JP2000273102A - Method for isolation and purification of chondroitin sulfate - Google Patents
Method for isolation and purification of chondroitin sulfateInfo
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- JP2000273102A JP2000273102A JP11075726A JP7572699A JP2000273102A JP 2000273102 A JP2000273102 A JP 2000273102A JP 11075726 A JP11075726 A JP 11075726A JP 7572699 A JP7572699 A JP 7572699A JP 2000273102 A JP2000273102 A JP 2000273102A
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- chondroitin sulfate
- aqueous solution
- separating
- purifying
- ultrafiltration
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Abstract
Description
【０００１】[0001]
【発明の属する技術分野】本発明は、コンドロイチン硫
酸を含む動物の結合組織、特にサケ（鮭）頭部の鼻軟骨
より抽出したコンドロイチン硫酸含有水溶液を限外ろ過
処理して連続的にコンドロイチン硫酸の濃縮と精製を行
ない、濃縮液からコンドロイチン硫酸粉末を得るコンド
ロイチン硫酸の分離精製方法に関する。本発明により分
離精製されるコンドロイチン硫酸は、医薬品原料、化粧
品原料、食品添加物等の工業製品に広く利用することが
できる。TECHNICAL FIELD The present invention relates to an ultrafiltration treatment of a chondroitin sulfate-containing aqueous solution extracted from animal chondroitin sulfate-containing connective tissue, in particular, nasal cartilage of salmon (salmon) head. The present invention relates to a method for separating and purifying chondroitin sulfate by performing concentration and purification to obtain chondroitin sulfate powder from a concentrate. The chondroitin sulfate separated and purified according to the present invention can be widely used for industrial products such as pharmaceutical raw materials, cosmetic raw materials, and food additives.
【０００２】[0002]
【発明の背景】孵化、稚魚養殖、放流技術により、近年
大量に生産されるサケは、冷凍、加工技術の進歩と相俟
って、有用な蛋白源として広く食されている。サケは北
海道において約１５万トン／年と大量に漁獲され、その
加工残骸である頭部や内臓も大量に排出されているが、
その一部がフィッシュミールの原料として利用されてい
る以外殆ど利用されておらず、その有効利用が水産加工
業者から強く望まれている。BACKGROUND OF THE INVENTION In recent years, salmon, which has been produced in large quantities by hatching, fry cultivation, and stocking techniques, has been widely eaten as a useful protein source along with advances in freezing and processing techniques. Salmon is caught in Hokkaido in large quantities of about 150,000 tons / year, and the head and internal organs, which are the remains of processing, are also emitted in large quantities.
Some of them are hardly used except as raw materials for fish meal, and their effective use is strongly desired by fishery processors.
【０００３】サケ頭部の鼻軟骨にはコンドロイチン硫酸
が含まれており、従来のコンドロイチン硫酸と比べ、硫
酸基分布が比較的ランダムな構造を持つ新規なコンドロ
イチン硫酸であることが、報告されているが（佐々木綾
子等，日本化学会北海道支部1998年夏期研究発表会講演
要旨集，２３頁）、これまで、工業的規模での分離精製
は全く行なわれていない。[0003] The nasal cartilage of the salmon head contains chondroitin sulfate, which has been reported to be a novel chondroitin sulfate having a structure in which the distribution of sulfate groups is relatively random as compared with conventional chondroitin sulfate. (Ayako Sasaki et al., Proceedings of the 1998 Summer Meeting of the Chemical Society of Japan Hokkaido Branch, p. 23), but no separation and purification on an industrial scale has been performed so far.
【０００４】コンドロイチン硫酸は、グルクロン酸とＮ
−アセチルガラクトサミンの二糖繰返し構造を基本と
し、そのＮ−アセチルガラクトサミンに硫酸基が結合し
た構造を有している。その分子量は原料及び調製法によ
り異なるが、数万〜３０万程度で、無味無臭の酸性多糖
類であり、現在使用されている主な原料であるサメのヒ
レや子牛の鼻中隔軟骨などから生産されたものが、その
生理活性、保水性、増粘性を生かして食品添加物や化粧
品等に利用されている。その生産量は年間約２００トン
程度であるが、サメのヒレは資源量が少ないこと、子牛
の鼻中隔軟骨は感染症（狂牛病）の危険性など原料の安
全性に問題があり、いずれも安価な原料を安定化して確
保することが困難な状況にある。[0004] Chondroitin sulfate is composed of glucuronic acid and N
-Has a structure in which a sulfate group is bonded to N-acetylgalactosamine based on a disaccharide repeating structure of -acetylgalactosamine. Its molecular weight varies depending on the raw material and the preparation method, but it is about tens of thousands to 300,000, and is a tasteless and odorless acidic polysaccharide, and is produced from shark fin, which is the main raw material currently used, and nasal septal cartilage of calf etc. What has been used is utilized for food additives, cosmetics, etc. by utilizing its physiological activity, water retention, and viscosity increase. Although its production is about 200 tons per year, shark fins have low resources and calf nasal septum cartilage has a problem with the safety of raw materials such as the risk of infection (mad cow disease). However, it is difficult to stabilize and secure inexpensive raw materials.
【０００５】従来、工業的に実施されている水溶液中の
コンドロイチン硫酸の分離精製法としては、アルコール
等の有機溶媒をコンドロイチン硫酸含有水溶液に添加す
ることによってコンドロイチン硫酸を沈析させる方法
（K. Meyer，E. Davidson et,al, Biochem. Biophys．A
cta., 21，506，1956）、第４級アンモニウムを添加し
て水難溶性の複合体として沈殿させ分離する方法（Meth
ods in Carbohydrate Chemistry, R．L．Whistler Acad
imic Press, New York 5, 38, 1965）及びその改良法
（特開平1-210401号）が開示されている。[0005] Conventionally, as a method of separating and purifying chondroitin sulfate in an aqueous solution, a method of precipitating chondroitin sulfate by adding an organic solvent such as an alcohol to an aqueous solution containing chondroitin sulfate (K. Meyer). , E. Davidson et, al, Biochem. Biophys. A
cta., 21, 506, 1956), a method of adding quaternary ammonium to precipitate as a poorly water-soluble complex and separating it (Meth
ods in Carbohydrate Chemistry, R. L. Whistler Acad
imic Press, New York 5, 38, 1965) and an improved method thereof (Japanese Patent Application Laid-Open No. 1-210401).
