JP4679138B2 - Method for producing highly purified mucopolysaccharide - Google Patents

Description

  The present invention relates to a method for producing mucopolysaccharides which are expected to be used in various fields 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 their raw materials include fish such as salmon and coral, other aquatic animals such as whales and sea cucumbers, and cattle, pigs, chickens, horses, etc. Bone, cartilage, skin, fish scales and the like extracted from terrestrial animals 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 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, maintenance of corneal transparency, prevention of infection, and the like are known. Development of a simple and inexpensive method for producing high-purity chondroitin sulfate has been demanded in order to sufficiently exert these physiological functions as active ingredients of foods, beverages, cosmetics or pharmaceuticals.

  In general, methods for producing mucopolysaccharide-containing extracts include alkali treatment methods that decompose and extract from raw materials such as bone, cartilage, and skin with an alkaline solution, neutral salt treatment methods that use a neutral salt solution, protease, pronase, etc. A method such as an enzyme method for treatment with a proteolytic enzyme can be used, and a treatment method combining these methods can also be used.

  By these treatments, the mucopolysaccharide and the core protein covalently bonded to the mucopolysaccharide are cleaved to obtain a mucopolysaccharide-containing extract. However, the mucopolysaccharide-containing extract contains a large amount of protein and its degradation product (hereinafter also referred to as protein). Mixing proteins and the like in mucopolysaccharides may interfere with use in the pharmaceutical and cosmetic fields. For example, when it is used as a pharmaceutical agent for injection, it tends to cause an allergic reaction when it is mixed with proteins and the like, and in cosmetic applications, it tends to cause problems such as browning with time.

  Therefore, it is difficult to obtain sufficient purity due to the remaining of proteins and the like only by these methods. In order to solve this problem, it has been proposed that the mucopolysaccharide-containing extract is subjected to alcohol fractionation treatment, ion exchange chromatography treatment, ultrafiltration, activated carbon treatment, or a combination of these methods.

  The treatment by ultrafiltration is a method of removing proteins and the like as separation targets as a permeate. In such ultrafiltration, water is lost together with proteins, etc., and the permeation amount tends to decrease as the viscosity of the filtered liquid gradually increases. To avoid this, it was lost. It is necessary to supply water corresponding to the minute (for example, refer to Patent Documents 1 and 2).

Japanese Unexamined Patent Publication No. 57-192401 JP 2000-273102 A

  However, it is difficult to completely remove proteins and the like even by the ultrafiltration described above, and even when a high-purity mucopolysaccharide is obtained by performing ultrafiltration for a long time, some amount of protein or the like is mixed. There were problems such as cases. Japanese Patent Application Laid-Open No. 57-192401 describes a method for obtaining chondroitin sulfate having such a high purity that it can be used as an injection solution. In this method, the pretreatment is carried out oxidatively using heavy metal or the like. Depolymerization was necessary and the operation was complicated.

  Accordingly, an object of the present invention is to provide a method for producing a high-purity mucos and so on substantially free of protein by a simple and low-cost method suitable for industrial production.

The present inventors have intensively studied to solve the above problems. As a result, by subjecting an aqueous solution of mucopolysaccharide containing protein or the like to ultrafiltration under specific conditions, the protein or the like can be removed very efficiently, and a high-purity mucopolysaccharide can be obtained. As a result of further investigation, the present invention has been completed. That is, the present invention performs ultrafiltration using a raw material aqueous solution containing a mucopolysaccharide and a protein and / or a degradation product thereof as a liquid to be filtered. In the method for producing a highly purified mucopolysaccharide comprising the step of separating the protein and / or its degradation product from the aqueous solution as a permeate, the OH ⁻ concentration is 0.01-1 mol as the raw material aqueous solution. / L become as both use one basic substance is blended, OH ^- concentration Hiro the aqueous solution basic substance is blended so that 0.01 to 1 mol / L It is further characterized in that to carry out while replenishing the liquid.

  By the method of the present invention, it is possible to easily obtain a high-purity mucopolysaccharide having a low content of protein or protein degradation product. As a result, it is possible to provide a variety of mucos and the like at low cost for uses requiring high purity such as medical use and cosmetic use.

