JP3963491B2 - Crystalline maltosyl glucoside, its production method and use - Google Patents

Description

【００４７】
表１の工程でゲル濾過溶出液として得られた精製酵素標品をポリアクリルアミドゲル（ゲル濃度７．５ｗ／ｖ％）を用いる電気泳動法で純度を検定したところ、蛋白バンドは単一であることが示され、得られた酵素標品は電気泳動的に単一な純度の高い標品であった。
【００４８】
【実験Ａ−３ 酵素の性質】
実験Ａ−２で得られた精製酵素標品をＳＤＳ−ポリアクリルアミドゲル（ゲル濃度１０ｗ／ｖ％）を用いる電気泳動法に供し、同時に泳動した分子量マーカー（日本バイオ・ラド・ラボラトリーズ株式会社製）と比較して本酵素の分子量を測定したところ、分子量約７７，０００乃至８７，０００ダルトンであった。
【００４９】
精製酵素標品をポリアクリルアミドゲル（２ｗ／ｖ％アンフォライン含有、スウエーデン国、ファルマシア・エルケイビー社製）を用いる等電点電気泳動法に供し、泳動後、ゲルのｐＨを測定して本酵素の等電点を求めたところ、等電点は約３．６乃至４．６であった。
【００５０】
本酵素活性に対する温度の影響、ｐＨの影響は活性測定方法に準じて調べた。結果を図１（温度の影響）、図２（ｐＨの影響）に示した。酵素の至適温度は、ｐＨ７．０、６０分間反応で、４０℃付近、至適ｐＨは、４０℃、６０分間反応で、約７．０であった。本酵素の温度安定性は、酵素溶液（５０ｍＭリン酸緩衝液を含む、ｐＨ７．０）を各温度に６０分間保持し、水冷した後、残存する酵素活性を測定することにより求めた。また、ｐＨ安定性は、本酵素を各ｐＨの５０ｍＭ緩衝液中で２５℃、１６時間保持した後、ｐＨを７に調整し、残存する酵素活性を測定することにより求めた。それぞれの結果を図３（温度安定性）、図４（ｐＨ安定性）に示した。本酵素の温度安定性は４０℃付近まで安定であり、ｐＨ安定性は約６乃至９であった。
【００５１】
【実験Ａ−４ 非還元性糖質の調製】
基質として、グルコース、マルトース、マルトトリオース、マルトテトラオース、マルトペンタオース、マルトヘキサオース、又はマルトヘプタオースの２０％水溶液を調製し、それぞれに実験Ａ−２で得られた精製酵素標品を基質固形物グラム当たり２単位の割合で加え、４０℃、ｐＨ７．０で４８時間作用させた後、脱塩し、ワコービーズ ＷＢ−Ｔ−３３０カラム（和光純薬工業株式会社製）を用いた高速液体クロマトグラフィーで反応生成物を分析した。高速液体クロマトグラフィーは、室温下で行い、溶離液として水を流速０．５ｍｌ／分で流し、示差屈折計ＲＩ−８０１２（東ソー株式会社製造）で分析した。その結果を表２に示す。
【００５２】
【表２】

【００５３】
表２の結果から明らかなように、反応物中には残存するそれぞれの基質と新たに生成したそれぞれの糖質ＰＩ、ＰＩＩ、ＰＩＩＩ、ＰＩＶ、ＰＶからなり、それ以外の糖質はほとんど検出されない。それぞれの生成率はグルコース重合度３のＰＩが比較的低いものの、グルコース重合度４以上のＰＩＩ、ＰＩＩＩ、ＰＩＶ、ＰＶは８５％以上の高い生成率であることが判明した。なお、グルコース、マルトースからは、新たな糖質を生成しないことが判明した。
【００５４】
それぞれの反応物から新たに生成した糖質を精製するため、脱色、脱塩、濃縮後、アルカリ金属型強酸性カチオン交換樹脂（ＸＴ−１０１６、Ｎａ⁺型、架橋度４％、東京有機化学工業株式会社製造）を用いたカラム分画を行った。樹脂を内径２．０ｃｍ、長さ１ｍのジャケット付きステンレス製カラム３本に充填し、直列につなぎ、カラム内温度を５５℃に維持しつつ、反応糖液を樹脂に対して５ｖ／ｖ％加え、これに５５℃の温水をＳＶ０．１３で流して分画し、新たに生成した糖質含量９７％以上の高純度画分を採取した。得られた高純度画分を真空乾燥し、それぞれ高純度糖質標品を調製した。基質原料に対する収率は、固形物換算で、それぞれＰＩで約９％、ＰＩＩで約６５％、ＰＩＩＩで約８２％、ＰＩＶで約８０％、ＰＶで約７７％であった。その純度は、それぞれＰＩで９７．５％、ＰＩＩで９８．６％、ＰＩＩＩで９９．５％、ＰＩＶで９８．４％、ＰＶで９８．４％であった。
【００５５】
またこれらの新たに生成した高純度糖質標品の還元力をソモギー・ネルソン法で測定し、ＤＥで表した。結果は表３にまとめた。
【００５６】
【表３】

【００５７】
表３の結果から明らかなように、いずれの標品にも僅かな還元力しか認めらなかった。その僅かな還元力は、その標品中に微量に混入、残存している基質由来の還元性マルトオリゴ糖に起因するものと推定され、新たに生成した糖質はいずれも実質的に非還元性であると判断される。
【００５８】
【実験Ａ−５ グルコアミラーゼによる酵素分解】
実験Ａ−４において調製した非還元性糖質標品、ＰＩ、ＰＩＩ、ＰＩＩＩ、ＰＩＶ又は、ＰＶのそれぞれ５０ｍｇを、５０ｍＭ酢酸緩衝液（ｐＨ４．５）１ｍｌに溶解し、１単位のグルコアミラーゼ（生化学工業株式会社製造）を加え、４０℃で６時間保ち、酵素分解した後、高速液体クロマトグラフィーで分解物を分析したところ、いずれの標品からも分解物としてグルコースとトレハロースのみが検出された。検出されたグルコース含量、トレハロース含量、その組成モル比の結果を表４に示す。
【００５９】
【表４】

【００６０】
表４の結果から明らかなように、グルコアミラーゼにより、非還元性糖質ＰＩはグルコース１分子とトレハロース１分子に分解され、非還元性糖質ＰＩＩはグルコース２分子とトレハロース１分子に分解され、非還元性糖質ＰＩＩＩはグルコース３分子とトレハロース１分子に分解され、非還元性糖質ＰＩＶはグルコース４分子とトレハロース１分子に分解され、非還元性糖質ＰＶはグルコース５分子とトレハロース１分子に分解されることが判明した。
【００６１】
また、グルコアミラーゼの反応特性を考慮すると、これら非還元性糖質の構造はトレハロース分子にグルコース分子がα−１，４結合、もしくはα−１，６結合で結合した糖質で、それぞれ、ＰＩはトレハロース１分子にグルコース１分子が結合したグルコース重合度３の非還元性糖質で、ＰＩＩはトレハロース１分子にグルコース２分子が結合したグルコース重合度４の非還元性糖質で、ＰＩＩＩはトレハロース１分子にグルコース３分子が結合したグルコース重合度５の非還元性糖質で、ＰＩＶはトレハロース１分子にグルコース４分子が結合したグルコース重合度６の非還元性糖質で、ＰＶはトレハロース１分子にグルコース５分子が結合したグルコース重合度７の非還元性糖質であると判断される。なお、同様に、非還元性糖質標品、ＰＩ、ＰＩＩ、ＰＩＩＩ、ＰＩＶ、又はＰＶにβ−アミラーゼを作用させたところ、非還元性糖質ＰＩ、ＰＩＩは分解されず、ＰＩＩＩはマルトースの１分子とＰＩの１分子に分解され、ＰＩＶはマルトースの１分子とＰＩＩの１分子に分解され、ＰＶはマルトースの２分子とＰＩの１分子に分解されることが判明した。
【００６２】
以上の結果から、本発明の非還元性糖質生成酵素による反応は、基質の低分子化及び高分子化を伴わない、換言すれば、グルコース重合度の変化を伴わない、分子内変換反応と判断され、また、この非還元性糖質生成酵素によって生成した非還元性糖質、ＰＩ、ＰＩＩ、ＰＩＩＩ、ＰＩＶ及びＰＶは、それぞれ、α−グルコシルトレハロース（別名、α−マルトシルグルコシド）、α−マルトシルトレハロース（別名、α−マルトトリオシルグルコシド）、α−マルトトリオシルトレハロース（別名、α−マルトテトラオシルグルコシド）、α−マルトテトラオシルトレハロース（別名、α−マルトペンタオシルグルコシド）及びα−マルトペンタオシルトレハロース（別名、α−マルトヘキサオシルグルコシド）で示されるα−グリコシルトレハロース（Ｇ_n−Ｔ：但し、Ｇはグルコース残基を意味し、ｎは１以上の整数を意味し、Ｔはα，α−トレハロースを意味する。）であると判断される。
【００６３】
【実験Ａ−６ 末端にトレハロース構造を有する非還元性糖質とトレハロースの調製】
澱粉部分分解物（松谷化学工業株式会社製造、商品名パインデックス＃４）４０重量部を水６０重量部に加熱溶解し、この溶液を４５℃、ｐＨ６．５に調整した後、実験Ａ−２の方法で調製した非還元性糖質生成酵素を還元性澱粉部分分解物ｇ当たり１単位の割合になるように加えて９６時間反応させ、末端にトレハロース構造を有する非還元性糖質を生成させ、次いで１００℃で１０分間加熱して、酵素を失活させた。本反応液を濃度約２０％まで希釈し、グルコアミラーゼ（ナガセ生化学工業株式会社製造、商品名グルコチーム）を澱粉部分分解物ｇ当たり１０単位加えて４０時間反応させ、次いで加熱して、酵素を失活させた。本溶液を、常法にしたがって、活性炭にて脱色し、イオン交換樹脂にて脱塩し、濃度約６０％に濃縮した。本糖液中には固形物当たり２９．５％のトレハロースを含有していた。この濃縮液を塩型強酸性カチオン交換樹脂（オルガノ株式会社販売、商品名ＣＧ６０００、Ｎａ型）が充填されたジャケット付きステンレス製カラムに、６０℃、ＳＶ ０．４でチャージし、トレハロース高含有画分を採取した。本高含有液は、固形物当たり約９０％のトレハロースを含有していた。本溶液を濃度約７５％に濃縮した後、助晶缶にとり、種晶としてトレハロース含水結晶を約２％加えて徐冷し、晶出率約４５％のマスキットを得た。本マスキットを乾燥塔上のノズルより１５０ｋｇ／ｃｍ²の高圧にて噴霧した。これと同時に８５℃の熱風を乾燥塔の上部より送風し、底部に設けた移送金網コンベア上に結晶粉末を補集し、コンベアの下より４５℃の温風を送りつつ、該粉末を乾燥塔外に徐々に移動させて、取り出した。この結晶粉末を熟成塔に充填して温風を送りつつ、１０時間熟成させ、結晶化と乾燥を完了し、トレハロース含水結晶粉末を得た。
【００６４】
【実験Ｂ−１ マルトシルグルコシドの調製】
純度９８％のマルトペンタオース（株式会社林原生物化学研究所製造）５重量部を水１０重量部に加熱溶解し、ｐＨ７．０、温度４０℃に調整し、これに実験Ａ−２の方法で調製した非還元性糖質生成酵素をマルトペンタオースグラム当たり２単位になるように加えた。４８時間反応させた後、１００℃で２０分間加熱して、酵素を失活させた。本溶液中には、マルトテトラオシルグルコシドを固形物当たり約８８％含有していた。
【００６５】
このマルトテトラオシルグルコシド含有溶液を、ｐＨ５．５、温度４０℃に調整し、β−アミラーゼ（ナガセ生化学工業株式会社製造、商品名＃１５００）を固形物グラム当たり２０単位になるように加えて、２４時間反応させた後、１００℃で２０分間加熱して、酵素を失活させた。本液には、マルトシルグルコシドを固形物当たり約５３％含有しており、その他に、マルトース、マルトトリオースが含まれていた。
【００６６】
本液は、常法にしたがって、活性炭にて脱色し、イオン交換樹脂（Ｈ型及びＯＨ型）にて脱塩し、固形物濃度約５０％に濃縮した。次いで、この濃縮液を、実験Ａ−６の方法に準じて、塩型強酸性カチオン交換樹脂を用いるカラムクロマトグラフィーにかけてマルトシルグルコシド高含有画分を採取した。本液は、固形物当たり約８１％のマルトシルグルコシドを含有していた。
【００６７】
更に、純度を高めるために、この溶液に、水酸化ナトリウムを０．１Ｎとなるように加え、１００℃で２時間加熱し、還元性糖質を分解した。この反応液は、常法にしたがって、脱色、脱塩し、固形物濃度約５０％に濃縮した。本液は、固形物当たり約９４％のマルトシルグルコシドを含有していた。採取されたマルトシルグルコシド高含有液を、更にオクタデシルシリカゲルを担体とした分取用液体クロマトグラフィー（株式会社ＹＭＣ製造、分取用カラムＹＭＣ−Ｐａｃｋ Ｒ−３５５−１５）にかけ、マルトシルグルコシド純度約９９％以上の高含有画分を採取した。
【００６８】
この画分を常法にしたがって、更に精製し、濃度７８％に濃縮し、ビーカーに入れ、４℃の冷室に放置したところ、結晶の析出が認められた。本結晶を遠心分離して集め、少量の水で洗浄し、純度９９．９％の含水結晶を得た。また、本品を１００℃で一夜減圧乾燥して、無水結晶を得た。
【００６９】
これら結晶の理化学的性質を調べたところ、次の通りであった。
（１）元素分析（無水物として）
含水結晶 Ｃ＝４２．４％ Ｈ＝６．８％
無水結晶 Ｃ＝４２．６％ Ｈ＝６．７％
理論値 Ｃ＝４２．８６％ Ｈ＝６．３９％
（２）質量分析（無水物として）
ＭＷ＝５０４（分子式Ｃ₁₈Ｈ₃₂０₁₆）
（３）水分
カールフィッシャー法にて測定
含水結晶 ９．９％（マルトシルグルコシド１モルに水３モル）
無水結晶 ０．３％
（４）融点
マルトシルグルコシドの含水結晶又は無水結晶を融点測定器に入れ、昇温速度を毎分１℃で融点を観察したところ、いずれの結晶も１８０℃で融解した。このことは、両結晶の融点が必ずしも同一であることを意味しているとは限らず、例えば、含水結晶の場合には、加熱昇温中に外観上変化することなく、含水結晶から無水結晶に変わり、この無水結晶の融解現象を観察していることが考えられる。
（５）比旋光度（無水物として）
含水結晶、無水結晶とも、
［α］_D ²⁰ ＋２０７．９°（ｃ＝５．０、Ｈ₂Ｏ）
（６）紫外線吸収スペクトル
含水結晶、無水結晶とも、水溶液にして測定すると特徴ある吸収は示さない。
（７）赤外線吸収スペクトル
含水結晶又は無水結晶の粉末２ｍｇをそれぞれ乾燥ＫＢｒ ２００ｍｇと攪拌、混合して透明なタブレットを作成し、赤外線吸収スペクトルを測定した。結果は、含水結晶、無水結晶ともほぼ同一のパターンを示した。図５に含水結晶の結果を示す。
（８）溶解度（無水物として）
含水結晶、無水結晶ともに、２５℃で水１００ｍｌに対し、約１６３ｇ溶解する。
（９）甘味度（無水物として）
マルトシルグルコシド２５％水溶液の甘味は、蔗糖１０％水溶液のそれとほぼ同等であることから、蔗糖の約４０％の甘味度である。なお、その甘味の質は、極めて優れている。
（１０）呈色反応
含水結晶、無水結晶ともアントロン−硫酸反応で緑色を呈し、フェーリング氏液還元反応、よう素反応は陰性。
（１１）構造（無水物として）
（ａ）１Ｎ−硫酸で加水分解するとＤ−グルコースのみを生成する。
（ｂ）グルコアミラーゼの作用により、グルコース１モルとトレハロース１モルを生成した。
（ｃ）炭素核磁気共鳴分析（¹³Ｃ−ＮＭＲ）の結果を図６に示す。
クラウス・ボック（ＫＬＡＵＳ ＢＯＣＫ）等が『アドバンス・イン・カーボハイドレート・ケミストリー・アンド・バイオケミストリー（Ａｄｖａｎｃｅｓ ｉｎ Ｃａｒｂｏｈｙｄｒａｔｅ Ｃｈｅｍｉｓｔｒｙ ａｎｄ Ｂｉｏｃｈｅｍｉｓｔｒｙ）』、第４２巻、第１９３乃至２２５頁（１９８４年）で報告している標準物質、α−Ｄ−グルコピラノース、α，α−トレハロース及びマルトオリゴ糖の化学シフトより、各炭素を帰属し、本物質は、Ｏ−α−Ｄ−グルコピラノシル−（１→４）−α−Ｄ−グルコピラノシル α−Ｄ−グルコピラノシドすなわちマルトシルグルコシドの構造を有していると判断される。
（１２）粉末Ｘ線回折