【０００６】しかしながら、上記方法で高純度のコンド
ロイチン硫酸を大量に得るためには、沈殿の生成及び洗
浄に多量の有機溶媒（アルコール等）が必要であり、タ
ンパク質等の爽雑物が同時に沈殿するためイオン交換樹
脂などを使用する必要がある。また、第４級アンモニウ
ム塩を用いる方法では、アンモニウム塩の製品への混入
を防ぐための分離工程が必要となる。However, in order to obtain a large amount of high-purity chondroitin sulfate by the above-mentioned method, a large amount of an organic solvent (alcohol or the like) is required for generation and washing of a precipitate, and contaminants such as proteins precipitate simultaneously. Therefore, it is necessary to use an ion exchange resin or the like. Further, the method using a quaternary ammonium salt requires a separation step for preventing the ammonium salt from being mixed into the product.
【０００７】[0007]
【発明が解決しようとする課題】したがって、本発明の
目的は、従来廃棄物として処理されている動物組織、特
に加工場で残骸として大量に生じるサケ頭部（鼻軟骨部
分）を原料としてコンドロイチン硫酸を大量に安価に供
給し得る技術を確立することにある。さらに、本発明の
目的は、動物組織、特にサケの鼻軟骨から抽出されるコ
ンドロイチン硫酸含有水溶液から、高純度の製品を効率
よく得るコンドロイチン硫酸の分離精製法を提供するこ
とにある。SUMMARY OF THE INVENTION Accordingly, an object of the present invention is to provide chondroitin sulfate from animal tissue which has been conventionally treated as waste, particularly salmon heads (nasal cartilage) which are produced in large quantities as debris in processing plants. It is an object of the present invention to establish a technology capable of supplying a large amount of inexpensively. It is a further object of the present invention to provide a method for separating and purifying chondroitin sulfate, which can efficiently produce a high-purity product from an aqueous solution containing chondroitin sulfate extracted from animal tissues, particularly salmon nasal cartilage.
【０００８】[0008]
【課題を解決するための手段】本発明者らは、サケの頭
部から採取した鼻軟骨中に含まれるコンドロイチン硫酸
以外の成分（蛋白質など）を予め分解処理して低分子化
した、夾雑物とコンドロイチン硫酸を含有する水溶液の
濃縮精製について検討した。濃縮精製法としては、径の
規定された限外ろ過膜面の細孔により高分子化合物から
低分子化合物までの膜の細孔の大小により、種々の大き
さの分子をこし分けることができ、食品産業分野で乳製
品、醤油、調味料等の製造工程において実用化されてい
る限外ろ過膜を用いる方法について鋭意検討した。その
結果、特定の分画分子量の限外ろ過膜を用い、多段連続
処理することにより、用途に対応した種々の純度のコン
ドロイチン硫酸が効率よく得られることを確認し、本発
明を完成するに至った。なお、本発明の方法はサケの鼻
軟骨を原料として確認されたものであるが、サケの鼻軟
骨に限られず、コンドロイチン硫酸を含む他の動物の各
種組織中のコンドロイチン硫酸の分離精製にも広く適用
できるものである。Means for Solving the Problems The present inventors have developed a contaminant obtained by subjecting components (such as proteins) other than chondroitin sulfate contained in nasal cartilage collected from the head of salmon to a low molecular weight treatment in advance. Concentration and purification of aqueous solution containing and chondroitin sulfate were studied. As the concentration purification method, by the size of the pores of the membrane from a high molecular compound to a low molecular compound by the pores of the ultrafiltration membrane surface of a defined diameter, it is possible to separate molecules of various sizes, We studied diligently the method of using an ultrafiltration membrane which has been put to practical use in the manufacturing process of dairy products, soy sauce, seasonings and the like in the food industry. As a result, it was confirmed that, by using an ultrafiltration membrane having a specific molecular weight cut-off and performing multi-stage continuous treatment, chondroitin sulfate having various purities corresponding to the intended use could be efficiently obtained, and the present invention was completed. Was. Although the method of the present invention has been confirmed using salmon nasal cartilage as a raw material, the method is not limited to salmon nasal cartilage and is widely used for separation and purification of chondroitin sulfate in various tissues of other animals including chondroitin sulfate. Applicable.
【０００９】すなわち、本発明は、 １）コンドロイチン硫酸を含む動物の組織をアルカリ処
理してコンドロイチン硫酸を含有する水溶液を得、この
水溶液を限外ろ過処理してコンドロイチン硫酸の濃縮と
精製を行なうことを特徴とするコンドロイチン硫酸の分
離精製方法、 ２）アルカリ処理後にタンパク分解酵素で処理してコン
ドロイチン硫酸を含有する水溶液を得、その水溶液を限
外ろ過処理する前記１に記載のコンドロイチン硫酸の分
離精製方法、 ３）限外ろ過処理により得られたコンドロイチン硫酸の
濃縮液を乾燥することによりコンドロイチン硫酸粉末を
得る前記１または２に記載のコンドロイチン硫酸の分離
精製方法、 ４）限外ろ過処理により得られたコンドロイチン硫酸の
濃縮液にエタノールを添加して生じたコンドロイチン硫
酸の沈殿を取得する前記１または２に記載のコンドロイ
チン硫酸の分離精製方法、 ５）動物の組織がサケの鼻軟骨である前記１乃至４のい
ずれかに記載のコンドロイチン硫酸の分離精製方法、 ６）限外ろ過処理を少なくとも２回行なう前記１乃至５
のいずれかに記載のコンドロイチン硫酸の分離精製方
法、および ７）限外ろ過処理に先立ち、コンドロイチン硫酸含有液
に水を添加する前記１乃至６いずれかに記載の分離精製
方法を提供するものである。That is, the present invention provides: 1) alkali treatment of an animal tissue containing chondroitin sulfate to obtain an aqueous solution containing chondroitin sulfate, and ultrafiltration of the aqueous solution for concentration and purification of chondroitin sulfate; 2) a method for separating and purifying chondroitin sulfate, 2) a treatment with alkali and a treatment with a proteolytic enzyme to obtain an aqueous solution containing chondroitin sulfate, and ultrafiltration treatment of the aqueous solution. 3) a method for separating and purifying chondroitin sulfate according to 1 or 2 above, wherein a chondroitin sulfate powder is obtained by drying a concentrated solution of chondroitin sulfate obtained by the ultrafiltration treatment; Chondroitin produced by adding ethanol to concentrated chondroitin sulfate concentrate 5. The method for separating and purifying chondroitin sulfate according to the above item 1 or 2, wherein a precipitate of sulfuric acid is obtained; 5) The method for separating and purifying chondroitin sulfate according to any one of the above items 1 to 4, wherein the animal tissue is salmon nasal cartilage; The above 1 to 5 wherein the ultrafiltration treatment is performed at least twice
7. The method for separating and purifying chondroitin sulfate according to any one of the above, and 7) the method for separating and purifying according to any one of the above 1 to 6, wherein water is added to the chondroitin sulfate-containing liquid prior to the ultrafiltration treatment. .