  The present invention performs ultrafiltration using a raw material aqueous solution containing a mucopolysaccharide and a protein and / or a degradation product thereof as a liquid to be filtered to obtain a high-purity mucopolysaccharide.

Although the said mucopolysaccharide to which the manufacturing method of this invention is applied is not specifically limited, A glycosamino glucan can be mentioned typically. 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, 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, an acid (-OSO ₃ H) or Na salt (-OSO ₃ Na), K salt (-OSO ₃ K), Ca salt (-OSO ₃ Ca) It may be an ionic structure such as) or a mixture thereof. When the mucopolysaccharide has a carboxyl group structure, an acid (—CO ₂ H), or a Na salt (—CO ₂ Na), a K salt (—CO ₂ K), a Ca salt (—CO _It may be an ionic structure such as ₂ Ca) or a mixture thereof.

  As described above, mucos are classified into bones, cartilage, skin, scales, etc. of fishes such as salmon and coral, other aquatic animals such as whales and sea cucumbers, and land animals such as cattle, pigs, chickens and horses ( Hereinafter, the mucopolysaccharide-containing tissue) is obtained in a state containing a large amount of protein or the like by subjecting it to an alkali treatment, an enzyme treatment or the like.

  The ultrafiltration in the production method of the present invention is carried out in order to remove the protein and the like from the mucopolysaccharide containing the protein and the like as described above to obtain a highly pure mucopolysaccharide. That is, proteins contained in the raw material composition to which the ultrafiltration in the present invention is applied (removed by ultrafiltration) were processed under what conditions, what kind of tissue was used as the mucopolysaccharide-containing tissue. It is decided by.

  As a target to which the production method of the present invention is applied, the origin is not limited as long as it is a mucopolymorphism containing such a protein or the like. In general, fish, particularly salmon, has a particularly high purity of the processed product, has low odor, and is easily supplied in a large amount and stably. Among them, cartilage such as nasal cartilage of salmon has an advantage that it has a large content of mucos and is not suitable for food and is easy to handle.

  A method for obtaining a raw material aqueous solution containing mucopolysaccharide and protein and / or a degradation product thereof by applying the ultrafiltration in the production method of the present invention from the mucopolysaccharide-containing tissue is not particularly limited. However, a typical method is specifically described as follows.

  In mucopolysaccharide-containing tissues such as bone, cartilage, and skin, the mucopolysaccharide exists in a state of being covalently bound to the core protein. In order to break this covalent bond and make mucopolysaccharide and core protein separable by ultrafiltration, which will be described later, an alkali treatment method in which mucopolysaccharide-containing tissue is decomposed and extracted with an alkaline solution, neutrality A known method such as a neutral salt treatment method for extraction with a salt solution, an enzyme method for treatment by adding a proteolytic enzyme such as protease, or a treatment method combining these methods may be applied. Among these methods, treatment by an alkali treatment method and an enzyme method is preferred, and enzyme treatment is particularly preferred. In the enzyme treatment, it is particularly preferable to use an alkaline protease. The mucopolysaccharide-containing tissues such as bone, cartilage, and skin to be used are preferably preliminarily removed of contaminants such as meat and fat by washing with water, washing with an organic solvent, and the like. These mucopolysaccharide-containing tissues are usually subjected to treatments such as cutting, crushing, adding water and pressurizing before treatment to increase the efficiency of the alkali treatment method and enzyme treatment.

  Depending on the treatment method and the type of mucopolysaccharide-containing tissue used, the liquid to be treated obtained by the treatment includes mucopolysaccharides, proteins and / or degradation products thereof, fat and muscle tissue, and other various contaminants. In general, it is obtained as an aqueous solution containing. When applying ultrafiltration in the production method of the present invention, it is preferable to remove such impurities as much as possible. In order to remove insoluble impurities, centrifugation, filtration using filter paper, filter cloth, filter sand, and the like can be performed. Furthermore, in order to remove turbid components that cannot be removed by centrifugation or filtration, deodorization, decolorization, degreasing, etc., filtration using a filter aid such as diatomaceous earth, activated carbon treatment, or the like can be performed. In addition, in the activated carbon treatment, if a large amount of activated carbon is used, the yield of mucopolysaccharide may be reduced. Therefore, when the component obtained by removing water from the aqueous solution to which the activated carbon treatment is applied is 100% by mass, activated carbon The amount is preferably 2.5% by mass or less.

