JP2009007322A - Method for purifying colored substance from molasses or blackstrap molasses, and colored substance purified by the same - Google Patents

Method for purifying colored substance from molasses or blackstrap molasses, and colored substance purified by the same Download PDF

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JP2009007322A
JP2009007322A JP2007236467A JP2007236467A JP2009007322A JP 2009007322 A JP2009007322 A JP 2009007322A JP 2007236467 A JP2007236467 A JP 2007236467A JP 2007236467 A JP2007236467 A JP 2007236467A JP 2009007322 A JP2009007322 A JP 2009007322A
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molasses
colored substance
colored
resin
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Kenichi Hatano
賢一 秦野
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Gunma University NUC
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    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
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    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
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Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for simply separating residual saccharide components and colored substances from molasses or blackstrap molasses and producing the colored substances in a good efficiency. <P>SOLUTION: This method for purifying the colored substances from the molasses or blackstrap molasses comprises a process of preparing a diluted molasses liquid by diluting the molasses or blackstrap molasses with water, a process of adjusting the pH of the diluted molasses liquid to 1-3 by adding an acid thereto, a process of separating the colored substance from the diluted molasses liquid by bringing the pH-adjusted molasses-diluted liquid in contact with a hydrophobic chromatographic resin, to adsorb the colored substance in the diluted liquid onto the resin and a process of recovering a liquid of eluted colored substance from the resin through bringing an aqueous alkaline solution in contact with the hydrophobic chromatographic resin adsorbing the colored substance. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

本発明は、砂糖の製造過程で結晶化する糖を回収した後の糖蜜又は廃糖蜜から残存糖分と着色物質とに分離して、着色物質を精製する方法及びこの方法により精製された着色物質、この物質に含まれるタンパク質加水分解酵素阻害剤に関するものである。   The present invention relates to a method for purifying a colored substance by separating residual sugar and a colored substance from molasses or waste molasses after recovering the sugar that crystallizes during the sugar production process, and a colored substance purified by this method, The present invention relates to a protein hydrolase inhibitor contained in this substance.

ブラジルや欧米で既に導入されているバイオマス由来のエタノール等のバイオ燃料は、自動車などの輸送機関から発生する炭酸ガス、ＮＯx、ＳＯxの削減が期待されている。 Biofuels such as ethanol derived from biomass that have already been introduced in Brazil and Europe and the United States are expected to reduce carbon dioxide, NO x , and SO x generated from transportation such as automobiles.

しかし、一方では、これまで行われてきているバイオ燃料としてのエタノール製造では、サトウキビ等の糖分を発酵原料としているため、将来的には食糧としての供給と競合するという問題を生じさせる可能性がある。このため、バイオマスとしての糖原料については従来よりもより高度なアプローチでの技術的対応が求められている。   However, on the other hand, in the production of ethanol as biofuel that has been carried out so far, sugar content such as sugarcane is used as a fermentation raw material, which may cause a problem of competing with supply as food in the future. is there. For this reason, technical measures with a more advanced approach than before are required for sugar raw materials as biomass.

例えば、砂糖の製造過程で発生する、糖蜜から砂糖を回収した後に残った廃糖蜜は、全世界で年間約３００万トン、日本では約５万トンが副産物として生じている。これらは産業廃棄物として、十分に有効活用されずに捨てられている現状がある。廃糖蜜は糖分以外の成分も含んだ粘状で黒褐色の液体である。現在、この廃糖蜜の一部は、家畜の飼料、ソースやタイヤの着色料に使用されているが付加価値が低い。   For example, waste molasses generated after sugar is recovered from molasses produced during the sugar production process is produced as a by-product of about 3 million tons annually worldwide and about 50,000 tons in Japan. There is the present condition that these are thrown away as industrial waste without being fully utilized effectively. Waste molasses is a viscous black-brown liquid containing components other than sugar. Currently, some of the molasses is used in livestock feeds, sauces and tire colorants, but its added value is low.

また、例えばビート廃糖蜜を、パン酵母の培養のための炭素源として使用することが知られている（例えば、特許文献１参照。）。しかしながら、廃糖蜜にはパン酵母の培養に適当でない成分を含むものもあり、実際には、必ずしもパン酵母培養に好適な廃糖蜜を常に入手できるとは限らないため、製造する工場の側としては出来れば使用したくないが、やむを得ず使用しているのが実情であった。   For example, it is known to use beet molasses as a carbon source for culturing baker's yeast (see, for example, Patent Document 1). However, some molasses contains components that are not suitable for baker's yeast culture, and in fact, it is not always possible to obtain molasses suitable for baker's yeast culture. I didn't want to use it if possible, but it was unavoidable.

また、廃糖蜜を希釈してｐＨ変化により固形物を自然沈降させて上澄液を分離する廃糖蜜の処理法が提案されている（例えば、特許文献２参照。）。またこの特許文献２では、上記処理法で得られた上澄み液をアミノ酸の発酵原料として用いる発酵生産法が開示されている。しかし、上記処理法では、廃糖蜜の有色物質との分離が十分でなく、分離効率は実用上満足できるレベルではなかった。   In addition, a treatment method for waste molasses has been proposed in which waste molasses is diluted and solids are naturally precipitated by pH change to separate the supernatant (for example, see Patent Document 2). Moreover, in this patent document 2, the fermentation production method using the supernatant liquid obtained by the said processing method as a fermentation raw material of an amino acid is disclosed. However, in the above treatment method, the molasses is not sufficiently separated from the colored substance, and the separation efficiency is not at a level that is practically satisfactory.

更に、上記特許文献２に示されるような沈降分離の方法とは別に、廃糖蜜液からの糖の分離回収にイオン交換クロマトによる方法も検討されてきているが、陰イオン交換樹脂の場合には特に樹脂に着色物質が強力に吸着して樹脂の再生が困難であり、陽イオン交換樹脂の場合では着色物質の分離はできないという問題点がある。
特開平１０−１３６９７５号公報（発明の詳細な説明の段落［０００２］〜［０００３］） 特許第３０４１９２１号公報（特許請求の範囲の請求項１及び請求項３］） In addition to the sedimentation separation method shown in Patent Document 2, ion separation chromatography has also been studied for separating and recovering sugar from waste molasses, but in the case of anion exchange resins, In particular, the coloring substance is strongly adsorbed on the resin and it is difficult to regenerate the resin. In the case of a cation exchange resin, the coloring substance cannot be separated.
JP-A-10-136975 (paragraphs [0002] to [0003] in the detailed description of the invention) Japanese Patent No. 3041921 (Claims 1 and 3 of Claims)

上記廃糖蜜をエタノール製造に利用することができれば、砂糖原料との競合を避けることができる。しかしながら、廃糖蜜には、糖分以外の成分であるヒドロキシメチルフルフラール（ＨＭＦ）などの発酵阻害物質が存在しているため、そのままでは廃糖蜜のエタノール製造への利用には都合が悪いと思われる。また発酵後の蒸留残渣はその着色度の高さからそのまま廃棄することができず、嫌気的バクテリア処理と好気的処理によって着色度を下げてから廃棄するなど非常にコストがかかっている。   If the above-mentioned molasses can be used for ethanol production, competition with sugar raw materials can be avoided. However, because molasses contains fermentation inhibitors such as hydroxymethylfurfural (HMF), which is a component other than sugar, it seems to be inconvenient for use of molasses in ethanol production. Moreover, the distillation residue after fermentation cannot be discarded as it is because of its high coloring degree, and it is very costly, such as discarding after reducing the coloring degree by anaerobic bacterial treatment and aerobic treatment.

