JP4580688B2 - Method for producing chondroitin sulfate - Google Patents

Description

  The present invention relates to a method for producing chondroitin sulfate, which 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 lubricating metabolism and fulfilling cell activation function It is presumed to be widely used as pharmaceuticals, cosmetics, and health foods. The physiological actions 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, chondroitin sulfate can be produced by decomposing raw materials such as bone, cartilage, and skin with an alkaline solution to extract mucopolysaccharide, a neutral salt treatment method with a neutral salt solution, protease, and pronase. A method such as an enzymatic method of treating with a proteolytic enzyme such as is known. Chondroitin sulfate can also be obtained by a treatment method combining these methods.

  In these treatments, chondroitin sulfate is extracted (extracted) from the raw material by cleaving the core protein covalently bound to chondroitin sulfate. However, the extract obtained by such treatment contains a large amount of protein and its degradation products. Mixing of protein and / or a degradation product thereof with chondroitin sulfate 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 the contaminants such as proteins and their degradation products from the extracts obtained by these methods so as to increase the purity of chondroitin sulfate. 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 chondroitin sulfate having a specific molecular weight range from a chondroitin sulfate composition 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 “protein and / or its degradation product” in ethanol is significantly higher than that of chondroitin sulfate, as a result, these contaminants can be separated and removed by this method. However, the operation for fractionation while changing the ethanol concentration is complicated and expensive, and is not suitable for industrial production. In the ethanol fractionation treatment, chondroitin sulfate does not readily precipitate even if ethanol is simply added to the aqueous solution of chondroitin sulfate, so that sodium acetate or calcium acetate is slightly added (usually 5% by weight for the purpose of promoting precipitation). % Or less) is added (see Non-Patent Document 1). Further, in the combination of ethanol precipitation recovery and ion exchange resin treatment (see Patent Document 1), the purity of chondroitin sulfate collected by precipitation with ethanol added to the extract aqueous solution containing chondroitin sulfate is generally low. In order to achieve high purity, it is essential to perform treatment using an ion exchange resin. Furthermore, in ultrafiltration processing (refer patent document 2), there exists a problem that an expensive installation is required.

  In addition, for the purpose of degreasing, deodorizing and decoloring the extract containing chondroitin sulfate, a method of treating the extract with 2.5% by weight of activated carbon has been proposed. Contains 10 to 40% by weight of collagen and 20 to 40% by weight of amino acid in addition to 20 to 40% by weight of chondroitin sulfate (see Patent Document 3), and high purity chondroitin sulfate is not obtained by the activated carbon treatment. .

Japanese Biochemical Society, New Chemistry Experiment Course 3 "Carbohydrate II", Tokyo Chemical Doujin, 1991, p. 28 JP 2001-231497 A JP 2000-273102 A JP 2003-299497 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.

  In addition, regarding chondroitin sulfate among mucopolysaccharides, it is said that the effects of physiological functions as described above depend on the amount of sulfate radicals contained in chondroitin sulfate (corresponding to the sulfur content), and the sulfur content is within a suitable range ( For example, 5.0 to 8.0% by weight, preferably 5.5 to 7.5% by weight) of an appropriate molecular weight (for example, 10,000 to 300,000, preferably 20,000 to 250,000). Things tend to be desirable. However, the molecular weight distribution of chondroitin sulfate contained in the extract obtained by the extraction method as described above is very wide (for example, chondroitin sulfate in the extract extracted from fish cartilage by enzymatic method usually has a molecular weight of 8000). With a molecular weight distribution of ~ 600,000). Therefore, in order to obtain chondroitin sulfate having a molecular weight as described above from the extract obtained by the extraction method, the ethanol fractionation method must be adopted, and there are problems in terms of operational complexity and cost. there were.

  Therefore, the present invention removes contaminants, low molecular weight and high molecular weight chondroitin sulfate from the extract obtained by the extraction method simply and at low cost, and makes chondroitin sulfate having a high purity and an appropriate molecular weight distribution efficient. It is an object of the present invention to provide a manufacturing method.