  Thus, the aqueous solution (crude aqueous solution) from which impurities are removed contains mucopolysaccharides, proteins and / or degradation products thereof, and ultrafiltration is performed by adding a basic substance to be described later in this state. However, since it is easy to handle during storage and transportation, and it is easy to prevent spoilage, etc., it is once solidified and powdered by methods such as spray drying (spray drying), evaporation drying, freeze drying, etc. It is common to use them. The solid obtained in this way may contain mucopolysaccharides, proteins and / or degradation products thereof, as well as salts, ash, other inorganic substances, and the like. Hereinafter, the solid matter obtained by removing such a solvent is referred to as “crude mucopolysaccharide”. Even when the solvent is not removed from the crude aqueous solution obtained by the above-described method and the solution is used in the next step in the state of the aqueous solution, the remaining component is also referred to as crude mucopolysaccharide if the solvent is removed.

  In addition, from the point that the effect of the present invention becomes remarkable, the target to which the ultrafiltration in the production method of the present invention is applied includes the protein and / or protein degradation product in the crude mucopolysaccharide as described above. It is preferable that the rate (content rate of protein etc.) is 10 to 90% by mass.

The production method of the present invention is that the raw material aqueous solution containing mucopolysaccharide and protein and / or protein degradation product obtainable by the above method is subjected to ultrafiltration under specific conditions. Has characteristics. That is, first, the above mucopolysaccharide, protein and / or protein degradation product (these are usually blended as components constituting the crude mucopolysaccharide), water, and an aqueous solution in which a basic substance is blended. To prepare. Here, the basic substance, OH ^- concentration needs to be blended so that 0.01 to 1 mol / L.

Mucopolysaccharides and proteins are relatively strongly bonded by hydrogen bonding in aqueous solution, and it is sufficient to perform ultrafiltration in a basic state with a neutral to OH ⁻ concentration of less than 0.01 mol / L. Cannot be separated. That, OH ^- concentration above hydrogen bonds are sufficiently broken by a 0.01 mol / L, now and mucopolysaccharides and proteins such behaves as a separate molecule, can be sufficient separation by ultrafiltration It becomes. On the other hand, when the OH ⁻ concentration is higher than 1 mol / L, the mucopolysaccharide is decomposed (for example, the sulfate ester is hydrolyzed), and the yield and quality are lowered. Even under strong acidic conditions, mucopolysaccharides are degraded very easily. Preferably, OH ^- concentration formulated a basic material so that 0.05 to 0.5 mol / L.

Furthermore, in the ultrafiltration in the production method of the present invention, proteins and the like are removed as a permeate and the mucopolysaccharide is concentrated. However, mucopolysaccharide has a high effect of increasing the viscosity of the solution, and as the concentration proceeds, the viscosity increases and it becomes difficult to filter. Thus, although water is added by an amount corresponding substantially permeate volume, OH ^- when the concentration resulting in added low water, separation of such mucopolysaccharides and proteins is difficult. On the other hand, if the OH ⁻ concentration is too high, the yield, quality, etc. of the mucopolysaccharide are reduced as described above. Therefore, it is necessary to use an aqueous solution in which a basic substance is blended so that the OH ⁻ concentration is 0.01 to 1 mol / L for the replenisher as a permeate.

The concentration of each component occupying in the raw material aqueous solution, OH ^- is not particularly limited except the concentration, but towards the concentration of mucopolysaccharides is high good efficiency, the viscosity of too high an aqueous solution filtered high difficult Therefore, it is preferable to prepare so that the density | concentration of mucopolysaccharide may be 0.2-60 mass%, and it is preferable to prepare so that it may become 0.5-30 mass%. The concentration of mucopolysaccharide in the crude mucopolysaccharide can be roughly determined by various methods as described later, and the blending ratio of each component is determined from the mucopolysaccharide concentration in the crude mucopolysaccharide. do it.

