JP3557287B2 - Thermostable non-reducing saccharide-forming enzyme, its production method and use - Google Patents

Description

【００４８】
表１の結果から明らかなように、本精製酵素は、グルコース重合度３以上の澱粉部分分解物であるマルトトリオース乃至マルトヘプタオースから、末端にトレハロース構造に有する非還元性糖質であるα−グルコシルトレハロース乃至α−マルトペンタオシルトレハロースを生成することが判明した。反応物中には残存するそれぞれの基質とグルコース重合度が変わることなく生成した非還元性糖質以外に、比較的少量の基質の加水分解物であるグルコースや低分子マルトオリゴ糖及びそれから生成される非還元性糖質が存在し、非還元性糖質生成作用以外にも、弱いながら加水分解作用を有することが判明した。また、本精製酵素によるそれぞれの基質からの非還元性糖質及び加水分解物により生成した還元性糖質の生成率は、マルトトリオースから３０．２％及び２７．６％で、マルトテトラオースから６５．４％及び１８．４％で、マルトペンタオース乃至マルトヘプタオースから約７４乃至７５％及び約２乃至３％であり、グルコース重合度が５以上のマルトオリゴ糖からは非還元性糖質を高い生成率で生成し、加水分解物の生成は僅か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。
【００４９】
【実験２−２ 分子量】
ユー・ケー・レムリが『ネーチャー（Ｎａｔｕｒｅ）』、第２２７巻、第６８０乃至６８５頁（１９７０年）に報告している方法に準じてＳＤＳ−ポリアクリルアミドゲル電気泳動したところ、本酵素は、分子量約６９，０００乃至７９，０００ダルトンに相当する位置に単一バンドを示した。なお、このときの分子量マーカーには、ミオシン（２００，０００ダルトン）、β−ガラクトシダーゼ（１１６，２５０ダルトン）、フォスフォリラーゼＢ（９７，４００ダルトン）、血清アルブミン（６６，２００ダルトン）及びオボアルブミン（４５，０００ダルトン）を使用した。
【００５０】
【実験２−３ 等電点】
精製耐熱性非還元性糖質生成酵素をポリアクリルアミドゲル（２％アンフォライン含有、ファルマシア・エルケービー社製）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５．４乃至６．４であった。
【００５１】
【実験２−４ 至適温度】
常法により、２０ｍＭ酢酸緩衝液（ｐＨ５．５）中で６０分間インキュベートする条件で試験したところ、図１に示すように、本酵素は、７５℃付近に至適温度を示した。
【００５２】
【実験２−５ 至適ｐＨ】
常法により、ｐＨの相違するマッキルヴェイン氏緩衝液中、６０℃で６０分間インキュベートする条件で試験したところ、図２に示すように、本酵素は、ｐＨ５．０乃至５．５付近に至適ｐＨを示した。
【００５３】
【実験２−６ 温度安定性】
常法により、１０ｍＭ燐酸緩衝液（ｐＨ７．０）中で６０分間インキュベートする条件で試験したところ、図３に示すように、本酵素は、８５℃付近まで安定であった。
【００５４】
【実験２−７ ｐＨ安定性】
常法により、ｐＨの相違するマッキルヴェイン氏緩衝液、又は炭酸ナトリウム−炭酸水素ナトリウム緩衝液中、２５℃で１６時間インキュベートする条件で試験したところ、図４に示すように、本酵素は、ｐＨ４．５乃至９．５付近まで安定であった。
【００５５】
【実験２−８ Ｎ末端アミノ酸配列】
実験１の方法で得られた精製耐熱性非還元性糖質生成酵素標品の一部をそれぞれ蒸留水に対して透析した後、蛋白量として約８０μｇをＮ末端アミノ酸配列分析用の試料とした。Ｎ末端アミノ酸配列は、アプライド・バイオシステムズ・ジャパン販売気相プロテイン・シーケンサ『４７３Ａ型』を用い、Ｎ末端から１０残基まで分析した。得られたＮ末端配列を次に示す。
【００５６】

【００５７】
【実験３ 他のスルフォロブス属微生物由来の耐熱性非還元性糖質生成酵素の調製】
スルフォロブス・アシドカルダリウス ＡＴＣＣ３３９０９に代えて、スルフォロブス・アシドカルダリウス ＡＴＣＣ４９４２６、スルフォロブス・ソルファタリカス ＡＴＣＣ３５０９１及びスルフォロブス・ソルファタリカス ＡＴＣＣ３５０９２を用いた以外は、実験１と同様にファーメンターで４２時間培養した。それぞれの培養液約１７０ｌから菌体を回収し、超音波処理し、その上清を硫安塩析、透析し、更にイオン交換カラムクロマトグラフィーと疎水カラムクロマトグラフィーにかけ、部分精製酵素標品を得、その性質を調べた。結果を表２にまとめた。
【００５８】
【表２】

【００５９】
また、これらの部分精製酵素を用いて、実験２−１の方法に従って、非還元性糖質の生成を調べたところ、いずれの酵素もスルフォロブス・アシドカルダリウス ＡＴＣＣ３３９０９由来の耐熱性非還元性糖質生成酵素の場合と同様に、グルコース重合度３以上から選ばれる還元性澱粉部分分解物から末端にトレハロース構造を有するグルコース重合度３以上から選ばれる非還元性糖質を生成することが判明した。