  The present inventors have intensively studied to solve the above problems. As a result, it is known that chondroitin sulfate with high purity and appropriate molecular weight distribution may be obtained when an extract extracted from fish cartilage by an enzymatic method is treated with activated carbon having a specific surface area. I came to get. Further investigations have been made based on this finding, and it has been found that in order to obtain such chondroitin sulfate with good reproducibility, the amount of activated carbon used must be within a specific range with respect to the extract. The present invention has been completed.

That is, the present invention
(1) Raw material aqueous solution preparation step of preparing an aqueous solution containing a raw material composition consisting of chondroitin sulfate having a molecular weight distribution of at least 8000 to 500,000 and protein and / or a degradation product thereof, and (2) the raw material aqueous solution, After contacting the activated carbon having a specific surface area of 1.2 to 1.6 m ² / g in an amount of 50 to 500% by weight with respect to the raw material composition contained in the raw material aqueous solution, the activated carbon and the aqueous solution are brought into contact with each other. A method for producing chondroitin sulfate having a molecular weight distribution with a molecular weight of 10,000 to 300,000 and having a purity of 95% by weight or more, comprising an activated carbon treatment step for separation.

  According to the production method of the present invention, chondroitin sulfate having a high purity and an appropriate molecular weight distribution is obtained by removing impurities, low molecular weight and high molecular weight chondroitin sulfate from an extract obtained by the extraction method in a simple and low cost manner. Can be efficiently manufactured.

  In the present invention, a molecular weight distribution having a molecular weight of 10,000 to 300,000, preferably 20,000 to 250,000 is preferably obtained from a raw material composition consisting of chondroitin sulfate having a molecular weight distribution of 8000 to 500,000 and a protein and / or a degradation product thereof. A chondroitin sulfate having a molecular weight distribution and a purity of 95% by weight or more is obtained. The chondroitin sulfate having a molecular weight distribution of at least 8000 to 500,000 is a mixture of chondroitin sulfates having different molecular weights, and the minimum molecular weight of the distribution is always less than 8000 when the molecular weight distribution is measured. , Which means that the highest molecular weight of the distribution always exceeds 500,000. In addition, chondroitin sulfate having a molecular weight distribution of 10,000 to 300,000 is a mixture of chondroitin sulfates having different molecular weights, and the mixture is substantially composed of chondroitin sulfate having a molecular weight of less than 10,000 and chondroitin sulfate having a molecular weight of more than 300,000. Means something that doesn't exist.

  The raw material composition used in the present invention can be obtained, for example, by cleaving a core protein covalently bound to chondroitin sulfate, which is a mucopolysaccharide, by treating a raw material derived from various raw animals with an alkali or an enzyme. Here, as the above-mentioned raw material animals, fish such as sea bream and bream; other aquatic animals such as whales, rays and sea cucumbers; or land animals such as cattle, pigs, chickens and horses can be used. It is preferable to use aquatic animals, particularly salmon, because the purity of the product is relatively high and the odor can be reduced. 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 it is easy to handle and has a high content of chondroitin sulfate, so that cartilage such as nasal cartilage is particularly useful. 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 for 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 chondroitin sulfate is extracted, chondroitin sulfate is neutralized from the raw material The neutral salt treatment method that extracts with liquid, the enzymatic method that extracts prodrolytic enzymes such as protease and pronase on the raw material, and extracts chondroitin sulfate, or a combination of these methods can be used without limitation, but chondroitin Among these methods, the treatment with an alkali treatment method and an enzyme method is preferred, and an enzyme treatment is particularly preferred because of the high recovery rate of sulfuric acid. By these treatments, an aqueous solution component containing chondroitin sulfate having a molecular weight distribution of at least a molecular weight of 8000 to 500,000 can be obtained. At this time, the cleaved protein and its degradation product (protein degradation product) are inevitably contained in the aqueous solution. The composition derived from the raw material obtained by such treatment is sometimes called a chondroitin sulfate-containing extract, but most of the composition is a raw material composition (that is, chondroitin sulfate having a molecular weight distribution of at least 8000 to 500,000 molecular weight). And protein and / or its degradation product).

