JP2005336330A - Method for producing highly purified mucopolysaccharide - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing a highly purified mucopolysaccharide by easily and economically removing foreign materials from a mucopolysaccharide-containing composition (raw material composition) containing foreign materials such as proteins, for example, sodium chondroitin sulfate extract liquid extracted e.g. from the nasal cartilage of salmon by alkali extraction method or enzyme method. <P>SOLUTION: The highly purified mucopolysaccharide is produced by preparing an aqueous solution containing a raw material composition containing mucopolysaccharide and protein and/or its decomposition product, sodium chloride and a salt such as sodium acetate and having a sodium chloride concentration of 8-60 wt.%, and mixing the aqueous solution with a water-soluble organic solvent such as ethanol to selectively precipitate the mucopolysaccharide. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a method for producing a mucopolysaccharide that is expected to have various uses in the fields of pharmaceuticals, cosmetics, health foods, food additives, feeds, and the like.

  Mucopolysaccharides are polysaccharides obtained from animals in a broad sense, and the raw materials include fish such as salmon and coral; other aquatic animals such as whales, rays and sea cucumbers; and cattle, pigs, chickens, Bone, cartilage, skin, fish scales and the like extracted from land animals such as horses are known. As a representative substance of mucopolysaccharide, glycosaminoglucan can be mentioned. Glycosaminoglucans generally exist as proteoglucans covalently bound to the core protein. Glycosaminoglucan is composed of repeating disaccharides, one of which is either D-glucosamine or D-galactosamine. As examples of glucosaminoglucan, hyaluronic acid, chondroitin sulfate, chondroitin, ketalan sulfate, heparin, heparin sulfate, dermatan sulfate and the like are known.

  Among them, chondroitin sulfate is a kind of mucopolysaccharide obtained from animal mucus secretions, cartilage, etc., which keeps water in animals and human body, thereby smoothing metabolism and fulfilling cell activation function It is presumed to be widely used as pharmaceuticals, cosmetics, and health foods. The physiological effects of chondroitin sulfate include extracellular fluid volume regulation and water metabolism, extracellular fluid ion migration and regulation, joint tissue smoothening, lipid serum clarification and blood coagulation inhibition, anti-inflammatory and anti-cancer effects. In addition, corneal transparency maintenance, infection prevention, etc. are known. Development of a simple and inexpensive method for producing high-purity chondroitin sulfate is required in order to fully exert these physiological functions as active ingredients of foods, beverages, cosmetics or pharmaceuticals.

  In general, mucopolysaccharides are processed by treating raw materials such as bone, cartilage, and skin, thereby cleaving the covalent bond of “core protein in which mucopolysaccharide and mucopolysaccharide are covalently bound” contained in these raw materials. It is manufactured by taking out (extracting). As a treatment method for cleaving the covalent bond of the core protein, an alkali treatment method using an alkaline solution, an enzyme method using a proteolytic enzyme such as protease or pronase, and the like are known. However, the mucopolysaccharide-containing extract extracted by these treatments contains a large amount of proteins and degradation products thereof. Mixing of proteins and / or degradation products thereof with mucopolysaccharides may hinder use particularly in the pharmaceutical and cosmetic fields. For example, when used as a pharmaceutical agent for injections, if these proteins are mixed, allergic reactions are likely to occur, and in cosmetic applications, problems such as browning with time are likely to occur.

  Therefore, it is necessary to remove contaminants such as proteins and their degradation products from the extracts obtained by these methods so as to increase the purity of the mucopolysaccharide. As methods for this purpose, ethanol fractionation, ion exchange chromatography, a combination of ethanol precipitation recovery and ion exchange resin treatment, ultrafiltration, and the like are known.

  Ethanol fractionation treatment is a method for obtaining mucopolysaccharides having a specific molecular weight range from mucopolysaccharide compositions having a broad molecular weight distribution, and is based on the fact that the solubility in ethanol varies depending on the molecular weight. . Since the solubility of “proteins and / or degradation products thereof” in ethanol is significantly higher than that of mucopolysaccharides, as a result, these contaminants can be separated and removed by this method (see Non-Patent Document 1). However, ethanol fractionation has a problem in terms of the total recovery rate of the recovered mucopolysaccharide. Further, in the combination of ethanol precipitation recovery and ion exchange resin treatment (see Patent Document 1), the purity of the mucopolysaccharide precipitated and recovered by adding ethanol to the mucopolysaccharide extract aqueous solution is generally low, so that the purity is further increased. In order to achieve this, it is essential to perform treatment using an ion exchange resin. Furthermore, in ultrafiltration processing (refer patent document 2), there existed a problem that an expensive installation was required.

Japanese Biochemical Society, New Chemistry Experiment Course 3 "Carbohydrate II", Tokyo Chemical Doujin, 1991, p. 28 JP 2001-231497 A JP 2000-273102 A

  Thus, a method for removing impurities from a mucopolysaccharide-containing extract containing impurities such as proteins and protein degradation products in a simple and low-cost manner to obtain a highly pure mucopolysaccharide has not been known so far. Therefore, an object of the present invention is to provide a method for removing impurities from a mucopolysaccharide-containing extract easily and at low cost, and to provide a method for efficiently producing high-purity mucopolysaccharide.

  The present inventors have intensively studied to solve the above problems. As a result, when a specific amount of sodium chloride is added to an aqueous solution of a raw material composition containing mucopolysaccharide and protein and / or a degradation product thereof, and then mixed with ethanol, the mucopolysaccharide is precipitated and recovered. The inventors have found that the purity of the recovered mucopolysaccharide is remarkably improved as compared with the case of the ethanol precipitation recovery method, and have completed the present invention.

  That is, the present invention is a method for producing a highly purified mucopolysaccharide by separating mucopolysaccharide from a raw material composition containing mucopolysaccharide and protein and / or a degradation product thereof, An aqueous solution preparation step of preparing an aqueous solution containing the raw material composition and sodium chloride and having a sodium chloride concentration of 8 to 60% by weight, and the aqueous solution obtained in the aqueous solution preparation step and a water-soluble organic solvent; And a precipitation step of selectively precipitating mucopolysaccharide.

  In the raw material composition, it is considered that “mucopolysaccharide” and “protein and / or its degradation product” are difficult to separate due to physical interaction such as molecular entanglement. Whereas the interaction is not weakened, in the production method of the present invention, the interaction is weakened by the salt present in an appropriate amount in the aqueous solution (for example, the molecular entanglement is released), and only the mucopolysaccharide is selectively used in the precipitation process. It is estimated that it is deposited.

  According to the production method of the present invention, for example, impurities can be removed easily and at low cost from mucopolysaccharide-containing extracts containing impurities such as proteins obtained from nasal cartilage of salmon by alkaline treatment or enzymatic method. Thus, it is possible to efficiently produce a high-purity mucopolysaccharide.

  The raw material composition used in the present invention is not particularly limited as long as it is a composition comprising mucopolysaccharide and protein and / or a degradation product thereof. For example, raw material materials derived from various raw animals are treated with an alkali or an enzyme. Thus, a composition (so-called mucopolysaccharide-containing extract) obtained by cleaving mucopolysaccharide and a core protein covalently bound to mucopolysaccharide can be used.