【００６０】
以下、本発明の非還元性糖質、それを含む低還元性糖質及びトレハロースの製造方法を実施例Ａで、非還元性糖質、それを含む低還元性糖質及び／又はトレハロースを含有せしめた組成物を実施例Ｂで示す。
【００６１】
【実施例Ａ−１】
スルフォロブス・アシドカルダリウス ＡＴＣＣ３３９０９を実験１の方法に準じて、ファーメンターで約４２時間培養した。培養後、ＳＦ膜を用いて菌体を濃縮し、更に遠心分離して菌体を回収した。実験３の方法に準じ、菌体を超音波処理し、その上清を硫安塩析、透析し、更にイオン交換カラムクロマトグラフィーと疎水カラムクロマトグラフィーを行い、比活性が約２０単位／ｍｇの部分精製酵素液（１８．０単位／ｍｌ）を得た。濃度６％の馬鈴薯澱粉乳を加熱糊化させた後、ｐＨ４．５、温度５０℃に調整し、これにイソアミラーゼ（株式会社林原生物化学研究所製）を澱粉グラム当り２５００単位の割合になるよう加え、２０時間反応させた。その反応液をｐＨ６．５に調整し、オートクレーブ（１２０℃）を１０分間行い、次いで６０℃に冷却し、これにノボ社製α−アミラーゼ『ターマミール６０Ｌ』を澱粉グラム当り３０単位の割合になるよう加え、２０時間反応させた。その反応液をオートクレーブ（１２０℃）を２０分間行った後、６５℃に冷却し、ｐＨを５．５に調整し、これに上記調製の耐熱性非還元性糖質生成酵素を澱粉グラム当たり１単位の割合になるよう加え、９６時間反応させた。その反応液を９７℃で３０分間保った後、冷却し、瀘過して得られる瀘液を、常法に従って、活性炭で脱色し、Ｈ型及びＯＨ型イオン交換樹脂により脱塩して精製し、更に濃縮して濃度約７０％のシラップを固形分当たり約９０％で得た。本品は、ＤＥ２４．６であって、非還元性糖質を固形分当り、α−グルコシルトレハロース １２．０％、α−マルトシルトレハロース ５．５％、α−マルトトリオシルトレハロース ２９．９％、α−マルトテトラオシルトレハロース １．５％、及びα−マルトペンタオシルトレハロース ２．２％を含有しており、温和で上品な甘味、適度の粘度、保湿性を有し、甘味料、呈味改良剤、品質改良剤、安定剤、賦形剤などとして、各種飲食物、化粧品、医薬品など各種組成物に有利に利用できる。
【００６２】
【実施例Ａ−２】
実施例Ａ−１の方法で得られた糖液を原糖液とし、非還元性糖質の含量を高めるため、東京有機化学工業株式会社製ナトリウム型強酸性カチオン交換樹脂『ＸＴ−１０１６』（架橋度４％）を用いたカラム分画を行った。樹脂を内径５．４ｃｍのジャケット付ステンレス製カラム４本に充填し、直列につなぎ樹脂層全長２０ｍとした。カラム内温度を５５℃に維持しつつ、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、グルコース及びマルトース高含有画分を除去し、非還元性糖質高含有画分を採取した。更に、精製、濃縮し、真空乾燥し、粉砕して、非還元性糖質高含有粉末を固形分当たり約６４％で得た。本品はＤＥ４．８であって、非還元性糖質を、固形物当り、α−グルコシルトレハロース １８．２％、α−マルトシルトレハロース ７．９％、α−マルトトリオシルトレハロース ４６．６％、α−マルトテトラオシルトレハロース ２．３％、及びα−マルトペンタオシルトレハロース ３．４％を含有しており、実施例Ａ−１と同様に、温和で上品な甘味、適度の粘度、保湿性を有し、甘味料、呈味改良剤、品質改良剤、安定剤、賦形剤などとして、各種飲食物、化粧品、医薬品など各種組成物に有利に利用できる。
【００６３】
【実施例Ａ−３】
３３％とうもろこし澱粉乳に最終濃度０．１％となるように炭酸カルシウムを加えた後、ｐＨ６．５に調整し、これにターマミール６０Ｌを澱粉グラム当たり０．２％になるよう加え、９５℃で１５分間反応させた。その反応液をオートクレーブ（１２０℃）を１０分間行った後、５５℃に冷却し、これに特開昭６３−２４０７８３号公報で開示されている株式会社林原生物化学研究所製マルトテオラオース生成アミラーゼを澱粉グラム当たり５単位の割合になるように加え、６時間反応させ、これに上田化学株式会社製α−アミラーゼ『α−アミラーゼ２Ａ』を澱粉グラム当り３０単位加え、更に６５℃で４時間反応させた。その反応液をオートクレーブ（１２０℃）を１０分間行い、次いで６５℃に冷却し、ｐＨを５．５に調整した後、これに実施例Ａ−１の方法で調製した耐熱性非還元性糖質生成酵素を澱粉グラム当り２単位の割合になるよう加え、４８時間反応させた。その反応液を９７℃で３０分間保った後、冷却し、瀘過して得られる瀘液を、常法に従って、活性炭で脱色し、Ｈ型及びＯＨ型イオン交換樹脂により脱塩して精製し、更に濃縮して濃度約７０％のシラップを固形物当り収率約９０％で得た。本品は、ＤＥ１７．１であって、非還元性糖質を、固形物当りα−グルコシルトレハロース ８．９％、α−マルトシルトレハロース ２９．３％、α−マルトトリオシルトレハロース ０．８％、α−マルトテトラオシルトレハロース ０．７％、及びα−マルトペンタオシルトレハロース ０．７％を含有しており、温和で上品な甘味、適度の粘度、保湿性を有し、甘味料、呈味改良剤、品質改良剤、安定剤、賦形剤などとして、各種飲食物、化粧品、医薬品など各種組成物に有利に利用できる。