【００１０】[0010]
【発明の実施の態様】以下、本発明を詳細に説明する。 ［コンドロイチン硫酸抽出原料］本発明で使用する動物
組織原料としては、コンドロイチン硫酸を比較的豊富に
含有する組織であれば制限はない。特に、従来、加工場
などで大量に生じ、利用価値のないものとして処理され
ている魚類、特にサケの頭部中の鼻軟骨が好ましく用い
られる。以下、サケの頭部（鼻軟骨）を例に挙げて説明
する。BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail. [Material for Extracting Chondroitin Sulfate] The material for animal tissue used in the present invention is not limited as long as it is a tissue containing chondroitin sulfate in a relatively abundant manner. In particular, fish, especially nasal cartilage in the head of salmon, which have conventionally been produced in large quantities at processing plants and the like and have been treated as having no use value, are preferably used. Hereinafter, the head of the salmon (nasal cartilage) will be described as an example.
【００１１】［前処理工程］サケ頭部より鼻軟骨を採取
し、これを細切（１〜５ｍｍ角程度）する。次いで、鼻
軟骨に含まれるタンパク質を分解するため、アルカリ処
理および、必要に応じて蛋白分解酵素処理する。すなわ
ち、まず、アルカリ水溶液（例えば、0.2〜0.4Ｎの苛性
ソーダ水溶液）中で３７℃乃至５０℃の温度で３０分〜
３時間処理した後、酢酸や塩酸等で中和し、不溶物をろ
過により除去する。次いで、ろ液のｐＨを中性付近に調
整して、蛋白分解酵素を添加して４０℃前後にて１時間
〜２時間処理した後、加熱により酵素を失活させる。冷
却後、遠心分離して、分解処理で生じた低分子化夾雑物
とコンドロイチン硫酸を含む上清を得る。この上清を次
工程の連続多段限外ろ過処理してコンドロイチン硫酸の
濃縮と精製を同時に行なう。[Pretreatment step] Nasal cartilage is collected from the salmon head and cut into small pieces (about 1 to 5 mm square). Next, in order to decompose the protein contained in the nasal cartilage, an alkali treatment and, if necessary, a proteolytic enzyme treatment are performed. That is, first, in an alkaline aqueous solution (for example, a 0.2 to 0.4 N aqueous solution of caustic soda) at a temperature of 37 ° C to 50 ° C for 30 minutes to
After treating for 3 hours, the mixture is neutralized with acetic acid, hydrochloric acid, or the like, and insolubles are removed by filtration. Next, the pH of the filtrate is adjusted to near neutrality, a protease is added, and the mixture is treated at about 40 ° C. for 1 hour to 2 hours, and then the enzyme is deactivated by heating. After cooling, centrifugation is performed to obtain a supernatant containing low molecular weight contaminants and chondroitin sulfate generated by the decomposition treatment. The supernatant is subjected to a subsequent multi-stage ultrafiltration treatment in the next step to simultaneously carry out concentration and purification of chondroitin sulfate.
【００１２】一実施例としてサケ頭部より鼻軟骨を採取
し、細切した鼻軟骨13.2ｋｇに最終濃度が0.2Ｎになる
ように0.4Ｎ水酸化ナトリウム水溶液を加え、３７℃で
２時間処理した後、酢酸で中和し、粗ろ過により不溶物
を除去した。ろ液のｐＨを7.0に調製し、プロテアーゼ
（天野製薬(株）製、商品名アマノＡ）を13.2ｇ(鼻軟骨
重量の0.1％）添加、３７℃で１時間処理し、８５℃で
５分間加熱失活させて、遠心分離後の上清２４リットル
（Ｌ）を得た。As an example, nasal cartilage was collected from salmon head, 0.4N sodium hydroxide aqueous solution was added to 13.2 kg of minced nasal cartilage to a final concentration of 0.2 N, and the mixture was treated at 37 ° C. for 2 hours. Thereafter, the mixture was neutralized with acetic acid, and insolubles were removed by coarse filtration. The pH of the filtrate was adjusted to 7.0, 13.2 g (0.1% of nasal cartilage weight) of protease (Amano Pharmaceutical Co., Ltd., trade name) was added, treated at 37 ° C. for 1 hour, and at 85 ° C. for 5 minutes. The mixture was inactivated by heating to obtain 24 liters (L) of the supernatant after centrifugation.