  Here, as the raw material animals, raw materials compositions that can be used such as fish such as sea breams, sea breams; other aquatic animals such as whales, rays, sea cucumbers; or land animals such as cows, pigs, chickens, horses, etc. It is preferable to use aquatic animals, particularly cocoons, because of their relatively high purity and low odor. In addition, the raw material is not particularly limited as long as it is a tissue containing a mucopolysaccharide component such as bone, cartilage, skin, fish scale, etc., but since it is easy to handle and has a high content of mucopolysaccharide, cartilage such as nasal cartilage is used. It is preferred to use. In addition, it is preferable to remove impurities such as meat and fat from the raw material used in advance by washing with water, washing with an organic solvent, or the like. These raw materials are usually improved in treatment efficiency by treatment such as cutting, pulverizing, adding water and pressurizing them before treatment.

  In addition, as a method of obtaining a raw material composition from a raw material, a conventionally employed method, that is, an alkali treatment method in which the raw material is decomposed with an alkaline solution and mucopolysaccharide is extracted, mucopolysaccharide is neutralized from the raw material. Neutral salt treatment method that extracts with liquid, enzymatic method that extracts protease by proteolytic enzymes such as protease and pronase on the raw material and extracts mucopolysaccharide, or a combination of these methods can be used without limitation. Among these methods, treatment by an alkali treatment method and an enzyme method is preferable, and enzyme treatment is particularly preferable because of a high polysaccharide recovery rate. By these treatments, the mucopolysaccharide and the core protein to which the mucopolysaccharide is covalently bonded can be cleaved to obtain an aqueous solution component containing the mucopolysaccharide. At this time, the cleaved protein and its degradation product (protein degradation product) are inevitably contained in the aqueous solution. The aqueous solution obtained by such treatment can be used as a raw material composition as it is, but the insoluble matter is removed by centrifugation, filtration or the like, and a filter aid may be used if necessary. It is preferable to remove turbid components, deodorize, decolorize, degrease, etc. by performing filtration, activated carbon treatment or the like. Furthermore, it is preferable to solidify and pulverize by spray drying (spray drying), evaporation drying, freeze drying, etc. from the ease of handling.

Examples of the mucopolysaccharide contained in the raw material composition include glycosaminoglucan, chitin, chitosan and the like. Glycosaminoglucans generally exist as proteoglucans covalently bound to the core protein. Glycosaminoglucan is composed of repeating disaccharides, and one of the disaccharides is composed of either D-glucosamine or D-galactosamine. Examples of glucosaminoglucan include hyaluronic acid, chondroitin sulfate, chondroitin, ketalan sulfate I and II, heparin, heparin sulfate and dermatan sulfate. Among these, hyaluronic acid, chondroitin sulfate, ketalan sulfate, and dermatan sulfate are preferable because of high purification efficiency, and chondroitin sulfate is particularly preferable. When the mucopolysaccharide has a sulfate ester group, the neutral structure (-OSO ₃ H), Na salt (-OSO ₃ Na), K salt (-OSO ₃ K), Ca salt (-OSO _It may be an ionic structure such as ₃ Ca) or a mixture thereof. Further, when the mucopolysaccharide has a carboxyl group structure, a neutral structure (—CO ₂ H), a Na salt (—CO ₂ Na), a K salt (—CO ₂ K), a Ca salt (— It may be an ionic structure such as CO ₂ Ca) or a mixture thereof.

  The content of mucopolysaccharide and protein and / or degradation product thereof in the raw material composition used in the present invention is not particularly limited, but the mucopolysaccharide content in the mucopolysaccharide-containing extract obtained by the method as described above is “ It is usually in the range of 10 to 90% by weight, expressed as the weight percentage of “mucopolysaccharide” based on the total weight of “mucopolysaccharide” and “protein and / or its degradation product”. In the present invention, a raw material composition having a mucopolysaccharide content in such a range can be used without any problem. In addition, the raw material composition may contain salts, ash, inorganic substances and the like as other components. These other components are preferably 0 to 500% by weight based on the total weight. When the raw material composition is an aqueous solution, the concentration of mucopolysaccharide in the aqueous solution is preferably 0.1 to 90% by weight.

  In the production method of the present invention, an aqueous solution preparation step of preparing an aqueous solution containing the raw material composition and sodium chloride and having a sodium chloride concentration of 8 to 60% by weight, and the aqueous solution obtained in this step and the water-soluble organic solvent The highly purified mucopolysaccharide is manufactured from the said raw material composition by including the precipitation process which mixes and selectively deposits mucopolysaccharide. It is the same as the conventional ethanol fractionation method and precipitation recovery method using ethanol in that it adds a water-soluble organic solvent such as ethanol to the raw material composition in aqueous solution to precipitate mucopolysaccharide. Is performed in the presence of a specific concentration of sodium chloride, it is possible to obtain effects that cannot be realized by these conventional methods. Hereinafter, these steps will be described.

  In the production method of the present invention, first, as an aqueous solution preparation step, an aqueous solution containing the raw material composition and sodium chloride and having a sodium chloride concentration of 8 to 60% by weight is prepared. When the salt concentration in the aqueous solution is less than 8% by weight, the purity of the mucopolysaccharide precipitated in the subsequent precipitation step does not increase. Further, when the salt concentration exceeds 60% by weight, the effect is low from the viewpoints of solubility, load on stirring power, economy and the like. From the viewpoint of economic efficiency and purification effect, the concentration of sodium chloride in the aqueous solution prepared in the aqueous solution preparation step is preferably 9 to 50% by weight, particularly 10 to 40% by weight. The concentration of mucopolysaccharide in the aqueous solution prepared by the aqueous solution preparation is not particularly limited, but is 0.2 to 60% by weight, particularly 0.5 to 30% by weight from the viewpoint of high purification efficiency. Is preferred. The pH of the aqueous solution is not particularly limited, but is preferably in the range of 1 to 5 (acidic) or 8 to 14 (alkaline) from the viewpoint of purification efficiency and prevention of spoilage.

  As salt, industrial salt, common salt, rock salt, salt salt, and various sodium chlorides can be used. Further, sodium hydroxide can be used by reacting sodium hydroxide, sodium hydrogen carbonate, or sodium carbonate with hydrochloric acid or hydrogen chloride in an aqueous solution. Furthermore, it is possible to use seawater.

  In addition, acid, alkali, other salts, and preservatives may be added to the aqueous solution prepared in the aqueous solution preparation step as necessary to improve purification efficiency and prevent the occurrence of fungi and mold (corruption). I can do it. For example, an acid is added in order to adjust the pH to 1 to 5, preferably 2 to 5. As the acid used at this time, organic carboxylic acids such as acetic acid, citric acid and tartaric acid, phosphoric acid and hydrochloric acid are used. Inorganic acids such as sulfuric acid can be used. Of these, acetic acid is particularly preferable.