エフ・エイチ・ストドーラ（Ｆ．Ｈ．Ｓｔｏｄｏｌａ）等が、『ジャーナル・オブ・ザ・アメリカン・ケミカル・ソサイエティー（Ｊｏｕｒｎａｌ ｏｆ ｔｈｅ Ａｍｅｒｉｃａｎ Ｃｈｅｍｉｃａｌ Ｓｏｃｉｅｔｙ）』、第７８巻、第２５１４乃至２５１８頁（１９５６年）に報告されている方法に準じて、結晶マルトシルグルコシドのＣｕＫα線を使用した粉末Ｘ線回折図形を測定した。含水結晶の結果を図７に、無水結晶の結果を図８に示した。図７、図８の結果より明らかなように含水結晶は粉末Ｘ線回折法における主な回折角（２θ）として７．９°、１３．２°、１７．２°及び２２．６°を有し、また、無水結晶は７．７°、１３．５°、１７．６°及び２３．６°を有する。
【００７０】
以上の事実より本発明の結晶は、従来全く知られていないマルトシルグルコシドの結晶であると判断される。
【００７１】
【実験Ｂ−２ 結晶マルトシルグルコシドと非晶質マルトシルグルコシドに及ぼす吸湿性とその影響】
含水結晶マルトシルグルコシド、無水結晶マルトシルグルコシド及び非晶質マルトシルグルコシドをそれぞれ、粉末状態で約１ｇずつポリエチレン製容器にとり、相対湿度９０．１％の高湿度雰囲気に放置して、経時的にそれらの水分を測定し、粉末の状態の変化を観察した。試料としては、含水結晶及び無水結晶は、実験Ｂ−１の方法で調製したものを用い、非晶質は、その含水結晶を脱イオン水で溶解し、真空乾燥して調製した。結果は、表５にまとめた。
【００７２】
【表５】

【００７３】
表５の結果から明らかなように、非晶質マルトシルグルコシドは、著しく吸湿して固結し、容易に流動性を失うことが判明した。これに対して、含水結晶は、吸湿が少なく、流動性を保持して、取扱い容易であることが判明した。また、無水結晶は、吸湿するものの、外観上変化なく、流動性を保持することから、無水結晶から含水結晶に変化して、安定しているものと考えられる。
【００７４】
【実験Ｂ−３ 急性毒性試験】
マウスを使用して、実験Ｂ−１の方法で調製したマルトシルグルコシドの含水結晶及び無水結晶をそれぞれに経口投与して急性毒性テストを行った。その結果、これら結晶マルトシルグルコシドは低毒性の物質で、投与可能な最大投与量においても死亡例は認められなかった。したがって、正確な値はいえないが、そのＬＤ５０値は、いずれも５０ｇ／ｋｇ以上であった。
【００７５】
以下、本発明の結晶マルトシルグルコシドの製造方法を実施例Ａで、その用途を実施例Ｂで示す。
【００７６】
【実施例Ａ−１】
純度９５％のマルトペンタオース（株式会社林原生物化学研究所製造）５０重量部を水２００重量部に加熱溶解し、ｐＨ７．０、温度４０℃に調整した後、実験Ａ−２の方法で調製した非還元性糖質生成酵素を、マルトペンタオースグラム当たり５単位になるように加え、２４時間反応させた。この反応液を加熱して、酵素を失活させた。本液には、マルトテトラオシルグルコシドを固形物当たり約８３％含有していた。本液を固形物濃度約５０％に濃縮し、冷室に放置し、マルトテトラオシルグルコシドの結晶を析出させ、濾過して、結晶マルトテトラオシルグルコシドを採取した。
【００７７】
本結晶マルトテトラオシルグルコシド１０重量部を水１００重量部に加熱溶解し、ｐＨ５．５、温度５０℃に調整し、β−アミラーゼ（ナガセ生化学工業株式会社製造、商品名＃１５００）を固形物グラム当たり２０単位になるように加えた。２４時間反応させた後、１００℃で２０分間加熱して、酵素を失活させた。本液にはマルトシルグルコシドを固形物当たり約５８％含有しており、その他にマルトースと少量のマルトトリオースが含まれていた。
【００７８】
本液は、常法にしたがって、活性炭にて脱色し、イオン交換樹脂（Ｈ型及びＯＨ型）にて脱塩し、固形物濃度約５０％に濃縮した。この濃縮液を塩型強酸性カチオン交換樹脂（オルガノ株式会社製造、商品名ＸＴ−１０１６、Ｎａ型）を充填したジャケット付きステンレス製カラム（５．４ｃｍ×４ｍを４本直列に接続）に、６０℃、ＳＶ０．４でチャージし、ついで、６０℃温水で溶出してマルトシルグルコシド高含有画分を採取した。本液は、固形物当たり約９２％のマルトシルグルコシドを含有していた。
【００７９】
本液は、常法にしたがって、活性炭にて脱色し、イオン交換樹脂（Ｈ型及びＯＨ型）にて脱塩し、固形物濃度約８０％に濃縮し、これを助晶缶にとり、結晶マルトシルグルコシドを種晶として２％添加し、徐冷しつつ、ゆっくり攪拌しながら助晶した。得られたマスキットは、バスケット型遠心機で分蜜し、少量の水で洗浄したのち、乾燥した。純度９９．４％の含水結晶マルトシルグルコシドを採取した。
【００８０】
本品は、実質的に吸湿性を示さず、取扱いが容易であり、甘味料、呈味改良剤、品質改良剤、安定剤、賦形剤などとして飲食物、化粧品、医薬品、成形物などの各種組成物に有利に利用できる。
【００８１】
【実施例Ａ−２】
炭酸カルシウム０．１％を含む濃度約２０％の馬鈴薯澱粉を、ｐＨ６．０に調整した後、α−アミラーゼ（ノボノルディスク インダストリー社製造、商品名ターマミール６０Ｌ）を澱粉固形物グラム当たり０．３％加え、９５乃至１００℃に加熱して、澱粉を糊化、液化した後、酵素を加熱失活させてＤＥ１９．５の液化液を得た。
【００８２】
本液をｐＨ６．２、温度４０℃に調整し、これに実験Ａ−２の方法で調製した非還元性糖質生成酵素を澱粉グラム当たり３単位、イソアミラーゼ（株式会社林原生物化学研究所製造）を１，０００単位の割合となるように加え、７２時間反応させた。この反応液を加熱して酵素を失活させた後、冷却し、ｐＨを５．５、温度５０℃に調整した後、β−アミラーゼ（ナガセ生化学工業株式会社製造、商品名＃１５００）を澱粉固形物グラム当たり５０単位になるように加え、２４時間反応させた。この反応液を加熱して、酵素を失活させた。本液には、マルトシルグルコシドを固形物当たり約２１％含有していた。
【００８３】
これを実施例Ａ−１と同様に精製、濃縮し、塩型強酸性カチオン交換樹脂を用いるカラムクロマトグラフィーにかけて、マルトシルグルコシド高含有画分を採取した。本液は、固形物当たり約８９％のマルトシルグルコシドを含有していた。
【００８４】
本液は常法にしたがって、活性炭にて脱色し、イオン交換樹脂（Ｈ型及びＯＨ型）にて脱塩し、固形物濃度約８５％に濃縮し、結晶マルトシルグルコシドを、種結晶として２％添加し、徐冷しつつゆっくり攪拌しながら、助晶した。得られたマスキットは、バスケット型遠心機で分蜜し、少量の水で洗浄した後、乾燥した。純度９８．１％の含水結晶マルトシルグルコシドを採取した。
【００８５】
本品は、実質的に吸湿性を示さず、取扱いが容易であり、甘味料、呈味改良剤、品質改良剤、安定剤、賦形剤などとして飲食物、化粧品、医薬品、成形物などの各種組成物に有利に利用できる。
【００８６】
【実施例Ａ−３】
澱粉部分分解物（松谷化学工業株式会社製造、商品名パインデックス＃４）を用いて、濃度２０％、ｐＨ５．５、温度４５℃に調整し、イソアミラーゼを澱粉部分分解物グラム当たり５００単位加え、２４時間反応し、澱粉由来のα−１，６結合を分解した。反応液を１００℃に加熱し、１０分間保った後、４０℃に冷却し、ｐＨを６．５に調整した。これに実験Ａ−２の方法で調製した非還元性糖質生成酵素を固形物グラム当たり５単位加え、４８時間反応させ、その反応液を１００℃に加熱し、１０分間保った後、冷却した。温度を５０℃に調整した後、β−アミラーゼを固形物グラム当たり５０単位、α−アミラーゼを１０単位となるように加え、２４時間反応させた後、１００℃で２０分間加熱し、酵素を失活させた。本液には、マルトシルグルコシドを固形物当たり約２４％含有していた。
【００８７】
これを実施例Ａ−１と同様に、精製、濃縮し、塩型強酸性カチオン交換樹脂を用いるカラムクロマトグラフィーにかけてマルトシルグルコシド高含有画分を採取した。本液は、固形物当たり約９１％のマルトシルグルコシドを含有していた。
【００８８】
本液は、常法にしたがって、活性炭にて脱色し、イオン交換樹脂（Ｈ型及びＯＨ型）にて脱塩し、固形物濃度約８５％に濃縮し、結晶マルトシルグルコシドを、種晶として３％添加し、ゆっくり攪拌しながら助晶し、晶出率約４５％のマスキットを得た。
【００８９】
本マスキットを乾燥塔上のノズルより１５０ｋｇ／ｃｍ²の高圧で噴霧した。 これと同時に８５℃の熱風を乾燥塔の上部より送風し、乾燥塔底部に設けた移送金網コンベア上に結晶粉末を捕集し、コンベアの下より４５℃の温風を送りつつ、該粉末を乾燥塔外に徐々に移動させて取りだした。この粉末結晶を熟成塔に充填し、温風を送りつつ、１０時間熟成させ、結晶化と乾燥を完了し、含水結晶マルトシルグルコシド含有粉末を得た。
【００９０】
本品は、実質的に吸湿性を示さず、取扱いが容易であり、甘味料、呈味改良剤、品質改良剤、安定化剤、賦形剤などとして飲食物、化粧品、医薬品、成形物などの各種組成物に有利に利用できる。
【００９１】
【実施例Ａ−４】
実施例Ａ−３と同様の方法で澱粉部分分解物にイソアミラーゼ、非還元性糖質生成酵素、次いで、β−アミラーゼとα−アミラーゼを作用させた後、分離、精製して得られたマルトシルグルコシド高含有液を濃度約８０％まで濃縮した。この濃縮液を助晶缶にとり、結晶マルトシルグルコシドを種晶として２％加え、ゆっくり攪拌しながら助晶し、次いで、バットに取り出し、ブロックを作製した。ブロックは、約２５℃で２日間静置し、熟成させた後、切削型粉砕機で粉砕し、流動乾燥し、分級し、含水結晶マルトシルグルコシド含有粉末を得た。
【００９２】
本品は、実質的に吸湿性を示さず、取扱いが容易であり、甘味料、呈味改良剤、品質改良剤、安定化剤、賦形剤などとして飲食物、化粧品、医薬品、成形物などの各種組成物に有利に利用できる。
【００９３】
【実施例Ａ−５】
実施例Ａ−１の方法で得た含水結晶マルトシルグルコシドを１００℃で一夜減圧乾燥して、水分約０．２％の無水結晶マルトシルグルコシドを得た。
【００９４】
本品は、室内に放置すると吸湿し、水分約１０％の含水結晶マルトシルグルコシドに容易に戻り安定化する。本無水結晶マルトシルグルコシドは、種晶として有利に用いることができる他、吸湿剤、脱水剤、化学原料などとして利用することも有利に用いることができる。更に、飲食物、化粧品、医薬品、成形物などの各種組成物に有利に用いることができる。
【００９５】
【実施例Ａ−６】
トレハロース１重量部及び澱粉部分分解物（松谷化学工業株式会社製造、商品名パインデックス＃４）１重量部を水３重量部に加熱溶解し、温度６５℃、ｐＨ６．０に調整し、これにバチルス・ステアロサーモフィラス由来のシクロマルトデキストリン・グルカノトランスフェラーゼ（株式会社林原生物化学研究所製造）を澱粉部分分解物グラム当たり１０単位加えて２４時間反応の後、酵素を加熱失活させた。次いで、温度５５℃に調整し、これにプルラナーゼ（株式会社林原生物化学研究所製造）及びβ−アミラーゼを澱粉部分分解物グラム当たりそれぞれ２５単位、５０単位加えて、２４時間反応させて、これら酵素を加熱失活させた。この反応液には、マルトシルグルコシドを固形物当たり約２５％含有していた。
【００９６】
これを実施例Ａ−１と同様に精製し、塩型強酸性カチオン交換樹脂を用いるカラムクロマトグラフィーにかけてマルトシルグルコシド高含有画分を得、これを脱色、脱塩し、濃縮、助晶し、バットに取り出してブロックを作製した。本ブロックを切削、乾燥、分級して、含水結晶マルトシルグルコシド含有粉末を原料当たり収率約１７％で得た。
【００９７】
本品は、実質的に吸湿性を示さず、取扱いが容易であり、甘味料、呈味改良剤、品質改良剤、安定剤、賦形剤などとして、飲食物、化粧品、医薬品、成形物などの各種組成物に有利に利用できる。
【００９８】
【実施例Ｂ−１ 甘味料】
実施例Ａ−１の方法で得た高純度含水結晶マルトシルグルコシド１重量部に、α−グリコシルステビオシド（東洋精糖株式会社販売、商品名αＧスイート）０．０５重量部を均一に混合し、顆粒成形機にかけて、顆粒状甘味料を得た。本品は、甘味の質が優れ、蔗糖の約２倍の甘味度を有し、甘味度当たりカロリーは、蔗糖の約１／２に低下している。本甘味料は、低カロリー甘味料として、カロリー摂取を制限している肥満者、糖尿病者などのための低カロリー飲食物などに対する甘味付けに好適である。また、本甘味料は、虫歯誘発菌による酸の生成が少なく、不溶性グルカンの生成も少ないことより、虫歯を抑制する飲食物などに対する甘味付けに好適である。
【００９９】
【実施例Ｂ−２ ハードキャンデー】
５５％蔗糖溶液１００重量部に実施例Ａ−３の方法で得た含水結晶マルトシルグルコシド含有粉末３０重量部を加熱混合し、次いで減圧下で水分２％未満になるまで加熱濃縮し、これにクエン酸１重量部及び適量のレモン香料と着色料とを混和し、常法にしたがって成形し、製品を得た。本品は、歯切れ、呈味良好で、蔗糖の晶出も起こらない高品質のハードキャンデーである。
【０１００】
【実施例Ｂ−３ いちごジャム】
生いちご１５０重量部、蔗糖６０重量部、マルトース２０重量部、実施例Ａ−４の方法で得た含水結晶マルトシルグルコシド粉末４０重量部、ペクチン５重量部及びクエン酸１重量部をなべで煮詰め、ビン詰して製品を得た。本品は、風味、色調とも良好なジャムである。
【０１０１】
【実施例Ｂ−４ クリームウエファース】
実施例Ａ−５の方法で得た高純度無水結晶マルトシルグルコシド２，０００重量部、ショートニング１，０００重量部、レシチン１重量部、レモンオイル１重量部、バニラオイル１重量部を、常法により配合して得たクリームを加温して４０乃至４５℃に保ちウエファースに挟んでクリームウエファースを製造した。本品は、クリームの溶け具合がなめらかで風味も良好である。
【０１０２】
【実施例Ｂ−５ 加糖練乳】
原乳１００重量部に実施例Ａ−２の方法で得た高純度含水結晶マルトシルグルコシド５重量部を溶解し、プレートヒーターで加熱殺菌し、次いで濃度約７０％に濃縮し、無菌状態で缶詰して製品を得た。本品は、温和な甘味で、風味もよく、乳幼児食品、フルーツ、コーヒー、ココア、紅茶などの調味用に有利に利用できる。
【０１０３】
【実施例Ｂ−６ 乳酸飲料】
脱脂乳１０重量部を８０℃で２０分間加熱殺菌した後、４０℃に冷却し、これにスターター０．３重量部を加えて約３７℃で１０時間醗酵させた。次いで、これをホモゲナイズした後、実施例Ａ−２の方法で得た高純度含水結晶マルトシルグルコシド５重量部及び異性化糖シラップ２重量部を加えて７０℃に保って殺菌した。これを冷却し、適量の香料を加え、ビン詰して製品を得た。本品は、風味、甘味が酸味とよく調和した高品質の乳酸飲料である。
【０１０４】
【実施例Ｂ−７ 粉末ジュース】