そこで、本発明は、以上のとおりの背景から、従来の問題点を解消し、廃糖蜜を更に高度に有効利用すること、より具体的には、廃糖蜜から簡便に残存する糖分と着色物質とを分離し、効率良く着色物質を得ることを可能とし、更に廃糖蜜から着色物質を精製した後に残った糖分はエタノール製造に利用することができ、廃糖蜜の有効利用を図ることができる、新しい技術手段を提供することを課題としている。   Therefore, the present invention eliminates the conventional problems from the background as described above, and more effectively uses the molasses, and more specifically, the sugar and coloring substances that simply remain from the molasses. It is possible to obtain a colored substance efficiently, and the sugar content remaining after purifying the colored substance from the molasses can be used for ethanol production, enabling the effective utilization of the molasses. The challenge is to provide technical means.

請求項１に係る発明は、糖蜜又は廃糖蜜を水に希釈して糖蜜希釈液を調製する工程と、糖蜜希釈液に酸を加えてｐＨ１〜３に調整する工程と、ｐＨ調整した糖蜜希釈液を疎水性クロマト樹脂と接触させて希釈液中の着色物質を樹脂に吸着させることにより糖蜜希釈液から着色物質を分離する工程と、着色物質を吸着した疎水性クロマト樹脂にアルカリ水溶液を接触させて樹脂から着色物質が溶離した液を得る工程とを含む糖蜜又は廃糖蜜からの着色物質の精製方法である。   The invention according to claim 1 includes a step of diluting molasses or waste molasses in water to prepare a molasses dilution, a step of adjusting acid to pH 1-3 by adding acid to the molasses dilution, and a pH-adjusted molasses dilution A step of separating the colored substance from the molasses diluted solution by contacting the colored substance in the diluent with the hydrophobic chromatographic resin, and contacting the aqueous alkaline solution with the hydrophobic chromatographic resin adsorbing the colored substance. And a step of obtaining a liquid from which a colored substance is eluted from a resin, and a method for purifying the colored substance from molasses or waste molasses.

請求項１に係る発明では、上記工程を経ることで、糖蜜又は廃糖蜜から簡便に残存する糖分と着色物質とを分離し、効率良く着色物質を得ることができる。   In the invention which concerns on Claim 1, by passing through the said process, the sugar content and coloring substance which remain easily from molasses or waste molasses can be isolate | separated, and a coloring substance can be obtained efficiently.

請求項２に係る発明は、請求項１に係る発明であって、糖蜜又は廃糖蜜が一番蜜、二番蜜、三番蜜、四番蜜、五番蜜及び六番蜜からなる群より選ばれた１種又は２種以上の糖蜜からの着色物質の精製方法である。   The invention according to claim 2 is the invention according to claim 1, wherein the molasses or waste molasses comprises the most honey, second honey, third honey, fourth honey, fifth honey and sixth honey. This is a method for purifying colored substances from one or more selected molasses.

請求項２に係る発明では、上記種類の糖蜜であれば、着色物質の精製が可能である。このうち、着色物質の含有割合が高い五番蜜や六番蜜の使用が特に好ましい。   In the invention which concerns on Claim 2, if it is the said kind of molasses, purification of a colored substance is possible. Among these, the use of fifth honey or sixth honey having a high content of coloring substances is particularly preferable.

請求項３に係る発明は、請求項１又は２に係る発明であって、疎水性クロマト樹脂がアンバーライト（登録商標）ＸＡＤ７ＨＰ樹脂（以下、本明細書ではＸＡＤ樹脂と略す。）である着色物質の精製方法である。   The invention according to claim 3 is the invention according to claim 1 or 2, wherein the hydrophobic chromatographic resin is Amberlite (registered trademark) XAD7HP resin (hereinafter abbreviated as XAD resin in this specification). This is a purification method.

請求項３に係る発明では、ＸＡＤ樹脂は、市場に流通している疎水性クロマト樹脂の中でも入手が容易であり、かつ入手コストも安いため、着色物質の吸着に使用するのに好適である。   In the invention according to claim 3, the XAD resin is suitable for use in adsorbing colored substances because it is easily available among the hydrophobic chromatographic resins distributed in the market and the acquisition cost is low.

請求項４に係る発明は、請求項１ないし３いずれか１項に記載の方法により精製され、３４００ｃｍ-1、１６５０ｃｍ-1及び１０５０ｃｍ-1にそれぞれ赤外吸収ピークを有する廃糖蜜からの着色物質である。 Invention, purified by the method according to any one of claims 1 to 3, colored substances from molasses with 3400 cm -1, respectively infrared absorption peaks at 1650 cm -1 and 1050 cm -1 according to claim 4 It is.

請求項４に係る発明では、上記ＩＲ吸収ピークは、フミン物質が有する赤外吸収ピークと一致するものであり、上記波長の吸収ピークを有すれば、フミン物質が有する特性と似ていると言える。   In the invention according to claim 4, the IR absorption peak coincides with the infrared absorption peak of the humic substance, and if it has the absorption peak of the wavelength, it can be said that it is similar to the characteristic of the humic substance. .

請求項５に係る発明は、請求項４記載の着色物質に含まれることを特徴とするタンパク質加水分解酵素阻害剤である。   The invention according to claim 5 is a protein hydrolase inhibitor, which is contained in the colored substance according to claim 4.

本発明の糖蜜又は廃糖蜜からの着色物質の精製方法によれば、糖蜜又は廃糖蜜から簡便に残存する糖分と着色物質とを分離し、効率良く着色物質を得ることができる。   According to the method for purifying a colored substance from molasses or molasses of the present invention, the remaining sugar and the colored substance can be easily separated from the molasses or molasses, and the colored substance can be obtained efficiently.

また、上記方法により精製された本発明の着色物質は、その特性から土壌改良剤、肥料、水質改良剤、凝集剤等としてだけでなく、パパイン阻害剤のようなタンパク質加水分解酵素阻害剤としても応用可能である。   Further, the colored substance of the present invention purified by the above method is not only used as a soil improver, fertilizer, water quality improver, flocculant, etc., but also as a protein hydrolase inhibitor such as a papain inhibitor. Applicable.

次に本発明を実施するための最良の形態を説明する。   Next, the best mode for carrying out the present invention will be described.

本発明でいう、着色物質の精製方法の出発原料となる「糖蜜」は、主としてサトウキビやビートからの砂糖の製造過程で結晶化した糖を回収した後に残ったものである。また、「廃糖蜜」は、主としてサトウキビやビートからの砂糖の製造工程で発生する最終の糖蜜（サトウキビでは六番蜜、ビートでは三番蜜を一般に指す）から砂糖を回収した後に残ったものであって、従来では殆どが産業廃棄物として扱われていたものである。ここで、「廃糖蜜」は少なくともサトウキビからのものとして考慮される。本発明で使用される糖蜜又は廃糖蜜は、一番蜜、二番蜜、三番蜜、四番蜜、五番蜜及び六番蜜からなる群より選ばれた１種又は２種以上の糖蜜が挙げられる。上記種類の糖蜜であれば、残存する糖分と着色物質とを別々に分離回収することができ、着色物質の精製が可能である。このうち、着色物質の含有割合が高い五番蜜や六番蜜の使用が特に好ましい。   The “molasses” used as a starting material in the method for purifying a colored substance in the present invention is a residue left after recovering sugar crystallized mainly in the process of producing sugar from sugarcane or beet. In addition, “waste molasses” is left over after recovering sugar from the final molasses (usually 6th honey in sugarcane and 3rd honey in beet) mainly produced in the sugar production process from sugarcane and beet. In the past, most of them were treated as industrial waste. Here, “molasses” is considered at least as from sugarcane. The molasses or molasses used in the present invention is one or more molasses selected from the group consisting of honey, honey, honey, honey, honey and honey. Is mentioned. If it is the said kind of molasses, the remaining saccharide | sugar content and a coloring substance can be isolate | separated separately, and a coloring substance can be refine | purified. Among these, the use of fifth honey or sixth honey having a high content of coloring substances is particularly preferable.