  In order to adjust the pH to 8 to 14, preferably 8 to 13, a base is added. As the base used at this time, it is preferable to use an alkali. Examples of the alkali include hydroxides such as sodium hydroxide and potassium hydroxide, carbonates such as sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate and calcium carbonate, tetramethylammonium hydroxide, tetraethylammonium hydroxide and the like. An organic hydroxide or the like can be used. Since the alkali has not only a function of adjusting pH but also an effect of increasing the purification efficiency of mucopolysaccharide, the aqueous solution preferably contains an alkali.

  As the antiseptic, any one that is generally used for anti-corruption and does not decompose or denature mucopolysaccharide and / or protein can be used without limitation. For example, alcohols such as ethanol and n-butanol, guanidines such as polyhexamethylene, reverse soaps such as benzalkonium chloride, surface active agents such as alkylpolyaminoethylglycine, and halogen-based disinfectants such as sodium hypochlorite Agents, peroxides such as hydrogen peroxide and peracetic acid can be used. The amount of these preservatives used may be appropriately adjusted depending on the type of mucopolysaccharide used, the processing temperature, and the like.

  The order in which various components are added in preparing the aqueous solution in the aqueous solution preparation step is not particularly limited, and may be added at once or divided into several times. For example, while stirring a powdered raw material composition and a reaction kettle containing water, salt is added in a solid state or dissolved in water, and other additives are added in the same manner and dissolved in water. Can be more suitably prepared. The obtained aqueous solution is 4 ° C. to 100 ° C., preferably 20 to 90 ° C., particularly preferably 30 to 70 ° C. for 10 minutes to 100 hours, preferably 30 minutes to 50 hours, particularly preferably in order to increase the purification efficiency. It is preferable to stir for 30 minutes to 24 hours and to cool and stand as needed. In addition, it is useful from the viewpoint of purification to perform filtration or the like at this stage to remove insoluble matters and turbidity. In addition, as water, it is suitable to use clean water or ion-exchange water.

  In the production method of the present invention, the aqueous solution obtained in the aqueous solution preparation step and the water-soluble organic solvent are mixed in the precipitation step to selectively precipitate mucopolysaccharide. Here, the water-soluble organic solvent means an organic solvent having a solubility in water at 25 ° C. of 50 g or more per 100 g of water. It is preferable to use an organic solvent that is miscible with water as the water-soluble organic solvent because the recovery rate of mucopolysaccharide can be increased. Examples of water-soluble organic solvents that can be suitably used include lower alcohols such as methanol, ethanol, isopropyl alcohol, and isobutyl alcohol; polyhydric alcohols such as ethylene glycol, diethylene glycol, propylene glycol, and glycerin; ketones such as acetone and methyl ethyl ketone; Examples thereof include dioxane and dimethyl sulfoxide. Although it may be selected appropriately according to the mucopolysaccharide to be purified in these solvents, methanol and ethanol are generally preferred and ethanol is particularly preferred from the viewpoint of purification efficiency. The water-soluble organic solvent may be anhydrous or water-containing, but from the viewpoint of efficiently depositing the mucopolysaccharide, the water-soluble organic solvent in the water-soluble organic solvent to be mixed is used. The content is preferably 80% by weight or more, particularly 90% by weight or more. When the water-soluble organic solvent is ethanol, 85 to 99% by weight is preferable and 92 to 97% by weight is particularly preferable from the viewpoint of solvent recovery efficiency and purification efficiency.

  Of course, it is possible in the process to recover the water-soluble organic solvent from the separated aqueous solution after separating the mucopolysaccharide precipitated in the precipitation step and the aqueous solution containing the water-soluble organic solvent, and to reuse this. By doing so, the manufacturing cost can be reduced. In this case, the use of a water-soluble organic solvent that may contain water is advantageous in terms of recovery and purification costs of the water-soluble organic solvent.

  The amount of the water-soluble organic solvent used in the precipitation step is based on the total weight of the aqueous solution prepared in the aqueous solution preparation step and the water-soluble organic solvent (which may contain water) from the viewpoint of the precipitation efficiency of mucopolysaccharide. It is preferably 20 to 80% by weight, particularly 25 to 75% by weight, and most preferably 30 to 70% by weight, expressed in terms of% by weight of the water-soluble organic solvent (not including water).

  There is no particular limitation on the method of mixing the aqueous solution obtained in the aqueous solution preparation step and the water-soluble organic solvent. For example, a method of adding the water-soluble organic solvent to the aqueous solution by batch treatment, A method of adding an aqueous solution therein, a method of flowing the aqueous solution and a water-soluble organic solvent from separate lines and mixing them in a reaction vessel or line, etc. can be suitably employed. Among these, batch-type treatment is preferable from the viewpoint of the ease of the treatment process, and a method of adding a water-soluble organic solvent to an aqueous solution from the viewpoint of high removal efficiency of proteins and / or degradation products thereof as main impurities. Particularly preferred.

  When adding a water-soluble organic solvent to the aqueous solution obtained in the aqueous solution preparation step, put the aqueous solution in a reaction vessel, stir while adjusting the temperature, add the water-soluble organic solvent, and continue stirring. Complete precipitation. In this method, the temperature of the mixed solution, the stirring conditions, and the addition rate of the water-soluble organic solvent greatly affect the purity and handling properties of the precipitate. In order to obtain a precipitate having high purity and good filterability, it is preferable that the temperature during stirring and addition of the water-soluble organic solvent is 4 to 60 ° C., particularly 20 to 40 ° C. The stirring is preferably performed at a shear rate of 0.5 to 100 (m / second), particularly preferably 0.7 to 50 (m / second). The addition rate of the water-soluble organic solvent is preferably 0.00001 to 500 (l / min), particularly 0.0001 to 400 (l / min). The stirring time after the addition of the water-soluble organic solvent varies depending on the liquid temperature, but is usually from 30 minutes to 12 hours, and preferably within 3 hours from the viewpoint of preventing spoilage. By performing precipitation under such conditions, it is possible to precipitate 90% by weight or more, preferably 95% by weight or more of the mucopolysaccharide contained in the raw material composition at a time.

  When adding the aqueous solution obtained in the aqueous solution preparation step to the water-soluble organic solvent as an alternative method of the batch type treatment, the water-soluble organic solvent is put in the reaction vessel in advance and the temperature is adjusted while stirring and the aqueous solution is added. And stirring may be continued to complete the precipitation. The conditions at this time are the same as those described above except that the liquid to be added is changed from the water-soluble organic solvent to the aqueous solution obtained in the aqueous solution preparation step.

  The mucopolysaccharide precipitated in the precipitation step can be separated and recovered from the liquid component by a normal separation method such as centrifugal filtration, overpressure filtration, reduced pressure filtration, decantation, filter press and the like. The purity of the mucopolysaccharide recovered in this way is high, but when obtaining a higher-purity mucopolysaccharide, the separated mucopolysaccharide is dissolved again in water and an aqueous solution (hereinafter referred to as a secondary aqueous solution). (The second aqueous solution adjusting step), and then mixing the obtained secondary aqueous solution with the water-soluble organic solvent to precipitate the highly purified mucopolysaccharide (second precipitation step). . The secondary aqueous solution may be prepared in the same manner as the aqueous solution preparation step. In the secondary aqueous solution, it is preferable to add an organic carboxylate such as sodium acetate so as to have a concentration of 0.1 to 5% by weight for the purpose of promoting precipitation of mucopolysaccharide. Moreover, the prepared secondary aqueous solution may be subjected to at least one treatment selected from ultrafiltration treatment, activated carbon treatment, ion exchange resin treatment and filtration treatment without being mixed with the water-soluble organic solvent. These processes will be described below.