  The water used for preparing the raw material aqueous solution is not particularly limited, and for example, any of industrial water, purified water, clean water, ion-exchanged water, ultrapure water, distilled water, and spring water may be used.

Basic substances include hydroxides such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, carbonates such as sodium carbonate, sodium hydrogen carbonate, potassium carbonate, potassium hydrogen carbonate, calcium carbonate, tetramethylammonium hydroxide, tetraethyl Examples thereof include organic hydroxides such as ammonium hydroxide. Among these, alkali metal hydroxides such as sodium hydroxide and potassium hydroxide are preferred from the viewpoints of ease of adjustment of OH ^- concentration, availability and economy, and influence when mixed into the final product. Sodium hydroxide is preferred. Since strong bases such as alkali metal hydroxides are completely dissociated in an aqueous solution, the OH ⁻ concentration in the raw material aqueous solution can be calculated from the amount of the strong base added to the raw material aqueous solution. That is, for example if the use of alkali metal hydroxides, as it is OH concentration of the hydroxide ^- can and concentration.

  Moreover, in addition to the said rough mucopolysaccharide, water, and a basic substance, you may mix | blend the following components with the raw material aqueous solution used for ultrafiltration as needed.

  An organic carboxylate, sodium chloride, or an organic solvent may be added for the purpose of inhibiting the decay during ultrafiltration or pretreatment described later, and assisting the effect of unraveling the mucopolysaccharide and protein. Examples of organic carboxylates include monovalent or divalent or higher organic carboxylic acid salts such as acetate, citric acid, tartaric acid, and malonic acid. From the viewpoint of solubility in water, acetic acid is particularly preferred. The salt of 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 by reacting acetic acid with sodium hydroxide, sodium hydrogen carbonate, sodium carbonate or the like in an aqueous solution. As the organic solvent, a water-soluble organic solvent such as methanol, ethanol, and acetone can be suitably used. In particular, ethanol is particularly preferable from the viewpoint of sterilization effect and mixing into a product. Moreover, you may add the above-mentioned salt and organic solvent individually or in combination of 2 or more types.

  The concentration of the organic carboxylate or salt is not particularly limited, but the concentration in the prepared raw material aqueous solution is usually 8 to 60% by mass, preferably for the purpose of enhancing the solubility and the effect of unraveling mucopolysaccharide and protein in alkali treatment. The content may be appropriately selected from the range of 9 to 50% by mass, particularly preferably 10 to 40% by mass.

  Further, as preservatives, alcohols such as n-butanol, guanidines such as polyhexamethylene, reverse soaps such as benzalkonium chloride, surface active agents such as alkylpolyaminoethylglycine, halogens such as sodium hypochlorite, etc. A system disinfectant, 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 according to the kind of mucopolysaccharide to be used, processing temperature, etc.

  The method for preparing the raw material aqueous solution in which each of the above components is dissolved is not particularly limited, but the crude mucopolysaccharide and the organic carboxylate salt and salt blended as necessary are dissolved in water, and a predetermined amount is added to this solution. A method of adding an aqueous solution containing the basic substance is preferable. If the temperature is low, the dissolution takes time, and if it is high, the hydrolysis of the mucopolysaccharide is promoted. Therefore, the temperature is usually −10 to 120 ° C., preferably −5 to 100 ° C., more preferably 0 to 80 ° C. Preferably, it may be appropriately selected from the range of about 10 to 60 ° C.

  In order to increase the effect of the basic substance that dissolves mucopolysaccharides and proteins before subjecting to ultrafiltration, the raw material aqueous solution in which the above components are blended is subjected to basic conditions as a pretreatment in the ultrafiltration step. It is preferable to stir for a certain time (that is, after adding the basic substance). If the stirring time is too long, modification of the mucopolysaccharide is promoted. Therefore, the stirring time is usually appropriately selected from the range of 0.01 to 100 hours, preferably 0.5 to 50 hours, and more preferably 1 to 24 hours. When the stirring temperature is too low, the effect of the alkali treatment is reduced, and the aqueous raw material solution is solidified. When the stirring temperature is too high, decomposition reactions such as evaporation of the solution and hydrolysis of the mucopolysaccharide are promoted. It may be appropriately selected from the range of about -120 ° C, preferably -5 to 100 ° C, more preferably 0 to 80 ° C, and particularly preferably about 10 to 60 ° C.