噴霧乾燥により製造したオレンジ果汁粉末３３重量部に対し、実施例Ａ−２の方法で得た高純度含水結晶マルトシルグルコシド５０重量部、蔗糖１０重量部、無水クエン酸０．６５重量部、リンゴ酸０．１重量部、Ｌ−アスコルビン酸０．１重量部、クエン酸ソーダ０．１重量部、プルラン０．５重量部、粉末香料適量をよく混合撹拌し、粉砕し微粉末にしてこれを流動層造粒機に仕込み、排風温度４０℃とし、これに、実施例Ａ−２の方法で得た高含有マルトシルグルコシド濃縮液をバインダーとしてスプレーし、３０分間造粒し、計量、包装して製品を得た。本品は、果汁含有率約３０％の粉末ジュースである。また、本品は異味、異臭がなく、吸湿固結も起こさず長期に安定であった。
【０１０５】
【実施例Ｂ−８ チョコレート】
カカオペースト４０重量部、カカオバター１０重量部、蔗糖２０重量部、実施例Ａ−１の方法で得た高純度含水結晶マルトシルグルコシド３０重量部を混合してレファイナーに通して粒度を下げた後、コンチェに入れて５０℃で２昼夜練り上げる。この間に、レシチン０．５重量部を加え充分に混和分散させた。次いで、温度調節機で３１℃に調節し、バターの固まる直前に型に流し込み、振動機でアワ抜きを行い、１０℃の冷却トンネルを２０分間くぐらせて固化させた。これを型抜きして包装し製品を得た。本品は、吸湿性がなく、色、光沢共によく、内部組織も良好で、口中でなめらかに溶け、上品な甘味とまろやかな風味を有する。
【０１０６】
【実施例Ｂ−９ チューインガム】
ガムベース３重量部を柔らかくなる程度に加熱溶融し、これに無水結晶マルチトール（株式会社林原商事販売 登録商標マビット）４重量部及び実施例Ａ−２の方法で得た高純度含水結晶マルトシルグルコシド３重量部とを加え、更に適量の香料と着色料とを混合し、常法にしたがって、ロールにより練り合わせ、成形、包装して製品を得た。本品は、テクスチャー、風味とも良好なチューインガムである。
【０１０７】
【実施例Ｂ−１０ カスタードクリーム】
コーンスターチ１００重量部、実施例Ａ−３の方法で得た含水結晶マルトシルグルコシド含有粉末１００重量部、マルトース（株式会社林原商事販売 登録商標サンマルト）８０重量部、蔗糖２０重量部及び食塩１重量部を充分に混合し、鶏卵２８０重量部を加えて撹拌し、これに沸騰した牛乳１，０００重量部を徐々に加え、更に、これを火にかけて撹拌を続け、コーンスターチが完全に糊化して全体が半透明になった時に火を止め、これを冷却して適量のバニラ香料を加え、計量、充填、包装して製品を得た。本品は、なめらかな光沢を有し、温和な甘味で美味である。
【０１０８】
【実施例Ｂ−１１ ういろうの素】
米粉９０重量部に、コーンスターチ２０重量部、実施例Ａ−６の方法で得た含水結晶マルトシルグルコシド含有粉末１２０重量部、プルラン４重量部を均一に混合してういろうの素を製造した。ういろうの素と適量の抹茶と水とを混練し、これを容器に入れて６０分間蒸し上げて抹茶ういろうを製造した。本品は、照り、口当たりも良好で、風味も良い。また、澱粉の老化も抑制され、日持ちもよい。
【０１０９】
【実施例Ｂ−１２ 即席コーンポタージュスープ】
α−化コーン粉末３０重量部、α−化小麦粉５重量部、α−化ポテトスターチ４重量部、α−化ワキシーコーンスターチ１２重量部、実施例Ａ−３の方法で得た含水結晶マルトシルグルコシド含有粉末８重量部、グルタミン酸ナトリウム５重量部、食塩８．５重量部、脱脂粉乳７重量部、オニオンパウダー０．５重量部を磨砕してよく混合した後、これにソルビタン脂肪酸エステル０．５重量部と植物性硬化油９重量部とを加熱融解したものを添加して混合し、更に、乳糖１０重量部を加えて混合し、これを実施例Ｂ−７と同様に流動層造粒機に仕込んで少量の水をスプレーし、造粒した後、７０℃の熱風で乾燥し、篩別して即席コーンポタージュスープを製造した。本品は、熱湯を注げば、容易に溶解、分散し、風味の優れたスープとなる。
【０１１０】
【実施例Ｂ−１３ べったら漬の素】
実施例Ａ−４の方法で得た含水結晶マルトシルグルコシド含有粉末４重量部、甘草製剤０．０５重量部、リンゴ酸０．００８重量部、グルタミン酸ナトリウム０．０７重量部、ソルビン酸カリウム０．０３重量部及びプルラン０．２重量部を均一に混合してべったら漬の素を製造した。大根３０ｋｇを常法にしたがって、食塩により下漬し、次いで蔗糖で中漬したものを、本べったら漬の素４ｋｇで調製した調味液に漬けてべったら漬を製造した。本品は、色、艶、香気ともに良好で、適度の甘味を有し、歯切れもよかった。また、本品は、酸敗しにくく長期間安定であった。
【０１１１】
【実施例 Ｂ−１４ 経管栄養剤】
実施例Ａ−１の方法で得た高純度含水結晶マルトシルグルコシド２０重量部、グリシン１．１重量部、グルタミン酸ナトリウム１重量部、乳酸カルシウム０．４重量部、炭酸マグネシウム０．１重量部、チアミン０．０１重量部及びリボフラビン０．０１重量部からなる配合物を調製する。この配合物２４ｇずつをラミネートアルミ製小包に充填し、ヒートシールして製品を得た。本品は、１袋分を約３３乃至５００ｍｌの水に溶解して栄養補給液とし、経管方法により、鼻腔、胃腸などへ投与して使用する。本品は、ヒトのみならず、家畜などへの非経口的栄養補給液としても有利に利用できる。
【０１１２】
【実施例Ｂ−１５ 経管栄養剤】
実施例Ａ−２の方法で得た高純度含水結晶マルトシルグルコシド５８０重量部、乾燥卵黄１９０重量部、脱脂粉乳２０９重量部、塩化ナトリウム４．４重量部、塩化カリウム１．８５重量部、硫酸マグネシウム４重量部、チアミン０．０１重量部、アスコルビン酸ナトリウム０．１重量部、ビタミンＥアセテート０．６重量部及びニコチン酸アミド０．０４重量部からなる配合物を調製する。この配合物２５ｇずつを、ラミネートアルミ製小包に充填し、ヒートシールして製品を得た。本品は、１袋分を約１５０乃至３００ｍｌの水に溶解して、栄養補給液とし、経管方法により鼻腔、食道、胃などへ投与して使用する。
【０１１３】
【実施例Ｂ−１６ 外傷治療用膏薬】
実施例Ａ−１の方法で得た高純度含水結晶マルトシルグルコシド３００重量部及び結晶マルトース２００重量部に、ヨウ素３重量部を溶解したメタノール５０重量部を加え混合し、更に１０％プルラン水溶液２００重量部を加えて混合し、適度の延び、付着性を示す外傷治療用膏薬を得た。本品は、治癒期間が短縮され、創面もきれいに治る。
【０１１４】
【実施例Ｂ−１７ インターフェロン液剤】
ヒト天然型インターフェロン−γ標品（株式会社林原生物学研究所製造、コスモ・バイオ株式会社販売）を、常法にしたがって、固定化抗ヒトインターフェロン−γ抗体カラムにかけ、該標品に含まれるヒト天然型インターフェロン−γを吸着させ、安定剤である牛血清アルブミンを素通りさせて除去し、次いで、ｐＨを変化させて、ヒト天然型インターフェロン−γを実施例Ａ−１の方法で得た高純度含水結晶マルトシルグルコシドを７％含有する生理食塩水を用いて溶出した。本液を精密濾過し、無菌的にバイアルビンに充填して、ｍｌ当たりヒト天然型インターフェロン−γを１０⁵単位含有する液剤を得た。本品は、１日当たり、大人 １乃至２０ｍｌ程度が経口的又は非経口的に投与され、ウイルス性疾患、アレルギー性疾患、リューマチ、糖尿病、悪性腫瘍などの治療に有利に利用できる。本品は、マルトシルグルコシドが安定剤として作用し、４℃又は２５℃で２０日間放置しても、その活性をよく維持している。
【０１１５】
【実施例Ｂ−１８ ツモア・ネクロシス・ファクター液剤】
ヒト天然型ツモア・ネクロシス・ファクター−α標品（株式会社林原生物化学研究所製造、コスモ・バイオ株式会社販売）を、常法にしたがって、固定化抗ヒトツモア・ネクロシス・ファクター−α抗体カラムにかけ、該標品に含まれるヒト天然型ツモア・ネクロシス・ファクター−αを吸着させ、安定剤である牛血清アルブミンを素通りさせて除去し、次いで、ｐＨを変化させて、ヒト天然型ツモア・ネクロシス・ファクター−αを実施例Ａ−１の方法で得た高純度含水結晶マルトシルグルコシドを１０％含有する生理食塩水を用いて溶出した。本液を精密濾過し、無菌的にバイアルビンに充填して、ｍｌ当たりヒト天然型ツモア・ネクロシス・ファクター−αを１０^４単位含有する液剤を得た。本品は、１日当たり、大人１乃至２０ｍｌ程度が経口的又は非経口的に投与され、ウイルス性疾患、アレルギー性疾患、リューマチ、糖尿病、悪性腫瘍などの治療に有利に利用できる。本品は、マルトシルグルコシドが安定剤として作用し、４℃又は２５℃で２０間放置しても、その活性をよく維持している。
【０１１６】
【実施例 Ｂ−１９ インターフェロン錠剤】
ヒト天然型インターフェロン−α標品（株式会社林原生物化学研究所製造、コスモ・バイオ株式会社販売）を、常法にしたがって、固定化抗ヒトインターフェロン−α抗体カラムにかけ、該標品に含まれるヒト天然型インターフェロン−αを吸着させ、安定剤である牛血清アルブミンを素通りさせて除去し、次いでｐＨを変化させて、ヒト天然型インターフェロン−αを実施例Ａ−１の方法で得た高純度含水結晶マルトシルグルコシドを５％含有する生理食塩水を用いて溶出した。本液を精密濾過し、約２０倍量の無水結晶マルトース粉末（株式会社林原商事販売、商品名ファイントースＴ）に加えて脱水、粉末化し、これを打錠機にて打錠し、１錠（約２００ｍｇ）当たりヒト天然型インターフェロン−αを約１５０単位含有する錠剤を得た。本品は、舌下錠などとして、１日当たり、大人１乃至１０錠程度が経口的に投与され、ウイルス性疾患、アレルギー性疾患、リューマチ、糖尿病、悪性腫瘍などの治療に有利に利用できる。とりわけ、近年、患者数の急増しているエイズ、肝炎などの治療剤として有利に利用できる。本品は、マルトシルグルコシドと共にマルトースが安定剤として作用し、室温で放置してもその活性を長期間よく維持する。
【０１１７】
【実施例Ｂ−２０ 糖衣錠】
重量１５０ｍｇの素錠を芯剤とし、これに実施例Ａ−２の方法で得た高純度含水結晶マルトシルグルコシド４５重量部、プルラン（平均分子量２０万）２重量部、水３０重量部、タルク２５重量部及び酸化チタン３重量部からなる下掛け液を用いて錠剤重量が約２３０ｍｇになるまで糖衣し、次いで、同じ含水結晶マルトシルグルコシド含有粉末６５重量部、プルラン１重量部及び水３４重量部からなる上掛け液を用いて糖衣し、更に、ロウ液で艶出しして光沢のある外観の優れた糖衣錠を得た。本品は、糖衣掛け時の作業性が優れているだけでなく、耐衝撃性にも優れており、高品質を長期間維持する。
【０１１８】
【実施例Ｂ−２１ 乳液】
ポリオキシエチレンベヘニルエーテル０．５重量部、テトラオレイン酸ポリオキシエチレンソルビトール１重量部、親油型モノステアリン酸グリセリン１重量部、ベヘニールアルコール０．５重量部、アボガド油１重量部、実施例Ａ−２の方法で得た高純度含水結晶マルトシルグルコシド３．５重量部、α−グリコシルルチン１重量部、ビタミンＥ及び防腐剤の適量を、常法にしたがって加熱溶解し、これに１，３−ブチレングリコール５重量部、カルボキシビニルポリマー０．１重量部及び精製水８５．３重量部を加え、ホモゲナイザーにかけ、乳化し、乳液を製造した。本品は、保湿性ある乳液で、日焼け止め、色白剤などとして有利に利用できる。
【０１１９】
【実施例Ｂ−２２ スキンクリーム】
モノステアリン酸ポリオキシエチレングリコール２重量部、自己乳化型モノステアリン酸グリセリン５重量部、α−グリコシルルチン２重量部、流動パラフィン１重量部、トリオクタン酸グリセリル１０重量部、実施例Ａ−１の方法で得た高純度含水結晶マルトシルグルコシド１重量部、マルチトール３重量部及び防腐剤の適量を、常法にしたがって加熱溶解し、これに１，３−ブチレングリコール５重量部及び精製水６６重量部を加え、ホモゲナイザーにかけて乳化し、更に、香料の適量を加えて撹拌混合し、クリームを製造した。本品は、伸びの良いクリームで、日焼け止め、美肌剤、色白剤などとして有利に使用できる。
【０１２０】
【実施例Ｂ−２３ 練歯磨】
第二リン酸カルシウム４５重量部、ラウリル硫酸ナトリウム１．５重量部、グリセリン２５重量部、ポリオキシエチレンソルビタンラウレート０．５重量部、実施例Ａ−２の方法で得た高純度含水結晶マルトシルグルコシド１５重量部、サッカリン０．０２重量部及び防腐剤０．０５重量部を水１３重量部と混合して練歯磨を得た。本品は、光沢、洗浄力も良好で、練歯磨として好適である。
【０１２１】
【実施例Ｂ−２４ 肥料杭】
配合肥料（Ｎ＝１４％、Ｐ₂Ｏ₅＝８％、Ｋ₂Ｏ＝１２％）、プルラン、実施例 Ａ−６の方法で得た含水結晶マルトシルグルコシド含有粉末、硫酸カルシウム及び水の割合を重量でそれぞれ７０、５、５、１５及び５として充分混合した後、押出機（Ｌ／Ｄ＝２０、圧縮比＝１．８、ダイスの口径＝３０ｍｍ）で８０℃に加熱して肥料杭を製造した。本品は、肥料用容器が不要で取扱い容易であり、全層施肥に適した強度を有し、更に配合割合を変えることにより肥料成分の溶出速度を調節できる。必要ならば、この肥料杭に植物ホルモン、農業用薬剤及び土壌改良剤等の混合も容易である。
【０１２２】
【発明の効果】
上記から明らかなように、本発明の結晶マルトシルグルコシドは、実質的に非吸湿性乃至難吸湿性で流動性良好であって取扱い易く、水に溶解し易い物質であり、適度な粘性を有し、良質な甘味を有している。また、結晶マルトシルグルコシドは、化学的に安定であり、褐変反応を起こし易いアミノ酸、オリゴペプチド、更には、有効成分、活性の失われ易い生理活性物質などを安定化し得る性質を有している。加えて、浸透圧調節性、賦活性、照り付与性、保湿性、他の糖の晶出防止、難発酵性、澱粉の老化防止性などの性質を具備している。これら諸性質は、飲食物、化粧品、医薬品、成形物など各種組成物の製造に有利に利用できる。
【０１２３】
したがって、本発明の結晶マルトシルグルコシドとその製造方法並びに用途の確立は、飲食物、化粧品、医薬品分野における産業的意義が極めて大きい。
【図面の簡単な説明】
【図１】リゾビウム・スピーシーズ Ｍ−１１由来の非還元性糖質生成酵素の活性に及ぼす温度の影響を示す図である。
【図２】リゾビウム・スピーシーズ Ｍ−１１由来の非還元性糖質生成酵素の活性に及ぼすｐＨの影響を示す図である。
【図３】リゾビウム・スピーシーズ Ｍ−１１由来の非還元性糖質生成酵素の温度安定性を示す図である。
【図４】リゾビウム・スピーシーズ Ｍ−１１由来の非還元性糖質生成酵素のｐＨ安定性を示す図である。
【図５】含水結晶マルトシルグルコシドの赤外線吸収スペクトルを示す図である。
【図６】マルトシルグルコシドの炭素核磁気共鳴分析結果を示す図である。
【図７】含水結晶マルトシルグルコシドの粉末Ｘ線回折図形を示す図である。
【図８】無水結晶マルトシルグルコシドの粉末Ｘ線回折図形を示す図である。[0001]
[Industrial application fields]
The present invention relates to a novel crystalline carbohydrate and a production method and use thereof, and particularly relates to crystalline maltosyl glucoside and a production method and use thereof.