(1) Ultrafiltration treatment As the treatment solution (secondary aqueous solution) used for ultrafiltration, it is suitable to use an aqueous solution containing 0.1 to 30% by weight, preferably 2 to 10% by weight of mucopolysaccharide. . A preservative, acid, and alkali may be added to the secondary aqueous solution for the purpose of preventing decay. The pH of the secondary aqueous solution is not particularly limited, but is preferably pH 8 to 14 or pH 1 to 5 from the viewpoint of preventing spoilage. The membrane used for ultrafiltration may be appropriately selected according to the molecular weight of the mucopolysaccharide. When the mucopolysaccharide is chondroitin sulfate, a membrane having a molecular weight cut off of 6000 or more is effective. However, in the case of chondroitin sulfate derived from straw, a membrane having a molecular weight cut off of 30,000 to 100,000 is preferable. It is particularly preferable to use 50,000 membranes in terms of purification efficiency.

  The operating procedure is to increase the purity of the mucopolysaccharide in the secondary aqueous solution by bringing the secondary aqueous solution into contact with the fractionation membrane under pressure and eluting the contents other than the mucopolysaccharide together with water. I can do it. The pressure at this time is usually 1 MPa or less. By this operation, the organic carboxylate can be removed, and a small amount of mixed proteins and / or degradation products thereof can also be removed.

  As an operation mode when performing ultrafiltration, an operation mode used for normal ultrafiltration treatment can be used without limitation. Examples include a batch system, a continuous system, a cascade system, and the like. As the batch system, a fed batch flow and a fed batch flow with a supply pump are used in addition to a normal batch operation flow.

  In the batch system, the secondary aqueous solution is once introduced into the stock solution tank and concentrated within a certain time. In the case of this system, mucopolysaccharide can be purified by adding water to the stock solution concentrated by ultrafiltration as necessary and repeatedly performing concentration treatment. Moreover, by using a continuous system, it is possible to continuously process the ultrafiltration stock solution and obtain a processing solution continuously.

(2) Activated carbon treatment Activated carbon used for the activated carbon treatment is available without any limitation. For example, PK, PKDA MESY / MRX, ELORIT, AZ0, DARCO, HYDRODACO 3000/4000, DARCO 12X20LI, DARCO12X20DC, PETRODARCO, DARCO MRX, GAC, GAC PLUS, GARCU VAPUR, GARCU VAPURE, GARCU CA, CN, CG, DARCO KB / KBB, S-51, S-51-HF, S-51-FF, PREMIUM darco, DARCO GFP, HDC / HDR / HDH, GRO SAFE, FM-1, DARCO TRS , DARCO FGD, SX, SX ULTRA, SA, D-10, PN, ZN, SA-SW, W, GL, HB PLUS and other powdered activated carbons, ROW, RO, ROX, R , R, R. Molded activated carbon and impregnated activated carbon such as EXTRA, SORBONORIT, GF 40/50, CNR, ROZ, RBAA, RBHG, RZN, RGM, etc., PICA granular activated carbon, spherical activated carbon, powdered activated carbon, Nippon Enviro Chemical's Molcybon , WHA, granular white birch (X2M, GM2X, GH2X, GHXUG, GS1X, GS3X, GTX, GTSX, G2X, GS2X, GAAX, MAC-W, GOC, GOX, GOX, APRC, TAC, MAC, XRC, NCC, SRCX) Functional activated carbon such as granular white birch (G2C, C2C, WH2C, W2C, WH5C, W5C, LGK-400, LGK-100, LH2C, KL, G2X, GH2X, WH2X, S2X, C2X, X7000H, X7100H, X700H- 3, X7100 Granular activated carbons such as H-3, LGK-700, DX7-3), X-7000, X-7100, X-7000-3, X-7100-3, white birch (C, M, A, P, PHC) , FAC-10), carborafine, strong white birch, refined white birch, refined white birch, special white birch, white birch DO-2, white birch DO-5, white birch DO-11, etc., powdered activated carbon, honeycomb carbo white birch, molded carbon, carbon Activated carbon products such as paper, white birch C-DC, carborafine DC, granular white birch DC, aldenite, and aldenite SP Granular activated carbon such as SG and SGP from Nimura Chemical Industry, TA, TS, TG, TM, etc. Granular activated carbon, S, FC, SA1000, K, A, KA, AC, M, P, IC, IP, CB, GB, GLP, CLP, W, etc. Powdered activated carbon, CG48B, CG48BR, CW1 0B, CW130A, CW130BR, CW130AR, CW480SZ, CW6100SZ, GL130A, GL240A, GM130A, GM240A, crushing activated carbon such as GMC, and the like. Since the activated carbon to be used varies depending on the type of mucopolysaccharide raw material composition, it may be appropriately selected. Moreover, you may use it in combination of 2 or more types. Among these activated carbons, powdered activated carbons are particularly preferable because it is easy to obtain higher-purity mucopolysaccharides. Examples of commercially available products include purified white birch, purified white birch 2, and SX ULTRA.

  If the amount of activated carbon used is too small, the effect will be drastically reduced, and if it is too much, mucopolysaccharide will be adsorbed and the yield will be reduced, and from the viewpoint of economy, mucopolysaccharide contained in the secondary aqueous solution, What is necessary is just to select suitably from 0.001-500 weight%, Preferably it is 0.01-400 weight%, More preferably, it is 0.1-100 weight%.

  The solvent for the activated carbon treatment is basically an aqueous solution, but may be a mixed solution with a water-soluble organic solvent. Specific examples of the water-soluble organic solvent include methanol, ethanol, 1-propyl alcohol, 2-propyl alcohol, 1-butyl alcohol, 2-butyl alcohol, isobutyl alcohol, t-butyl alcohol and other alcohols, acetonitrile, etc. Nitriles, ethers such as tetrahydrofuran, and ketones such as acetone and methyl ethyl ketone. As the ratio of the water-soluble organic solvent to water, mucopolysaccharide is precipitated when the amount of the water-soluble organic solvent is too much. Therefore, the water-soluble organic solvent is usually used at 50% by weight or less. The concentration of the mucopolysaccharide in the secondary aqueous solution is not particularly limited, but is generally appropriately selected from the range of 0.1 to 30% by weight. It is preferable that the pH of the secondary aqueous solution is adjusted to 3 to 11, particularly 4 to 10, and contacted with activated carbon.

  The activated carbon treatment is carried out by bringing activated carbon into contact with the secondary aqueous solution. At this time, the contact may be performed under atmospheric pressure, overpressure, or reduced pressure, and further, inert such as nitrogen, helium, argon, etc. You may process in any atmosphere of gasification and air | atmosphere. The contact is usually performed in a batch system, and the order of addition is not particularly limited. When the liquidity at the time of contact is too low, the aqueous solution coagulates. When the liquidity is high, mucopolysaccharide is denatured. Therefore, it is usually −20 to 100 ° C., preferably −10 to 90 ° C., more preferably −5 to 80 ° C. is there. If the contact time is too short, the effect is not manifested, and if it is too long, mucopolysaccharide is modified, and therefore it is usually 0.1 to 24 hours.

