JP4915888B2 - Method for producing dermatan sulfate from pork skin extract - Google Patents

Description

【００１２】
【化２】

デルマタン硫酸の繰り返し二糖（括弧内の構造）には、硫酸基とカルボキシル基（いずれも波付下線で示す）が一個ずつ含まれ、これがキトサン分子のアミノ基（波付下線）と電気的に結合する可能性がある。
【００１３】
すなわち、キトサンの括弧内の二糖と、デルマタン硫酸の括弧内の二糖単位が相対して結合すると考えられ、前者、キトサンの括弧内二糖の式量３２２と、後者の式量４３５から、両者の理論的結合比は１：１．３５となり、１００ｇのキトサンは１３５ｇのデルマタン硫酸を吸着できることになる。
【００１４】
通常得られるキトサンは、キチンの混在によりキトサン自身の純度が６０〜８０％であることや、反応液中で必ずしも理論上の結合が起こらないことなどから、１００ｇのキトサンに対するデルマタン硫酸の最大結合量は、２５〜５０ｇ程度と考えられる。
【００１５】
実験的に純度が約８０％のキトサンを使用し、ｐＨ５で吸着実験を行ったところ、純品換算で４０ｍｇのキトサンは、少なくとも１０ｍｇのデルマタン硫酸をほぼ１００％吸着することが分かった。この量は上記推定吸着量の下限値であるが、吸着条件をさらに検討すれば、さらに良好な結果が得られるものと考える。
ｂ）吸着至適ｐＨの検討
キトサンがコンドロイチン硫酸を吸着する至適ｐＨを、コンドロイチン硫酸Ｃを用いて調べたところ、ｐＨ７以下ではほぼ１００％吸着されるが、ｐＨ８では１６％が未吸着となり、ｐＨ９では１００％非吸着であった。
【００１６】
吸着ｐＨが５以下では、吸着剤であるキトサン分子が不安定になり、ｐＨが１０以上では被吸着物質であるコンドロイチン硫酸の分解が起こるおそれがあるので、実際の吸着は主としてｐＨ５で行うことにした。
ｃ）他成分の混在による吸着能の差異
デルマタン硫酸含有試料液にキトサン微粉末を加えて吸着させるとき、コンドロイチン硫酸が純粋に近い溶液であっても、豚皮エキス水解物などのような夾雑物の多い溶液を用いても、溶液のｐＨを５に維持し、他種の陰電荷物質の存在がない状態であれば、キトサンの吸着能はあまり影響されない。
２．脱着条件の検討
キトサンに吸着されたコンドロイチン硫酸は、ｐＨ９．０のリン酸緩衝液を加えて脱着する。この場合、脱着されるコンドロイチン硫酸量は吸着量の６０〜８０％であるが、これ以上アルカリ側の緩衝液を用いても、脱着されるコンドロイチン硫酸量は増加せず、その上、ｐＨ１０以上の緩衝液では、アルカリによるムコ多糖分解作用が起こり、かえって悪い影響が現れる。
【００１７】
そこで脱着ｐＨは９．０とし、塩化ナトリウムを添加することにより、イオン強度を大きくして脱着を容易にすることとし、Ｍ／１５リン酸緩衝液（ｐＨ９．０）に塩化ナトリウムを２Ｍの濃度に加えたものを脱着液とした。
【００１８】
【実施例】
（実施例１）
濃厚シロップ状の豚皮エキス（Ｐｏｒｋ Ｓｋｉｎ Ｌｉｑｕｉｄ、以下ＰｋＳＬと略）９５．５ｇを秤取し、これを沸騰している精製水２００ｍｌ中に攪拌しながら注入し、加え終わってからさらに３０分間、引き続きよく攪拌する。
【００１９】
この液を１０℃に冷却し、１０，０００ｒｐｍ、１０℃で２５分遠心した後、上清をとり、これを５０℃に加温した後、攪拌しつつ１／１０容量のＭ／１５リン酸緩衝液および２．５Ｎ ＮａＯＨを加えてｐＨを７．５とする。さらに３０分間、攪拌を続けた後、アルカラーゼ、プロナーゼなどの蛋白分解酵素の適量を加え、その酵素の至適作用ｐＨおよび温度で１６〜２０時間加温反応させ、エキス中に含まれる蛋白部分を分解する。
【００２０】
反応後、液を１００℃、１５分間加熱して酵素作用を停止させた後、１０℃以下に冷却し、１０，０００ｒｐｍ、１０℃、１５分間遠心して上清を集める。この液には原エキス中のコンドロイチン硫酸の大部分が含まれる。この上清の一部を取り、ウロン酸法によるデルマタン硫酸量を測定しておく。
【００２１】