[0002]
[Prior art]
Maltotriose is a trisaccharide represented by the formula: O-α-D-glucopyranosyl- (1 → 4) -O-α-D-glucopyranosyl- (1 → 4) -D-glucopyranose. It has long been known as a sweet saccharide contained in the This carbohydrate is characterized by low sweetness and is used in compositions such as various beverages, processed foods, and favorite foods.
[0003]
However, since maltotriose is a reducing sugar, it has a drawback that it easily undergoes browning reaction with proteins, amino acids and the like contained in beverages, processed foods, and favorite foods, and is susceptible to alteration and deterioration. Therefore, the present inventors have intensively studied to eliminate this drawback, and have previously formed a trehalose structure from a reduced starch partial degradation product having a glucose polymerization degree of 3 or more described in Japanese Patent Application No. 5-349216. Non-reducing from maltotriose by the action of an enzyme that produces a non-reducing saccharide having the same (in this specification, this enzyme may be simply referred to as “non-reducing saccharide-forming enzyme”). It has been found to produce sex maltosyl glucoside. However, this non-reducing carbohydrate maltosyl glucoside powder is amorphous and physically unstable, and it is desired to establish more stable crystals.
[0004]
On the other hand, regarding maltosyl glucoside itself, for example, Von Werner Fischer et al., “Hoppe-Seiler's Zeitshirisfurt Physiology” 350, 1137 to 1147 (1969), the method of extracting from microbial cells, Shinkiti Koto et al., “Britin of Chemical Society of Reported in Japan (Bulletin of Chemical Society of Japan), Vol. 59, pp. 411-414 (1986), and also by Hans Peter Beh The preparation method is known by the chemical synthesis method reported by Cell (Hans Peter Wesel) et al. In “Helvetica Chimica Acta”, Vol. 74, pages 682 to 695 (1991). Although there is no description about the crystal, it is not yet known.
[0005]
[Problems to be solved by the invention]
The present invention intends to provide a method for establishing crystalline maltosyl glucoside, a method for producing crystalline maltosyl glucoside, and its use.
[0006]
[Means for Solving the Problems]
In order to solve the above problems, the present inventors have continued intensive studies. As a result, non-reducing saccharide-forming enzyme is allowed to act on maltopentaose, which is a partial degradation product of reducing starch, to convert maltopentaose into non-reducing maltotetraosyl glucoside, and then β- The liquid containing maltosyl glucoside obtained by the action of amylase was subjected to column chromatography using a salt-type strongly acidic cation exchange resin, and the liquid containing high maltosyl glucoside obtained by purification was concentrated and stored in a cold room. The present inventors have found a phenomenon in which crystals are precipitated, further elucidated that the crystals are crystalline maltosyl glucosides, established new crystalline maltosyl glucosides, established production methods and uses thereof, and completed the present invention. . At the same time, the presence of water-containing crystals and anhydrous crystals as crystalline maltosyl glucoside was elucidated, and the production method and use thereof were established, thereby completing the present invention.
[0007]
As a method for producing crystalline maltosyl glucoside, any method capable of producing the present crystal may be used. Usually, this is crystallized and collected from a solution containing maltosyl glucoside.
[0008]
In order to produce maltosyl glucoside, a chemical synthesis method may be used, but generally, a biochemical method using a glycosyltransferase or a non-reducing saccharide-forming enzyme is advantageous.
[0009]
As a method using a glycosyltransferase, for example, a glycosyltransferase is allowed to act on an aqueous solution containing trehalose and α-glucosyl saccharide, and for example, maltosyl glucoside, maltotriosyl glucoside, maltotetraosyl glucoside, etc. α-D-oligoglucosyl glucoside may be produced. If necessary, β-amylase or β-amylase and a starch debranching enzyme such as pullulanase or isoamylase are allowed to act on the maltosyl glucoside. It can also be advantageously carried out.
[0010]
In this case, the α-glucosyl saccharide may be any saccharide that can transfer one or more glucose residues to trehalose by α-1,4 linkage by the glycosyltransferase used. For example, starch 1 selected from saccharides having an α-glucosyl bond, such as gelatinized starch, liquefied starch, soluble starch, amylose, amylopectin, partial degradation product of reducing starch, sugar transfer product, cyclodextrin, dextrin, maltooligosaccharide, sucrose Species or two or more saccharides are appropriately employed.
[0011]
Usually, when the glycosyltransferase is cyclomaltodextrin glucanotransferase (EC 2.4.1.19) or α-amylase (EC 3.2.1.1), for example, starch, gelatinized starch, Amylopectin, amylose, reductive starch partial degradation product, cyclodextrin, dextrin, malto-oligosaccharide, etc. are suitable. In the case of α-glucosidase, malto-oligo such as maltose, maltotriose, maltotetraose, maltopentaose, etc. Sugar, dextrin, sucrose and the like are preferred.
[0012]
In order to produce maltosylglucoside using a non-reducing saccharide-forming enzyme, the enzyme may be allowed to act on high-purity maltotriose, but in general, a reduction with a glucose polymerization degree of 3 or more is possible. A non-reducing saccharide-forming enzyme is allowed to act on the partially degraded starch, and a non-reducing saccharide having a trehalose structure at the terminal, for example, α- such as maltosyl glucoside, maltotriosyl glucoside, maltotetraosyl glucoside, etc. D-oligoglucosyl glucoside may be generated. In this method, as in the case of using glycosyltransferase, if necessary, the starch debranching enzyme can be allowed to act together with β-amylase or β-amylase to increase the content of maltosylglucoside. .
[0013]
The reductive starch partial decomposition product may be anything that can generate maltosyl glucoside by a non-reducing saccharide-forming enzyme, or can generate a saccharide containing a maltosyl glucoside structure. Gelatinized starch, liquefied starch, soluble starch, amylopectin, amylose, dextrin, partially reduced product of reduced starch of DE1 to 40, maltooligosaccharide having a glucose polymerization degree of 3 or more can be advantageously used.
[0014]
As the non-reducing saccharide-forming enzyme used in the present invention, for example, as previously described in the specification of Japanese Patent Application No. 5-349216, the present inventors have already established 1-3 1-3 Higashi, Tsukuba City, Ibaraki Prefecture. Rhizobium sp. M-11 “Accession Number, JIKOH Jojo No. 4130 (FERM) deposited at the Institute of Biotechnology and Industrial Technology Research Institute of the Ministry of International Trade and Industry BP-4130) ”, Arthrobacter sp. Q36“ Accession Number, FERM BP-4316 ”, and other known microorganisms such as Brevibacterium herovolum ATCC 11822, Flavo Bacteria Aquatile erium aquatile IFO3772, Micrococcus luteus IFO3064, Micrococcus roseus ATCC 186, Curtobacterium citreum Ic 15231 Microorganisms capable of producing non-reducing saccharide-forming enzymes such as (Terrabacter tumescens) IFO12960 can be cultured in a nutrient medium, and this culture can be collected and used. If necessary, it can be appropriately purified by known methods. You may use it.
[0015]
The non-reducing saccharide-forming enzyme thus obtained generates a non-reducing saccharide having a trehalose structure at the end from one or more reducing starch partial degradation products selected from a glucose polymerization degree of 3 or more. Have the effect of
[0016]
These enzyme reaction conditions can be adopted as long as the target enzyme reaction proceeds. In general, the substrate concentration is about 1 to 50 w / w% (hereinafter, unless otherwise specified, w The reaction temperature is about 20 to 80 ° C., the reaction pH is about 4 to 10, and the reaction time is about 1 to 100 hours. Enzymes used in these reactions may be immobilized by a known method such as a carrier binding method, a crosslinking method, or a comprehensive method, if necessary, and used for continuous reactions or repeatedly in batch reactions. is there.
[0017]
Enzymatic reactions as described above usually contain about 1 to 67% maltosylglucoside per solid. In order to produce crystalline maltosyl glucoside from this reaction solution, generally, a contaminated substance is separated and purified to obtain a liquid containing a high amount of maltosyl glucoside, which is concentrated and crystallized crystal maltosyl glucoside is crystallized. It is advantageous to collect.
[0018]
As the separation and purification method, for example, a yeast fermentation method, a membrane filtration method, a fractional precipitation method, an alkali treatment method, a chromatography method and the like can be appropriately employed. In particular, by the column chromatography using a salt-type strongly acidic cation exchange resin disclosed in JP-A-58-23799, JP-A-59-148794, etc., contaminating saccharides are removed and maltosyl glucoside is increased. A method of collecting the contained liquid can be advantageously carried out. At this time, it is optional to adopt any of the known fixed bed method, moving bed method, and simulated moving bed method.
[0019]
In order to crystallize maltosylglucoside from the solution containing high maltosylglucoside thus obtained, appropriate conditions are selected depending on the type of crystal. As crystallization conditions, it is a supersaturated solution of maltosyl glucoside, and crystal maltosyl glucoside may be precipitated, and the production method of maltosyl glucoside itself is not limited.
[0020]
Specifically, maltosyl glucoside having a purity of 60% or more is set to a concentration of about 65 to 95% or more, and the temperature of the solution is such that the solution does not freeze and is below the melting point of the crystal. For example, in the case of water-containing crystals, a solution having a water content of about 10% or more is about 10 to 90 ° C, and in the case of anhydrous crystals, a solution having a water content of less than about 10% is about 90 to 150 ° C. Can be crystallized. Anhydrous crystals can also be easily produced by drying the hydrous crystals under reduced pressure or by heating. In addition, in order to adjust the supersaturation degree, viscosity, etc. of the solution at the time of crystallization, for example, it is optional to coexist methanol, ethanol, acetone or the like.
[0021]
The crystallization method usually involves taking a relatively high temperature supersaturated maltosyl glucoside-containing solution in an auxiliary crystal can, and allowing seed crystals to coexist in this, preferably 0.1 to 20%, and slowly cooling while slowly stirring. , Promote crystallization and make a mass kit.
[0022]
The method for producing crystalline maltosyl glucoside from the crystallized mass kit is not limited as long as crystalline maltosyl glucoside can be collected.For example, a known method such as a honey method, a block grinding method, a fluidized granulation method, or a spray drying method can be used. That's fine.
[0023]
For example, the honey method is usually a method of separating the crystalline maltosyl glucoside and honey by applying a basket-type centrifuge, and if necessary, it is easy to spray and wash the crystal with a small amount of cold water. Suitable for producing higher purity crystalline maltosyl glucoside. The other three methods do not separate nectar, so that the resulting crystalline product does not show an improvement in purity but has a high product yield. Therefore, in the case of this product, the crystalline product usually contains sugars derived from raw materials or production processes, such as trehalose, glucose, maltose, maltotriose, maltotetraose, etc. in addition to maltosylglucoside. It is.
[0024]
In the case of the spray drying method, usually, a mass kit having a crystallization rate of about 25 to 60% is sprayed from a nozzle with a high-pressure pump, dried at a temperature at which the crystal powder does not melt, for example, hot air of about 60 to 100 ° C. If dried and aged with warm air, non-hygroscopic or hardly hygroscopic crystal-containing powder can be easily produced.
[0025]
The block pulverization method is usually performed by allowing a mass kit having a crystallization rate of about 10 to 60% to stand for about 0.5 to 5 days to crystallize and solidify the whole into a block shape, which is then pulverized or cut. If crushed and dried, non-hygroscopic and hardly hygroscopic crystal-containing powder can be easily produced.
[0026]
In addition, a maltosyl glucoside-containing aqueous solution is heated and concentrated to a moisture content of less than 5% according to a conventional method to obtain a supersaturated maltosyl glucoside-containing solution in a molten state, and seed crystals are mixed into this solution at a temperature below the melting point of the crystal. It is also optional to produce a non-hygroscopic or hardly hygroscopic crystal-containing solid by forming it into various shapes such as powder, granules, rods, plates, cubes, and the like.
[0027]
The crystalline maltosyl glucoside thus obtained varies in its non-hygroscopicity somewhat depending on the purity of the maltosyl glucoside, but is substantially non-hygroscopic, fluid, and has a concern about adhesion and sticking. It is easy to handle with little, and material and human expenses required for management such as packaging, transportation and storage can be greatly reduced.
[0028]
The crystalline maltosyl glucoside of the present invention is a substantially non-hygroscopic or hardly hygroscopic powder, and has high heat resistance and good stability. , Chewing gum, instant juice, instant soup, granules, excipients such as tablets, bulking agents, powder bases, etc., for example, various compositions such as food, drinks, cosmetics, pharmaceuticals, molded products, as well as reagents, chemical industry It can be advantageously used in various applications such as raw materials.