  For the removal of activated carbon from the treatment liquid after the activated carbon treatment, a normal separation method is used without limitation. For example, centrifugal filtration, overpressure filtration, reduced pressure filtration, decantation, filter press and the like can be mentioned. In general, a filter aid such as diatomaceous earth is used as a filter aid, and filtration is performed by pressure filtration, vacuum filtration, or filter press. The filter aid may be added to the aqueous solution, added to the filter, or a combination of both. Furthermore, it is more preferable to subject the aqueous solution after filtration to 0.1-1.0 μm microfiltration (membrane filter, pole filter).

(3) Ion exchange resin treatment When performing the ion exchange treatment, it is preferable to use a secondary aqueous solution to which an anti-corrosion agent is added from the viewpoint of anti-corruption. The mucopolysaccharide concentration in the secondary aqueous solution is preferably 0.1 to 30% by weight, particularly 0.5 to 25% by weight. Treatment with a cation exchange resin can remove proteins and / or degradation products thereof, salts, and heavy metals if necessary. Metal ions and proteins and / or degradation products thereof are bound to the proton-type exchanger resin, and free mucopolysaccharide is present in the filtrate or eluent. The treatment temperature is generally 0 to 50 ° C., but 10 to 25 ° C. is preferable from the viewpoints of purification efficiency, resin regeneration, prevention of corruption, and the like. As a treatment method, a batch method by stirring in an ion exchange resin, or a continuous or discontinuous column method can be used. As the cation exchange resin, a strongly acidic resin is preferable, and examples thereof include Dowex X50, Amberlite IR120, PK216, and the like. The amount of the ion exchange resin used is 1 to 50% by weight, preferably 5 to 40% by weight, based on 1 part by weight of the mucopolysaccharide in the secondary aqueous solution.

(4) Filtration treatment When turbidity is observed in the aqueous solution containing the mucopolysaccharide raw material composition before and after the treatments (1) to (3), the turbidity component is added to further improve the effect of the treatment. In order to remove, it is preferable to perform a filtration treatment. As the filtration treatment, a usual treatment method is used without any limitation. For example, centrifugal filtration, overpressure filtration, reduced pressure filtration, decantation, filter press and the like can be mentioned. In general, a filter aid such as diatomaceous earth is used as a filter aid, and filtration is performed by pressure filtration, vacuum filtration, or filter press. The filter aid may be added to the aqueous solution, added to the filter, or a combination of both. Further, it is more preferable to subject the aqueous solution after filtration to 0.1-1.0 μm microfiltration (membrane filter, pole filter).

  After performing such a secondary treatment as necessary, a more highly purified mucopolysaccharide can be obtained by a method such as separating the precipitated mucopolysaccharide or removing the solvent from the treated liquid.

  The mucopolysaccharide separated and recovered by a method such as filtration after the precipitation step (the first precipitation step and the second precipitation step), or the mucopolysaccharide separated after the treatments (1) to (3) above, Since it contains a water-soluble organic solvent, it is preferable to perform a drying treatment. As the drying process, a normal process is used without any limitation. For example, drying under reduced pressure, warm air drying, humidity drying, air drying, shelf drying may be mentioned, and drying may be performed by a shelf type, or may be performed by rotating like a conical dryer. What is necessary is just to select suitably according to the kind of mucopolysaccharide obtained. If the temperature of the drying operation is too low, the drying time will be prolonged, and if it is too high, the mucopolysaccharide is denatured. Therefore, it is usually from -10 to 120 ° C, preferably from 0 to 110 ° C, more preferably from 10 to 100 ° C. Select as appropriate.

  As a method for directly obtaining a mucopolysaccharide solid without performing the above drying treatment, freeze drying and spray drying are used without any limitation. As the concentration of the aqueous solution during lyophilization, the concentration of mucopolysaccharide is preferably 50% by weight or less, the freezing temperature is −5 ° C. or less, and the degree of vacuum is 700 mmHg or less. On the other hand, the concentration of the aqueous solution during spray drying may be in the range of 1 to 40% by weight, and the drying temperature may be appropriately selected in the range of 50 to 200 ° C. By the above-described operation, it is possible to obtain a mucopolysaccharide solid.

  The solid of mucopolysaccharide obtained by the drying treatment is classified by pulverizing the solid, if necessary. Any known pulverization / classification treatment may be used without any limitation. As crushing methods, jaw crusher, gyratory crusher, cone crusher, hammer crusher, shredder, roll crusher, hammer mill, disintegrator, cutter mill, disk mill, pin mill, stamp mill, fret mill, rod mill, roller mill, table mill, Ring roll mill, ring roll mill, erotic fall mill, turbo mill, screen mill, centrifugal classification mill, vertical jet mill, micronizer jet mill, collision jet mill, pot mill, tube mill, conical mill, radial aluminum, Examples thereof include a tower type pulverizer, a circular vibration mill, a helical vibration mill, a planetary pulverizer, a sand mill, a colloid mill, a mortar, a stone mill, and a pharmaceutical laboratory.

  On the other hand, as a classification method, a known sieving method is used without any limitation. Illustrative examples include gravity flow, mechanical forced flow, vibration flow, and air entrainment flow. What is necessary is just to select suitably the size of the mesh of the sieve to be used from the range of 3.5-635 mesh.

  Further, the purity of mucopolysaccharide purified by the method of the present invention and analysis of various substances contained as impurities can be performed by the following methods.

1) Mucopolysaccharide Analyzes are made by a method according to the structure of mucopolysaccharide. For example, in the case of glucosaminoglucan such as chondroitin sulfate, hyaluronic acid, dermatan sulfate, etc., it can be analyzed by uronic acid analysis which is one of the constituent units. As the uronic acid analysis method, the carbazole method can be generally used. In the case of mucopolysaccharides having sulfate groups such as chondroitin sulfate, heparin, dermatan sulfate, etc., sulfate group determination methods such as barium sulfate turbidimetry and rosin acid method can be used. When the characteristic peak of the mixed impurity is clear and can be separated from the signal of the target mucopolysaccharide, the purity can be analyzed also in the proton nuclear magnetic resonance spectrum ( ¹ H-NMR). Purity analysis can be performed by gel permeation chromatography (GPC).

2) Protein and / or degradation product thereof A ninhydrin method that utilizes degradation of a protein in water and coloration of the produced amino acid by a ninhydrin reaction can be used. Also, the Kjeldahl method, which quantifies all nitrogen as ammonium ions by ignition with sulfuric acid, and a reaction that forms a complex salt with divalent copper on the strongly alkaline side when two or more peptide bonds exist in close proximity A burette method using a phenol reagent, a lorry method which is a color reaction derived from an aromatic amino acid in a phenol reagent and protein, a UV method using an aromatic amino acid content as an index, an excess acid (or basic) dye added Analyze proteins in general, such as by forming an insoluble salt with protein, precipitating, measuring the amount of unreacted dye with a spectrophotometer, and determining the amount of protein from the calculated amount of bound dye Method can be used. Among these, an analysis method corresponding to the mucopolysaccharide to be produced may be adopted, but the ninhydrin method can be preferably used because it is generally less susceptible to interfering substances and has high detection sensitivity. In addition, if the characteristic peak of the protein and / or its degradation product is clear and separable from the signal of the desired mucopolysaccharide, it is possible to analyze the mixed amount in the proton nuclear magnetic resonance spectrum ( ¹ H-NMR). Is possible.