【００１３】［連続多段限外ろ過処理］図１に、連続多
段限外ろ過処理に使用した限外ろ過装置の概要を示す。
図中、１は限外ろ過膜であり、抽出液２をタンク４に収
納し高圧ポンプ５により限外ろ過膜１に圧送供給する。
この場合、上記で得られた上清の抽出液をそのまま限外
ろ過膜に供給すると、濃縮を目的とするコンドロイチン
硫酸が透過液側に抜けて歩留まりが悪くなることが判明
した。そこで、上清に対して水（水道水）３を添加した
ものを限外ろ過膜に供給する。水の添加量はコンドロイ
チン硫酸が透過液側に抜けなくなる量であるが、上清に
対して大旨同量添加すればよい。[Continuous Multi-stage Ultrafiltration Treatment] FIG. 1 shows an outline of an ultrafiltration apparatus used for the continuous multi-stage ultrafiltration treatment.
In the drawing, reference numeral 1 denotes an ultrafiltration membrane, which stores an extract 2 in a tank 4 and feeds it to the ultrafiltration membrane 1 by a high-pressure pump 5.
In this case, it was found that if the extract of the supernatant obtained above was supplied to the ultrafiltration membrane as it was, chondroitin sulfate for the purpose of concentration would escape to the permeate side and the yield would be poor. Thus, the supernatant obtained by adding water (tap water) 3 to the supernatant is supplied to the ultrafiltration membrane. The amount of water added is such that chondroitin sulfate does not escape to the permeate side, but may be added to the supernatant in roughly the same amount.
【００１４】使用する限外ろ過膜は、原料液に含まれる
コンドロイチン硫酸の分子量を考慮して選定すればよい
が、サケ鼻軟骨に含まれるコンドロイチン硫酸の場合は
その分子量が２万以上（およそ３万〜３０万）であるこ
とから、分画分子量２万の限外ろ過膜を使用すればよ
い。供給液の平均操作圧は、使用する限外ろ過膜の耐圧
性と限外ろ過膜を通過する分画分子量以上の成分を含む
濃縮液（６）の排出量および分画分子量以下の分子およ
び水からなる透過液（７）の透過量のバランスをみて決
められるが、大旨２〜３ｋｇ／ｃｍ2である。The ultrafiltration membrane to be used may be selected in consideration of the molecular weight of chondroitin sulfate contained in the raw material liquid. In the case of chondroitin sulfate contained in salmon nasal cartilage, the molecular weight is 20,000 or more (about 3 000). Therefore, an ultrafiltration membrane having a molecular weight cutoff of 20,000 may be used. The average operating pressure of the feed solution is determined by the pressure resistance of the ultrafiltration membrane to be used, the discharge amount of the concentrated solution (6) containing a component having a molecular weight of not less than the fraction passing through the ultrafiltration membrane, and molecules and water having a molecular weight of not more than the fractionated molecular weight. It is determined in view of the balance of the amount of the permeated liquid (7) consisting of, but is approximately 2 to 3 kg / cm 2 .
【００１５】上記抽出液２４リットル（Ｌ）（コンドロ
イチン硫酸濃度約１７ｇ／Ｌ，コンドロイチン酸純度約
３０％。なお、コンドロイチン硫酸の分析は液の一部を
採取し、ガランボス法（Galambos JT 1967 The reactio
n of carbazole with carbohydrates 1. Effect of bor
ate and sulfamate on the carbazole color of sugar
s. Anal. Biochem. 19:119-132）にてグルクロン酸の量
を測定してコンドロイチン硫酸濃度を定量した。純度は
固形物に対するコンドロイチン硫酸の割合，以下同様）
に対して、水２４Ｌを添加した処理液を、５０℃，平均
圧力２ｋｇ／ｃｍ2，１０Ｌ／分の条件で処理し、透過
液約37.0Ｌ（濃縮率4.4倍）を得た（第１ステージの濃
縮）。24 liters (L) of the above extract (chondroitin sulfate concentration: about 17 g / L, chondroitin acid purity: about 30%. For analysis of chondroitin sulfate, a part of the solution was collected and analyzed by the Galanbos method (Galambos JT 1967 The reactio).
n of carbazole with carbohydrates 1.Effect of bor
ate and sulfamate on the carbazole color of sugar
s. Anal. Biochem. 19: 119-132), the amount of glucuronic acid was measured to quantify the chondroitin sulfate concentration. Purity is the ratio of chondroitin sulfate to solids, and so on)
Then, a treatment liquid to which 24 L of water was added was treated under the conditions of 50 ° C., an average pressure of 2 kg / cm 2 , and 10 L / min to obtain about 37.0 L of permeate (concentration ratio 4.4 times) (first stage). Concentration).
【００１６】濃縮液中のコンドロイチン硫酸濃度は31.8
ｇ／Ｌ（純度は63.5％）、透過液中のコンドロイチン硫
酸濃度は0.2ｇ／Ｌ（純度は1.2％）であった。次いで、
透過液量と同量の水道水を添加混合し、図１のタンク４
から処理液を供給して同様に第２ステージの限外ろ過処
理を行なった。透過液量は36.4Ｌで、濃縮液中のコンド
ロイチン硫酸濃度は32.5ｇ／Ｌ（純度は88.4％）、透過
液中にはコンドロイチン硫酸濃度は認められなかった。The concentration of chondroitin sulfate in the concentrate is 31.8
g / L (purity: 63.5%), and the concentration of chondroitin sulfate in the permeate was 0.2 g / L (purity: 1.2%). Then
The same amount of tap water as the amount of permeate was added and mixed.
, And a second stage ultrafiltration treatment was similarly performed. The amount of the permeate was 36.4 L, the concentration of chondroitin sulfate in the concentrate was 32.5 g / L (purity: 88.4%), and no concentration of chondroitin sulfate was found in the permeate.
【００１７】さらに、透過液量と同量の水道水を添加し
て、同様に第３ステージ目の限外ろ過処理を行なった。
透過液量は35.6Ｌで、濃縮液12.0Ｌを得た。濃縮液中の
コンドロイチン硫酸（ＣＳ）濃度は31.6ｇ／Ｌ（純度は
98.6％）、透過液中にはコンドロイチン硫酸は認められ
なかった。Furthermore, the same amount of tap water as the amount of the permeate was added, and a third stage ultrafiltration treatment was similarly performed.