  By subjecting the aqueous raw material solution prepared as described above to ultrafiltration as a filtrate to be filtered, aqueous solutions of proteins, protein degradation products, basic substances, etc. pass through the ultrafiltration membrane, and the target mucopolysaccharide Is concentrated in the circulating solution of ultrafiltration.

  The ultrafiltration membrane used for the ultrafiltration is not particularly limited as long as the mucopolysaccharide to be concentrated does not pass through, but the protein or the like passes through, and is appropriately limited depending on the type of mucopolysaccharide. Just choose. In general, those having a molecular weight cutoff of 10,000 to 200,000 can be used, and an ultrafiltration membrane having a molecular weight cutoff of 20,000 to 50,000 is preferred.

  The pressure of the ultrafiltration operation in the present invention may be performed under atmospheric pressure or under pressure within the range of the ultrafiltration device to be used, and in an atmosphere of air, compressed air, nitrogen, argon, helium, etc. You can go. In order to improve the filtration rate, it is usually performed under a pressure of about 0.01 to 0.5 MPa. If the temperature at the time of ultrafiltration is high, the mucopolysaccharide aqueous solution will decay and the mucopolysaccharide will be decomposed. If the temperature is low, the viscosity of the mucopolysaccharide aqueous solution will rise and the ultrafiltration permeate will decrease significantly. 100 degreeC is preferable and about 10-60 degreeC is more preferable.

As described above, in addition to proteins and the like, water is lost as a permeate during ultrafiltration, which increases the concentration of mucopolysaccharide and increases the concentration of the liquid to be filtered. It is necessary to carry out while replenishing an amount of liquid corresponding to the minute. In the present invention, as the liquid to be replenished at this time, an aqueous solution in which a basic substance is blended so that the OH ⁻ concentration is 0.01 to 1 mol / L (preferably 0.05 to 0.5 mol / L) is used. . The water and the basic substance in this case are the same as those described in the preparation of the raw material aqueous solution, and a sodium hydroxide aqueous solution is particularly preferable. Further, the aqueous solution may contain an organic solvent, a salt, a basic substance, and a preservative as mixed in the raw material aqueous solution described above, and most preferably, the raw aqueous solution obtained by removing crude mucopolysaccharides. It is an aqueous solution in which the same kind of components are blended at the same concentration.

  The replenishment amount of the additional liquid to the liquid to be filtered is not particularly limited, but usually the same amount as that lost as the permeate is replenished as needed. The replenishment may be performed continuously, or when the permeated liquid amount reaches a certain amount, the procedure of replenishing the corresponding amount all at once may be repeated several times. When the amount of permeated liquid reaches a certain amount and is replenished as a whole, it may be appropriately determined based on the degree of decrease in permeation rate, but generally 5 to 50% of the liquid to be filtered prepared as a raw material aqueous solution. It is preferable to carry out the process every time about 10 to 33% is lost as the permeate.

  By continuing ultrafiltration while replenishing the basic aqueous solution as described above, proteins and the like contained in the raw material aqueous solution are gradually removed as a permeate. Depending on the purpose of obtaining the highly purified mucopolysaccharide finally obtained, the ultrafiltration usually detects proteins and the like in the permeate. Do it until it runs out. That is, the end of the ultrafiltration in the present invention is any one of the permeated liquid such as ninhydrin color test, color test such as xanthoprotein test, chromatography such as gel permeation chromatography, absorption spectrum such as UV and IR. Or by a combined test and can be determined by the absence of detection of protein and protein degradation products.

  In this way, an aqueous solution of mucopolysaccharide substantially free of protein or the like can be obtained. The obtained aqueous solution of mucopolysaccharides may be used for various purposes as it is, but considering the storage stability of mucopolysaccharides and the harm to the living body, the pH of the aqueous solution should be once adjusted to near neutrality. Is preferred. The pH adjustment range is usually set to pH = 5-10. Examples of the method include a method of adding an acid such as acetic acid and hydrochloric acid, a method of adding ultrafiltration with water not containing a basic substance, and a method of adding a buffering substance or a mixture. When neutralizing by adding an acid, the mucopolysaccharide is neutralized with a carboxylic acid to prevent the neutralized salt from being precipitated together with the mucopolysaccharide when the mucopolysaccharide is solidified by the precipitation method described later. It is particularly preferable to neutralize with acetic acid. In addition, there may be a substance that precipitates due to neutralization. In such a case, it may be removed by centrifugation or filtration under conditions that allow an aqueous solution of mucopolysaccharide to pass through.