上清液に精製水を加え約１０倍に希釈した後、その容量の１／１０容のＭ／１０リン酸緩衝液（ｐＨ５）を加えてｐＨを５．０とし、これに微粉状のキトサンを添加し、１０℃以下に保って約３０分攪拌を続けた後、４℃、１２，０００ｒｐｍで２０分遠心して沈殿を集め、Ｍ／１００リン酸緩衝液（ｐＨ５）を加えて沈殿を洗浄した後、ｐＨ９．０の塩化ナトリウム加リン酸緩衝液を加えてデルマタン硫酸を脱着し、遠心して上清を集める。この操作を反復して得られた上清を合一し、凍結乾燥すれば粗デルマタン硫酸が得られる。収量は使用したＰｋＳＬ重量の約０．３％であった。文献（Ｃｉｆｏｎｅｌｌｉら：Ｂｉｏｃｈｅｍ．Ｐｒｅｐ．，１２（５）１９６８）によれば、脂肪をアセトン抽出して除去した豚皮から得られるデルマタン硫酸量は、０．２０〜０．２４％と記載されているから、上述の方法によりＰｋＳＬ中のデルマタン硫酸は、ほぼ完全に抽出できたものと考えられる。
【００２２】
（実施例２）
豚皮エキスＰｋＳＬ１ｋｇを秤取し、これを沸騰中の精製水２リットル中に小量ずつ、攪拌しながら添加する。加え終わってから、さらに１０分間沸騰させ、エキス中に含まれる蛋白質を十分変性させる。この時、突沸現象が起こりやすいから、激しく攪拌を続ける。
【００２３】
その後、液温が５０度に低下するまで水冷し、これにＭ／１５リン酸水素二ナトリウム液を、液容量の１／２０量を攪拌しながら添加した後、ｐＨを７．５になるように調整する。
【００２４】
添加後５０℃に保温しつつさらに３０分間、ゆるく攪拌を続け、反応液のｐＨをチェックし、必要あれば２Ｎ水酸化ナトリウム、あるいは２Ｎ塩酸を加えてｐＨ７．５となるようにする。
【００２５】
この液に蛋白分解酵素としてアルカラーゼ２，４Ｌ（液状）を加えてよく攪拌した後、５５℃の恒温槽に入れ、１８〜２０時間保温し、豚皮エキス中の蛋白を十分分解する。酵素液の添加量は、酵素の純度と豚皮エキス中に蛋白含量により一定ではないから、予備実験により必要量を確かめる。蛋白分解酵素は他の種類（プロナーゼ等）でもよいが、蛋白分解が不十分であるときはムコ多糖の収得量が低下し、かつ、ブタの蛋白（主としてコラーゲン）が最終製品に混入することになるから、好ましくない。
【００２６】
酵素で消化した後、反応液を加熱沸騰させるか、または反応液中に過熱水蒸気を通じて１００〜１１０℃に加熱し、約２０分間この温度に保つことにより、酵素蛋白を十分変性させる。その後冷水により１０℃以下に冷却し、シャープレス等の遠心機（５，０００Ｇ以上）により遠心分離して上清を集める。３層に分離するときは、上層は脂肪分画、下層は変性蛋白層であるから、中層のみを集める。消化酵素としてアルカラーゼを使用したときは、褐色やや混濁した液が得られる。
【００２７】
遠心上清を集め、混和して全体を均一にした後一部を採取し、ウロン酸法によりデルマタン硫酸量を測定し、吸着に必要とするキトサン量を決めておく。合一上清に１０倍量の精製水をくわえて攪拌、希釈し、リン酸緩衝液を加えてｐＨ５．０とし、計算量のキトサンを加えて１０℃で約３０分攪拌した後遠心して沈殿を集め、前の沈殿と合わせてＭ／１００リン酸緩衝液を加えて攪拌洗浄し、再度遠心して沈殿を取る。
【００２８】
沈殿にｐＨ９の塩化ナトリウム加リン酸緩衝液を添加し、３７℃に加温攪拌してデルマタン硫酸を溶出し、遠心して上清を集める。これを凍結乾燥すれば塩化ナトリウムとリン酸ナトリウムの混入した粗デルマタン硫酸が得られる。デルマタン硫酸としての収量は、出発原料のＰｋＳＬに対して０．２３％であった。
【００２９】
この粗製品から純デルマタン硫酸を製造するときは、アルコール分別沈殿法、あるいはカラムクロマト法を用いる必要があるが、前者の場合においてもエタノール使用量は一般の抽出法に比べると、著しく少量である。
【００３０】
【発明の効果】
この発明によれば、高価なエタノールは製造の最終過程においてのみ少量使用すれば済むので、製造原価を大幅に切り下げ、従来品よりも純度の高い製品を、より安価に市場に提供することができ、同時に従来その処理に困っていた豚皮の効果的なリサイクルも図ることができる。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for producing dermatan sulfate from mucopolysaccharide, particularly pig skin.