[0029]
Crystalline maltosyl glucoside changes in physicochemical properties such as melting point and specific optical rotation due to differences in purity. Among these, melting | fusing point falls as the purity of maltosyl glucoside falls, and the range of melting | fusing point becomes wide. For example, the melting point of crystalline maltosyl glucoside having a purity of 88.6% is 138 to 146 ° C. Therefore, the crystalline maltosyl glucoside may be used by appropriately selecting its purity as required.
[0030]
The crystalline maltosyl glucoside of the present invention is soluble in water and has a good quality and moderate sweetness. Moreover, it is digested and absorbed by ingestion and used as a calorie source. Furthermore, it can be used as a sweetener that does not easily cause caries because it is not easily fermented by caries-inducing bacteria. In addition, since it is a good quality sweetener and excipient, it can be advantageously used as a sugar coating for tablets in combination with a binder such as pullulan and hydroxyethyl starch. In addition, it has properties such as body imparting property, shimmering property, moisture retention, viscosity, heat resistance, acid resistance, non-reducing property or low reducing property.
[0031]
These properties possessed by crystalline maltosyl glucoside can be advantageously used for the production of foods, tastes, feeds, foods and other foods, and various compositions such as cosmetics, pharmaceuticals and molded products.
[0032]
The crystalline maltosyl glucoside of the present invention can be used as it is as a seasoning for sweetening. If necessary, for example, flour, glucose, maltose, sucrose, isomerized sugar, honey, maple sugar, sorbitol, dihydrochalcone, stevioside, α-glycosyl stevioside, rebaudioside, glycyrrhizin, L-aspartyl-L-phenylalanine methyl ester, It may be used in combination with one or more suitable amounts of other sweeteners such as saccharin, glycine, alanine, etc., and if necessary, mixed with a bulking agent such as dextrin, starch, lactose, etc. Can also be used.
[0033]
In addition, crystalline maltosyl glucoside powder product is a substantially non-hygroscopic powder, has high heat resistance and good stability, so as an extender, excipient, powder base, etc. If necessary, it is optional to mix with other extenders, excipients, binders, etc., and form into various shapes such as granules, spheres, short bars, plates, cubes and tablets. In addition, the present powder product can be advantageously replaced with a part of flours such as wheat flour, corn grits, starch and the like, for example, for use in confectionery materials, bread making materials, cereal materials and the like.
[0034]
In addition, the sweetness of crystalline maltosyl glucoside matches well with other substances having other tastes such as acidity, salt to taste, astringency, umami, and bitterness, and has high acid resistance and heat resistance. Can be advantageously used for sweetening, taste improvement, and quality improvement. For example, soy sauce, powdered soy sauce, miso, powdered miso, moromi, hashio, sprinkle, mayonnaise, dressing, vinegar, three cups vinegar, powdered sushi vinegar, Chinese food, tentsuyu, noodle soup, sauce, ketchup, grilled meat sauce, curry roux, stew It can be advantageously used as various seasonings such as Nomoto, soup, dashi, compound seasoning, mirin, new mirin, table sugar, coffee sugar. In addition, various Japanese sweets such as rice crackers, hail, rice cakes, rice cakes, manju, oirou, chanterelles, sheep mushrooms, water sheep rice cakes, brocade balls, jellies, castellas, rice balls, bread, biscuits, crackers, cookies, pie Pudding, butter cream, custard cream, cream puff, waffle, sponge cake, donut, chocolate, chewing gum, caramel, candy and other Western confectionery, ice cream, sherbet and other ice confectionery, fruit syrup pickled, syrup such as honey, Paste such as flower paste, peanut paste, fruit paste, spread, fruit such as jam, marmalade, syrup pickles, sugar cane, processed foods of vegetables, pickles such as Fukujin pickles, bedara pickles, thousand pieces pickles, pickles, pickles, etc. , Chinese cabbage pickles Pickled vegetables, meat products such as ham and sausage, fish meat ham, fish sausage, fish products such as kamaboko, chikuwa, tempura, sea urchin, squid salted salt, vinegar kombu, sakisume, fugumirin dried and other delicacies , Paste, wild vegetable, sardine, small fish, shellfish, etc., boiled beans, potato salad, side dish foods such as konbu rolls, dairy products, fish meat, livestock meat, fruit, vegetable bottling, canned food, synthetic Alcoholic beverages such as liquor, western liquor, coffee, cocoa, juice, carbonated beverages, soft drinks such as lactic acid beverages, lactic acid bacteria beverages, pudding mix, hot cake mix, instant food such as instant soup, instant soup, baby food, treatment It can be advantageously used for sweetening various foods and drinks such as foods and drinks, improving taste, and improving quality.
[0035]
Moreover, it can also be used in order to improve the palatability of feed, feed, etc. for domestic animals, poultry, and other domestic animals such as bees, sharks, and fish. In addition, tobacco, toothpaste, lipstick, lip balm, internal medicine, troche, tablets, liver oil drop, mouth freshener, mouth fragrance, mouthwash, various solids, pastes, liquids, etc. It can be advantageously used as a sweetener for use in the mouth, as a taste improver, a taste improver, and further as a quality improver.
[0036]
In addition, crystalline maltosyl glucoside is a stabilizer for soaps, skin creams, body shampoos, hair creams, lip balms, skin beautifiers, hair restorers, osmotic pressure regulators, excipients, moisturizing regulators, viscosity modifiers. It can be advantageously used in cosmetic production as an agent, quality improver and the like.
[0037]
In addition, physiologically active substances such as interferon-α, interferon-β, interferon-γ, tsumore necrosis factor-α, tsumore necrosis factor-β, lymphotoxin, macrophage migration inhibitory factor, colony stimulating factor, transfer factor , Cytokines such as interleukin 2, insulin, growth hormone, prolactin, erythropoietin, follicle stimulating hormone, BCG vaccine, Japanese encephalitis vaccine, measles vaccine, polio vaccine, seedling seedling, tetanus toxoid, hub antitoxin, human immunity Globulin and other vaccines, penicillin, erythromycin, chloramphenicol, tetracycline, streptomycin, kanamycin sulfate and other antibiotics, thiamine, riboflavin Vitamins such as L-ascorbic acid, liver oil, carotenoids, ergosterol, tocopherol, enzymes such as lipase, esterase, urokinase, protease, glucanase, ginseng extract, suppon extract, chlorella extract, propolis, royal jelly extracts, viruses , Active ingredients such as lactic acid bacteria, bifidobacteria, yeast and other active fungi, and active stabilizers, and can be advantageously used in pharmaceutical production as osmotic pressure regulators, excipients, tube feeding nutrients, syrups, etc. . The method of adding the crystalline maltosyl glucoside of the present invention to various compositions such as foods and drinks, cosmetics, pharmaceuticals, and molded products as described above may be included in the process until the composition is completed. Known methods such as mixing, kneading, dissolving, melting, dipping, infiltration, spraying, coating, coating, spraying, pouring, solidification, and crystallization are appropriately selected. The amount to be contained varies depending on the composition, but generally, an amount of 0.1% or more, preferably 0.5% or more is suitable as the crystalline maltosyl glucoside. The composition thus obtained can be used not only for foods and drinks, cosmetics, pharmaceuticals, and molded products that are used orally or parenterally, but also for example, daily necessities, agricultural, forestry and fishery products, and chemical industry products. Have a wide range of uses.
[0038]
Next, the present invention will be described in more detail by experiments.
[0039]
First, in Experiment A, the production, purification, and properties of an enzyme from Rhizobium sp. M-11 as an example of a non-reducing saccharide-forming enzyme will be described, and then from a reduced starch partial degradation product using the enzyme. A preparation example of a non-reducing carbohydrate having a trehalose structure at the end and trehalose will be described. Next, in Experiment B, preparation examples of the crystalline maltosyl glucoside of the present invention and its physicochemical properties will be described.
[0040]
[Experiment A-1 Production of non-reducing carbohydrate-forming enzyme from Rhizobium sp. M-11]
It consists of maltose 2.0 w / v%, peptone 0.5 w / v%, yeast extract 0.1 w / v%, disodium phosphate 0.1 w / v%, monopotassium phosphate 0.1 w / v% and water. The liquid medium was adjusted to pH 7.0. About 100 ml of this medium is placed in a 500 ml Erlenmeyer flask, sterilized by autoclaving at 120 ° C. for 20 minutes, cooled, inoculated with Rhizobium species M-11 (FERM BP-4130), and incubated at 27 ° C. and 130 rpm for 24 hours. The culture was used as a seed culture solution.
[0041]
A fermenter with a volume of 30 l is charged with about 20 l of medium having the same composition as in the case of seed culture, sterilized and cooled to a temperature of 30 ° C., then inoculated with 1 v / v% of the seed culture solution, temperature 30 ° C., pH 6.0 The culture was aerated and agitated for about 24 hours while maintaining at ˜8.0. The enzyme activity of the culture was about 1.5 units / ml. A portion of the culture solution is collected and centrifuged to separate the cells and the culture supernatant, and the cells are suspended in 50 mM phosphate buffer (pH 7.0) in the same volume as the original culture solution. Then, the enzyme activity of the cell suspension and the culture supernatant was measured. The cell suspension had about 0.6 units / ml of enzyme activity, and the culture supernatant had about An enzyme activity of 0.9 units / ml was observed.
[0042]
The non-reducing carbohydrate purified enzyme activity was measured by adding 1 ml of enzyme solution to 4 ml of maltopentaose 1.25 w / v% (50 mM phosphate buffer, pH 7.0) as a substrate, and reacting at 40 ° C. for 60 minutes. Thereafter, the reaction is stopped by heating at 100 ° C. for 10 minutes, the reaction solution is accurately diluted 10 times with deionized water, and the reducing power of the diluted solution is measured by the Somogy Nelson method. As a control, the same measurement is performed using an enzyme solution inactivated by heating at 100 ° C. for 10 minutes in advance. Using the above measurement method, the amount of enzyme that reduces the reducing power corresponding to 1 μmole of maltopentaose per minute was defined as 1 unit.
[0043]
[Experiment A-2 Enzyme Purification]
About 18 liters of the culture solution obtained in Experiment A-1 was treated with an ultrahigh pressure microbial cell disruption device minilab (manufactured by Dainippon Pharmaceutical Co., Ltd.), and the microbial cells contained were crushed. The treatment solution was centrifuged (10,000 rpm, 30 minutes) to obtain about 16 l of supernatant. Ammonium sulfate was dissolved in the liquid so that the degree of saturation was 0.2, and the mixture was allowed to stand at 4 ° C. for 1 hour, and then centrifuged (10,000 rpm, 30 minutes) to recover the supernatant.
[0044]
Further, ammonium sulfate was dissolved in the solution so as to have a saturation degree of 0.6, and allowed to stand at 4 ° C. for 24 hours, and then centrifuged (10,000 rpm, 30 minutes) to recover ammonium sulfate salted-out. The obtained ammonium sulfate salted-out product was dissolved in 10 mM phosphate buffer (pH 7.0), dialyzed against the same buffer for 24 hours, and centrifuged (10,000 rpm, 30 minutes) to remove insoluble matter. Excluded. The dialysate (360 ml) was divided into two portions and subjected to ion exchange column chromatography (gel amount: 300 ml) using DEAE-Toyopearl gel (manufactured by Tosoh Corporation).
[0045]
The enzyme was adsorbed on DEAE-Toyopearl gel and eluted from the column with the same buffer containing sodium chloride. The obtained enzyme activity fraction is dialyzed against the same buffer containing 2M ammonium sulfate, the dialyzed solution is centrifuged (10,000 rpm, 30 minutes) to remove insoluble matters, and the resulting supernatant is butyltoyopearl. Hydrophobic column chromatography (gel amount: 300 ml) using 650 gel (manufactured by Tosoh Corporation) was performed. The adsorbed enzyme was eluted from the column with an ammonium sulfate 2M to 0M linear gradient, and the enzyme activity fraction was recovered. Subsequently, gel filtration chromatography (gel amount: 300 ml) using Toyopearl HW-55 resin (manufactured by Tosoh Corporation) was performed, and the enzyme activity fraction was recovered. Table 1 shows the amount of enzyme activity, specific activity, and yield in each step of purification.
[0046]
[Table 1]

[0047]
When the purity of the purified enzyme preparation obtained as a gel filtration eluate in the step of Table 1 was tested by electrophoresis using polyacrylamide gel (gel concentration 7.5 w / v%), the protein band was single. As a result, the enzyme preparation obtained was an electrophoretically single preparation with high purity.
[0048]
[Experiment A-3 Properties of enzyme]
The purified enzyme preparation obtained in Experiment A-2 was subjected to electrophoresis using SDS-polyacrylamide gel (gel concentration: 10 w / v%), and simultaneously migrated molecular weight marker (manufactured by Nippon Bio-Rad Laboratories, Inc.) The molecular weight of the present enzyme was measured in comparison with the molecular weight of about 77,000 to 87,000 daltons.
[0049]
The purified enzyme preparation was subjected to isoelectric focusing using a polyacrylamide gel (containing 2 w / v% ampoline, Sweden, Pharmacia El Kavy), and after electrophoresis, the pH of the gel was measured. When the isoelectric point was determined, the isoelectric point was about 3.6 to 4.6.
[0050]
The effects of temperature and pH on the enzyme activity were examined according to the activity measurement method. The results are shown in FIG. 1 (effect of temperature) and FIG. 2 (effect of pH). The optimum temperature of the enzyme was about 7.0 ° C. when reacted at pH 7.0 for 60 minutes, and the optimum pH was about 7.0 after reacting at 40 ° C. for 60 minutes. The temperature stability of the enzyme was determined by measuring the remaining enzyme activity after holding the enzyme solution (containing 50 mM phosphate buffer, pH 7.0) at each temperature for 60 minutes, cooling with water. The pH stability was determined by maintaining the enzyme in a 50 mM buffer solution at each pH at 25 ° C. for 16 hours, adjusting the pH to 7, and measuring the remaining enzyme activity. The respective results are shown in FIG. 3 (temperature stability) and FIG. 4 (pH stability). The temperature stability of this enzyme was stable up to around 40 ° C., and the pH stability was about 6 to 9.
[0051]
[Experiment A-4 Preparation of non-reducing carbohydrate]
As substrates, prepare 20% aqueous solutions of glucose, maltose, maltotriose, maltotetraose, maltopentaose, maltohexaose, or maltoheptaose, and prepare the purified enzyme preparations obtained in Experiment A-2 for each. Added at a rate of 2 units per gram of substrate solids, allowed to act at 40 ° C. and pH 7.0 for 48 hours, desalted, and used a Wako Beads WB-T-330 column (manufactured by Wako Pure Chemical Industries, Ltd.). The reaction product was analyzed by high performance liquid chromatography. The high performance liquid chromatography was performed at room temperature, water was flowed as an eluent at a flow rate of 0.5 ml / min, and analysis was performed with a differential refractometer RI-8012 (manufactured by Tosoh Corporation). The results are shown in Table 2.
[0052]
[Table 2]

[0053]
As is apparent from the results in Table 2, each of the remaining substrates and newly generated carbohydrates PI, PII, PIII, PIV, and PV are hardly detected in the reaction product. . Each of the production rates was found to have a high production rate of 85% or more for PII, PIII, PIV and PV having a glucose polymerization degree of 4 or more, although the PI with a glucose polymerization degree of 3 was relatively low. It has been found that no new carbohydrate is produced from glucose or maltose.