  The aqueous solution of chondroitin sulfate-containing extract obtained by the above treatment can be used as a raw material aqueous solution as it is or after adjusting the amount of water, but it is necessary to remove insoluble matter by centrifugation, filtration, etc. Depending on the filter, filtration may be used, removal of turbid components, deodorization, decolorization, degreasing by performing activated carbon treatment using a small amount of activated carbon of about 2.5% by weight on the extract and extract Etc. are suitable. Furthermore, for ease of handling, it is preferable to temporarily solidify and powder by a method such as spray drying (spray drying), evaporation drying, freeze drying, etc., and dissolve in water when necessary to obtain a raw material aqueous solution.

  The chondroitin sulfate contained in the raw material composition has a molecular weight distribution of at least a molecular weight of 8000 to 500,000, but the ratio of the total molecular weight of less than 10,000 and the molecular weight of more than 300,000 in the entire chondroitin sulfate is usually 1 to 1. 30% by weight, preferably 5 to 25% by weight. Further, the content of chondroitin sulfate and protein and / or degradation product thereof in the raw material composition used in the present invention is not particularly limited, but the content of chondroitin sulfate in the extract obtained by the above-described method is “chondroitin” It is usually in the range of 10 to 90% by weight, expressed as the weight percent of “chondroitin sulfate” based on the total weight of “sulfuric acid” and “protein and / or its degradation product”. In the present invention, a raw material composition having such a composition range can be used without any problem.

  The molecular weight distribution of chondroitin sulfate can be known by gel permeation chromatography (GPC) analysis. GPC analysis can be suitably performed, for example, under the following conditions. Detector: differential refractometer, column: two SHODEX OHPAK SB-806MHQ linked (stationary phase), column temperature: 40 ° C., mobile phase: sodium nitrite 0.1 mol / L, flow rate: 1.0 ml / min.

  In the production method of the present invention, the raw material aqueous solution containing the raw material composition is treated with activated carbon. At this time, the concentration of chondroitin sulfate in the aqueous solution from the viewpoint of treatment efficiency is not economical if it is too thin, and if the concentration is too high, it becomes a highly viscous aqueous solution and insufficient contact with the activated carbon in the subsequent process, It is suitable that the range is 0.2 to 60% by weight, preferably 0.5 to 30% by weight of the aqueous solution prepared by chondroitin sulfate. In the raw material aqueous solution, it is not necessary that all components of the raw material composition are dissolved, and a part thereof may be in a suspended state. In addition to the raw material composition, the raw material aqueous solution may contain salts, ash, inorganic substances, and the like. These other components are preferably 0 to 500% by weight based on the total weight.

  The raw material aqueous solution can be prepared by adding water when the raw material composition is a solid such as a powder. In addition, when the raw material composition is an aqueous solution of chondroitin sulfate-containing extract obtained by the extraction treatment, the raw material aqueous solution may be prepared by adjusting the amount of water by adding water or evaporating the water. Good.

In the production method of the present invention, it is preferable to perform an alkali pretreatment before subjecting the raw material aqueous solution to the activated carbon treatment step. By the alkali pretreatment, physical interactions with proteins and / or degradation product thereof in the raw material composition is solved hog (hereinafter, also referred to this effect the dispersion effect), obtained after treatment with activated carbon The purity of the chondroitin sulfate produced is further improved. The alkali pretreatment is carried out by adding an alkali to the raw material aqueous solution, adjusting the pH of the aqueous solution to 8 to 13, preferably 8.5 to 13, and holding it for a predetermined time. Examples of alkalis that can be suitably used at this time 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. And organic hydroxides such as tetraethylammonium hydroxide. Of these, sodium hydroxide and potassium hydroxide are preferred, and sodium hydroxide is particularly preferred from the standpoint of availability and economy. The amount of alkali added should be selected so that