[0002]
[Prior art]
When pigs were slaughtered, especially in the Kanto region and beyond, the skin was not distributed with meat, but was treated as an unnecessary product peeled off from the meat. As a result, pig skin can only be used as a raw material for glue, used as feed, or discarded, and its effective use has been desired.
[0003]
By the way, it is known that dermatan sulfate (chondroitin sulfate B) is extracted from pig skin, and it is well known that dermatan sulfate can be a useful substance for the human body. Therefore, it is conceivable to use pig skin as a raw material for dermatan sulfate.
[0004]
In general, when extracting mucopolysaccharides such as dermatan sulfate from animal tissues such as pig skin, digestive enzymes are added to the ground tissue to degrade proteins, and a large amount of ethanol is added thereto to precipitate mucopolysaccharides. A method of further purifying this by a chromatographic method or the like is used.
[0005]
[Problems to be solved by the invention]
However, this method requires a very large amount of ethanol compared to the obtained mucopolysaccharide, and it is not easy to recover and reuse ethanol after use. For this reason, the price of ethanol, which is a high rate of taxable goods, is passed on to the product, so that the price of the purified mucopolysaccharide is necessarily high. On the other hand, methanol or methanol-free duty-free ethanol that acts as a highly toxic substance in the human body may cause toxic methanol to remain in the final product because the product is used as a medical, cosmetic, or food additive. That is dangerous.
[0006]
[Means for Solving the Problems]
The present invention was created in view of the above problems of the prior art, and an object thereof is to provide a method for extracting dermatan sulfate from pig skin at a low cost and in a safe manner.
[0007]
That is, the present invention is characterized in that a cationic substance is added to a pork skin extract to adsorb dermatan sulfate, and after washing to remove impurities, dermatan sulfate is extracted with a weak alkaline salt solution. In this invention, the pork skin extract is obtained by grinding the pork skin and then treating with a proteolytic enzyme. Moreover, chitosan is disclosed as a cationic substance.
[0008]
DETAILED DESCRIPTION OF THE INVENTION
1. Examination of adsorption conditions The principle of the present invention is to add chitosan insoluble in water with a polymer compound with a strong positive charge to an aqueous solution of chondroitin sulfate such as dermatan sulfate which is a polymer compound with a strong negative charge. The purpose is to bind and separate dermatan sulfate on the chitosan molecule surface.
[0009]
The charge of dermatan sulfate and chitosan varies depending on the pH. On the acidic side, dermatan sulfate is negatively charged and dissolved as an acid. On the alkaline side, it loses positive charge and exists as an alkali salt such as sodium salt. Dermatan sulfate has a sulfate group and a carboxyl group. However, since the molecule as a whole is a weak acid, it has a low ionization degree in an alkaline state, and actually behaves as a neutral salt. Therefore, it is not adsorbed by a positively charged substance such as chitosan on the alkali side.
[0010]
On the other hand, at an acidic pH, it exists as a negatively charged acid, so that it is electrically attracted to the positively charged chitosan and is adsorbed on the molecular surface.
a) Examination of the adsorption capacity of chitosan The structures of chitosan (A) and dermatan sulfate (B) are considered as follows: chitosan is the same molecule, dermatan sulfate is two different monosaccharide derivative molecules, Each is a long polymer.
[0011]
[Chemical 1]

[0012]
[Chemical formula 2]

The repetitive saccharide of dermatan sulfate (structure in parentheses) contains one sulfate group and one carboxyl group (both indicated by wavy underline), which is electrically connected to the amino group (wavy underline) of the chitosan molecule. May combine.