本発明の着色物質の精製方法では、先ず、前述した糖蜜又は廃糖蜜を水に希釈して糖蜜希釈液を調製する。ここで糖蜜希釈液を調製するのは、後に続く工程での取扱いを容易にするためである。糖蜜や廃糖蜜は、水による希釈が任意の割合で可能である。希釈率は、糖蜜や廃糖蜜の全重量に対し、７倍以上、更には８〜１０倍の水希釈が一般的な条件として考慮される。希釈率が低いほど着色物質の精製コストが下がるため好ましいが、希釈率が低すぎる、例えば５倍以下の希釈率の場合では、粘性が高く、効率的な着色物質の精製が難しくなる。この希釈調整した糖蜜希釈液のｐＨはおおよそ５〜６程度である。   In the method for purifying a colored substance of the present invention, first, the molasses or waste molasses described above is diluted in water to prepare a molasses dilution. The reason why the molasses diluted solution is prepared here is to facilitate handling in subsequent steps. Molasses and molasses can be diluted with water in any proportion. As for the dilution rate, water dilution of 7 times or more, and further 8 to 10 times the total weight of molasses and waste molasses is considered as a general condition. The lower the dilution rate, the lower the purification cost of the colored material, which is preferable. However, when the dilution rate is too low, for example, 5 times or less, the viscosity is high and it is difficult to efficiently purify the colored material. The pH of the diluted molasses diluted solution is about 5-6.

次いで、この糖蜜希釈液に酸を加えてｐＨ１〜３に調整する。ｐＨ調整に使用する酸は、どのような種類の酸を使用してもよい。例えば、塩酸、硫酸、硝酸などの鉱酸や有機酸を使用することができる。糖蜜希釈液を上記ｐＨに調整する理由は、着色物質のカルボキシル基などの酸性解離基の負電荷を無くすためである。   Subsequently, an acid is added to this molasses diluted solution, and it adjusts to pH 1-3. Any kind of acid may be used as the acid used for pH adjustment. For example, mineral acids such as hydrochloric acid, sulfuric acid, and nitric acid, and organic acids can be used. The reason for adjusting the molasses diluted solution to the above pH is to eliminate the negative charge of acidic dissociation groups such as carboxyl groups of the coloring substance.

また、ｐＨ調整した糖蜜希釈液は、濁りを生じているため、疎水性クロマト樹脂との接触の前に、遠心分離処理してもよい。この遠心分離処理によって分離させた固形沈殿成分を除去することで、得られた上澄み液の透明度が高まり、本発明の着色物質の精製方法の各工程で溶離してくる液が、完全に透明になる。遠心分離条件としては１００００〜２００００ｒｐｍの超遠心分離処理が特に好ましい。   In addition, since the diluted molasses solution adjusted for pH is turbid, it may be subjected to a centrifugal separation treatment before contact with the hydrophobic chromatographic resin. By removing the solid precipitate components separated by this centrifugation treatment, the transparency of the obtained supernatant is increased, and the liquid eluted in each step of the colored substance purification method of the present invention is completely transparent. Become. As centrifugation conditions, ultracentrifugation treatment at 10,000 to 20,000 rpm is particularly preferred.

次に、ｐＨ調整した糖蜜希釈液を疎水性クロマト樹脂と接触させて希釈液中の帯電していない着色物質を樹脂に吸着させることにより糖蜜希釈液から着色物質を分離する。疎水性クロマト樹脂と糖蜜希釈液との接触方法は、疎水性クロマト樹脂をカラムに詰め、カラムの上方から糖蜜希釈液を一定の流量で通液させることにより行われる。糖蜜希釈液をカラムに通液して、カラム中の疎水性クロマト樹脂と接触させることで、希釈液中の着色物質が樹脂に吸着され、樹脂に吸着されない糖分等はカラムから排出され、回収される。この工程により糖蜜希釈液中の着色物質が疎水性クロマト樹脂に高効率で吸着する。カラムに通液した後に素通り画分として回収された糖溶液は、製糖工場の結晶化工程に再度循環させるか、エタノール発酵の原料として利用してもよい。   Next, the colored substance is separated from the molasses diluted solution by bringing the pH-adjusted molasses diluted solution into contact with the hydrophobic chromatographic resin to adsorb the uncharged colored substances in the diluted solution to the resin. The contact method between the hydrophobic chromatoresin and the molasses diluent is performed by filling the column with the hydrophobic chromatoresin and allowing the molasses dilute to flow through the column at a constant flow rate. By passing the molasses diluted solution through the column and bringing it into contact with the hydrophobic chromatographic resin in the column, the colored substances in the diluted solution are adsorbed on the resin, and sugars etc. that are not adsorbed on the resin are discharged from the column and collected. The By this step, the colored substance in the molasses diluted solution is adsorbed on the hydrophobic chromatographic resin with high efficiency. The sugar solution collected as a flow-through fraction after passing through the column may be recycled again to the crystallization process of the sugar factory or used as a raw material for ethanol fermentation.

使用される疎水性クロマト樹脂は、従来の技術で使用されていたイオン交換樹脂とは異なった特性を示し、官能基を持たないＭＲ構造（Macro Reticular structure，巨大網目構造）を持つものとして特徴を有している。疎水性クロマト樹脂としては、ＸＡＤ樹脂、ＯＤＳ（OctaDecylSilyl）、ＲＥＳＯＵＲＣＥ ＲＰＣ等が挙げられるが、これらの種類に限定されるものではない。なお、ＸＡＤ樹脂は、スチレン又はアクリルとジビニルベンゼンの共重合体であって、ＭＲ構造のイオン交換樹脂に類似した白色の不透明球状粒子である。イオン交換樹脂と異なり官能基をもたないので化学的に極めて安定している。また、ＯＤＳは、シリカゲル担体にオクタデシルシリル基を化学結合した充填剤であり、この充填剤がカラムに詰められてＯＤＳカラムを形成する。ＲＥＳＯＵＲＣＥ ＲＰＣは、ポリスチレン／ジビニルベンゼン製ビーズがカラムに詰められたものである。このうち、ＸＡＤ樹脂が、市場に流通している疎水性クロマト樹脂の中でも入手が容易であり、かつ入手コストも安いため、着色物質の吸着に使用するのに好適である。   Hydrophobic chromatographic resins used have characteristics different from those of ion exchange resins used in the prior art, and are characterized as having an MR structure (Macro Reticular structure) without functional groups. Have. Hydrophobic chromatographic resins include XAD resin, ODS (OctaDecylSilyl), RESOURCE RPC, etc., but are not limited to these types. The XAD resin is a copolymer of styrene or acrylic and divinylbenzene, and is white opaque spherical particles similar to an ion exchange resin having an MR structure. Unlike ion exchange resins, it has no functional groups and is therefore extremely chemically stable. ODS is a packing material in which an octadecylsilyl group is chemically bonded to a silica gel carrier, and this packing material is packed in a column to form an ODS column. RESOURCE RPC is a polystyrene / divinylbenzene bead packed in a column. Among these, XAD resins are suitable for use in adsorbing colored substances because they are easily available among the hydrophobic chromatographic resins on the market and are inexpensive to obtain.

なお、使用する疎水性クロマト樹脂は粒状であることが好ましく、その中間径は一般的には０.３５〜０.９０ｍｍの範囲にあることが好ましい。   In addition, it is preferable that the hydrophobic chromatography resin to be used is a granular form, and it is preferable that the intermediate diameter is generally in the range of 0.35 to 0.90 mm.

着色物質を吸着させる接触処理においては、例えば、疎水性クロマト樹脂にＸＡＤ樹脂を使用する場合、一般的には次のような条件が好適に採用される。なお、一般にクロマト樹脂の使用方法として、カラムへの送液温度を上げる、またはカラム自体を加温するなど行って分離能を上げることがよく試みられるが、本発明の方法では加温しなくても十分な分解能を得ることができる。   In the contact treatment for adsorbing the coloring substance, for example, when an XAD resin is used as the hydrophobic chromatographic resin, generally, the following conditions are preferably employed. In general, as a method of using a chromatographic resin, it is often attempted to increase the resolution by increasing the liquid feeding temperature to the column or heating the column itself. However, the method of the present invention does not require heating. Sufficient resolution can be obtained.