3) Sodium chloride The amount of chloro ions and sodium ions can be analyzed by ion exchange chromatography. The concentration can be determined from the turbidity by silver nitrate titration. In addition, quantification from sodium ions is possible by atomic absorption or ICP-MAS.

4) Sulfur content Based on the Japanese Pharmacopoeia general test method, it can be analyzed by oxygen flask combustion method.

5) Microbial analysis method Although it is possible by a publicly known method, for example, a microbial limit test method viable cell count test (membrane filter method) based on the Japanese Pharmacopoeia general test method can be used.

6) Water-soluble organic solvent content It can be measured by gas chromatography (GC). It is also possible to perform quantification based on a characteristic signal derived from a water-soluble organic solvent also by proton nuclear magnetic resonance spectrum ( ¹ H-NMR).

  Applications of the mucopolysaccharide obtained by the production method of the present invention include pharmaceuticals, cosmetics, foods, beverages, seasonings, feeds and the like. Of these uses, pharmaceuticals and cosmetics will be described in detail below.

  The medicament containing the highly purified mucopolysaccharide of the present invention as an active ingredient (hereinafter referred to as the medicament of the present invention) has physiological functions, for example, physiological functions of chondroitin sulfate include smooth joint tissue, fat Examples thereof include pharmaceuticals utilizing serum clarification action, blood coagulation inhibitory action, anti-inflammatory action, and anti-cancer action.

  In the case of producing a pharmaceutical comprising the mucopolysaccharide purified by the method of the present invention as an active ingredient, for example, the mucopolysaccharide may be combined with a known pharmaceutical carrier. In general, the preparation is generally prepared by blending the highly purified mucopolysaccharide of the present invention with a pharmaceutically acceptable liquid or solid carrier and, if necessary, a solvent, a dispersant, an emulsifier, and a buffer. , Stabilizers, excipients, binders, disintegrants, lubricants, etc., solids such as tablets, granules, powders, powders, capsules, etc., liquids such as normal solutions, suspensions, emulsions, etc. It can be. Moreover, it can be made into a dry product that can be made into a liquid material by adding an appropriate carrier before use.

  The pharmaceutical carrier can be selected according to the above administration form and dosage form. In the case of an oral preparation, for example, starch, lactose, sucrose, mannitol, carboxymethylcellulose, corn starch, inorganic salt, etc. are used. In preparation of the oral preparation, a binder, a disintegrant, a surfactant, a lubricant, a fluidity promoter, a corrigent, a colorant, a fragrance and the like can be further added.

  On the other hand, in the case of a parenteral agent, the highly purified mucopolysaccharide of the present invention according to a conventional method, distilled water for injection as a diluent, physiological saline, aqueous glucose solution, vegetable oil for injection, sesame oil, peanut oil, It can be prepared by dissolving or suspending in tide oil, corn oil, propylene glycol, polyethylene glycol or the like, and adding a bactericidal agent, stabilizer, isotonic agent, soothing agent, etc. as necessary.

  The medicament thus obtained can be administered by an appropriate administration route according to the preparation form. There is no particular limitation on the administration method, and it can be used for internal use, external use and injection. Injections can be administered, for example, intravenously, intramuscularly, subcutaneously, intradermally, and external preparations include suppositories. In addition to oral administration as it is, it can be added to any food or drink and taken daily.

  In addition, other uses and cosmetics of the highly purified mucopolysaccharide obtained by the production method of the present invention may be mentioned. The cosmetics referred to here include, for example, lotions, emulsions, creams, packs, bath preparations, facial cleansers, bath soaps and bath detergents. A cosmetic containing mucopolysaccharide as an active ingredient has known physiological functions as a cosmetic material possessed by mucopolysaccharide, such as an effect of improving skin moisture retention and elasticity, and an effect of preventing skin aging. When mucopolysaccharide is used as a cosmetic, it can be produced according to a conventional method.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not limited to these Examples.

Production Example 1 Preparation of Mucopolysaccharide raw material composition Nasal cartilage was collected from the heel and pulverized. Next, twice the amount of ion-exchanged water was added to the solid content to adjust the pH to 9, and 0.2% by weight of proteolytic enzyme (alkaline protease) was added, followed by treatment at around 50 ° C. for 1-2 hours. Thereafter, the enzyme was inactivated by heating to 90 ° C. After cooling, the mixture was centrifuged, and 0.2% by weight of activated carbon was added and stirred. Next, a filter aid (diatomaceous earth, radiolite 300) was added and filtered, and then the filtrate was dried using a spray dryer to obtain a slightly yellow powder. When the composition containing the obtained mucopolysaccharide was analyzed, it was found that the purity of chondroitin sulfate was about 43%, and the protein and / or its degradation product was about 55% by Kjeldahl analysis.

Example 1
A meniscus stirring blade (radius 5 cm) connected to a three-one motor (35 W), a thermometer, and a condenser were attached to a 1000 ml four-necked flask. To this, 200 ml of ion-exchanged water, 50.0 g of a mucopolysaccharide raw material composition {mucopolysaccharide (sodium chondroitin sulfate) 43 wt% (21.5 g), protein 55 wt% (27.5 g), salt 2 wt% (1 0.0g) and NMR purity 43%} were added and dissolved at 40 ° C. with stirring (shear rate 2 m / sec). Further, 23.4 g of sodium chloride was added and dissolved at 50 ° C. (the concentration of sodium chloride at this point was 8.6% by weight). Thereafter, 22.0 g of a 4 wt% aqueous sodium hydroxide solution (0.08 g of sodium hydroxide, 21.92 g of ion-exchanged water) was added, and the mixture was stirred at 50 ° C. for 2 hours. Next, the mixture was cooled to 22 ° C., 1.32 g of acetic acid was added, 28.5 g of sodium chloride was further added, and the mixture was dissolved at 22 ° C. (the concentration of sodium chloride at this point was 16.0% by weight). After dissolution, 513.5 g of 95 wt% ethanol (ethanol 487.8 g, ion-exchanged water 25.7 g) was added dropwise over 2 hours with vigorous stirring (shear rate 2 m / sec) at 30 ° C. or lower. After dropping, the mixture was further stirred for 1 hour, and the precipitate was separated by filtration under reduced pressure. The obtained wet body was 105.3 g, and when a shelf-type vacuum drying was performed at 50 ° C., 48.7 g of a dry body was obtained. As a result of GPC, IEC (ion chromatography) and automatic amino acid analysis (ninhydrin method), the content of sodium chondroitin sulfate was 21.5 g, and 7.2 g of protein / protein degradation product and 20.0 g of sodium chloride were included as impurities. It was. Further, the obtained chondroitin sulfate was subjected to a nuclear magnetic resonance spectrum (NMR) measurement, a microbial limit test method viable cell count test (membrane filter method), and an oxygen flask combustion method (sulfur) based on the Japanese Pharmacopoeia general test method. The results are shown in Table 1.