[0054]
In order to purify newly produced carbohydrates from each reaction product, after decolorization, desalting and concentration, alkali metal type strongly acidic cation exchange resin (XT-1016, Na⁺Column fractionation using a mold, a crosslinking degree of 4%, manufactured by Tokyo Organic Chemical Industry Co., Ltd.). The resin is packed in three stainless steel columns with an inner diameter of 2.0 cm and a length of 1 m, connected in series, and the reaction sugar solution is added to the resin at 5 v / v% while maintaining the temperature in the column at 55 ° C. This was fractionated by flowing warm water at 55 ° C. at SV 0.13, and a newly produced high-purity fraction having a carbohydrate content of 97% or more was collected. The obtained high-purity fractions were vacuum-dried to prepare high-purity carbohydrate preparations, respectively. The yield based on the substrate material was about 9% for PI, about 65% for PII, about 82% for PIII, about 80% for PIV, and about 77% for PV, respectively, in terms of solid matter. The purity was 97.5% for PI, 98.6% for PII, 99.5% for PIII, 98.4% for PIV, and 98.4% for PV.
[0055]
The reducing power of these newly produced high-purity carbohydrate preparations was measured by the Somogy Nelson method and expressed in DE. The results are summarized in Table 3.
[0056]
[Table 3]

[0057]
As is apparent from the results in Table 3, only a slight reducing power was observed in any of the samples. The slight reducing power is presumed to be attributable to the reducing malto-oligosaccharide derived from the substrate mixed in and remaining in the sample in a trace amount, and any newly produced carbohydrate is substantially non-reducing. It is judged that.
[0058]
[Experiment A-5 Enzymatic degradation with glucoamylase]
50 mg each of the non-reducing carbohydrate preparation, PI, PII, PIII, PIV or PV prepared in Experiment A-4 was dissolved in 1 ml of 50 mM acetate buffer (pH 4.5), and 1 unit of glucoamylase ( Seikagaku Co., Ltd.) was added and kept at 40 ° C. for 6 hours. After enzymatic degradation, the degradation product was analyzed by high performance liquid chromatography. As a result, only glucose and trehalose were detected as degradation products from any sample. It was. Table 4 shows the results of the detected glucose content, trehalose content, and compositional molar ratio.
[0059]
[Table 4]

[0060]
As is apparent from the results in Table 4, glucoamylase decomposes non-reducing carbohydrate PI into one glucose molecule and one trehalose molecule, and non-reducing carbohydrate PII into two glucose molecules and one trehalose molecule. Non-reducing carbohydrate PIII is decomposed into 3 glucose molecules and 1 trehalose molecule, non-reducing carbohydrate PIV is decomposed into 4 glucose molecules and 1 trehalose molecule, and non-reducing carbohydrate PV is 5 glucose molecules and 1 trehalose molecule. It was found that it was decomposed.
[0061]
Further, considering the reaction characteristics of glucoamylase, the structure of these non-reducing carbohydrates is a carbohydrate in which glucose molecules are linked to trehalose molecules with α-1,4 bonds or α-1,6 bonds. Is a non-reducing saccharide with a glucose polymerization degree 3 in which one molecule of glucose is bound to one trehalose molecule, PII is a non-reducing saccharide with a glucose polymerization degree 4 in which two molecules of glucose are bound to one trehalose molecule, and PIII is a trehalose A non-reducing saccharide with a glucose polymerization degree of 5 consisting of 3 molecules of glucose bonded to one molecule, PIV is a non-reducing saccharide with a glucose polymerization degree of 6 having 4 molecules of glucose bonded to 1 molecule of trehalose, and PV is a molecule of trehalose. It is judged that this is a non-reducing carbohydrate having a glucose polymerization degree of 7 and having 5 glucose molecules bound thereto. Similarly, when β-amylase was allowed to act on a non-reducing carbohydrate preparation, PI, PII, PIII, PIV, or PV, the non-reducing carbohydrate PI, PII was not degraded, and PIII was maltose. It was found that 1 molecule was decomposed into 1 molecule of PI, PIV was decomposed into 1 molecule of maltose and 1 molecule of PII, and PV was decomposed into 2 molecules of maltose and 1 molecule of PI.
[0062]
From the above results, the reaction by the non-reducing saccharide-forming enzyme of the present invention does not involve lowering the molecular weight and increasing the molecular weight of the substrate, in other words, an intramolecular conversion reaction without changing the degree of glucose polymerization. Non-reducing carbohydrates, PI, PII, PIII, PIV and PV produced by this non-reducing carbohydrate-forming enzyme are α-glucosyl trehalose (also known as α-maltosyl glucoside), α, respectively. -Maltosyl trehalose (also known as α-maltotriosyl glucoside), α-maltotriosyl trehalose (also known as α-maltotetraosyl glucoside), α-maltotetraosyl trehalose (also known as α-maltopentaosyl glucoside) ) And α-maltopentaosyl trehalose (also known as α-maltohexaosyl glucoside) Source (G_n-T: However, G means a glucose residue, n means an integer of 1 or more, and T means α, α-trehalose. ).
[0063]
[Experiment A-6 Preparation of Trehalose and Non-reducing Carbohydrate Having Trehalose Structure at Terminal]
After dissolving 40 parts by weight of a partially decomposed starch (manufactured by Matsutani Chemical Industry Co., Ltd., trade name: Paindex # 4) in 60 parts by weight of water and adjusting the solution to 45 ° C. and pH 6.5, Experiment A-2 The non-reducing saccharide-forming enzyme prepared by the above method was added at a rate of 1 unit per g of reduced starch partial degradation product and reacted for 96 hours to produce a non-reducing saccharide having a trehalose structure at the end. Then, the enzyme was inactivated by heating at 100 ° C. for 10 minutes. This reaction solution is diluted to a concentration of about 20%, glucoamylase (manufactured by Nagase Seikagaku Co., Ltd., trade name Glucozyme) is added for 10 units per g of partially decomposed starch, reacted for 40 hours, and then heated, Was deactivated. This solution was decolorized with activated carbon, desalted with an ion exchange resin, and concentrated to a concentration of about 60% according to a conventional method. The sugar solution contained 29.5% trehalose per solid. This concentrated liquid was charged at 60 ° C. and SV 0.4 to a jacketed stainless steel column packed with a salt-type strongly acidic cation exchange resin (Sold by Organo Co., Ltd., trade name: CG6000, Na type). Minutes were collected. The high content liquid contained about 90% trehalose per solid. After concentrating this solution to a concentration of about 75%, it was placed in an auxiliary crystal can, and about 2% of a trehalose hydrous crystal was added as a seed crystal and slowly cooled to obtain a mass kit having a crystallization rate of about 45%. This mass kit is 150kg / cm from the nozzle on the drying tower.²Sprayed at high pressure. At the same time, hot air of 85 ° C. is blown from the top of the drying tower, the crystal powder is collected on a transfer wire mesh conveyor provided at the bottom, and hot air of 45 ° C. is sent from the bottom of the conveyor while the powder is dried. It was gradually moved out and removed. This crystal powder was packed in an aging tower and aged for 10 hours while sending warm air, and crystallization and drying were completed to obtain a water-containing trehalose crystal powder.
[0064]
[Experiment B-1 Preparation of maltosyl glucoside]
5 parts by weight of 98% pure maltopentaose (manufactured by Hayashibara Biochemical Laboratories Co., Ltd.) was dissolved in 10 parts by weight of water and adjusted to pH 7.0 and temperature of 40 ° C. The prepared non-reducing saccharide-forming enzyme was added to 2 units per maltopentaosegram. After reacting for 48 hours, the enzyme was inactivated by heating at 100 ° C. for 20 minutes. The solution contained about 88% maltotetraosyl glucoside per solid.
[0065]
This maltotetraosyl glucoside-containing solution is adjusted to pH 5.5 and a temperature of 40 ° C., and β-amylase (manufactured by Nagase Seikagaku Corporation, trade name # 1500) is added so as to be 20 units per gram of solid matter. Then, after reacting for 24 hours, the enzyme was inactivated by heating at 100 ° C. for 20 minutes. This liquid contained about 53% of maltosyl glucoside per solid, and additionally contained maltose and maltotriose.
[0066]
This solution was decolorized with activated carbon according to a conventional method, desalted with an ion exchange resin (H type and OH type), and concentrated to a solid concentration of about 50%. Subsequently, this concentrated solution was subjected to column chromatography using a salt-type strongly acidic cation exchange resin in accordance with the method of Experiment A-6, and a fraction containing a high content of maltosylglucoside was collected. This solution contained about 81% maltosyl glucoside per solid.
[0067]
Further, in order to increase the purity, sodium hydroxide was added to this solution to a concentration of 0.1 N, and the mixture was heated at 100 ° C. for 2 hours to decompose the reducing saccharide. This reaction solution was decolorized and desalted according to a conventional method, and concentrated to a solid concentration of about 50%. This solution contained about 94% maltosyl glucoside per solid. The collected maltosyl glucoside-rich solution is further subjected to preparative liquid chromatography (manufactured by YMC Co., Ltd., preparative column YMC-Pack R-355-15) using octadecyl silica gel as a carrier, and the purity of maltosyl glucoside is about A high content fraction of 99% or more was collected.
[0068]
This fraction was further purified according to a conventional method, concentrated to a concentration of 78%, placed in a beaker, and left in a cold room at 4 ° C., whereupon precipitation of crystals was observed. The crystals were collected by centrifugation and washed with a small amount of water to obtain hydrous crystals having a purity of 99.9%. The product was dried under reduced pressure at 100 ° C. overnight to obtain anhydrous crystals.
[0069]
When the physicochemical properties of these crystals were examined, they were as follows.
(1) Elemental analysis (as anhydride)
Hydrous crystal C = 42.4% H = 6.8%
Anhydrous crystal C = 42.6% H = 6.7%
Theoretical value C = 42.86% H = 6.39%
(2) Mass spectrometry (as anhydride)
MW = 504 (molecular formula C₁₈H₃₂0₁₆)
(3) Moisture
Measured by Karl Fischer method
Water-containing crystals 9.9% (1 mol of maltosylglucoside and 3 mol of water)
Anhydrous crystals 0.3%
(4) Melting point
When water-containing crystals or anhydrous crystals of maltosylglucoside were placed in a melting point measuring device and the melting point was observed at a heating rate of 1 ° C. per minute, all the crystals melted at 180 ° C. This does not necessarily mean that the melting points of both crystals are the same. For example, in the case of a water-containing crystal, the water-containing crystal is changed from the water-containing crystal to the anhydrous crystal without any change in appearance during heating and heating. It is considered that the melting phenomenon of the anhydrous crystals is observed.
(5) Specific rotation (as anhydride)
Both hydrous and anhydrous crystals
[Α]_D ²⁰  + 207.9 ° (c = 5.0, H₂O)
(6) UV absorption spectrum
Neither hydrous crystals nor anhydrous crystals show characteristic absorption when measured in aqueous solution.
(7) Infrared absorption spectrum
A transparent tablet was prepared by stirring and mixing 2 mg of powder of hydrous crystal or anhydrous crystal with 200 mg of dry KBr, and the infrared absorption spectrum was measured. The results showed almost the same pattern for both hydrous crystals and anhydrous crystals. FIG. 5 shows the results of the water-containing crystal.
(8) Solubility (as anhydride)
About 163 g of both hydrous crystals and anhydrous crystals are dissolved in 100 ml of water at 25 ° C.
(9) Sweetness (as anhydride)
Since the sweetness of the 25% aqueous solution of maltosylglucoside is almost the same as that of the 10% aqueous solution of sucrose, it is about 40% sweetness of sucrose. The quality of sweetness is extremely excellent.
(10) Color reaction
Both hydrous and anhydrous crystals are green in anthrone-sulfuric acid reaction, and Ferring's liquid reduction reaction and iodine reaction are negative.
(11) Structure (as anhydride)
(A) When hydrolyzed with 1N-sulfuric acid, only D-glucose is produced.
(B) By the action of glucoamylase, 1 mol of glucose and 1 mol of trehalose were produced.
(C) Carbon nuclear magnetic resonance analysis (¹³The results of (C-NMR) are shown in FIG.
Klaus Bock et al., “Advanced in Carbohydrate Chemistry and Biochemistry”, Vol. 42, pp. 193-225 (1984) From the chemical shifts of the standard substance, α-D-glucopyranose, α, α-trehalose and maltooligosaccharide, and this substance is O-α-D-glucopyranosyl- (1 → 4) -α -D-glucopyranosyl It is judged to have the structure of α-D-glucopyranoside, that is, maltosylglucoside.
(12) Powder X-ray diffraction
F. H. Stodola et al., “Journal of the American Chemical Society”, Vol. 78, pages 2514-2518 (1956). The powder X-ray diffraction pattern using CuKα rays of crystalline maltosyl glucoside was measured according to the method reported in the above. The result of the water-containing crystal is shown in FIG. 7, and the result of the anhydrous crystal is shown in FIG. As is apparent from the results of FIGS. 7 and 8, the water-containing crystal has 7.9 °, 13.2 °, 17.2 ° and 22.6 ° as main diffraction angles (2θ) in the powder X-ray diffraction method. In addition, anhydrous crystals have 7.7 °, 13.5 °, 17.6 ° and 23.6 °.
[0070]
From the above facts, the crystal of the present invention is judged to be a maltosylglucoside crystal that has never been known.
[0071]
[Experiment B-2 Hygroscopicity and its effect on crystalline maltosyl glucoside and amorphous maltosyl glucoside]
About 1 g each of water-containing crystalline maltosyl glucoside, anhydrous crystalline maltosyl glucoside and amorphous maltosyl glucoside in a powder state are taken in a polyethylene container and left in a high humidity atmosphere with a relative humidity of 90.1%. Their water content was measured and the change in the state of the powder was observed. As the sample, the water-containing crystal and the anhydrous crystal were prepared by the method of Experiment B-1, and the amorphous was prepared by dissolving the water-containing crystal with deionized water and vacuum drying. The results are summarized in Table 5.
[0072]
[Table 5]

[0073]
As is apparent from the results in Table 5, it was found that amorphous maltosyl glucoside absorbs moisture significantly and solidifies, and easily loses fluidity. On the other hand, it has been found that the water-containing crystal has a low moisture absorption, retains fluidity, and is easy to handle. In addition, although anhydrous crystals absorb moisture, they retain fluidity with no change in appearance, and thus are considered to be stable by changing from anhydrous crystals to water-containing crystals.
[0074]
[Experiment B-3 Acute toxicity test]
Using mice, the acute toxicity test was conducted by orally administering the water-containing crystals and anhydrous crystals of maltosylglucoside prepared by the method of Experiment B-1. As a result, these crystalline maltosyl glucosides are low-toxic substances, and no deaths were observed even at the maximum dose that could be administered. Therefore, although the exact value cannot be said, the LD50 value was 50 g / kg or more in any case.
[0075]
Hereinafter, the production method of the crystalline maltosyl glucoside of the present invention is shown in Example A, and its use is shown in Example B.
[0076]
Example A-1
Prepared by the method of Experiment A-2 after heating and dissolving 50 parts by weight of 95% pure maltopentaose (manufactured by Hayashibara Biochemical Laboratories Co., Ltd.) in 200 parts by weight of water and adjusting the pH to 7.0 and the temperature to 40 ° C. The non-reducing saccharide-forming enzyme was added to 5 units per maltopentaosegram and allowed to react for 24 hours. This reaction solution was heated to inactivate the enzyme. This solution contained about 83% maltotetraosyl glucoside per solid. This liquid was concentrated to a solid concentration of about 50% and left in a cold room to precipitate maltotetraosyl glucoside crystals, which were filtered to collect crystalline maltotetraosyl glucoside.
[0077]
10 parts by weight of this crystalline maltotetraosyl glucoside is dissolved by heating in 100 parts by weight of water, adjusted to pH 5.5 and temperature of 50 ° C., and β-amylase (manufactured by Nagase Seikagaku Corporation, trade name # 1500) is solid. Added to 20 units per gram. After reacting for 24 hours, the enzyme was inactivated by heating at 100 ° C. for 20 minutes. This liquid contained about 58% maltosylglucoside per solid, and contained maltose and a small amount of maltotriose.