糖蜜希釈液の供給速度：約８ｃｍ3／ｃｍ2・ｍｉｎ
糖蜜希釈液の液温：１０〜２８℃
糖蜜希釈液のｐＨ：１〜３
次に、着色物質を吸着した疎水性クロマト樹脂にアルカリ水溶液を接触させて樹脂から着色物質が溶離した液を得る。疎水性クロマト樹脂とアルカリ水溶液との接触は、前工程での糖蜜希釈液の通液を終え、水洗した後、カラムにアルカリ水溶液を一定の流量で通液することにより行われる。この工程では疎水性クロマト樹脂から着色物質が完全に脱離するので、カラム再生が非常に容易である。疎水性クロマト樹脂からの着色物質の溶離にアルカリ水溶液を用いるのは、疎水性相互作用で吸着していた着色物質をアルカリ条件にして酸性解離基に負電荷を持たせ樹脂との相互作用力を失わせるためである。使用するアルカリ水溶液としては、ＮａＯＨやＫＯＨ、Ｃａ(ＯＨ)2等が挙げられる。アルカリ水溶液は、例えば、０.０１〜０.１Ｍの濃度で用いることが好適である。水溶液濃度が下限値未満では、上記の理由により着色物質が溶離されないという不具合を生じる。また、溶離する際のアルカリ水溶液の液温は、室温（１０〜２８℃）が好ましい。 The feed rate of molasses diluted solution: about 8cm 3 / cm 2 · min
Molasses dilution liquid temperature: 10-28 ° C
Molasses dilution pH: 1-3
Next, an aqueous alkaline solution is brought into contact with the hydrophobic chromatographic resin that has adsorbed the coloring substance to obtain a liquid in which the coloring substance is eluted from the resin. The contact between the hydrophobic chromatographic resin and the aqueous alkali solution is performed by passing the molasses diluted solution in the previous step, washing with water, and then passing the aqueous alkaline solution through the column at a constant flow rate. In this step, since the coloring substance is completely desorbed from the hydrophobic chromatographic resin, the column regeneration is very easy. The alkaline aqueous solution is used for elution of the colored substance from the hydrophobic chromatographic resin because the colored substance adsorbed by the hydrophobic interaction is subjected to an alkaline condition so that the acidic dissociation group has a negative charge and the interaction force with the resin is increased. This is to make it disappear. Examples of the alkaline aqueous solution to be used include NaOH, KOH, Ca (OH) 2 and the like. The alkaline aqueous solution is preferably used at a concentration of 0.01 to 0.1 M, for example. When the concentration of the aqueous solution is less than the lower limit value, a problem that the colored substance is not eluted for the above-described reason occurs. Further, the temperature of the alkaline aqueous solution at the time of elution is preferably room temperature (10 to 28 ° C.).

以上述べたように、上記工程を経ることで、糖蜜又は廃糖蜜から簡便に残存糖分と着色物質とに分離して、効率良く着色物質を得ることができる。   As described above, the colored substance can be efficiently obtained by separating the molasses or waste molasses into the residual sugar and the colored substance simply by passing through the above steps.

なお、得られた着色物質が溶離した液について、着色物質を精製する場合には、例えば、着色物質が溶離した液をｐＨ３以下の酸性に調整し、疎水性クロマト樹脂に通液して着色物質を吸着させ、アルカリ水溶液での溶離のプロセスを行うことにより達成される。   In addition, when purifying a colored substance with respect to the liquid eluted from the obtained colored substance, for example, the liquid eluted from the colored substance is adjusted to an acidic pH of 3 or less, and is passed through a hydrophobic chromatographic resin to give the colored substance. Is achieved by adsorbing and elution with an aqueous alkaline solution.

また、得られた着色物質が溶離した液を凍結乾燥してもよい。着色物質が溶離した液を凍結乾燥して着色物質を粉末状態とすることで、運搬や保管などをする際の取扱いが容易になる。着色物質が溶離した液を凍結乾燥した粉末は、雲母のような形状でキラキラと金属光沢を示す。   Moreover, you may freeze-dry the liquid which the obtained colored substance eluted. By lyophilizing the liquid from which the colored substance has been eluted to make the colored substance powder, handling during transportation and storage becomes easy. The powder obtained by freeze-drying the liquid eluted from the colored substance has a mica-like shape and glitter and metallic luster.

上記精製方法により得られる本発明の着色物質は、その強力なイオン交換能、長波長域から短波長側にむかって単調に上昇する吸収曲線、そして赤外（ＩＲ）吸収スペクトルから、フミン物質にその特性が酷似していることが明らかとなった。ここでいうフミン物質は、一般的には「腐植物質」と呼ばれるものであって、植物などが土壌中の微生物によって複雑に分解、重合されて生成する最終生成物である。このフミン物質は高温高圧などの物理的作用を与えることで、石炭や石油になるといわれている。フミン物質はどのような反応機構を経ることで生成されるか未だ解明されていないため、その構造及び特性が類似している本発明の着色物質を研究、解析することによって、フミン物質の誕生過程の解明が期待される。フミン物質の代表的なものとしてはフミン酸やフルボ酸などが存在する。   The colored substance of the present invention obtained by the above purification method is converted into a humic substance from its strong ion exchange capacity, an absorption curve that increases monotonously from the long wavelength region toward the short wavelength side, and an infrared (IR) absorption spectrum. It became clear that the characteristics were very similar. The humic substance here is generally called “humic substance”, and is a final product that is produced by complex decomposition and polymerization of plants and the like by microorganisms in the soil. This humic substance is said to become coal and oil by giving physical action such as high temperature and high pressure. Since the reaction mechanism of humic substances is not yet elucidated, the birth process of humic substances is studied by analyzing and analyzing the colored substances of the present invention whose structures and properties are similar. The elucidation of is expected. Typical examples of humic substances include humic acid and fulvic acid.

本発明の着色物質は、３４００ｃｍ-1、１６５０ｃｍ-1及び１０５０ｃｍ-1にそれぞれ赤外吸収ピークを有する。３４００ｃｍ-1の吸収ピークは、水素結合に関与しているＯ−Ｈ振動に、１６５０ｃｍ-1の吸収ピークは、Ｃ＝Ｏと共役している芳香族Ｃ＝Ｃ振動に、１０５０ｃｍ-1の吸収ピークは、アルコール又はエステルのＣ−ＯＨ振動にそれぞれ帰属されている。上記赤外吸収ピークは、フミン物質が有する赤外吸収ピークと一致するものであり、本発明の着色物質とフミン物質の特性が類似することを裏付けるデータの一つとなる。 Coloring materials of the present invention, respectively 3400 cm -1, 1650 cm -1 and 1050 cm -1 with an infrared absorption peak. Absorption peak of 3400 cm -1 is the O-H vibration involved in hydrogen bonding, absorption peaks of 1650 cm -1 is the aromatic C = C vibrations are C = O and conjugate, absorption of 1050 cm -1 The peaks are attributed to the C-OH vibration of the alcohol or ester, respectively. The infrared absorption peak coincides with the infrared absorption peak of the humic substance, and is one of data supporting the similar characteristics of the colored substance and the humic substance of the present invention.

また本発明の着色物質は、パパインの阻害活性を有する。パパイン（papain, ＥＣ.３.４.２２.２）とは、タンパク質加水分解酵素（プロテアーゼ）の中のシステインプロテアーゼに分類される酵素であって、植物由来のプロテアーゼとしては最も研究が進んでいるものの一つである。なお、本発明の着色物質は、アスパラギン酸プロテアーゼ、セリンプロテアーゼ、メタロプロテアーゼに対して阻害活性を有しておらず、システインプロテアーゼに分類される酵素のみの阻害活性を有する点で、特異的な性質を示す。   The colored substance of the present invention has papain inhibitory activity. Papain (papain, EC 3.4.22.2) is an enzyme that is classified as a cysteine protease in protein hydrolase (protease), and is the most researched as a plant-derived protease. One of things. The colored substance of the present invention has a specific property in that it has no inhibitory activity against aspartic proteases, serine proteases, metalloproteases, and has inhibitory activity only for enzymes classified as cysteine proteases. Indicates.