Comparative Example 1
A meniscus stirring blade (radius 5 cm) connected to a three-one motor (35 W), a thermometer, and a condenser were attached to a 1000 ml four-necked flask. To this, 200 ml of ion-exchanged water, 50.0 g of a mucopolysaccharide raw material composition {mucopolysaccharide (sodium chondroitin sulfate) 43 wt% (21.5 g), protein 55 wt% (27.5 g), salt 2 wt% (1 0.0g) and NMR purity 43%} were added and dissolved at 40 ° C. with stirring (shear rate 2 m / sec). Further, 8.2 g of sodium chloride was added and dissolved at 50 ° C. (the concentration of sodium chloride at this time was 3.2% by weight). Thereafter, 22.0 g of a 4 wt% aqueous sodium hydroxide solution (0.08 g of sodium hydroxide, 21.92 g of ion-exchanged water) was added, and the mixture was stirred at 50 ° C. for 2 hours. Next, the mixture was cooled to 22 ° C., 1.32 g of acetic acid was added, 6.0 g of sodium chloride was further added, and the mixture was dissolved at 22 ° C. (the concentration of sodium chloride at this time was 6.2% by weight). After dissolution, 513.5 g of 95 wt% ethanol (ethanol 487.8 g, ion-exchanged water 25.7 g) was added dropwise over 2 hours with vigorous stirring (shear rate 2 m / sec) at 30 ° C. or lower. After dropping, the mixture was further stirred for 1 hour, and the precipitate was separated by filtration under reduced pressure. The obtained wet body was 85.3 g, and when a shelf-type vacuum drying was performed at 50 ° C., 34.7 g of a dry body was obtained. As a result of GPC, IEC, and automatic amino acid analysis (ninhydrin method), it was 21.3 g of chondroitin sulfate, 21.3 g of protein / protein degradation product, and 10.0 g of sodium chloride. Furthermore, a nuclear magnetic resonance spectrum (NMR) measurement, a microbial limit test method based on the Japanese Pharmacopoeia general test method, a viable cell count test (membrane filter method), and an oxygen flask combustion method (sulfur) were performed. The results are shown in Table 2.

Example 2
23.3 g of wet body obtained in the same manner as in Example 1 (4.76 g of sodium chondroitin sulfate) was dissolved in 350 g of ion-exchanged water and microfiltered with a 0.45 μm membrane filter. Using the company's ultrafiltration device (UHP-76K) equipped with Advantech polysulfone ultrafiltration membrane (molecular weight cut off 50,000), the obtained filtrate was subjected to a nitrogen pressure of 0.2 MPa and a liquid temperature of 20 ° C. Ultrafiltration was performed for 20 hours. At this time, when the aqueous solution in the apparatus reached 30 g, ion-exchanged water was added so that the total amount became 350 g, and this was repeated. The total amount of ion-exchanged water added was 1500 g. The amount of the aqueous solution in the apparatus at the end of the ultrafiltration was 125 g, and this was transferred to a 4-neck 500 ml flask. The flask was equipped with a meniscus stirring blade (radius 5 cm) connected to a three-one motor (35 W), a thermometer and a condenser, and 2 g of sodium acetate was added and dissolved while stirring. After dissolution, the temperature was cooled to 5 ° C., the shear rate was adjusted to 10 m / sec, and 238 g of 95 wt% ethanol (226 g of ethanol, 12 g of ion-exchanged water) was added dropwise over 2 hours. After completion of the dropwise addition, the mixture was further aged for 1 hour, and then the precipitate was separated by filtration under reduced pressure. Furthermore, it was washed twice with 50 g of 95 wt% ethanol (ethanol 47.5 g, ion-exchanged water 2.5 g).

  The obtained wet body was 10 g, and was subjected to shelf-type vacuum drying at 50 ° C. to obtain 4.7 g of a dry body. As a result of GPC, IEC, and automatic amino acid analysis (ninhydrin method), it was 4.6 g of sodium chondroitin sulfate, 0.06 g of protein / protein degradation product, and sodium acetate was not detected. Further, the NMR purity at this time was 100%. Furthermore, when the oxygen flask combustion method (sulfur) based on the Japanese Pharmacopoeia general test method was performed, it was 5.9 weight% of sulfur.

Example 3
23.3 g of wet matter (4.76 g of sodium chondroitin sulfate) obtained in the same manner as in Example 1 was dissolved in 125 g of ion-exchanged water and microfiltered with a 0.45 μm membrane filter. The filtrate obtained by the above microfiltration was added to a 1.5 cm × 20 cm column packed with Mitsubishi Chemical Corporation cation exchange resin PK216, and the column treatment was performed for 1 hour. Further, the resin was washed with 20 ml of ion exchange water. The aqueous solution obtained after the cation exchange resin treatment was 150 g together with the washing liquid, and this was transferred to a 4-neck 500 ml flask. Thereafter, 0.38 g of sodium hydroxide was added for neutralization. A meniscus stirring blade (radius 5 cm), a thermometer and a condenser connected to a three-one motor (35 W) were attached to this flask, and the pH was adjusted to 5.5 with 0.5 g of acetic acid while stirring. Thereafter, 2 g of sodium acetate was further added and dissolved. After dissolution, the temperature was cooled to 5 ° C., the shear rate was adjusted to 0.7 m / sec, and 285 g of 95 wt% ethanol (271 g of ethanol, 14 g of ion-exchanged water) was added dropwise over 2 hours. After completion of the dropwise addition, the mixture was further aged for 1 hour, and then the precipitate was separated by filtration under reduced pressure. Furthermore, it was washed twice with 50 g of 95 wt% ethanol (ethanol 47.5 g, ion-exchanged water 2.5 g).

  The obtained wet body was 10 g, and was subjected to shelf-type vacuum drying at 50 ° C. to obtain 4.7 g of a dry body. As a result of GPC, IEC, and automatic amino acid analysis (ninhydrin method), sodium chondroitin sulfate was 4.6 g, protein / protein degradation product was 0.04 g, and sodium acetate was not detected. Further, the NMR purity at this time was 100%. Furthermore, when the oxygen flask combustion method (sulfur) based on the Japanese Pharmacopoeia general test method was performed, it was 5.9 weight% of sulfur.