The amount of the permeate was 35.6 L, and 12.0 L of the concentrate was obtained. The chondroitin sulfate (CS) concentration in the concentrate is 31.6 g / L (purity is
98.6%), and chondroitin sulfate was not found in the permeate.
【００１８】以上の結果の流れを図２に示し、各ステー
ジの前後の抽出液と濃縮液の分析結果を表１に示す。FIG. 2 shows the flow of the above results, and Table 1 shows the results of analysis of the extract and concentrate before and after each stage.
【００１９】[0019]
【表１】 処理段階 ＣＳ濃度 ＣＳ純度 ｇ／ｌ ％ 処理前 抽出液 16.8 30.4 第１ステージ 濃縮液 31.8 63.5 透過液 0.2 1.2 第２ステージ 濃縮液 32.5 88.4 透過液 0 − 第３ステージ 濃縮液 31.6 98.6 透過液 0 − [Table 1] Processing stage CS concentration CS purity g / l% pretreatment extract 16.8 30.4 first stage concentrate 31.8 63.5 Permeate 0.2 1.2 The second stage concentrate 32.5 88.4 permeate 0 - third stage concentrate 31.6 98.6 permeate 0 -
【００２０】表１の膜処理前後の各処理液のコンドロイ
チン硫酸濃度と純度の分析結果から明らかなように、各
ステージで透過液へのコンドロイチン硫酸の流出が極め
て小さく、コンドロイチン硫酸の純度は、処理前の抽出
液で30.4％であったが、各ステージごとに上昇し、ステ
ージ３では９８％以上まで精製され、これによって得ら
れた濃縮液をそのまま乾燥するか、あるいは有機溶剤、
好ましくはエタノールを添加して生じたコンドロイチン
硫酸の沈殿を取得するかして高純度のコンドロイチン硫
酸を得ることができる。As is clear from the analysis results of the chondroitin sulfate concentration and the purity of each treatment solution before and after the membrane treatment in Table 1, the outflow of chondroitin sulfate into the permeate at each stage was extremely small, and the purity of chondroitin sulfate was Although it was 30.4% in the previous extract, it increased in each stage, and was purified to 98% or more in stage 3. The concentrated solution obtained by this was dried as it was, or
Preferably, high-purity chondroitin sulfate can be obtained by obtaining a precipitate of chondroitin sulfate generated by adding ethanol.
【００２１】[0021]
【発明の効果】本発明は、サケ頭部の鼻軟骨よりコンド
ロイチン硫酸をアルカリで抽出し、必要に応じてタンパ
ク分解酵素により抽出液中に共存するタンパク質を低分
子化して得られたコンドロイチン硫酸を含有する水溶液
から、限外ろ過処理を繰り返すことにより、共存するペ
プチド等の低分子爽雑物を水と共に除去し、水溶液の濃
縮と同時にコンドロイチン硫酸の精製を、大量かつ効率
的に行なう方法を提供したものである。水溶液を濃縮、
精製することにより、濃縮液をそのまま乾燥するか、あ
るいは従来に比べてはるかに少ないエタノール等の有機
溶媒を用いてコンドロイチン硫酸を沈殿させることによ
りコンドロイチン硫酸を得ることができる。さらにイオ
ン交換樹脂等を使用せずとも高純度のコンドロイチン硫
酸を得ることができ、コストの低減が図られる。Industrial Applicability According to the present invention, chondroitin sulfate obtained by extracting chondroitin sulfate from a nasal cartilage of a salmon head with an alkali and, if necessary, reducing the protein coexisting in the extract with proteolytic enzyme is obtained. A method for removing large amounts of low-molecular contaminants, such as coexisting peptides, together with water by repeating ultrafiltration treatment from the contained aqueous solution, and providing a method for concentrating the aqueous solution and simultaneously purifying chondroitin sulfate in a large amount and efficiently. It was done. Concentrate the aqueous solution,
By purifying, the chondroitin sulfate can be obtained by drying the concentrated liquid as it is, or by precipitating chondroitin sulfate using a much smaller amount of an organic solvent such as ethanol than before. Furthermore, high-purity chondroitin sulfate can be obtained without using an ion exchange resin or the like, and cost can be reduced.
【００２２】サケは北海道において約１５万トン／年と
大量に漁獲され、その加工残骸である頭部（鼻軟骨約１
０％を含む）は約１万５千トン排出され、コンドロイチ
ン硫酸を大量に製造する上で、安価で非常に安定した原
料である。さらに、サケ頭部の鼻軟骨には他の動物組織
由来のものに見られるコンドロイチン硫酸以外の酸性多
糖体（ヒアルロン酸、デルマタン酸、ヘパラン硫酸な
ど）が殆ど共存していないことが確認されている。Salmon is caught in Hokkaido in a large amount of about 150,000 tons / year, and its remnants, the head (about 1 nose cartilage).
(Including 0%) is discharged at about 15,000 tons, and is an inexpensive and very stable raw material for producing large quantities of chondroitin sulfate. Furthermore, it has been confirmed that there is almost no coexistence of acidic polysaccharides (such as hyaluronic acid, dermatanic acid, heparan sulfate) other than chondroitin sulfate found in other animal tissues in the nasal cartilage of the salmon head. .
【００２３】本発明では、膜処理のステージを選択する
ことで、低純度（例えば、食品添加物用）から高純度
（医薬用）のコンドロイチン硫酸を任意に製造すること
が可能である。また、精製濃縮液にエタノールなどの有
機溶剤を添加する操作を行なわずに濃縮液をそのまま乾
燥することによって、コンドロイチン硫酸の粉末を得る
ことができる。In the present invention, it is possible to arbitrarily produce low-purity (for example, for food additives) to high-purity (for pharmaceuticals) chondroitin sulfate by selecting the stage of membrane treatment. Further, a powder of chondroitin sulfate can be obtained by drying the concentrated liquid as it is without performing an operation of adding an organic solvent such as ethanol to the purified concentrated liquid.
【図１】 限外ろ過装置の概要を示す。FIG. 1 shows an outline of an ultrafiltration apparatus.