[0013]
That is, it is considered that the disaccharide in the parenthesis of chitosan and the disaccharide unit in the parenthesis of dermatan sulfate are bound to each other. From the former, the formula amount 322 of the chitosan bracket in parentheses and the latter formula amount 435, The theoretical binding ratio between them is 1: 1.35, and 100 g of chitosan can adsorb 135 g of dermatan sulfate.
[0014]
The chitosan obtained usually has chitosan purity of 60 to 80% due to the mixture of chitin, and theoretical binding does not always occur in the reaction solution. Therefore, the maximum binding amount of dermatan sulfate to 100 g of chitosan Is considered to be about 25 to 50 g.
[0015]
Experimentally, when chitosan having a purity of about 80% was used and an adsorption experiment was conducted at pH 5, it was found that 40 mg of chitosan adsorbed almost 100% of at least 10 mg of dermatan sulfate in terms of pure product. This amount is the lower limit value of the estimated adsorption amount, but it is considered that better results can be obtained if the adsorption conditions are further examined.
b) Examination of optimum pH for adsorption When the optimum pH at which chitosan adsorbs chondroitin sulfate was examined using chondroitin sulfate C, almost 100% was adsorbed at pH 7 or lower, but 16% was unadsorbed at pH 8. At pH 9, it was 100% non-adsorbed.
[0016]
If the adsorption pH is 5 or less, the adsorbent chitosan molecule becomes unstable, and if the pH is 10 or more, the adsorbed substance chondroitin sulfate may be decomposed. Therefore, actual adsorption is mainly performed at pH 5. did.
c) Difference in adsorbability due to mixing of other components When chitosan fine powder is added to a sample solution containing dermatan sulfate and adsorbed, impurities such as pig skin extract hydrolyzate, even if the solution is nearly pure chondroitin sulfate Even if a solution with a large amount of the solution is used, the adsorption ability of chitosan is not significantly affected as long as the pH of the solution is maintained at 5 and there is no other kind of negatively charged substance.
2. Examination of Desorption Conditions Chondroitin sulfate adsorbed on chitosan is desorbed by adding pH 9.0 phosphate buffer. In this case, the amount of chondroitin sulfate to be desorbed is 60 to 80% of the adsorbed amount, but the amount of chondroitin sulfate to be desorbed does not increase even when an alkaline buffer solution is used. In the buffer solution, the mucopolysaccharide decomposition action by alkali occurs, and the adverse effect appears.
[0017]
Therefore, the desorption pH is set to 9.0, and sodium chloride is added to increase the ionic strength to facilitate desorption. Sodium chloride is added to the M / 15 phosphate buffer (pH 9.0) at a concentration of 2M. What was added to was used as the desorption liquid.
[0018]
【Example】
Example 1
Thick syrupy pork skin extract (Park Skin Liquid, hereinafter abbreviated as PkSL) is weighed in 95.5 g and poured into 200 ml of boiling purified water while stirring. Continue to stir well.
[0019]
After cooling this solution to 10 ° C. and centrifuging at 10,000 rpm and 10 ° C. for 25 minutes, the supernatant was taken, heated to 50 ° C., and then stirred with 1/10 volume of M / 15 phosphoric acid. Buffer and 2.5N NaOH are added to bring the pH to 7.5. After further stirring for 30 minutes, an appropriate amount of a proteolytic enzyme such as alcalase or pronase was added, and the mixture was allowed to warm for 16 to 20 hours at the optimum pH and temperature of the enzyme, and the protein portion contained in the extract was Decompose.
[0020]
After the reaction, the solution is heated at 100 ° C. for 15 minutes to stop the enzyme action, then cooled to 10 ° C. or lower, centrifuged at 10,000 rpm, 10 ° C. for 15 minutes, and the supernatant is collected. This solution contains most of the chondroitin sulfate in the original extract. A part of this supernatant is taken and the amount of dermatan sulfate by the uronic acid method is measured in advance.
