronic acid in order to calculate the amount of proteoglycan. Moreover, using a high-speed liquid chromatography (column TSK-GEL G4000PWXL, produced by Shimadzu Corporation) was determined molecular weight of proteoglycan.

As a result of these analyses it was found that the dry residue contains 12,76% protein, 20.4% of the ash components, 66,84% carbohydrates and 0,0% lipids. Based on the description of the Patent document 1 mass fraction of the crustal protein proteoglycan around 7.0%. Thus, the estimated purity of proteoglycan of the present invention according to estimates by 89.8% ((carbohydrates×1,07+lipids)/(carbohydrates+proteins+lipids)×100=(66,84×1,07+0,0)/(66,84+12,76+0,0)×100=89,8%). In addition, it was shown that the molecular weight of proteoglycan about 1250000 Dalton.

1. The method of obtaining of proteoglycan, which includes stages:
immersion biological sample containing proteoglycan, in 0,0025-0,05 N. alkaline solution at 0-10°C, a solution after immersion; and allocation of proteoglycan from the resulting solution.

2. The method of receiving proteoglycan according to claim 1 where the alkaline solution is a solution of salts of alkaline metal.

3. The method of receiving proteoglycan according to claim 1, where the biological sample containing proteo the douping, is cartilage tissue, or skin of fish, shellfish, bird, or mammal.

4. The method of receiving proteoglycan according to claim 3, where the biological sample containing proteoglycan is cartilaginous tissue of fish, bird or mammal or skin mollusk.



 

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