【図２】 本発明の実施例のフロー図である。FIG. 2 is a flowchart of an embodiment of the present invention.
１ 限外ろ過膜 ２ 抽出液 ３ 水 ４ タンク ５ 高圧ポンプ ６ 濃縮液 ７ 透過液 ８ 圧力計 ９ 熱交換器 １０ 流量計 １１ バルブ DESCRIPTION OF SYMBOLS 1 Ultrafiltration membrane 2 Extract 3 Water 4 Tank 5 High pressure pump 6 Concentrate 7 Permeate 8 Pressure gauge 9 Heat exchanger 10 Flow meter 11 Valve
─────────────────────────────────────────────────────
────────────────────────────────────────────────── ───
【手続補正書】[Procedure amendment]
【提出日】平成１２年３月１５日（２０００．３．１
５）[Submission date] March 15, 2000 (200.3.1.
5)
【手続補正１】[Procedure amendment 1]
【補正対象書類名】明細書[Document name to be amended] Statement
【補正対象項目名】特許請求の範囲[Correction target item name] Claims
【補正方法】変更[Correction method] Change
【補正内容】[Correction contents]
【特許請求の範囲】[Claims]
【手続補正２】[Procedure amendment 2]
【補正対象書類名】明細書[Document name to be amended] Statement
【補正対象項目名】０００１[Correction target item name] 0001
【補正方法】変更[Correction method] Change
【補正内容】[Correction contents]
【０００１】[0001]
【発明の属する技術分野】本発明は、コンドロイチン硫
酸を含む動物の結合組織、具体的には魚類（例えば、サ
ケ（鮭））頭部の鼻軟骨より抽出したコンドロイチン硫
酸含有水溶液を限外ろ過処理して連続的にコンドロイチ
ン硫酸の濃縮と精製を行ない、濃縮液からコンドロイチ
ン硫酸粉末を得るコンドロイチン硫酸の分離精製方法に
関する。本発明により分離精製されるコンドロイチン硫
酸は、医薬品原料、化粧品原料、食品添加物等の工業製
品に広く利用することができる。[0001] The present invention relates to an ultrafiltration treatment of an aqueous solution containing chondroitin sulfate extracted from the connective tissue of an animal containing chondroitin sulfate, specifically, nasal cartilage of the head of fish (eg, salmon). The present invention relates to a method for separating and purifying chondroitin sulfate by continuously performing concentration and purification of chondroitin sulfate to obtain chondroitin sulfate powder from the concentrate. The chondroitin sulfate separated and purified according to the present invention can be widely used for industrial products such as pharmaceutical raw materials, cosmetic raw materials, and food additives.
【手続補正３】[Procedure amendment 3]
【補正対象書類名】明細書[Document name to be amended] Statement
【補正対象項目名】０００７[Correction target item name] 0007
【補正方法】変更[Correction method] Change
【補正内容】[Correction contents]
【０００７】[0007]
【発明が解決しようとする課題】したがって、本発明の
目的は、従来廃棄物として処理されている魚類頭部の鼻
軟骨、特に加工場で残骸として大量に生じるサケ頭部
（鼻軟骨部分）を原料としてコンドロイチン硫酸を大量
に安価に供給し得る技術を確立することにある。さら
に、本発明の目的は、魚類頭部の鼻軟骨、特にサケの鼻
軟骨から抽出されるコンドロイチン硫酸含有水溶液か
ら、高純度の製品を効率よく得るコンドロイチン硫酸の
分離精製法を提供することにある。SUMMARY OF THE INVENTION Accordingly, an object of the present invention is to reduce the nasal cartilage of a fish head, which has been conventionally treated as waste, and particularly the salmon head (nasal cartilage part) generated in large quantities as debris in a processing plant. An object of the present invention is to establish a technology capable of supplying a large amount of chondroitin sulfate as a raw material at low cost. It is a further object of the present invention to provide a method for separating and purifying chondroitin sulfate from a chondroitin sulfate-containing aqueous solution extracted from nasal cartilage of a fish head, particularly salmon nasal cartilage, to efficiently obtain a high-purity product. .
【手続補正４】[Procedure amendment 4]
【補正対象書類名】明細書[Document name to be amended] Statement
【補正対象項目名】０００８[Correction target item name] 0008
【補正方法】変更[Correction method] Change
【補正内容】[Correction contents]
【０００８】[0008]
【課題を解決するための手段】本発明者らは、魚類（例
えば、サケ）の頭部から採取した鼻軟骨中に含まれるコ
ンドロイチン硫酸以外の成分（蛋白質など）を予め分解
処理して低分子化した、夾雑物とコンドロイチン硫酸を
含有する水溶液の濃縮精製について検討した。濃縮精製
法としては、径の規定された限外ろ過膜面の細孔により
高分子化合物から低分子化合物までの膜の細孔の大小に
より、種々の大きさの分子をこし分けることができ、食
品産業分野で乳製品、醤油、調味料等の製造工程におい
て実用化されている限外ろ過膜を用いる方法について鋭
意検討した。その結果、特定の分画分子量の限外ろ過膜
を用い、多段連続処理することにより、用途に対応した
種々の純度のコンドロイチン硫酸が効率よく得られるこ
とを確認し、本発明を完成するに至った。なお、本発明
の方法はサケの鼻軟骨を原料として確認されたものであ
るが、サケの鼻軟骨に限られず、コンドロイチン硫酸を
含む他の魚類の鼻軟骨中のコンドロイチン硫酸の分離精
製にも広く適用できるものである。Means for Solving the Problems The present inventors have previously degraded components (such as proteins) other than chondroitin sulfate contained in nasal cartilage collected from the head of fish (eg, salmon) to reduce low molecular weight. The concentration and purification of the aqueous solution containing contaminants and chondroitin sulfate were investigated. As the concentration purification method, by the size of the pores of the membrane from a high molecular compound to a low molecular compound by the pores of the ultrafiltration membrane surface of a defined diameter, it is possible to separate molecules of various sizes, We studied diligently the method of using an ultrafiltration membrane which has been put to practical use in the manufacturing process of dairy products, soy sauce, seasonings and the like in the food industry. As a result, it was confirmed that, by using an ultrafiltration membrane having a specific molecular weight cut-off and performing multi-stage continuous treatment, chondroitin sulfate having various purities corresponding to the intended use could be efficiently obtained, and the present invention was completed. Was. Although the method of the present invention has been confirmed using salmon nasal cartilage as a raw material, the method is not limited to salmon nasal cartilage and is widely used for separation and purification of chondroitin sulfate in nasal cartilage of other fish including chondroitin sulfate. Applicable.