[0021]
After adding purified water to the supernatant and diluting it about 10 times, 1/10 volume of M / 10 phosphate buffer (pH 5) is added to adjust the pH to 5.0, which is finely powdered chitosan. The mixture was kept at 10 ° C. or lower and stirred for about 30 minutes, and then centrifuged at 4 ° C. and 12,000 rpm for 20 minutes to collect the precipitate. M / 100 phosphate buffer (pH 5) was added to wash the precipitate. Thereafter, pH 9.0 sodium chloride phosphate buffer is added to desorb dermatan sulfate, and the supernatant is collected by centrifugation. The supernatant obtained by repeating this operation is combined and freeze-dried to obtain crude dermatan sulfate. The yield was about 0.3% of the PkSL weight used. According to the literature (Cifonelli et al .: Biochem. Prep., 12 (5) 1968), the amount of dermatan sulfate obtained from pig skin from which fat has been extracted by acetone extraction is described as 0.20 to 0.24%. Therefore, it is considered that dermatan sulfate in PkSL was almost completely extracted by the above-described method.
[0022]
(Example 2)
1 kg of pig skin extract PkSL is weighed and added in small amounts to 2 liters of boiling purified water while stirring. After the addition is complete, boil for an additional 10 minutes to sufficiently denature the protein contained in the extract. At this time, bumping is likely to occur, so vigorous stirring is continued.
[0023]
Thereafter, the solution is cooled with water until the temperature drops to 50 ° C., and M / 15 disodium hydrogenphosphate solution is added to this while stirring 1/20 of the solution volume so that the pH becomes 7.5. Adjust to.
[0024]
After the addition, keep stirring at 50 ° C. and continue stirring gently for another 30 minutes to check the pH of the reaction solution, and if necessary, add 2N sodium hydroxide or 2N hydrochloric acid to pH 7.5.
[0025]
After adding alcalase 2,4L (liquid) as a proteolytic enzyme to this solution and stirring well, it is placed in a thermostatic bath at 55 ° C. and kept warm for 18 to 20 hours to sufficiently decompose the protein in the pork skin extract. The amount of enzyme solution added is not constant depending on the purity of the enzyme and the protein content in the pork skin extract, so the necessary amount is confirmed by preliminary experiments. Proteolytic enzymes may be of other types (such as pronase), but if proteolysis is insufficient, the yield of mucopolysaccharide will be reduced and porcine protein (mainly collagen) will be mixed into the final product. Therefore, it is not preferable.
[0026]
After digestion with the enzyme, the reaction solution is heated to boiling, or heated to 100-110 ° C. through superheated steam in the reaction solution, and kept at this temperature for about 20 minutes to sufficiently denature the enzyme protein. Thereafter, the mixture is cooled to 10 ° C. or lower with cold water, and centrifuged with a centrifuge (5,000 G or more) such as a sharp press to collect the supernatant. When separating into three layers, since the upper layer is a fat fraction and the lower layer is a denatured protein layer, only the middle layer is collected. When alcalase is used as a digestive enzyme, a slightly turbid liquid is obtained.
[0027]
Centrifuge supernatants are collected and mixed to make the whole uniform, then a part is collected, and the amount of dermatan sulfate is measured by the uronic acid method to determine the amount of chitosan required for adsorption. Add 10 times the amount of purified water to the combined supernatant, stir and dilute, add phosphate buffer to pH 5.0, add the calculated amount of chitosan, stir at 10 ° C for about 30 minutes, then centrifuge to precipitate The M / 100 phosphate buffer is added together with the previous precipitate, washed with stirring, and centrifuged again to obtain the precipitate.
[0028]
Add pH 9 sodium chloride phosphate buffer to the precipitate, heat and stir to 37 ° C. to elute dermatan sulfate, centrifuge and collect the supernatant. If this is freeze-dried, crude dermatan sulfate mixed with sodium chloride and sodium phosphate is obtained. The yield as dermatan sulfate was 0.23% based on the starting material PkSL.
[0029]
When pure dermatan sulfate is produced from this crude product, it is necessary to use an alcohol fractional precipitation method or a column chromatography method. Even in the former case, the amount of ethanol used is extremely small compared to the general extraction method. .
[0030]
【Effect of the invention】
According to the present invention, since a small amount of expensive ethanol needs to be used only in the final manufacturing process, the manufacturing cost can be greatly reduced, and a product having a higher purity than the conventional product can be provided to the market at a lower cost. At the same time, it is possible to effectively recycle pigskin, which has been in trouble in the past.

Claims (2)

A method for producing dermatan sulfate from pork skin extract, comprising adding chitosan to pork skin extract to adsorb dermatan sulfate, washing to remove impurities, and then extracting dermatan sulfate with weak alkaline salt solution . The method for producing dermatan sulfate from a pork skin extract according to claim 1, wherein the pork skin extract is obtained by grinding the pork skin and then treating with a proteolytic enzyme.