パパインは、パパイアから見つかったことからこの名前が付けられたが、その触媒残基はシステインとヒスチジンで、システイン残基のチオール基の硫黄原子がペプチド結合のカルボニル炭素に求核攻撃を行うことからタンパク質やペプチドの加水分解が始まる。そして、基質特異的に、塩基性アミノ酸、グリシン及びロイシンと続くアミノ酸とのペプチド結合を切断する。   Papain was named because it was found in papaya, but its catalytic residues are cysteine and histidine, and the sulfur atom of the thiol group of the cysteine residue makes a nucleophilic attack on the carbonyl carbon of the peptide bond. Protein and peptide hydrolysis begins. Then, the peptide bond between the basic amino acid, glycine and leucine and the subsequent amino acid is cleaved in a substrate-specific manner.

阻害剤としては、ロイペプチンなどのペプチド系阻害剤や、システイン残基（チオール基）修飾試薬（水銀化合物など）が知られているが、本発明の着色物質を使用することで、新しいタイプの阻害剤が提供されることになる。   As inhibitors, peptide inhibitors such as leupeptin and cysteine residue (thiol group) modifying reagents (mercury compounds, etc.) are known. By using the coloring substances of the present invention, a new type of inhibitor is used. An agent will be provided.

以上述べたように、本発明の着色物質は、パパイン阻害剤のようなタンパク質加水分解酵素阻害剤として応用することが可能であり、抗ウイルス剤、抗害虫剤等として、医薬、農薬等への応用が期待される。   As described above, the coloring substance of the present invention can be applied as a protein hydrolase inhibitor such as a papain inhibitor, and can be applied to drugs, agricultural chemicals, etc. Application is expected.

また、本発明の着色物質は、フミン酸やフルボ酸等のフミン物質と類似した特性を有するので、フミン酸やフルボ酸等と同様に、土壌改良剤、肥料、水質改良剤、凝集剤等として広範囲への応用が期待できる。   In addition, since the coloring substance of the present invention has characteristics similar to those of humic substances such as humic acid and fulvic acid, it can be used as a soil conditioner, fertilizer, water quality improver, flocculant, etc., like humic acid and fulvic acid. Application to a wide range can be expected.

次に本発明の実施例を詳しく説明する。   Next, embodiments of the present invention will be described in detail.

＜実施例１＞
関西精糖株式会社から提供されたサトウキビの廃糖蜜（六番蜜）２５ｍｌを水に希釈して７倍希釈液を調製した。この希釈液に塩酸を加えてｐＨ３に調整した。ｐＨ調整した希釈液を遠心分離（２００００ｒｐｍ、１０分間）し上澄みを回収した。 <Example 1>
A 7-fold diluted solution was prepared by diluting 25 ml of sugarcane molasses (six honey) provided by Kansai Seika Co., Ltd. in water. The diluted solution was adjusted to pH 3 by adding hydrochloric acid. The diluted solution whose pH was adjusted was centrifuged (20,000 rpm, 10 minutes), and the supernatant was collected.

次に、疎水性クロマト樹脂としてＸＡＤ樹脂を用意し、ＸＡＤ樹脂をカラムに充填した。また、ｐＨが３に調整されたイオン交換水、０.０５ＭのＮａＯＨをそれぞれ用意した。ＸＡＤ樹脂が充填されたカラムの上方から上澄み液（１７５ｍｌ）を通じ、続いて、イオン交換水（１３５ｍｌ）を通じ、次に、ＮａＯＨ（１３５ｍｌ）を通じた後、更にイオン交換水（２７０ｍｌ）を通じた。カラムへの通液操作は、膨潤ＸＡＤ樹脂を１５０ｍｌ（ゲル高２６ｃｍ）、流速を８ｃｍ3／ｃｍ2・ｍｉｎ、１フラクションの容量を５.５ｍｌとした。なお、このカラム通液操作は全て室温で行った。 Next, an XAD resin was prepared as a hydrophobic chromatography resin, and the column was packed with the XAD resin. Also, ion-exchanged water whose pH was adjusted to 3 and 0.05M NaOH were prepared. From the top of the column filled with XAD resin, the supernatant (175 ml) was passed, followed by ion exchange water (135 ml), then NaOH (135 ml), and then further ion exchange water (270 ml). The operation of passing through the column was 150 ml of swollen XAD resin (gel height: 26 cm), the flow rate was 8 cm 3 / cm 2 · min, and the volume of the fraction was 5.5 ml. All the column flow operations were performed at room temperature.

＜評価１＞
実施例１のカラムを通液した液について、色度に関しては波長２８０ｎｍの吸収により、糖濃度に関してはフェノール・硫酸法によって定量して評価した。フェノール・硫酸法は、サンプル溶液１５０μｌに５％フェノール液１５０μｌ、濃硫酸７５０μｌの順で加え、１０分放置後直ちに２６℃の水溶液中で１５分間保温した後に、４８５ｎｍの吸光度を測定した。標準試料としてスクロースを用いて検量線を作成した。 <Evaluation 1>
The liquid passed through the column of Example 1 was evaluated by quantifying the chromaticity by absorption at a wavelength of 280 nm and the sugar concentration by the phenol / sulfuric acid method. In the phenol / sulfuric acid method, 150 μl of 5% phenol solution and 750 μl of concentrated sulfuric acid were added to 150 μl of the sample solution in this order, and the mixture was allowed to stand for 10 minutes and immediately kept in an aqueous solution at 26 ° C. for 15 minutes. A calibration curve was prepared using sucrose as a standard sample.

図１（ａ）にカラムを通液した液のＡ280と糖濃度の関係を、図１（ｂ）にカラムを通液した液のＡ280とｐＨの関係を示す。なお、図１（ａ）中の実線は波長２８０ｎｍの吸収を、破線は糖濃度を示す。図１（ｂ）中の実線は波長２８０ｎｍの吸収を、破線はｐＨを示す。 The relationship between A 280 and sugar concentration of the solution was passed through the column in FIG. 1 (a), shows the A 280 and pH of the relationship of the liquid was passed through the column in FIG. 1 (b). In addition, the continuous line in Fig.1 (a) shows absorption of wavelength 280nm, and a broken line shows sugar concentration. The solid line in FIG. 1B indicates absorption at a wavelength of 280 nm, and the broken line indicates pH.

図１（ａ）から明らかなように、フラクションＮｏ．１０〜６０程度で糖濃度に関してピークが形成され、フラクションＮｏ．９０〜１１０程度でＡ280のメインピークが形成された。また、図１（ｂ）から明らかなように、フラクションＮｏ．１〜８０程度まではｐＨ３程度を推移し、フラクションＮｏ．８０を越えたあたりからｐＨが上昇し、フラクションＮｏ．１１０前後でｐＨ１３にまで達した。この結果から、ｐＨ上昇とＡ280のメインピークとが一致しており、アルカリによって着色物質が樹脂から溶離されていることが確認された。なお、結果は示さないが、廃糖蜜中の全糖成分の約９０％を占めるスクロースを用いて同様の実験を行った結果、スクロースがＸＡＤ樹脂に全く吸着されずに素通りして溶離してきた。このことから、Ａ280のメインピークの部分には糖は含まれていないことが判り、糖とその他の着色物質が完全に分離できたことが確認できた。 As is clear from FIG. A peak with respect to the sugar concentration is formed at about 10 to 60. A 280 main peak was formed at about 90-110. Further, as is clear from FIG. From about 1 to about 80, the pH is about 3 and the fraction No. The pH increased from around 80 and the fraction No. The pH reached 13 at around 110. This result was consistent and the main peak of the pH increase and A 280, the coloring material by alkali is eluted from the resin was confirmed. In addition, although a result is not shown, as a result of having conducted the same experiment using sucrose which occupies about 90% of all the sugar components in molasses, sucrose was eluted without being adsorbed by the XAD resin at all. Therefore, the portion of the main peak of A 280 know that does not include sugar, sugar and other coloring material was confirmed that could be completely separated.