Example 4
A meniscus stirring blade (radius 5 cm) connected to a three-one motor (35 W), a thermometer, and a condenser were attached to a four-neck 500 ml flask, and 125 g of ion-exchanged water was injected. Further, 23.3 g of wet matter obtained in the same manner as in Example 1 (4.76 g of sodium chondroitin sulfate) was dissolved while stirring at room temperature, and 4 g of activated carbon / purified white birch 2 manufactured by Nippon Enviro Chemical Co., Ltd. was added. Thereafter, the mixture was stirred at room temperature for 2 hours. After stirring, 10 g of diatomaceous earth was pre-fed into a vacuum filter and filtered, and washed with 20 g of ion-exchanged water. The filtrate was then microfiltered with a 0.45 μm membrane filter. Further, a meniscus stirring blade (radius 5 cm) connected with a three-one motor (35 w) to a four-necked 500 ml flask, a thermometer, and a condenser were attached, and 125 g of the filtrate was transferred. 2 g of sodium acetate was dissolved in the aqueous solution. After dissolution, the temperature was cooled to 5 ° C. and the shear rate was adjusted to 1 m / second, and then 95 wt% ethanol (ethanol 226 g, ion-exchanged water 12 g) was added dropwise over 2 hours. After completion of the dropwise addition, the mixture was further aged for 1 hour, and then the precipitate was separated by filtration under reduced pressure. Furthermore, it was washed twice with 50 g of 95 wt% ethanol (ethanol 47.5 g, ion-exchanged water 2.5 g).

  The obtained wet body was 10 g, and was subjected to shelf-type vacuum drying at 50 ° C. to obtain 4.7 g of a dry body. As a result of GPC, IEC, and automatic amino acid analysis (ninhydrin method), it was 4.6 g of sodium chondroitin sulfate, 0.05 g of protein / protein degradation product, and sodium acetate was not detected. Further, the NMR purity at this time was 100%. Furthermore, when the oxygen flask combustion method (sulfur) based on the Japanese Pharmacopoeia general test method was performed, it was 5.9 weight% of sulfur.

Example 5
23.3 g of wet body obtained in the same manner as in Example 1 (4.76 g of sodium chondroitin sulfate) was dissolved in 350 g of ion-exchanged water and microfiltered with a 0.45 μm membrane filter. Using the company's ultrafiltration device (UHP-76K) equipped with Advantech polysulfone ultrafiltration membrane (molecular weight cut off 50,000), the filtrate was subjected to ultrafiltration for 20 hours under the conditions of nitrogen pressure 0.2 MPa and 20 ° C. went. At this time, when the aqueous solution in the apparatus reached 30 g, ion-exchanged water was added so that the total amount became 350 g, and this was repeated. The total amount of ion-exchanged water added was 1500 g. The amount of the aqueous solution in the apparatus at the end of the ultrafiltration was 125 g.

  The aqueous solution was transferred by attaching a meniscus stirring blade (5 cm radius), a thermometer and a condenser with a three-one motor (35 w) connected to a four-necked 500 ml flask. After adding 4 g, the mixture was stirred at room temperature for 2 hours. After stirring, 10 g of diatomaceous earth was pre-fed into a vacuum filter and filtered, and washed with 20 g of ion-exchanged water. The filtrate was then microfiltered with a 0.45 μm membrane filter. Further, a meniscus stirring blade (radius 5 cm) connected with a three-one motor (35 w) to a four-necked 500 ml flask, a thermometer, and a condenser were attached, and 125 g of the filtrate was transferred. 2 g of sodium acetate was dissolved in the aqueous solution. After dissolution, the temperature was cooled to 5 ° C. and the shear rate was adjusted to 1 m / second, and then 95 wt% ethanol (ethanol 226 g, ion-exchanged water 12 g) was added dropwise over 2 hours. After completion of the dropwise addition, the mixture was further aged for 1 hour, and then the precipitate was separated by filtration under reduced pressure. Furthermore, it was washed twice with 50 g of 95 wt% ethanol (ethanol 47.5 g, ion-exchanged water 2.5 g).

  The obtained wet body was 10 g, and was subjected to shelf-type vacuum drying at 50 ° C. to obtain 4.7 g of a dry body. As a result of GPC, IEC, and automatic amino acid analysis (ninhydrin method), 4.6 g of sodium chondroitin sulfate, 0.01 g of protein / protein degradation product, and sodium acetate were not detected. Further, the NMR purity at this time was 100%. Furthermore, when the oxygen flask combustion method (sulfur) based on the Japanese Pharmacopoeia general test method was performed, it was 5.9 weight% of sulfur.

Example 6
A meniscus stirring blade (radius 5 cm) connected to a three-one motor (35 W), a thermometer, and a condenser were attached to a 1000 ml four-necked flask. To this, 200 ml of ion-exchanged water, 12.8 g of marine chondroitin-40 (MC-40) manufactured by Japan Barrier Free Co., Ltd. (42% by weight of sodium chondroitin sulfate (5.4 g), 46% by weight of protein (5.9 g), etc. 11.3 wt% (1.5 g) and NMR purity 44} were added and dissolved at 40 ° C. with stirring. Further, 32.8 g of sodium chloride was added and dissolved at 50 ° C. (the concentration of sodium chloride at this point was 13.4% by weight). Thereafter, 22.0 g of a 4 wt% aqueous sodium hydroxide solution (0.08 g of sodium hydroxide, 21.92 g of ion-exchanged water) was added, and the mixture was stirred at 50 ° C. for 2 hours. Next, the mixture was cooled to 22 ° C., 1.32 g of acetic acid was added, 40.0 g of sodium chloride was further added, and the mixture was dissolved at 22 ° C. (the concentration of sodium chloride at this point was 23.6% by weight). After dissolution, 513.5 g of 95% by weight ethanol (ethanol 487.8 g, ion-exchanged water 25.7 g) was added dropwise over 2 hours with vigorous stirring (shear rate 5 m / sec) at 30 ° C. or lower. After dropping, the mixture was further stirred for 1 hour, and the precipitate was separated by filtration under reduced pressure. The obtained wet body was 50 g, and a shelf-type vacuum drying was performed at 50 ° C. to obtain 5.4 g of a dry body. As a result of GPC, IEC, and automatic amino acid analysis (ninhydrin method), it was 5.2 g of sodium chondroitin sulfate, 1.3 g of protein / protein degradation product, and 0.1 g of sodium acetate. Furthermore, a nuclear magnetic resonance spectrum (NMR) measurement, a microbial limit test method based on the Japanese Pharmacopoeia general test method, a viable cell count test (membrane filter method), and an oxygen flask combustion method (sulfur) were performed. The results are shown in Table 3.

Example 7
The same treatment as in Example 1 was performed except that ethanol was changed to methanol. The results are shown in Table 4.

Claims (2)

A method for producing a highly purified mucopolysaccharide by separating mucopolysaccharide from a mucopolysaccharide and a raw material composition containing protein and / or a degradation product thereof, wherein the raw material composition is used. An aqueous solution preparation step of preparing an aqueous solution containing the raw material composition and sodium chloride and having a salt concentration of 8 to 60% by weight; and the aqueous solution obtained in the aqueous solution preparation step and a water-soluble organic solvent are mixed, A method comprising a precipitation step of selectively precipitating polysaccharides. A separation step of separating the mucopolysaccharide precipitated in the precipitation step and an aqueous solution containing a water-soluble organic solvent, a second aqueous solution preparation step of preparing an aqueous solution by dissolving the mucopolysaccharide separated in the separation step in water, And further comprising a step of subjecting the aqueous solution obtained in the second aqueous solution preparation step to at least one treatment selected from ultrafiltration treatment, activated carbon treatment, ion exchange resin treatment and filtration treatment. The method described.