  In the alkali pretreatment, chondroitin sulfate may be denatured when stored as an alkaline aqueous solution having a pH as described above for a long time, so the alkali pretreatment time is 0.001 to 100 hours, preferably 0.01 to It needs to be 50 hours, more preferably 0.01 to 12 hours. The liquid temperature in the alkali pretreatment may be appropriately selected from the range of −10 to 120 ° C., preferably −5 to 100 ° C., and more preferably 0 to 80 ° C. from the viewpoint of the dispersion effect. The alkali pretreatment is stopped (terminated) by adding an acid after elapse of a predetermined time and adjusting the pH of the aqueous solution to 5 to 10, preferably 5 to 8. At this time, when a strong alkali such as sodium hydroxide is used as the alkali, it is preferable to add an acid in an equivalent amount. Examples of the acid to be added after the alkali treatment include organic carboxylic acids such as acetic acid, citric acid and tartaric acid, and inorganic acids such as phosphoric acid, hydrochloric acid and sulfuric acid.

  In the alkali treatment, an organic carboxylate or salt may be added to the raw material aqueous solution for the purpose of assisting the dispersion effect. As the salt of organic carboxylic acid, monovalent or divalent or higher organic carboxylic acid such as acetate, citrate, tartaric acid, malonic acid and the like can be used. From the viewpoint of solubility in water, acetate is particularly preferred. Is preferred. As the salt counter ion (cationic species), sodium ion, potassium ion, calcium ion, barium ion, and the like can be used, and sodium ion is particularly preferable in terms of purification efficiency. Sodium acetate can be mentioned as a particularly suitable organic carboxylate. Further, sodium acetate may be procured at the time of use (to form a salt on the spot) by reacting acetic acid with sodium hydroxide, sodium bicarbonate, sodium carbonate or the like in an aqueous solution. Moreover, you may add the above-mentioned organic carboxylate and sodium chloride individually or in combination of 2 or more types. The concentration of the organic carboxylate or sodium chloride during the alkali treatment is not particularly limited, but the concentration in the prepared aqueous solution is usually 8 to 60% by weight, preferably 9 to 50% by weight for the purpose of enhancing the solubility and the dispersion effect in the alkali treatment. Particularly preferred is 10 to 40% by weight. The addition of alkali, organic carboxylate, and sodium chloride in the alkali pretreatment also has the effect of preventing the occurrence of fungi, mold, etc. (rot) in addition to the dispersion effect.

  Further, a preservative may be added during the alkali pretreatment. Examples of preservatives that can be suitably used include alcohols such as ethanol and n-butanol, guanidines such as polyhexamethylene, reverse soaps such as benzalkonium chloride, surfactants such as alkylpolyaminoethylglycine, hypochlorite Halogen-based disinfectants such as sodium acid, peroxides such as hydrogen peroxide and peracetic acid can be used. What is necessary is just to adjust the usage-amount of these preservatives suitably with process temperature etc.

  The alkali pretreatment method (addition order of each additive, etc.) is not particularly limited, and can be suitably performed by the following method, for example. That is, first, a predetermined amount of raw material composition powder and water are put in a reaction kettle, respectively, and then the raw material composition is dissolved or suspended while stirring to prepare a raw material aqueous solution. Next, an organic carboxylate or sodium chloride is added to the raw material aqueous solution to dissolve or suspend them, and then the alkali is added as it is or as an aqueous solution. Then, after sufficient stirring and alkali pretreatment for a predetermined time, an acid may be added to adjust the pH.

In the production method of the present invention, in order to obtain chondroitin sulfate having a molecular weight distribution with a molecular weight of 10,000 to 300,000 and a purity of 95% by weight or more from a raw material aqueous solution, preferably a raw material aqueous solution subjected to alkali pretreatment, The treatment is performed with activated carbon having a specific surface area of 1.2 to 1.6 m ² / g in an amount of 50 to 500% by weight based on the raw material composition contained in the raw material aqueous solution. When activated carbon with a specific surface area outside the above range is used, it becomes difficult to obtain chondroitin sulfate having an initial molecular weight distribution, and when the amount of activated carbon used deviates from the above range, the molecular weight distribution is controlled. It becomes difficult not only to obtain chondroitin sulfate having the desired purity. From the viewpoint of the effect, the amount of activated carbon used in the activated carbon treatment step is preferably 50 to 500% by weight, particularly 70 to 450% by weight based on the above criteria.