  The (neutral) aqueous solution of mucopolysaccharides substantially free of proteins and the like thus obtained may be used as it is for various applications. However, in order to facilitate handling, it is further separated from water and solid. It is preferable to use a product. Examples of the method for separating water from water include a method of volatilizing and removing water directly from an aqueous solution (direct drying method) and a method of adding a water-soluble organic solvent to an aqueous solution to precipitate mucopolysaccharide (precipitation method).

  Examples of the method for removing the water directly from the aqueous solution by volatilization include freeze drying (freeze drying) and spray drying (spray drying). As the concentration of the aqueous solution during lyophilization, the concentration of mucopolysaccharide is preferably 50% by mass 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 mass, and the drying temperature may be appropriately selected in the range of 50 to 200 ° C.

  In the method of directly removing moisture from the aqueous solution as described above, the obtained solid content includes the basic substance used in the ultrafiltration, the salt neutralized with the basic substance, the organic carboxylate, or the like. May be included in large amounts. When used in applications where such components are problematic, it is preferable to employ a precipitation method. The water-soluble organic solvent used in the precipitation method is not particularly limited as long as the target mucopolysaccharide does not dissolve, but alcohols are preferably used in terms of efficiency (hereinafter, precipitation methods using alcohol are used). Also called alcohol precipitation).

  As the alcohol used for the alcohol precipitation, known alcohols can be used without any limitation. Illustrative examples 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 them, methanol and ethanol are preferable from the viewpoint of easy drying. Ethanol is particularly preferred because of its low toxicity.

  Although the temperature at the time of the alcohol precipitation varies depending on the kind of mucopolysaccharide to be precipitated, it is usually -20 to 70 ° C, preferably -10 to 60 ° C, more preferably -5 to 55 ° C. Good.

  For the contact between the alcohol and the aqueous solution, the aqueous solution may be dropped into the alcohol or the alcohol may be dropped into the aqueous solution. However, from the viewpoint of workability, a method of adding the alcohol to the aqueous solution is preferable. In the alcohol precipitation, 100% alcohol is not necessarily used, and alcohol containing water such as 95% by mass ethanol may be used.

  At the time of alcohol precipitation, an aqueous solution in which the concentration of mucopolysaccharide in the aqueous solution containing mucopolysaccharide obtained by ultrafiltration is adjusted to be in the range of 0.01 to 10% by mass is used for the ease of precipitation. And good. Moreover, what is necessary is just to determine the quantity of the alcohol to add suitably according to the kind and density | concentration of mucopolysaccharide, and generally it is about 40-400 mass% with respect to aqueous solution.

  When the alcohol and the aqueous solution are brought into contact with each other, the aqueous solution is preferably stirred, and the stirring speed may be adjusted so that the shearing speed of the stirring blade is 0.7 to 50 m / second. The dropping rate of the alcohol is preferably adjusted so that the dropping rate is in the range of 0.0001 to 40 L / min.

  Moreover, in order to increase the precipitation rate of mucopolysaccharide in alcohol precipitation, it is good to mix | blend various salts in aqueous solution. The salts are preferably not precipitated during the precipitation of alcohol, carboxylates are preferred, and acetates are more preferred from the viewpoint of toxicity and the like. Particularly preferred is an alkali metal salt of acetic acid, and the most preferred salt is sodium acetate. When sodium acetate is blended as a salt, it is preferably added and dissolved so that its concentration is 0.1 to 5% by mass. In addition, when the raw material aqueous solution at the time of ultrafiltration is adjusted or neutralized with acetic acid after ultrafiltration, the mucopolysaccharide aqueous solution may contain a larger amount of acetate than the above range. is there. In this case, it is preferable to adjust the concentration of sodium acetate to 0.1 to 5% by mass by adding water.