【手続補正５】[Procedure amendment 5]
【補正対象書類名】明細書[Document name to be amended] Statement
【補正対象項目名】０００９[Correction target item name] 0009
【補正方法】変更[Correction method] Change
【補正内容】[Correction contents]
【０００９】すなわち、本発明は、 １）魚類の鼻軟骨をアルカリ処理してコンドロイチン硫
酸を含有する水溶液を得、これに水を添加して希釈し限
外ろ過により濃縮する工程を複数回繰り返すことにより
コンドロイチン硫酸の精製を行なうことを特徴とするコ
ンドロイチン硫酸の分離精製方法。 ２）アルカリ処理後にタンパク分解酵素で処理してコン
ドロイチン硫酸を含有する水溶液を得、その水溶液を限
外ろ過処理する前記１に記載のコンドロイチン硫酸の分
離精製方法。 ３）限外ろ過処理により得られたコンドロイチン硫酸の
濃縮液を乾燥することによりコンドロイチン硫酸粉末を
得る前記１または２に記載のコンドロイチン硫酸の分離
精製方法。 ４）限外ろ過処理により得られたコンドロイチン硫酸の
濃縮液にエタノールを添加して生じたコンドロイチン硫
酸の沈殿を取得する前記１または２に記載のコンドロイ
チン硫酸の分離精製方法。 ５）魚類の鼻軟骨がサケの鼻軟骨である前記１乃至４の
いずれかに記載のコンドロイチン硫酸の分離精製方法。 ６）分画分子量２万の限外ろ過膜を用いて限外ろ過処理
を行なう前記１乃至５のいずれかに記載のコンドロイチ
ン硫酸の分離精製方法。 ７）コンドロイチン硫酸を含有する水溶液をタンク内に
て水で希釈し、得られた希釈液を高圧ポンプを用いて限
外ろ過装置に送出し、得られた濃縮液を前記タンクに循
環することによりコンドロイチン硫酸の多段分離精製操
作を連続的に行なう前記１〜６のいずれかに記載のコン
ドロイチン硫酸の分離精製方法。That is, the present invention provides: 1) repeating a plurality of times the step of alkali-treating fish nasal cartilage to obtain an aqueous solution containing chondroitin sulfate, adding water thereto, diluting the solution, and concentrating the solution by ultrafiltration; A method for separating and purifying chondroitin sulfate, comprising purifying chondroitin sulfate by the method described above. 2) The method for separating and purifying chondroitin sulfate according to 1 above, wherein an aqueous solution containing chondroitin sulfate is obtained by treating with an protease after alkali treatment, and the aqueous solution is subjected to ultrafiltration treatment. 3) The method for separating and purifying chondroitin sulfate according to 1 or 2 above, wherein a chondroitin sulfate powder is obtained by drying a concentrated solution of chondroitin sulfate obtained by the ultrafiltration treatment. 4) The method for separating and purifying chondroitin sulfate according to 1 or 2 above, wherein a precipitate of chondroitin sulfate generated by adding ethanol to a concentrated solution of chondroitin sulfate obtained by ultrafiltration is obtained. 5) The method for separating and purifying chondroitin sulfate according to any one of 1 to 4 above, wherein the nasal cartilage of fish is nasal cartilage of salmon. 6) The method for separating and purifying chondroitin sulfate according to any one of 1 to 5, wherein the ultrafiltration treatment is performed using an ultrafiltration membrane having a molecular weight cutoff of 20,000. 7) By diluting an aqueous solution containing chondroitin sulfate with water in a tank, sending the obtained diluent to an ultrafiltration device using a high-pressure pump, and circulating the obtained concentrate to the tank. 7. The method for separating and purifying chondroitin sulfate according to any one of the above 1 to 6, wherein a multi-stage separation and purification operation of chondroitin sulfate is performed continuously.
【手続補正６】[Procedure amendment 6]
【補正対象書類名】明細書[Document name to be amended] Statement
【補正対象項目名】００１０[Correction target item name] 0010
【補正方法】変更[Correction method] Change
【補正内容】[Correction contents]
【００１０】[0010]
【発明の実施の態様】以下、本発明を詳細に説明する。 ［コンドロイチン硫酸抽出原料］本発明で使用する動物
組織原料としては、コンドロイチン硫酸を比較的豊富に
含有する組織のうち、従来、加工場などで大量に生じ、
利用価値のないものとして処理されている魚類、特にサ
ケの頭部中の鼻軟骨が好ましく用いられる。以下、サケ
の頭部（鼻軟骨）を例に挙げて説明する。BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail. [Material for extracting chondroitin sulfate] As a raw material for animal tissue used in the present invention, among tissues containing relatively abundant chondroitin sulfate, conventionally, a large amount is produced in processing plants and the like.
Nasal cartilage in the head of fish, especially salmon, which has been treated as having no utility, is preferably used. Hereinafter, the head of the salmon (nasal cartilage) will be described as an example.
Claims (7)
アルカリ処理してコンドロイチン硫酸を含有する水溶液
を得、この水溶液を限外ろ過処理してコンドロイチン硫
酸の濃縮と精製を行なうことを特徴とするコンドロイチ
ン硫酸の分離精製方法。1. An chondroitin sulfate-containing animal tissue is alkali-treated to obtain an aqueous solution containing chondroitin sulfate, and the aqueous solution is subjected to ultrafiltration to concentrate and purify the chondroitin sulfate. Separation and purification method.