なお、図１（ａ）及び図１（ｂ）から、既にその結果は推定されるが、カラムに通液するアルカリ水溶液をＫＯＨに変更した場合についても同様の評価を行った。その結果、図示しないが、アルカリ水溶液にＮａＯＨを使用した場合と同一のクロマトグラムを示した。この結果から、アルカリ水溶液としてＫＯＨを使用する場合についても、問題なく本発明の方法により着色物質を精製できることが確認された。   Although the result is already estimated from FIGS. 1 (a) and 1 (b), the same evaluation was performed when the alkaline aqueous solution passed through the column was changed to KOH. As a result, although not shown, the same chromatogram as when NaOH was used for the alkaline aqueous solution was shown. From this result, it was confirmed that the colored substance can be purified by the method of the present invention without problems even when KOH is used as the alkaline aqueous solution.

また、上記関西精糖株式会社から提供されたサトウキビの廃糖蜜の他に、新東日本精糖株式会社から提供されたサトウキビの廃糖蜜（六番蜜）、関門精糖株式会社から提供されたサトウキビの廃糖蜜（六番蜜）をそれぞれ用いて上記と同様の方法を行い、評価したが、どれも殆ど同じクロマトグラムを示した。   In addition to sugarcane waste molasses provided by Kansai Seika Co., Ltd., sugarcane molasses (six honey) provided by Shinto Nippon Seika Co., Ltd., sugarcane waste molasses provided by Kanmon Seika Co., Ltd. (Six honey) was used to evaluate and evaluate the same method as above, but all showed almost the same chromatogram.

＜評価２＞
次に、溶離した吸着画分（着色物質）として、実施例１のカラムを通液した液のうち、フラクションＮｏ．９４〜１０２を回収した。この回収したサンプルを凍結乾燥し、その一部を用いて赤外吸収スペクトルを測定した。赤外吸収スペクトル測定では、先ず、凍結乾燥したサンプル２ｍｇとＫＢｒ２００ｍｇをメノウ乳鉢と乳棒を用いて十分に粉砕し、粉砕物を錠剤成型機で円板状に成型することにより、サンプルの錠剤を作製した。次に、ＪＡＳＣＯ Ａ−７０２型分光計（日本分光社製）を用いて、サンプルの錠剤における赤外吸収スペクトルを測定した。 <Evaluation 2>
Next, as the eluted adsorbed fraction (colored substance), the fraction No. 94-102 were recovered. The collected sample was freeze-dried, and an infrared absorption spectrum was measured using a part of the sample. In infrared absorption spectrum measurement, 2 mg of lyophilized sample and 200 mg of KBr are first sufficiently pulverized using an agate mortar and pestle, and the pulverized product is molded into a disk shape by a tablet molding machine to produce a sample tablet. did. Next, the infrared absorption spectrum of the sample tablet was measured using a JASCO A-702 spectrometer (manufactured by JASCO Corporation).

その結果を図２に示す。なお、図２の「Fumic acid」とある赤外吸収スペクトルは、和光純薬株式会社より市販されているフミン酸のスペクトルを、図２の「Molasses」とある赤外吸収スペクトルは、今回測定したフラクションＮｏ．９４〜１０２のスペクトルを示す。   The result is shown in FIG. Note that the infrared absorption spectrum “Fumic acid” in FIG. 2 is the spectrum of humic acid commercially available from Wako Pure Chemical Industries, Ltd., and the infrared absorption spectrum “Molasses” in FIG. Fraction No. 94-102 spectra are shown.

図２から明らかなように、着色物質の赤外吸収スペクトルは、３４００ｃｍ-1、１６５０ｃｍ-1及び１０５０ｃｍ-1にそれぞれ吸収ピークを示した。３４００ｃｍ-1の吸収ピークは、水素結合に関与しているＯ−Ｈ振動を、１６５０ｃｍ-1の吸収ピークは、Ｃ＝Ｏと共役している芳香族Ｃ＝Ｃ振動を、１０５０ｃｍ-1の吸収ピークは、アルコール又はエステルのＣ−ＯＨ振動をそれぞれ表している。これらはフミン酸に特徴的な吸収ピークであることが報告されている。この結果から、得られた赤外吸収ピークは、フミン物質のそれらと類似した位置に現れることが確認された。 As apparent from FIG. 2, the infrared absorption spectrum of the coloring material, 3400 cm -1, showing respectively the absorption peaks at 1650 cm -1 and 1050 cm -1. Absorption peak of 3400 cm -1 is the O-H vibration involved in hydrogen bonding, absorption peaks of 1650 cm -1 is an aromatic C = C vibrations are C = O and conjugate, absorption of 1050 cm -1 The peaks represent the C-OH vibration of the alcohol or ester, respectively. These are reported to be absorption peaks characteristic of humic acid. From this result, it was confirmed that the obtained infrared absorption peak appeared at a position similar to those of the humic substance.

＜評価３＞
以前の予備的な研究から廃糖蜜から精製される着色物質には、パパイン阻害活性が存在することが判っていたが、この着色物質がパパイン以外のプロテアーゼ阻害活性を有しているか否かを確認した。本研究では各クラスの代表的なプロテアーゼである、ペプシン（アスパラギン酸）、パパイン（システイン）、キモトリプシン（セリン）、サーモライシン（金属）について阻害活性を測定した。パパインを除く各プロテアーゼの阻害活性測定方法は、下記にその概略を示す。 <Evaluation 3>
From previous preliminary studies, it was found that colored substances purified from molasses have papain inhibitory activity, but it is confirmed whether this colored substance has protease inhibitory activity other than papain. did. In this study, inhibitory activity was measured for pepsin (aspartic acid), papain (cysteine), chymotrypsin (serine), and thermolysin (metal), which are representative proteases of each class. The method for measuring the inhibitory activity of each protease excluding papain is outlined below.

キモトリプシンの阻害活性測定では、緩衝溶液に２０ｍＭ酢酸ナトリウム（ｐＨ４.５）を用い、基質にp-nitrophenyl benzyloxycarbonyl-L-phenylalaninateを用いた。サーモライシンの阻害活性測定では、緩衝溶液に５０ｍＭ酢酸ナトリウム（１０ｍＭ塩化カルシウム、ｐＨ５.０）を用い、基質にN-[3-(2-furyl)acrytoyl]-Gly-L-Leu amideを用いた。更にペプシンの阻害活性測定では、基質に２.５％ヘモグロビン（０.１Ｍ酢酸ナトリウム、ｐＨ３.８）を用い、反応停止剤に５％トリクロロ酢酸を用いた。   In measuring the inhibitory activity of chymotrypsin, 20 mM sodium acetate (pH 4.5) was used as a buffer solution, and p-nitrophenyl benzyloxycarbonyl-L-phenylalaninate was used as a substrate. In the measurement of the inhibitory activity of thermolysin, 50 mM sodium acetate (10 mM calcium chloride, pH 5.0) was used as a buffer solution, and N- [3- (2-furyl) acrytoyl] -Gly-L-Leuamide was used as a substrate. Further, in the measurement of the inhibitory activity of pepsin, 2.5% hemoglobin (0.1M sodium acetate, pH 3.8) was used as a substrate, and 5% trichloroacetic acid was used as a reaction terminator.