The activated carbon used in the activated carbon treatment step is industrially or as a reagent if its specific surface area is 1.20 to 1.60 m ² / g, preferably 1.25 to 1.55 m ² / g. What can be used without limitation. Examples of activated carbon that can be used in the present invention include PK, PKDA MESY / MRX, ELORIT, AZ0, DARCO, HYDRODACO 3000/4000, DARCO 12X20LI, DARCO12X20DC, PETRODACO, DARCO MRX, GAC, GAC PLUS, DAR PLUS, DARCO Crushed activated carbons such as GCN, C-GRANULAR, CA, CN, CG, DARCO KB / KBB, S-51, S-51-HF, S-51-FF, PREMIUM DARCO, DARCO GFP, HDC / HDR / HDH, Powdered activated carbon such as GRO SAFE, FM-1, DARCO TRS, DARCO FGD, SX, SX ULTRA, SA, D-10, PN, ZN, SA-SW, W, GL, HB PLUS , ROW, RO, ROX, RB, 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 2, special white birch, white birch DO-2, white birch DO-5, white birch DO-11, etc., powdered activated carbon, honeycomb carbon 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, can CW130A, CW130BR, CW130AR, CW480SZ, CW6100SZ, GL130A, GL240A, GM130A, GM240A, be given a crushing activated carbon such as GMC. Of these activated carbons, those having a specific surface area satisfying the above conditions may be appropriately selected and used.

  Among these activated carbons, 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, white birch (C, M, A, P, PHC , FAC-10), carborafine, strong white rabbit, refined white rabbit, purified white rabbit 2, special white rabbit, white rabbit DO-2, white rabbit DO-5, white rabbit DO-11, S, FC, SA1000, K, A, KA, AC , M, P, IC, IP, CB, GB, GLP, CLP, W, etc., powdered activated carbon, granular white birch (G2C, C2C, WH2C, W2C, WH5C, W5C, LGK-400, LGK-100, LH2C, KL, G2X, GH2X, WH2X, S2X, C2X, X7000H, X7100H, X700H-3, X7100H-3, LGK-700, DX7-3), X-7000, X Granular activated carbons such as -7100, X-7000-3, and X-7100-3 are preferred, and among them, powdered activated carbon purified white birch and purified white birch-2 are particularly preferred.

  The activated carbon treatment may be performed by separating the activated carbon from the treated aqueous solution, after mixing and stirring the raw material aqueous solution, preferably, the alkaline aqueous solution and the predetermined amount of activated carbon. . If the contact time between the raw material aqueous solution and the activated carbon is too short, the effect is not exhibited, and if it is too long, it is not economical, so it is usually 0.01 to 100 hours, preferably 0.1 to 24 hours, and more preferably 0. It is good to select suitably from the range of 2 to 10 hours. Moreover, the contact temperature of the raw material aqueous solution at the time of contact may be appropriately selected from the range of −10 to 120 ° C., preferably −5 to 100 ° C., more preferably 0 to 80 ° C. in consideration of operability and efficiency.

  Separation methods such as centrifugal filtration, overpressure filtration, vacuum filtration, decantation, filter press, etc. can be adopted as a method for separating activated carbon from an aqueous solution after activated carbon treatment. It is preferable to separate by pressure filtration using a filter aid, vacuum filtration or filter press. The filter aid may be added to the aqueous solution, may be added to the filter, or both may be combined. Furthermore, it is more preferable to subject the aqueous solution after filtration to 0.1-1.0 μm microfiltration (membrane filter, pole filter). Here, the removed protein and / or its degradation product and the activated carbon adsorbed with low molecular weight or high molecular weight chondroitin sulfate should be effectively used as fertilizer for paddy field, field, orchard, forest, flower field, vegetable field. Can do.