  The solid of mucopolysaccharide precipitated by the alcohol precipitation operation can be obtained by a normal solid-liquid separation operation. Illustrative examples include centrifugal filtration, overpressure filtration, reduced pressure filtration, decantation, filter press and the like. Further, the separated solid may be washed with alcohol, alcohol / water mixed solution or the like.

  Since the solid substance isolated by the precipitation method such as alcohol precipitation as described above contains water or a water-soluble organic solution, it is preferable to further perform a drying treatment. Further, the solid obtained by the direct drying method can be further dried as necessary.

  As the drying treatment, a known treatment 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 is prolonged, and if it is too high, the mucopolysaccharide is denatured. Therefore, the temperature is usually −10 to 120 ° C., preferably 0 to 110 ° C., more preferably 10 to 100 ° C. Select as appropriate.
When drying under reduced pressure, the degree of reduced pressure is appropriately selected within the range of 0 to 700 mmHg.

  The solid material of the mucopolysaccharide obtained by the drying treatment can be pulverized and further classified as 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.

  The mucopolysaccharide thus obtained is a high-purity mucopolysaccharide with a low content of protein and its degradation products, and is particularly suitable for pharmaceutical and cosmetic applications.

  In the production method of the present invention, analysis of the purity of various components in raw materials, intermediate products, final obtained products, and the like can be performed by the following method.

(1) Mucopolysaccharide Analyzed by a method according to the structure of the target 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 is possible by gel permeation chromatography (GPC).

(2) Protein and its degradation product The ninhydrin method can be used which utilizes the fact that protein is decomposed in water and the resulting amino acid is colored by the ninhydrin reaction.

  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 peaks of proteins and their degradation products are clear and can be separated from the signal of the desired mucopolysaccharide, it is possible to analyze the amount of contamination in the proton nuclear magnetic resonance spectrum ( ¹ H-NMR). is there.

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

(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 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 quantify by proton nuclear magnetic resonance spectrum ( ¹ H-NMR) based on the characteristic signal derived from the water-soluble organic solvent.

  Although there is no restriction | limiting in particular as a use of the mucopolysaccharide obtained by the manufacturing method of this invention, A pharmaceutical, cosmetics, a foodstuff, a drink, a seasoning, feed etc. are mentioned.

  The highly purified mucopolysaccharide obtained by using the production method of the present invention is particularly suitable for pharmaceutical and cosmetic applications in that it can easily take advantage of the fact that it contains very little protein or protein degradation products. For example, the physiological functions of chondroitin sulfate include pharmaceuticals utilizing smoothing of joint tissues, lipid serum clarification action, blood coagulation inhibition action, anti-inflammatory action, and anti-cancer action. In the case of use as a pharmaceutical product, a known formulation form may be appropriately employed depending on the administration form. The administration form may be a known form, for example, internal use, external use and injection. The injection can be administered, for example, intravenously, intramuscularly, subcutaneously, intracutaneously, etc. The external preparation may be a suppository. It can also be added to any food or drink and taken daily.

  When used in pharmaceutical applications, the highly purified mucopolysaccharide obtained by the production method of the present invention is used as an active ingredient, and this may be combined with a known pharmaceutical carrier. In general, the preparation is prepared by blending mucopolysaccharide with a pharmaceutically acceptable liquid or solid carrier and, if necessary, solvent, dispersant, emulsifier, buffer, stabilizer, excipient. In addition, a binder, a disintegrant, a lubricant, and the like can be added to form a solid agent such as a tablet, granule, powder, powder, and capsule, and a liquid agent such as a normal solution, suspension, and emulsion. 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 parenteral preparations, mucopolysaccharides are added in accordance with conventional methods, such as distilled water for injection, physiological saline, aqueous glucose solution, vegetable oil for injection, sesame oil, peanut oil, tide oil, corn oil, propylene glycol. It can be prepared by dissolving or suspending in polyethylene glycol or the like and adding a bactericidal agent, stabilizer, tonicity agent, soothing agent, etc., if necessary.