阻害活性測定方法は、測定セルに酵素溶液とサンプル溶液を加え、最後に基質溶液を加えることによって反応を開始させた。活性測定は全て２４℃で行った。阻害率（％）の計算方法に関しては、パパインとキモトリプシンが３１７ｎｍ、サーモライシンが３４５ｎｍ、ペプシンが２８０ｎｍの吸光度の経時変化を解析して求めた。   In the inhibitory activity measurement method, an enzyme solution and a sample solution were added to a measurement cell, and finally a substrate solution was added to initiate the reaction. All activity measurements were performed at 24 ° C. The method for calculating the inhibition rate (%) was determined by analyzing the change in absorbance over time of 317 nm for papain and chymotrypsin, 345 nm for thermolysin, and 280 nm for pepsin.

結果として、解析サンプルはパパイン以外のプロテアーゼへの阻害活性を示さなかった。このことから、本発明の廃糖蜜からの着色物質は、特定の酵素を阻害する特異的な構造を持っていると考えられる。   As a result, the analysis sample showed no inhibitory activity on proteases other than papain. From this, it is considered that the colored substance from the molasses of the present invention has a specific structure that inhibits a specific enzyme.

＜評価４＞
着色物質のパパイン阻害活性を更に精製することを目的として、ゲル濾過クロマトグラフィーを行った。樹脂としてセファデックス Ｇ−５０を用い、カラム体積は５２５ｍｌ（ゲル高９０ｃｍ）、流速を２ｃｍ3／ｃｍ2・ｍｉｎ、１フラクションの容量を５.５ｍｌとした。なお、このカラム通液操作は全て室温で行った。送液は、緩衝液ではなくｐＨが９.５に調整されたイオン交換水を用いた。 <Evaluation 4>
Gel filtration chromatography was performed for the purpose of further purifying the papain inhibitory activity of the colored substance. Sephadex G-50 was used as the resin, the column volume was 525 ml (gel height 90 cm), the flow rate was 2 cm 3 / cm 2 · min, and the fraction volume was 5.5 ml. All the column flow operations were performed at room temperature. For the liquid transfer, ion-exchanged water whose pH was adjusted to 9.5 was used instead of the buffer solution.

パパインの阻害活性測定では、１００ｍＭリン酸ナトリウム緩衝溶液（２ｍＭ ＥＤＴＡ、１ｍＭ ＤＴＴ、ｐＨ６.３）を用い、基質にN-CBZ-L-lysine-p-nitrophenylesterを用いた。   In the measurement of papain inhibitory activity, 100 mM sodium phosphate buffer solution (2 mM EDTA, 1 mM DTT, pH 6.3) was used, and N-CBZ-L-lysine-p-nitrophenylester was used as a substrate.

阻害活性測定方法は、測定セルに緩衝液（６５２μｌ）、酵素溶液（１６μｌ）、サンプル（１６μｌ）及び基質（１６μｌ）をこの順に加えることによって反応を開始させた。阻害率（％）の計算方法に関しては、酵素によって切断遊離されたp-nitrophenolの吸光度（３４０ｎｍ）の経時変化を傾きＸとし、コントロールから得られた傾きＹから、計算式（Ｙ−Ｘ）÷Ｙ×１００によって求めた。   In the inhibitory activity measurement method, the reaction was started by adding a buffer solution (652 μl), an enzyme solution (16 μl), a sample (16 μl) and a substrate (16 μl) in this order to the measurement cell. Regarding the calculation method of the inhibition rate (%), the change with time in the absorbance (340 nm) of p-nitrophenol cleaved and released by the enzyme is assumed to be a slope X, and from the slope Y obtained from the control, the calculation formula (Y−X) ÷ It calculated | required by Yx100.

図３にＡ280とパパイン阻害活性の関係を示す。なお、図３での実線は波長２８０ｎｍの吸収を、破線は阻害活性を示す。図３から、フラクションＮｏ．２０〜９０程度にかけて阻害活性が確認された。このパパインに対して強い阻害が見られたフラクションＮｏ．３０〜５０を回収し、凍結乾燥して解析用サンプルとした。 FIG. 3 shows the relationship between A 280 and papain inhibitory activity. The solid line in FIG. 3 indicates absorption at a wavelength of 280 nm, and the broken line indicates inhibitory activity. From FIG. Inhibitory activity was confirmed over about 20-90. Fraction No. in which strong inhibition against papain was observed. 30 to 50 were collected and lyophilized to prepare samples for analysis.

次に、着色物質の溶液中の分子量を測定するため、実施例１で行ったＸＡＤ樹脂に吸着した着色物質（Ｘ）、この評価４で行ったゲル濾過クロマトグラフィーでの阻害活性画分（Ｇ１）、そして、この評価４で行った活性が非常に弱い着色物質（フラクション６２〜７８：Ｇ２）を、それぞれ３５０ｍｇ／ｍｌの濃度に調整し、DynaPro（Wyatt Technology Corp.）により、動的光散乱測定を行った。なお、動的光散乱測定は、５０ｍＭ酢酸ナトリウム緩衝液（２ｍＭ ＥＤＴＡ、１ｍＭ ＤＴＴ、ｐＨ４.５）中で行った。   Next, in order to measure the molecular weight of the colored substance in the solution, the colored substance (X) adsorbed on the XAD resin performed in Example 1, and the inhibitory activity fraction (G1) in gel filtration chromatography performed in this evaluation 4 were used. ) And coloring substances (fractions 62 to 78: G2) having a very weak activity performed in this evaluation 4 were adjusted to a concentration of 350 mg / ml, respectively, and dynamic light scattering was performed by DynaPro (Wyatt Technology Corp.). Measurements were made. The dynamic light scattering measurement was performed in a 50 mM sodium acetate buffer (2 mM EDTA, 1 mM DTT, pH 4.5).

結果として、これらは単分散サンプルではなかったが、Ｘの分子量は約９２５０、Ｇ１の分子量は約１５５８０、そしてＧ２の分子量は約３２２０と見積もられた。同じ着色物質でもパパイン阻害活性を有するＧ１の分子量が、阻害活性が弱いＧ２の分子量に比べて約５倍も大きいという、非常に興味深い結果が得られた。   As a result, these were not monodisperse samples, but the molecular weight of X was estimated to be about 9250, the molecular weight of G1 was about 15580, and the molecular weight of G2 was estimated to be about 3220. A very interesting result was obtained that the molecular weight of G1 having papain inhibitory activity even with the same colored substance was about 5 times larger than the molecular weight of G2 having weak inhibitory activity.

次に、サンプルＧ１とサンプルＧ２のパパインに対する阻害様式と阻害定数を決定するため、基質の濃度と阻害剤の濃度を変数として各条件の反応速度を測定した。そして、Dixonプロットを作成してパパインに対する阻害定数を求めた。このときの条件は、５０ｍＭ酢酸ナトリウム（２ｍＭ ＥＤＴＡ、１ｍＭ ＤＴＴ、ｐＨ４.５）を緩衝液として用いた。他は上記パパイン阻害活性測定条件と同様である。   Next, in order to determine the inhibition mode and inhibition constant for papain in Sample G1 and Sample G2, the reaction rate under each condition was measured using the substrate concentration and the inhibitor concentration as variables. A Dixon plot was created to determine the inhibition constant for papain. As the conditions at this time, 50 mM sodium acetate (2 mM EDTA, 1 mM DTT, pH 4.5) was used as a buffer. Others are the same as the above-mentioned papain inhibitory activity measurement conditions.