  Chondroitin sulfate contained in an aqueous solution obtained by separating activated carbon by the method as described above has a molecular weight distribution of 10,000 to 300,000, preferably 2 to 280,000. Moreover, the protein and / or its degradation product contained in the aqueous solution is usually 0.1% by weight or less based on chondroitin sulfate. Therefore, chondroitin sulfate recovered from the aqueous solution has a relatively high sulfate group content, so that it is not only expected to have a high physiological function but also has extremely high purity. Chondroitin sulfate obtained by the method of the present invention can be called a high-purity product, and is widely used in the fields of pharmaceuticals, other foods, and cosmetics.

  As a method for separating and recovering the target chondroitin sulfate as a solid from the separated aqueous solution, drying methods such as freeze-drying (freeze-drying) and spray-drying (spray-drying) can also be applied. In order to efficiently remove acids, alkalis, organic carboxylates, salt, preservatives, etc. contained in the recovered aqueous solution, the aqueous solution and a poor solvent such as alcohol are mixed to precipitate chondroitin sulfate. A precipitation method of separating and recovering after the separation is suitably employed.

  As the poor solvent used at this time, it is preferable to use an alcohol having low solubility of chondroitin sulfate and water solubility. Examples of alcohols that can be suitably used include lower alcohols such as methanol, ethanol, isopropyl alcohol, and isobutyl alcohol, and polyhydric alcohols such as ethylene glycol, diethylene glycol, propylene glycol, and glycerin. Among these, methanol and ethanol are preferable. Ethanol is particularly preferred.

  In mixing the poor solvent such as alcohol and the aqueous solution, the aqueous solution may be dropped into the poor solvent or the poor solvent may be dropped into the aqueous solution, but the latter is preferable from the viewpoint of workability. Further, at the time of mixing, the concentration of chondroitin sulfate in the solution is preferably adjusted to be in the range of 0.01 to 10% by weight because chondroitin sulfate is likely to precipitate and the recovery rate is increased. Furthermore, in order to increase the deposition rate, sodium acetate may be added and dissolved so as to be 0.1 to 5% by weight. At this time, when sodium acetate is used in the alkaline pretreatment in the previous step and is present in the aqueous solution, the amount of water is adjusted so that the concentration of sodium acetate is 0.1 to 5% by weight. That's fine. Moreover, what is necessary is just to select the quantity of the poor solvent to be used so that the density | concentration of the poor solvent after mixing with a poor solvent may be 30 to 80 weight%. The liquid temperature when precipitating chondroitin sulfate from the mixed solution is preferably -20 to 70 ° C, particularly preferably -10 to 60 ° C, and most preferably -5 to 55 ° C. Furthermore, when precipitating chondroitin sulfate, it is preferable to stir the mixed solution, and the stirring speed may be adjusted so that the shear speed of the stirring blade is 0.7 to 50 m / sec.

  The chondroitin sulfate precipitated by such a method can be recovered by general solid-liquid separation operations such as centrifugal filtration, overpressure filtration, vacuum filtration, decantation, and filter press. Further, the separated solid may be washed with alcohol or a mixed solution of alcohol / water. The solid chondroitin sulfate isolated as described above is powdered by drying, pulverizing and classifying (for example, 3.5 to 635 mesh) as necessary.

  As a drying method, a known drying method such as reduced pressure drying, warm air drying, humidity conditioning drying, air drying, shelf drying can be applied without particular limitation, and even if it is dried on a shelf, it is rotated like a conical dryer. And may be dried. The drying temperature is preferably −10 to 120 ° C., particularly 10 to 100 ° C. from the viewpoint of drying efficiency and chondroitin sulfate stability.