  In addition, mucopolysaccharides are known to have effects such as skin moisturizing and elasticity improving effects, skin aging preventing effects, and the like, and are used as active ingredients in cosmetics. The mucopolysaccharide highly purified by the production method of the present invention can also be used in cosmetics as an active ingredient for obtaining such an effect. For example, lotions, emulsions, creams, packs, bath preparations, facial cleansers, bath soaps or bath detergents can be used.

  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 is added to the solid content to adjust the pH to about neutral, 0.2% by mass of proteolytic enzyme (alkaline protease) is added, and the temperature is around 50 ° C. for 1 to 2 hours. After the treatment, the enzyme was inactivated by heating to 90 ° C. After cooling, the mixture was centrifuged and 0.2% by mass 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, the NMR purity of chondroitin sulfate was about 43% by mass, the protein and its degradation products were about 55% by mass and about 2% by mass of sodium chloride by Kjeldahl analysis. It turns out that there is. This was used as the crude mucopolysaccharide used in Example 1 and Comparative Example 1.

Example 1
A 3L four-necked flask was equipped with a meniscus stirring blade (radius 5 cm) connected to a three-one motor (35w), a thermometer, and a condenser. To this, 190 ml of ion-exchanged water and 10.0 g of crude mucopolysaccharide were added and dissolved while stirring at 40 ° C. (shear rate 2 m / sec). Further, 10.0 g of an 8.4% by mass aqueous sodium hydroxide solution (0.8 g of sodium hydroxide, 9.2 g of ion-exchanged water) was added and stirred at 40 ° C. for 2 hours to obtain a raw material aqueous solution.

  Next, it cooled to 22 degreeC, and ultrafiltered for 40 hours, adding 0.1 mol / L sodium hydroxide aqueous solution at any time using the thing of the ultrafiltration membrane 50000 (made by Advantech).

  The aqueous solution (concentrated solution) obtained by ultrafiltration was neutralized with acetic acid (pH = 5.5), and then subjected to 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 260 g of 95% by mass ethanol (247 g of ethanol, 13 g of ion-exchanged water) 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 10 g of 95 mass% ethanol. The obtained wet body was 6.0 g, and when a shelf-type vacuum drying was performed at 50 ° C., 3.4 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), it was 3.4 g of sodium chondroitin sulfate, no protein / protein degradation product was detected, and no sodium acetate was detected. Furthermore, when the chondroitin sulfate sodium purity was 98% and the oxygen flask combustion method (sulfur) was performed by nuclear magnetic resonance spectrum (NMR) measurement, the sulfur content was 5.9%.

Comparative Example 1
An aqueous raw material solution was prepared in the same manner as in Example 1, and ultrafiltration was performed for 40 hours in the same manner except that ion-exchanged water was added as needed instead of the aqueous sodium hydroxide solution. Since the obtained concentrated liquid was almost neutral, neutralization with acetic acid was not performed, and membrane filter filtration and alcohol precipitation were performed in the same manner as in Example 1. The obtained wet body was 6.00 g, and shelf-type vacuum drying was performed at 50 ° C. to obtain 3.6 g of a dry body.

As a result of gel permeation chromatography (GPC), ion exchange chromatography (IEC), and automatic amino acid analysis (ninhydrin method), 3.4 g of sodium chondroitin sulfate, 1.4 g of protein / protein degradation product, and sodium acetate were not detected. Furthermore, when the chondroitin sulfate sodium purity was 70% and the oxygen flask combustion method (sulfur) was performed by nuclear magnetic resonance spectrum (NMR) measurement, the sulfur content was 4.5%.

Claims (2)

A step of separating the protein and / or its degradation product as a permeate from the aqueous solution by performing ultrafiltration using an aqueous raw material solution containing mucopolysaccharide and protein and / or its degradation product as a filtrate. including a process for the preparation of highly purified mucopolysaccharides, as the raw material aqueous solution OH ^- both use those concentrations basic substance such that 0.01 to 1 mol / L is blended, OH ^- The method as described above, which is carried out while replenishing the liquid to be filtered with an aqueous solution containing a basic substance so as to have a concentration of 0.01 to 1 mol / L. The method according to claim 1, further comprising a step of adding alcohol to an aqueous solution containing mucopolysaccharide obtained by performing ultrafiltration to precipitate the mucopolysaccharide.