結果として、図４に示したDixonプロットより、サンプルＧ１とサンプルＧ２ともに４種類の阻害剤濃度の直線がＹ軸より左側の領域で交差しているので、混合的にパパインを阻害（混合阻害）することが判った。また、Dixonプロットにより、Ｇ１サンプルの阻害定数は５.０１×１０-5Ｍ、Ｇ２サンプルのそれは１.０８×１０-3Ｍと見積もられた。一般的な阻害剤の阻害定数の範囲は１０-7〜１０-4であることが知られており、本発明の着色物質Ｇ２は、阻害剤としては非常に弱い部類に属することが判った。 As a result, from the Dixon plot shown in FIG. 4, the sample G1 and the sample G2 have four types of inhibitor concentration lines crossing in the region on the left side of the Y axis, so that papain is mixedly inhibited (mixed inhibition). I found out that Further, according to the Dixon plot, the inhibition constant of the G1 sample was estimated to be 5.01 × 10 −5 M, and that of the G2 sample was estimated to be 1.08 × 10 −3 M. It is known that the range of inhibition constants of general inhibitors is 10 −7 to 10 −4 , and it was found that the colored substance G2 of the present invention belongs to a very weak class as an inhibitor.

最後に総括として、フェノール・硫酸法により関西精糖株式会社提供の廃糖蜜では平均７９３ｇ／ｌの糖が含まれていることが判った。２５ｍｌの廃糖蜜より平均２０.９ｇ（ＸＡＤカラム処理の素通り糖画分での値）の糖を回収できることが明らかとなり、回収率でいうとほぼ１００％という高い値が得られた。一方で着色物質Ｘは、廃糖蜜２５ｍｌから平均で１.５６ｇ回収することができた。カラム中の未溶離の着色物質は考慮していないが、それでもクロマトグラムの面積積分計算からＸ画分の面積は全体の約８０％であることが判り、つまり素通り糖画分の色素除去率は８０％以上ということが判った。   Finally, as a summary, it was found by the phenol / sulfuric acid method that the molasses provided by Kansai Seika Co., Ltd. contained an average of 793 g / l of sugar. It was revealed that an average of 20.9 g of sugar (value in the pass-through sugar fraction of the XAD column treatment) can be recovered from 25 ml of molasses, and a high value of almost 100% was obtained in terms of the recovery rate. On the other hand, 1.56 g of coloring substance X was recovered on average from 25 ml of molasses. Although the uneluted colored substance in the column is not taken into account, the area integration calculation of the chromatogram still shows that the area of the X fraction is about 80% of the total, that is, the dye removal rate of the passing sugar fraction is It was found that it was over 80%.

本発明の着色物質は、前述した用途だけに限らず、健康飲料水や化粧品、サプリメント、調味料、脱臭剤などの用途や、止血剤、解熱剤、炎症防止剤、筋肉更正剤といった医薬品への用途や、染料、陶器、電池、泥水などといった新素材開発材料への用途にも適用できる。   The coloring substance of the present invention is not limited to the above-described uses, but is also used for health drinks, cosmetics, supplements, seasonings, deodorizers, and medicines such as hemostatic agents, antipyretic agents, anti-inflammatory agents, and muscle repair agents. It can also be applied to new material development materials such as dyes, ceramics, batteries, and muddy water.

図１（ａ）は、評価１における、カラムを通液した液のＡ280と糖濃度の関係を示す図である。図１（ｂ）は、評価１における、カラムを通液した液のＡ280とｐＨの関係を示す図である。FIG. 1 (a) is a graph showing the relationship between A 280 of the liquid passed through the column and the sugar concentration in Evaluation 1. FIG. FIG. 1B is a diagram showing the relationship between A 280 and pH of the liquid passed through the column in Evaluation 1. 図２は、評価２における着色物質及びフミン物質の赤外吸収スペクトル図である。FIG. 2 is an infrared absorption spectrum diagram of the coloring substance and the humic substance in Evaluation 2. 図３は、評価４におけるゲル濾過クロマトグラフィー後の着色物質のＡ280とパパイン阻害活性の関係を示す図である。FIG. 3 is a graph showing the relationship between A 280 of a colored substance after gel filtration chromatography and papain inhibitory activity in Evaluation 4. 図４は、評価４におけるＧ１サンプル及びＧ２サンプルのパパインに対する阻害様式と阻害定数の関係を示す図である。FIG. 4 is a diagram showing the relationship between the inhibition mode and inhibition constant for papain in the G1 sample and G2 sample in Evaluation 4.

Claims (5)

糖蜜又は廃糖蜜を水に希釈して糖蜜希釈液を調製する工程と、
前記糖蜜希釈液に酸を加えてｐＨ１〜３に調整する工程と、
前記ｐＨ調整した糖蜜希釈液を疎水性クロマト樹脂と接触させて希釈液中の着色物質を前記樹脂に吸着させることにより前記糖蜜希釈液から前記着色物質を分離する工程と、
前記着色物質を吸着した疎水性クロマト樹脂にアルカリ水溶液を接触させて前記樹脂から前記着色物質が溶離した液を得る工程と
を含む糖蜜又は廃糖蜜からの着色物質の精製方法。 A step of diluting molasses or waste molasses in water to prepare a molasses dilution;
Adjusting the pH to 1 to 3 by adding an acid to the molasses dilution;
Separating the colored substance from the molasses diluted solution by bringing the pH-adjusted molasses diluted solution into contact with a hydrophobic chromatographic resin and adsorbing the colored substance in the diluted solution to the resin;
A method for purifying a colored substance from molasses or waste molasses, comprising: contacting a hydrophobic chromatographic resin adsorbing the colored substance with an alkaline aqueous solution to obtain a liquid from which the colored substance is eluted from the resin. 糖蜜又は廃糖蜜が一番蜜、二番蜜、三番蜜、四番蜜、五番蜜及び六番蜜からなる群より選ばれた１種又は２種以上の糖蜜である請求項１記載の着色物質の精製方法。   The molasses or molasses is one or more molasses selected from the group consisting of honey, honey, honey, honey, honey and honey. A method for purifying colored substances. 疎水性クロマト樹脂がアンバーライト（登録商標）ＸＡＤ７ＨＰ樹脂である請求項１又は２記載の着色物質の精製方法。   The method for purifying a colored substance according to claim 1 or 2, wherein the hydrophobic chromatographic resin is Amberlite (registered trademark) XAD7HP resin. 請求項１ないし３いずれか１項に記載の方法により精製され、３４００ｃｍ-1、１６５０ｃｍ-1及び１０５０ｃｍ-1にそれぞれ赤外吸収ピークを有する糖蜜又は廃糖蜜からの着色物質。 Coloring substances from claims purified by the method of to claim 1, wherein in 3 any one, 3400 cm -1, molasses or molasses having respective infrared absorption peaks at 1650 cm -1 and 1050 cm -1. 請求項４記載の着色物質に含まれることを特徴とするタンパク質加水分解酵素阻害剤。   A protein hydrolase inhibitor, which is contained in the colored substance according to claim 4.
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CN101774834B (en) * 2009-02-27 2011-12-28 张志元 Plant nutritional agent
JP2012219228A (en) * 2011-04-12 2012-11-12 Masatsugu Yamashita Water-soluble black pigment
JP2013043988A (en) * 2011-08-26 2013-03-04 Toshi Kakugyo Kk Urinary calculus removing agent
CN104016758A (en) * 2014-05-07 2014-09-03 广西地源之本肥业有限公司 Method for preparing special-purpose eucalyptus organic compound fertilizer by utilizing fermenting waste
CN114098082A (en) * 2021-12-01 2022-03-01 南京财经大学 Preparation method and application of active ingredients in brown sugar

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101774834B (en) * 2009-02-27 2011-12-28 张志元 Plant nutritional agent
JP2012219228A (en) * 2011-04-12 2012-11-12 Masatsugu Yamashita Water-soluble black pigment
JP2013043988A (en) * 2011-08-26 2013-03-04 Toshi Kakugyo Kk Urinary calculus removing agent
CN104016758A (en) * 2014-05-07 2014-09-03 广西地源之本肥业有限公司 Method for preparing special-purpose eucalyptus organic compound fertilizer by utilizing fermenting waste
CN114098082A (en) * 2021-12-01 2022-03-01 南京财经大学 Preparation method and application of active ingredients in brown sugar

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