[0078]
This solution was decolorized with activated carbon according to a conventional method, desalted with an ion exchange resin (H type and OH type), and concentrated to a solid concentration of about 50%. This concentrated liquid was placed in a jacketed stainless steel column (5.4 cm × 4 m connected in series of 4) filled with a salt type strongly acidic cation exchange resin (manufactured by Organo Corporation, trade name: XT-1016, Na type). The fraction was charged at 40 ° C. and SV 0.4, and then eluted with warm water at 60 ° C. to collect a fraction containing a high content of maltosylglucoside. This solution contained about 92% maltosyl glucoside per solid.
[0079]
This solution is decolorized with activated carbon, deionized with ion exchange resin (H type and OH type), and concentrated to a solid concentration of about 80% according to a conventional method. Silglucoside was added as a seed crystal at 2%, and assisted crystallization was carried out with slow stirring while slowly cooling. The obtained mass kit was honeyed with a basket-type centrifuge, washed with a small amount of water, and then dried. Water-containing crystalline maltosyl glucoside having a purity of 99.4% was collected.
[0080]
This product does not substantially exhibit hygroscopicity, is easy to handle, and is used as a sweetener, taste improver, quality improver, stabilizer, excipient, etc. for foods, drinks, cosmetics, pharmaceuticals, molded products, etc. It can be advantageously used in various compositions.
[0081]
Example A-2
Potato starch having a concentration of about 20% containing 0.1% calcium carbonate was adjusted to pH 6.0, and then α-amylase (manufactured by Novo Nordisk Industries, trade name Termamil 60 L) was added at 0.3 per gram starch solids. % And heated to 95 to 100 ° C. to gelatinize and liquefy the starch, and then heat deactivate the enzyme to obtain a liquefied liquid of DE19.5.
[0082]
This solution was adjusted to pH 6.2, temperature 40 ° C., and 3 units of non-reducing carbohydrate-forming enzyme prepared by the method of Experiment A-2 per gram of starch, isoamylase (manufactured by Hayashibara Biochemical Laboratories Co., Ltd.) ) Was added at a rate of 1,000 units and allowed to react for 72 hours. The reaction solution is heated to inactivate the enzyme, then cooled, adjusted to pH 5.5 and a temperature of 50 ° C., and then β-amylase (manufactured by Nagase Seikagaku Corporation, trade name # 1500) is added. Added to 50 units per gram starch solids and allowed to react for 24 hours. This reaction solution was heated to inactivate the enzyme. This liquid contained about 21% of maltosyl glucoside per solid.
[0083]
This was purified and concentrated in the same manner as in Example A-1, and subjected to column chromatography using a salt-type strongly acidic cation exchange resin to collect a fraction containing high maltosylglucoside. This solution contained about 89% maltosyl glucoside per solid.
[0084]
This solution is decolorized with activated carbon according to a conventional method, desalted with ion exchange resins (H type and OH type), concentrated to a solid concentration of about 85%, and crystalline maltosyl glucoside as a seed crystal. % Was added and crystallization was carried out while slowly cooling and slowly stirring. The obtained mass kit was honeyed with a basket-type centrifuge, washed with a small amount of water, and then dried. Water-containing crystalline maltosylglucoside having a purity of 98.1% was collected.
[0085]
This product does not substantially exhibit hygroscopicity, is easy to handle, and is used as a sweetener, taste improver, quality improver, stabilizer, excipient, etc. for foods, drinks, cosmetics, pharmaceuticals, molded products, etc. It can be advantageously used in various compositions.
[0086]
Example A-3
Using starch partial decomposition product (manufactured by Matsutani Chemical Industry Co., Ltd., trade name: Paindex # 4), adjusting the concentration to 20%, pH 5.5, temperature 45 ° C, adding 500 units of isoamylase per gram of starch partial decomposition product For 24 hours to decompose starch-derived α-1,6 bonds. The reaction solution was heated to 100 ° C. and kept for 10 minutes, and then cooled to 40 ° C. to adjust the pH to 6.5. To this, 5 units of non-reducing saccharide-forming enzyme prepared by the method of Experiment A-2 was added per gram of solid matter, reacted for 48 hours, the reaction solution was heated to 100 ° C., kept for 10 minutes, and then cooled. . After adjusting the temperature to 50 ° C., add β-amylase to 50 units per gram of solids and α-amylase to 10 units, react for 24 hours, and then heat at 100 ° C. for 20 minutes to lose the enzyme. I made it live. This liquid contained about 24% maltosylglucoside per solid.
[0087]
This was purified and concentrated in the same manner as in Example A-1, and subjected to column chromatography using a salt-type strongly acidic cation exchange resin to collect a fraction containing a high content of maltosylglucoside. This solution contained about 91% maltosyl glucoside per solid.
[0088]
This solution is decolorized with activated carbon according to conventional methods, desalted with ion exchange resins (H type and OH type), concentrated to a solid concentration of about 85%, and crystalline maltosyl glucoside is used as a seed crystal. 3% was added, and crystallization was carried out with slow stirring to obtain a mass kit having a crystallization rate of about 45%.
[0089]
This mass kit is 150kg / cm from the nozzle on the drying tower.²Sprayed at high pressure. At the same time, hot air at 85 ° C. is blown from the top of the drying tower, the crystal powder is collected on a transfer wire mesh conveyor provided at the bottom of the drying tower, and hot powder at 45 ° C. is sent from below the conveyor while Gradually moved out of the drying tower. This powder crystal was packed in an aging tower and aged for 10 hours while sending warm air to complete crystallization and drying to obtain a hydrous crystalline maltosylglucoside-containing powder.
[0090]
This product does not substantially exhibit hygroscopicity, is easy to handle, and is used as a sweetener, taste improver, quality improver, stabilizer, excipient, etc. It can be advantageously used in various compositions.
[0091]
Example A-4
Malt obtained by allowing isoamylase, non-reducing saccharide-forming enzyme, then β-amylase and α-amylase to act on the partially degraded starch in the same manner as in Example A-3, followed by separation and purification The liquid containing high sylglucoside was concentrated to a concentration of about 80%. This concentrated solution was taken in an auxiliary crystal can, 2% of crystalline maltosyl glucoside was added as a seed crystal, and the auxiliary crystal was agitated while slowly stirring, and then taken out into a vat to prepare a block. The block was allowed to stand at about 25 ° C. for 2 days and aged, and then pulverized with a cutting pulverizer, fluidized and classified to obtain hydrous crystalline maltosylglucoside-containing powder.
[0092]
This product does not substantially exhibit hygroscopicity, is easy to handle, and is used as a sweetener, taste improver, quality improver, stabilizer, excipient, etc. It can be advantageously used in various compositions.
[0093]
Example A-5
The hydrous crystalline maltosyl glucoside obtained by the method of Example A-1 was dried under reduced pressure at 100 ° C. overnight to obtain anhydrous crystalline maltosyl glucoside having a water content of about 0.2%.
[0094]
This product absorbs moisture when left indoors and easily returns to water-containing crystalline maltosylglucoside with a water content of about 10% for stabilization. The anhydrous crystalline maltosyl glucoside can be advantageously used as a seed crystal, and can also be advantageously used as a hygroscopic agent, a dehydrating agent, a chemical raw material, or the like. Furthermore, it can be advantageously used in various compositions such as foods and drinks, cosmetics, pharmaceuticals, and molded products.
[0095]
Example A-6
1 part by weight of trehalose and 1 part by weight of a partially decomposed starch (manufactured by Matsutani Chemical Industry Co., Ltd., trade name: Paindex # 4) are heated and dissolved in 3 parts by weight of water, adjusted to a temperature of 65 ° C. and pH 6.0. Cyclomaltodextrin glucanotransferase derived from Bacillus stearothermophilus (manufactured by Hayashibara Biochemical Laboratories Co., Ltd.) was added at 10 units per gram of the partially decomposed starch and reacted for 24 hours, and then the enzyme was inactivated by heating. . Next, the temperature was adjusted to 55 ° C., and pullulanase (manufactured by Hayashibara Biochemical Laboratories Co., Ltd.) and β-amylase were added to each unit of 25 units and 50 units per gram of the partially decomposed starch and reacted for 24 hours. Was inactivated by heating. This reaction solution contained about 25% maltosylglucoside per solid.
[0096]
This was purified in the same manner as in Example A-1, and subjected to column chromatography using a salt-type strongly acidic cation exchange resin to obtain a fraction containing a high content of maltosylglucoside, which was decolorized, desalted, concentrated, and promoted. The block was made by taking it out into a bat. This block was cut, dried, and classified to obtain a hydrous crystalline maltosylglucoside-containing powder with a yield of about 17% per raw material.
[0097]
This product does not substantially exhibit hygroscopicity and is easy to handle, and as a sweetener, taste improver, quality improver, stabilizer, excipient, etc., food, drinks, cosmetics, pharmaceuticals, molded products, etc. It can be advantageously used in various compositions.
[0098]
Example B-1 Sweetener
To 1 part by weight of the high-purity water-containing crystalline maltosyl glucoside obtained by the method of Example A-1, 0.05 part by weight of α-glycosyl stevioside (sold by Toyo Seika Co., Ltd., trade name: αG sweet) is uniformly mixed to obtain granules. A granular sweetener was obtained through a molding machine. This product has excellent sweetness quality, has about twice the sweetness of sucrose, and the calorie per sweetness is about 1/2 that of sucrose. This sweetener is suitable as a low-calorie sweetener for sweetening low-calorie foods and drinks for obese, diabetics and the like who restrict caloric intake. Further, the present sweetener is suitable for sweetening foods and the like that suppress dental caries because it produces less acid by caries-inducing bacteria and produces less insoluble glucan.
[0099]
[Example B-2 Hard Candy]
30 parts by weight of water-containing crystalline maltosylglucoside-containing powder obtained by the method of Example A-3 was heated and mixed with 100 parts by weight of 55% sucrose solution, and then heated and concentrated under reduced pressure until the water content was less than 2%. 1 part by weight of citric acid and an appropriate amount of lemon flavor and colorant were mixed and molded according to a conventional method to obtain a product. This product is a high quality hard candy that is crisp, tastes good and does not cause sucrose crystallization.
[0100]
[Example B-3 Strawberry jam]
Stew 150 parts by weight raw strawberries, 60 parts by weight sucrose, 20 parts by weight maltose, 40 parts by weight hydrous crystalline maltosylglucoside powder obtained by the method of Example A-4, 5 parts by weight pectin and 1 part by weight citric acid. Bottled to get the product. This product is a jam with good flavor and color.
[0101]
[Example B-4 Cream wafer]
2,000 parts by weight of high-purity anhydrous crystalline maltosyl glucoside obtained by the method of Example A-5, 1,000 parts by weight of shortening, 1 part by weight of lecithin, 1 part by weight of lemon oil, and 1 part by weight of vanilla oil The cream obtained by mixing was heated and kept at 40 to 45 ° C., and sandwiched between wafers to produce a cream wafer. This product has a smooth cream melt and good flavor.
[0102]
[Example B-5 Sweetened condensed milk]
5 parts by weight of the high-purity water-containing crystalline maltosylglucoside obtained by the method of Example A-2 is dissolved in 100 parts by weight of raw milk, sterilized by heating with a plate heater, then concentrated to a concentration of about 70%, and canned in aseptic conditions. And got the product. This product has a mild sweet taste and good flavor, and can be advantageously used for seasoning infant foods, fruits, coffee, cocoa, tea, and the like.
[0103]
[Example B-6 Lactic acid beverage]
10 parts by weight of skim milk was sterilized by heating at 80 ° C. for 20 minutes, cooled to 40 ° C., 0.3 parts by weight of a starter was added thereto, and fermented at about 37 ° C. for 10 hours. Next, after homogenizing this, 5 parts by weight of high-purity water-containing crystalline maltosylglucoside obtained by the method of Example A-2 and 2 parts by weight of isomerized sugar syrup were added and sterilized by maintaining at 70 ° C. This was cooled, an appropriate amount of fragrance was added, and bottled to obtain a product. This product is a high quality lactic acid beverage whose flavor and sweetness are in good harmony with acidity.
[0104]
[Example B-7 powdered juice]
For 33 parts by weight of orange juice powder produced by spray drying, 50 parts by weight of high-purity hydrous crystal maltosylglucoside obtained by the method of Example A-2, 10 parts by weight of sucrose, 0.65 parts by weight of anhydrous citric acid, apple 0.1 part by weight of acid, 0.1 part by weight of L-ascorbic acid, 0.1 part by weight of sodium citrate, 0.5 part by weight of pullulan, and appropriate amount of powdered fragrance are mixed and stirred well, pulverized into a fine powder. Charged to fluidized bed granulator, exhaust air temperature set to 40 ° C, sprayed with high concentration maltosylglucoside concentrate obtained by the method of Example A-2 as binder, granulated for 30 minutes, weighed and packaged And got the product. This product is a powdered juice with a fruit juice content of about 30%. In addition, this product had no off-flavors and off-flavors, and did not cause moisture absorption and consolidation, and was stable for a long time.
[0105]
[Example B-8 Chocolate]
After mixing 40 parts by weight of cocoa paste, 10 parts by weight of cocoa butter, 20 parts by weight of sucrose, and 30 parts by weight of high-purity water-containing crystal maltosyl glucoside obtained by the method of Example A-1, the particle size is reduced by passing through a refiner Put in a conche and knead at 50 ° C for 2 days. During this period, 0.5 parts by weight of lecithin was added and sufficiently mixed and dispersed. Next, the temperature was adjusted to 31 ° C. with a temperature controller, poured into a mold just before the butter solidified, drained with a vibrator, and passed through a 10 ° C. cooling tunnel for 20 minutes to solidify. This was punched and packaged to obtain a product. This product has no hygroscopicity, good color and gloss, good internal structure, melts smoothly in the mouth, and has an elegant sweetness and mellow flavor.
[0106]
[Example B-9 Chewing gum]
3 parts by weight of gum base is heated and melted to a degree of softening, and 4 parts by weight of anhydrous crystalline maltitol (registered trademark Mabito, Hayashibara Shoji Co., Ltd.) and the high purity water-containing crystalline maltosyl glucoside obtained by the method of Example A-2 3 parts by weight was added, and an appropriate amount of fragrance and colorant were mixed, and kneaded by a roll, molded and packaged according to a conventional method to obtain a product. This product is a chewing gum with good texture and flavor.
[0107]
[Example B-10 Custard cream]
100 parts by weight of corn starch, 100 parts by weight of the hydrous crystalline maltosylglucoside-containing powder obtained by the method of Example A-3, 80 parts by weight of maltose (registered trademark Sanmalto, Hayashibara Shoji Co., Ltd.), 20 parts by weight of sucrose and 1 part by weight of sodium chloride Mix well, add 280 parts by weight of chicken egg, stir in, gradually add 1,000 parts by weight of boiled milk, and continue to stir over heat, until the corn starch is completely gelatinized and the whole When it became translucent, the fire was turned off, cooled, added with an appropriate amount of vanilla flavor, weighed, filled, and packaged to obtain a product. This product has a smooth luster, mild sweetness and deliciousness.
[0108]
Example B-11 Uiro Element
Rice bran was produced by uniformly mixing 90 parts by weight of rice flour with 20 parts by weight of corn starch, 120 parts by weight of hydrous crystalline maltosylglucoside-containing powder obtained by the method of Example A-6, and 4 parts by weight of pullulan. Ui-no-Moto, an appropriate amount of Matcha and water were kneaded, and the mixture was placed in a container and steamed for 60 minutes to produce Matcha Uiro. This product has good shine, mouthfeel, and good flavor. Moreover, the aging of starch is suppressed and the shelf life is good.