  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, Elo Fall Mill, Turbo Crusher, Screen Mill, Centrifugal Classification Mill, Vertical Jet Crusher, Micronizer Jet Crusher, Collision Jet Crusher, Pot Mill, Tube Mill, Conical Mill, Radial Known pulverization methods such as a mill, a tower pulverizer, a circular vibration mill, a helical vibration mill, a planetary pulverizer, a sand mill, a colloid mill, a mortar, a stone mortar, and a pharmaceutical laboratory can be applied without particular limitation. Further, as a classification method, a sieving method such as a gravity flow method, a mechanical forced flow method, an oscillating flow method, an air flow accompanying flow method, or the like can be applied. What is necessary is just to select the size of a mesh suitably from the range of 3.5-635 mesh.

Thus chondroitin sulfate obtained has a molecular weight of 10,000 to 300,000, preferably not only has a molecular weight distribution of 20,000 to 280,000, protein and / or purity less content of the degradation product Is 95% by weight or more, preferably 97% by weight or more, and is particularly suitable for pharmaceutical and cosmetic applications.

The amount of each component contained the raw material composition and not pure product chondroitin sulfuric acid which may be seen free can know by the following analytical methods, determining the purity of chondroitin sulfate on the basis of these results can do.

(1) Chondroitin sulfate It can be analyzed by uronic acid analysis which is one of the constituent units of chondroitin sulfate. As the uronic acid analysis method, the carbazole method can be generally used. Since chondroitin sulfate has a sulfate group, a sulfate group determination method such as a barium sulfate turbidimetric method or a rhodizonate method can be used.

If the characteristic peaks of the mixed impurities are clear and can be separated from the signal of chondroitin sulfate, the purity can be analyzed also in proton nuclear magnetic resonance spectrum ( ¹ H-NMR). Purity analysis is also possible by gel permeation chromatography (GPC).

  The sulfur content of chondroitin can be analyzed by an oxygen flask combustion method based on the Japanese Pharmacopoeia general test method.

(2) Protein and / or degradation product thereof A ninhydrin method that utilizes degradation of 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 The method can be used. Among these analytical methods, the ninhydrin method is preferably used because it is generally less susceptible to interfering substances and has high detection sensitivity.

In addition, when the characteristic peak of protein and / or its degradation product is clear and can be separated from the signal of chondroitin sulfate, it is possible to analyze the mixed amount in proton nuclear magnetic resonance spectrum ( ¹ H-NMR) It is.

(3) Organic carboxylate salt It can be analyzed by ion exchange chromatography. It is also possible to perform quantification by proton nuclear magnetic resonance spectrum ( ¹ H-NMR) based on the characteristic signal derived from the organic carboxylate.

(4) Microbial analysis method Although it is possible by a 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.

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

  Applications of the chondroitin sulfate of the present invention include pharmaceuticals, cosmetics, daily necessities, foods, beverages, seasonings, feeds and the like. Examples of the drug include drugs utilizing smoothing of joint tissues, lipid serum clarification action, blood coagulation inhibitory action, anti-inflammatory action, and anti-cancer action. Examples of cosmetics include lotions, emulsions, creams, and packs. Examples of daily necessities include bath preparations, facial cleansers, bath soaps, and bath detergents.

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

Production Example Preparation of Extract Containing Raw Material Composition (Extract containing Chondroitin Sulfate) Nasal cartilage was collected from the heel and pulverized. Next, twice the amount of ion-exchanged water is added to the solid content to adjust the pH to near neutrality, and 0.2 wt% of proteolytic enzyme (alkaline protease) is added. After the treatment, 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, after adding a filter aid (diatomaceous earth, radiolite 300) and filtering, the filtrate was dried using a spray dryer to obtain a chondroitin sulfate-containing extract as a slightly yellow powder. When the obtained chondroitin sulfate-containing extract was analyzed, it was found that the chondroitin sulfate purity was about 43% and the protein and / or its degradation product was about 55% by Kjeldahl analysis. Also, detector: differential refractometer, column: SHODEX OHPAK SB-806MHQ connected in two (stationary phase), column temperature: 40 ° C., mobile phase: sodium nitrite 0.1 mol / L, flow rate: 1.0 ml / min As a result of GPC measurement under the above conditions, the molecular weight distribution was 8000 to 580,000, and the proportion of the total molecular weight of 300,000 or more was 24% by weight. Hereinafter, when simply abbreviated as GPC, this condition is meant.