理してコンドロイチン硫酸を含有する水溶液を得、その
水溶液を限外ろ過処理する請求項１に記載のコンドロイ
チン硫酸の分離精製方法。2. The method for separating and purifying chondroitin sulfate according to claim 1, wherein an aqueous solution containing chondroitin sulfate is obtained by treating with an protease after alkali treatment, and the aqueous solution is subjected to ultrafiltration treatment.
チン硫酸の濃縮液を乾燥することによりコンドロイチン
硫酸粉末を得る請求項１または２に記載のコンドロイチ
ン硫酸の分離精製方法。3. The method for separating and purifying chondroitin sulfate according to claim 1, wherein a chondroitin sulfate powder is obtained by drying the concentrated solution of chondroitin sulfate obtained by the ultrafiltration treatment.
チン硫酸の濃縮液にエタノールを添加して生じたコンド
ロイチン硫酸の沈殿を取得する請求項１または２に記載
のコンドロイチン硫酸の分離精製方法。4. The method for separating and purifying chondroitin sulfate according to claim 1, wherein a precipitate of chondroitin sulfate produced by adding ethanol to the concentrated solution of chondroitin sulfate obtained by the ultrafiltration treatment is obtained.
１乃至４のいずれかに記載のコンドロイチン硫酸の分離
精製方法。5. The method for separating and purifying chondroitin sulfate according to claim 1, wherein the animal tissue is salmon nasal cartilage.
求項１乃至５のいずれかに記載のコンドロイチン硫酸の
分離精製方法。6. The method for separating and purifying chondroitin sulfate according to claim 1, wherein the ultrafiltration treatment is performed at least twice.
硫酸含有液に水を添加する請求項１乃至６いずれかに記
載の分離精製方法。7. The method according to claim 1, wherein water is added to the chondroitin sulfate-containing solution before the ultrafiltration treatment.
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|---|---|---|---|
| JP07572699A JP3278629B2 (en) | 1999-03-19 | 1999-03-19 | Method for separating and purifying chondroitin sulfate |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP07572699A JP3278629B2 (en) | 1999-03-19 | 1999-03-19 | Method for separating and purifying chondroitin sulfate |
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| Publication Number | Publication Date |
|---|---|
| JP2000273102A true JP2000273102A (en) | 2000-10-03 |
| JP3278629B2 JP3278629B2 (en) | 2002-04-30 |
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ID=13584573
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP07572699A Expired - Fee Related JP3278629B2 (en) | 1999-03-19 | 1999-03-19 | Method for separating and purifying chondroitin sulfate |
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| JP (1) | JP3278629B2 (en) |
Cited By (17)
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| KR20010070901A (en) * | 2001-06-18 | 2001-07-27 | 강헌수 | manufacturing method for chondroitin sulfate and its content |
| WO2003066683A1 (en) * | 2002-02-05 | 2003-08-14 | Nippon Barrier Free Co., Ltd. | Mucopolysaccharides and process for producing the same |
| WO2004039994A1 (en) * | 2002-11-01 | 2004-05-13 | Nippon Barrier Free Co., Ltd. | Sodium chondroitin sulfate, chondroitin sulfate-containing material and processes for producing the same |
| WO2004044009A1 (en) * | 2002-11-13 | 2004-05-27 | Cargill, Incorporated | Isolating chondroitin sulfate |
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| JP2005255727A (en) * | 2004-03-09 | 2005-09-22 | Tokuyama Corp | Method for producing highly purified mucopolysaccharide |
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| CN112481338A (en) * | 2020-12-22 | 2021-03-12 | 荣成万盈水产科技有限公司 | Preparation method of squid chondroitin sulfate glycoprotein |
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| WO2021081999A1 (en) * | 2019-11-01 | 2021-05-06 | 南京汉欣医药科技有限公司 | Low-molecular-weight chondroitin sulfate and preparation method therefor |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| KR20010070901A (en) * | 2001-06-18 | 2001-07-27 | 강헌수 | manufacturing method for chondroitin sulfate and its content |
| WO2003066683A1 (en) * | 2002-02-05 | 2003-08-14 | Nippon Barrier Free Co., Ltd. | Mucopolysaccharides and process for producing the same |
| JP2003299497A (en) * | 2002-02-05 | 2003-10-21 | Masayoshi Kachi | Mucopolysaccharide and method for producing the same |
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| JP2004149736A (en) * | 2002-11-01 | 2004-05-27 | Nippon Barrier Free:Kk | Chondroitin sodium sulfate, chondroitin sulfate-containing substance, and method for producing them |
| WO2004044009A1 (en) * | 2002-11-13 | 2004-05-27 | Cargill, Incorporated | Isolating chondroitin sulfate |
| JP2004189825A (en) * | 2002-12-10 | 2004-07-08 | Biochem:Kk | Method for producing sodium chondroitin sulfate |
| JP2005255727A (en) * | 2004-03-09 | 2005-09-22 | Tokuyama Corp | Method for producing highly purified mucopolysaccharide |
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| JPWO2012099216A1 (en) * | 2011-01-19 | 2014-06-30 | 国立大学法人弘前大学 | Large-scale preparation of proteoglycans |
| JP6071558B2 (en) * | 2011-01-19 | 2017-02-01 | 国立大学法人弘前大学 | Large-scale preparation of proteoglycans |
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| JP2016138061A (en) * | 2015-01-28 | 2016-08-04 | 一丸ファルコス株式会社 | High molecular sugar chain compound in which specific molecular binding unit is present at terminal portion in molecule, and use thereof |
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| CN106674380A (en) * | 2017-02-09 | 2017-05-17 | 高焕顺 | Preparation method of new chondroitin sulfate |
| US11572421B2 (en) | 2019-11-01 | 2023-02-07 | Nanjing Hanxin Pharmaceutical Technology Co., Ltd. | Low molecular weight chondroitin sulfate, composition, preparation method and use thereof |
| CN112481338A (en) * | 2020-12-22 | 2021-03-12 | 荣成万盈水产科技有限公司 | Preparation method of squid chondroitin sulfate glycoprotein |
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