[0109]
[Example B-12 Instant Corn Potage Soup]
30 parts by weight of α-ized corn powder, 5 parts by weight of α-ized wheat flour, 4 parts by weight of α-ized potato starch, 12 parts by weight of α-ized waxy corn starch, hydrous crystal maltosyl glucoside obtained by the method of Example A-3 After containing 8 parts by weight of powder, 5 parts by weight of sodium glutamate, 8.5 parts by weight of sodium chloride, 7 parts by weight of skim milk powder and 0.5 parts by weight of onion powder, they were mixed well, and then sorbitan fatty acid ester 0.5 What was heated and melted by weight and 9 parts by weight of vegetable hardened oil was added and mixed. Further, 10 parts by weight of lactose was added and mixed, and this was fluidized bed granulator as in Example B-7. The mixture was sprayed with a small amount of water, granulated, dried with hot air at 70 ° C., and sieved to produce instant corn potage soup. If hot water is poured into this product, it will dissolve and disperse easily, resulting in a flavorful soup.
[0110]
[Example B-13 Bettakarazuke-no-moto]
4 parts by weight of water-containing crystalline maltosylglucoside-containing powder obtained by the method of Example A-4, 0.05 parts by weight of licorice preparation, 0.008 parts by weight of malic acid, 0.07 parts by weight of sodium glutamate, 0. When 03 parts by weight and 0.2 parts by weight of pullulan were uniformly mixed, a pickle was prepared. According to a conventional method, 30 kg of radish was submerged with sodium chloride and then soaked with sucrose, and then soaked in a seasoning solution prepared with 4 kg of the main marinade. This product was good in color, gloss and aroma, had moderate sweetness, and was crisp. In addition, this product was resistant to rancidity and was stable for a long time.
[0111]
[Example B-14 Tube feeding agent]
20 parts by weight of high-purity water-containing crystalline maltosyl glucoside obtained by the method of Example A-1, 1.1 parts by weight of glycine, 1 part by weight of sodium glutamate, 0.4 parts by weight of calcium lactate, 0.1 parts by weight of magnesium carbonate, A formulation consisting of 0.01 parts by weight thiamine and 0.01 parts by weight riboflavin is prepared. Each 24 g of this blend was filled into a laminated aluminum parcel and heat sealed to obtain a product. This product is dissolved in about 33 to 500 ml of water to make a nutritional supplement, and administered to the nasal cavity, gastrointestinal tract, etc. by the tube method. This product can be advantageously used not only as a human but also as a parenteral nutritional supplement for livestock.
[0112]
[Example B-15 Tube feeding agent]
580 parts by weight of high-purity water-containing crystalline maltosyl glucoside obtained by the method of Example A-2, 190 parts by weight of dried egg yolk, 209 parts by weight of skim milk powder, 4.4 parts by weight of sodium chloride, 1.85 parts by weight of potassium chloride, sulfuric acid A formulation comprising 4 parts by weight of magnesium, 0.01 parts by weight of thiamine, 0.1 parts by weight of sodium ascorbate, 0.6 parts by weight of vitamin E acetate and 0.04 parts by weight of nicotinamide is prepared. Each 25 g of this blend was filled into a laminated aluminum package and heat sealed to obtain a product. This product is used by dissolving 1 bag in about 150 to 300 ml of water to give a nutritional supplement and administering it to the nasal cavity, esophagus, stomach, etc. by the tube method.
[0113]
[Example B-16 Plaster for treatment of trauma]
50 parts by weight of methanol in which 3 parts by weight of iodine was dissolved and mixed with 300 parts by weight of high-purity hydrous crystalline maltosylglucoside and 200 parts by weight of crystalline maltose obtained by the method of Example A-1, and further mixed with 200% pullulan aqueous solution 200 Part by weight was added and mixed to obtain a plaster for the treatment of trauma showing moderate extension and adhesion. This product shortens the healing period and heals the wound surface cleanly.
[0114]
[Example B-17 Interferon Solution]
Human natural interferon-γ preparation (manufactured by Hayashibara Biological Laboratories Co., Ltd., sold by Cosmo Bio Inc.) is applied to an immobilized anti-human interferon-γ antibody column according to a conventional method, and humans contained in the preparation High purity obtained by adsorbing natural interferon-γ, passing through and removing bovine serum albumin as a stabilizer, and then changing the pH to obtain human natural interferon-γ by the method of Example A-1. Elution was performed using physiological saline containing 7% of hydrous crystal maltosylglucoside. This solution is microfiltered, aseptically filled into vials, and 10 natural human interferon-γ per ml.^FiveA liquid containing the unit was obtained. About 1 to 20 ml of adult is orally or parenterally administered per day, this product can be advantageously used for the treatment of viral diseases, allergic diseases, rheumatism, diabetes, malignant tumors and the like. In this product, maltosyl glucoside acts as a stabilizer and maintains its activity well even when left at 4 ° C. or 25 ° C. for 20 days.
[0115]
[Example B-18 Tsumore Necrosis Factor Solution]
Human natural type Tsumore Necrosis factor-α preparation (manufactured by Hayashibara Biochemical Laboratories Co., Ltd., sold by Cosmo Bio Inc.) is applied to an immobilized anti-Human Tsumore Necrosis factor-α antibody column according to a conventional method, Adsorbs human natural tsumoa necrosis factor-α contained in the preparation, and passes bovine serum albumin as a stabilizer.RNext, physiological saline containing 10% of high-purity water-containing crystalline maltosyl glucoside obtained by the method of Example A-1 by changing the pH and obtaining human natural type Tsumor necrosis factor-α by the method of Example A-1. And eluted. This solution is microfiltered, aseptically filled into vials, and human natural tsumoa necrosis factor-α is 10 per ml.⁴A liquid containing the unit was obtained. This product is administered orally or parenterally to about 1 to 20 ml of adult per day, and can be advantageously used for the treatment of viral diseases, allergic diseases, rheumatism, diabetes, malignant tumors and the like. In this product, maltosyl glucoside acts as a stabilizer and maintains its activity well even after standing at 4 ° C or 25 ° C for 20 minutes.
[0116]
[Example B-19 interferon tablet]
Human natural interferon-α preparation (manufactured by Hayashibara Biochemical Laboratories Co., Ltd., Cosmo Bio Co., Ltd.) is applied to an immobilized anti-human interferon-α antibody column according to a conventional method, and humans contained in the preparation High purity water-containing water obtained by adsorbing natural interferon-α, passing through and removing bovine serum albumin as a stabilizer, and then changing the pH to obtain human natural interferon-α by the method of Example A-1. Elution was performed using physiological saline containing 5% of crystalline maltosyl glucoside. This solution is finely filtered, added to about 20 times the amount of anhydrous crystalline maltose powder (Hayashibara Shoji Co., Ltd., trade name: Finetose T), dehydrated and powdered, and tableted with a tableting machine. A tablet containing about 150 units of human natural interferon-α per (about 200 mg) was obtained. This product can be used advantageously as a sublingual tablet for the treatment of viral diseases, allergic diseases, rheumatism, diabetes, malignant tumors, etc. In particular, it can be advantageously used as a therapeutic agent for AIDS, hepatitis, etc., whose number of patients is rapidly increasing in recent years. In this product, maltose acts as a stabilizer together with maltosyl glucoside and maintains its activity well for a long time even when left at room temperature.
[0117]
[Example B-20 Dragee]
A core tablet of 150 mg in weight is used as a core, and 45 parts by weight of high-purity water-containing crystalline maltosylglucoside obtained by the method of Example A-2, 2 parts by weight of pullulan (average molecular weight 200,000), 30 parts by weight of water, talc Using an undercoat liquid consisting of 25 parts by weight and 3 parts by weight of titanium oxide, sugar coating is carried out until the tablet weight reaches about 230 mg, then 65 parts by weight of the same water-containing crystalline maltosylglucoside-containing powder, 1 part by weight of pullulan and 34 parts by weight of water The sugar-coated tablets were coated with a top part liquid and further polished with a wax liquid to obtain a sugar-coated tablet with an excellent glossy appearance. This product not only has excellent workability when applied to sugar, but also has excellent impact resistance and maintains high quality for a long time.
[0118]
[Example B-21 Latex]
0.5 parts by weight of polyoxyethylene behenyl ether, 1 part by weight of polyoxyethylene sorbitol tetraoleate, 1 part by weight of glyceryl monostearate, 0.5 parts by weight of behenyl alcohol, 1 part by weight of avocado oil A high-purity hydrous crystal maltosyl glucoside obtained by the method of Example A-2, 3.5 parts by weight, α-glycosyl rutin 1 part by weight, vitamin E and an appropriate amount of preservatives were dissolved by heating according to a conventional method. , 3-butylene glycol 5 parts by weight, 0.1 part by weight of carboxyvinyl polymer and 85.3 parts by weight of purified water were added, emulsified and emulsified to produce an emulsion. This product is a moisturizing emulsion that can be advantageously used as a sunscreen, whitening agent, and the like.
[0119]
[Example B-22 skin cream]
2 parts by weight of polyoxyethylene glycol monostearate, 5 parts by weight of glyceryl monostearate, 2 parts by weight of α-glycosyl rutin, 1 part by weight of liquid paraffin, 10 parts by weight of glyceryl trioctanoate, the method of Example A-1 1 part by weight of high-purity water-containing crystalline maltosyl glucoside obtained in 1 above, 3 parts by weight of maltitol and an appropriate amount of preservatives were dissolved by heating in accordance with a conventional method, and 5 parts by weight of 1,3-butylene glycol and 66 parts by weight of purified water Part was added, emulsified with a homogenizer, and an appropriate amount of a fragrance was added and mixed with stirring to produce a cream. This product is a cream with good elongation and can be advantageously used as a sunscreen, skin cleansing agent, whitening agent and the like.
[0120]
[Example B-23 toothpaste]
45 parts by weight of dibasic calcium phosphate, 1.5 parts by weight of sodium lauryl sulfate, 25 parts by weight of glycerin, 0.5 parts by weight of polyoxyethylene sorbitan laurate, high-purity water-containing crystalline maltosyl glucoside obtained by the method of Example A-2 Toothpaste was obtained by mixing 15 parts by weight, 0.02 part by weight of saccharin and 0.05 part by weight of preservative with 13 parts by weight of water. This product has good gloss and detergency and is suitable as a toothpaste.
[0121]
[Example B-24 Fertilizer pile]
Compound fertilizer (N = 14%, P₂O_Five= 8%, K₂O = 12%), pullulan, water-containing crystalline maltosylglucoside-containing powder obtained by the method of Example A-6, calcium sulfate and water after mixing sufficiently as 70, 5, 5, 15 and 5 respectively by weight A fertilizer pile was manufactured by heating to 80 ° C. with an extruder (L / D = 20, compression ratio = 1.8, die diameter = 30 mm). This product does not require a fertilizer container, is easy to handle, has strength suitable for full-layer fertilization, and can adjust the elution rate of fertilizer components by changing the blending ratio. If necessary, this fertilizer pile can be easily mixed with plant hormones, agricultural chemicals, soil conditioners and the like.
[0122]
【The invention's effect】
As is clear from the above, the crystalline maltosyl glucoside of the present invention is a substance that is substantially non-hygroscopic or hardly hygroscopic, has good fluidity, is easy to handle, is easily dissolved in water, and has an appropriate viscosity. And has a good sweetness. In addition, crystalline maltosyl glucoside is chemically stable and has the property of stabilizing amino acids and oligopeptides that are prone to browning reactions, as well as active ingredients and physiologically active substances that easily lose their activity. . In addition, it has properties such as osmotic pressure controllability, activation, irradiability, moisture retention, prevention of crystallization of other sugars, difficulty of fermenting, and anti-aging of starch. These various properties can be advantageously used in the production of various compositions such as foods and drinks, cosmetics, pharmaceuticals, and molded products.
[0123]
Therefore, the establishment of the crystalline maltosyl glucoside of the present invention, its production method, and its use has great industrial significance in the fields of food and drink, cosmetics, and pharmaceuticals.
[Brief description of the drawings]
FIG. 1 is a graph showing the effect of temperature on the activity of a non-reducing saccharide-forming enzyme derived from Rhizobium species M-11.
FIG. 2 is a graph showing the influence of pH on the activity of a non-reducing saccharide-forming enzyme derived from Rhizobium species M-11.
FIG. 3 is a graph showing the temperature stability of a non-reducing saccharide-forming enzyme derived from Rhizobium species M-11.
FIG. 4 is a graph showing the pH stability of a non-reducing saccharide-forming enzyme derived from Rhizobium species M-11.
FIG. 5 is an infrared absorption spectrum of hydrous crystal maltosyl glucoside.
FIG. 6 shows the results of carbon nuclear magnetic resonance analysis of maltosyl glucoside.
FIG. 7 shows a powder X-ray diffraction pattern of hydrous crystal maltosyl glucoside.
FIG. 8 is a diagram showing a powder X-ray diffraction pattern of anhydrous crystalline maltosyl glucoside.

Claims (8)

  Crystalline α-maltosyl α-glucoside.   The crystalline α-maltosyl according to claim 1, which is in the form of a hydrous crystal having 7.9 °, 13.2 °, 17.2 ° and 22.6 ° as main diffraction angles (2θ) in the powder X-ray diffraction method. α-Glucoside.   The crystalline α-maltosyl according to claim 1, in the form of anhydrous crystals having 7.7 °, 13.5 °, 17.6 ° and 23.6 ° as main diffraction angles (2θ) in powder X-ray diffraction method α-Glucoside.   Crystallized crystals of an aqueous saccharide solution containing α-maltosyl α-glucoside having a purity of 60 w / w% or more and having a concentration of about 65 w / w% or more in the presence of seed crystals of crystalline α-maltosyl α-glucoside A method for producing crystalline α-maltosyl α-glucoside, which comprises collecting α-maltosyl α-glucoside.   α-maltosyl α-glucoside is derived from trehalose and starch, gelatinized starch, liquefied starch, soluble starch, amylose, amylopectin, reducing starch partial degradation product, starch sugar transfer product, cyclodextrin, dextrin, maltooligosaccharide, and sucrose A glycosyltransferase is allowed to act on an aqueous solution containing one or two or more selected carbohydrates, or a hydrolase is allowed to act after the glycosyltransferase is allowed to act, or a glucose polymerization degree of 3 or more. A non-reducing saccharide-forming enzyme that generates a non-reducing saccharide having a trehalose structure at the end from a reducing starch partial decomposition product having a glucose polymerization degree of 3 or more is allowed to act on the reducing starch partial decomposition product or non-reducing thereof 5. The crystal α according to claim 4, wherein the crystal α is produced by the action of a hydrolase after the action of a functional sugar-forming enzyme. Method of manufacturing a maltosyl α- glucoside.   The method for producing crystalline α-maltosyl α-glucoside according to claim 5, wherein the hydrolase is β-amylase, or β-amylase and starch debranching enzyme.   Trehalose and one or more selected from starch, gelatinized starch, liquefied starch, soluble starch, amylose, amylopectin, reducing starch partial degradation product, starch saccharide transfer product, cyclodextrin, dextrin, maltooligosaccharide, and sucrose Or a hydrolase after allowing the glycosyltransferase to act on an aqueous solution containing the saccharides of the above, or a reduced starch partial degradation product having a glucose polymerization degree of 3 or more, A non-reducing saccharide-forming enzyme that produces a non-reducing saccharide having a trehalose structure at the end from a reduced starch partial degradation product having a glucose polymerization degree of 3 or more is allowed to act, or the non-reducing saccharide-forming enzyme is allowed to act. An aqueous solution containing α-maltosyl α-glucoside obtained by allowing a hydrolase to act after treatment with alkali, or chroma A method for producing crystalline α-maltosyl α-glucoside, characterized by subjecting to a topography to obtain a liquid containing a high amount of α-maltosyl α-glucoside, concentrating, crystallizing and collecting.   A saccharide aqueous solution having a concentration of about 65 w / w% or more containing α-maltosyl α-glucoside having a purity of 60 w / w% or more is crystallized in the presence of a seed crystal of crystalline α-maltosyl α-glucoside according to claim 1. A method for purifying α-maltosyl α-glucoside.

Images