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 3 liter (l) four-necked flask. To this, 1900 ml of ion-exchanged water, 100.0 g of chondroitin sulfate-containing extract obtained in the above production example {43% by weight of chondroitin sulfate (43.0 g), 55% by weight of protein (55.0 g), 2% by weight of salt ( 2.0 g); NMR purity 43% of chondroitin sulfate} was added and dissolved at 40 ° C. with stirring (shear rate 2 m / sec). Further, 100.0 g of an 8.4% by weight aqueous sodium hydroxide solution (8.4 g of sodium hydroxide, 91.6 g of ion-exchanged water) was added, and the mixture was stirred at 40 ° C. for 2 hours. Next, it was cooled to 22 ° C. and 12.6 g of acetic acid was added. Thereafter, 200 g of activated carbon (refined birch 2, specific surface area 1.44 m ² / g) was added and stirred at 25 ° C. for 2 hours (activated carbon treatment step). After completion of the stirring, diatomaceous earth (Radiolite 300) was used as a filter aid, and the activated carbon was removed by filtration under reduced pressure, followed by precision pressure filtration with a 0.45 μm membrane filter. After cooling the filtrate to 5 ° C., the stirring speed was adjusted so that the shear rate was 2 m / sec, and 2600 g of 95 wt% ethanol (ethanol 2470 g, ion-exchanged water 130 g) was added dropwise over 2 hours. After dropping, the mixture was further stirred at 10 ° C. or lower for 1 hour, and the precipitate was separated by filtration under reduced pressure. Furthermore, it was washed twice with 100 g of 95% ethanol (ethanol 95 g, ion-exchanged water 5 g). The obtained wet body was 64.7 g, and when a shelf-type vacuum drying was performed at 50 ° C., 25.8 g of a dry body was obtained. As a result of gel permeation chromatography (GPC), ion exchange chromatography (IEC), and automatic amino acid analysis (ninhydrin method), 25.8 g of sodium chondroitin sulfate, no protein / protein degradation product was detected, and sodium acetate was not detected. 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 1.

Comparative Example 1
In the same manner as in Example 1 except that the amount of activated carbon used in the activated carbon treatment step was 20.0 g, 35.8 g of a dried product was obtained. Table 2 shows the analysis results of the obtained dried product.

Comparative Example 2
40.8 g of a dried product was obtained in the same manner as in Example 1, except that the activated carbon used in the activated carbon treatment step (specific surface area 0.90 m ² / g) was used. Table 3 shows the analysis results of the obtained dried product.

Example 2
In the same manner as in Example 1 except that the activated carbon used in the activated carbon treatment step and the amount used were changed to 100 g of purified white birch (specific surface area 1.30 m ² / g), 25.8 g of a dried product was obtained. Table 4 shows the analysis results of the obtained dried product.

Claims (2)

(1) Raw material aqueous solution preparation step of preparing an aqueous solution containing a raw material composition consisting of chondroitin sulfate having a molecular weight distribution of at least 8000 to 500,000 and protein and / or a degradation product thereof, and (2) the raw material aqueous solution, After contacting the activated carbon having a specific surface area of 1.2 to 1.6 m 2 / g in an amount of 50 to 500% by weight with respect to the raw material composition contained in the raw material aqueous solution, the activated carbon and the aqueous solution are separated. A method for producing chondroitin sulfate having a molecular weight distribution of 10,000 to 300,000 and having a purity of 95% by weight or more, characterized by comprising an activated carbon treatment step. 2. The method according to claim 1, further comprising an alkali pretreatment step of maintaining the aqueous raw material solution at a pH of 8 to 13 for 0.01 to 12 hours as a pretreatment step of the activated carbon treatment step.