【００６４】
【実施例Ａ−４】
実施例のＡ−３の方法で得られた糖液を原糖液とし、本液のα−マルトシルトレハロースの含量を高めるため、分画用樹脂として、ダウケミカル社販売マグネシウム型強酸性カチオン交換樹脂『ダウエックス５０Ｗ×４』を用いた以外は、実施例Ａ−２の方法に従ってカラムクロマトグラフィーを行い、α−マルトシルトレハロース高含有画分を採取した。更に、精製、濃縮し、噴霧乾燥して、非還元性糖質高含有粉末を固形物当たり収率約４１％で得た。本品は、非還元性糖質を固形物当り、α−グルコシルトレハロース １０．９％、α−マルトシルトレハロース ６１．３％、α−マルトトリオシルトレハロース １．０％含有しており、そのＤＥは、２．５を示し、極めて還元性が少なく、実施例Ａ−３と同様に、温和で上品な甘味、適度の粘度、保湿性を有し、甘味料、呈味改良剤、品質改良剤、安定剤、賦形剤などとして、各種飲食物、化粧品、医薬品など各種組成物に有利に利用できる。
【００６５】
【実施例Ａ−５】
松谷化学工業株式会社製澱粉部分分解物『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６５℃、ｐＨ５．５に調整した後、実施例Ａ−１に方法で調製した耐熱性非還元性糖質生成酵素を澱粉部分分解物グラム当り１単位の割合になるように加えて、９６時間反応させ、次いで９７℃に３０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、ナガセ生化学工業株式会社製グルコアミラーゼ『グルコチーム』を澱粉部分分解物グラム当り１０単位加えて１０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、常法に従って、活性炭で脱色し、イオン交換樹脂により脱塩し、濃度約６０％に濃縮した。本糖液中には固形物当り３０．１％のトレハロースを含有していた。分画用樹脂として、オルガノ株式会社販売ナトリウム型強酸性カチオン交換樹脂『ＣＧ６０００』を用いた以外は、実施例Ａ−２の方法に従ってカラムクロマトグラフィーを行い、トレハロース高含有画分を採取した。本高含有液は、固形物当り約９７％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約２％を加えて徐冷し、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／ｃｍ^２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。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、各種飲食物、化粧品、医薬品など各種組成物に有利に利用できる。
【００６６】
【実施例Ａ−６】
スルフォロバス・ソルファタリカス ＡＴＣＣ３５０９１を実験３の方法に準じて、ファーメンターで約４２時間培養した。培養後、ＳＦ膜を用いて菌体を濃縮し、更に遠心分離して菌体を回収し、超音波処理し、その上清を硫安塩析、透析し、更にイオン交換カラムクロマトグラフィーと疎水カラムクロマトグラフィーを行い、比活性が約１８単位／ｍｇの部分精製酵素液（１９．０単位／ｍｌ）を得た。３０％とうもろこし澱粉乳を用いて、実施例Ａ−３の方法に準じて、ターマミール６０Ｌ、次いでマルトテトラオース生成アミラーゼ（株式会社林原生物化学研究所製）及びα−アミラーゼ２Ａを作用させ、オートクレーブ（１２０℃）処理し、次いで、６５℃に冷却し、これに上記の方法で調製した耐熱性非還元性糖質生成酵素を澱粉グラム当たり２単位になるように加え、６４時間反応させた。次いで、９７℃に３０分間加熱して酵素を失活させた。本反応液を、実施例Ａ−５の方法に準じて、グルコチームを作用させ、脱色、脱塩して、濃度約６０％に濃縮した。本糖液中には、固形物当たり約２３％のトレハロースを含有していた。本糖液を実施例Ａ−５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を行い、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９５％のトレハロースを含有していた。本溶液を蒸発釜にとり、減圧下で煮詰め、水分４．０％のシラップとし、助晶機に移し、これに種晶として無水結晶トレハロースをシラップ固形分当たり１％加え、９５℃で５分間撹拌助晶し、次いで、アルミ製バットに取り出し、１００℃で６時間晶出熟成させてブロックを調製した。次いで、本ブロックを切削機にて粉砕し、流動乾燥して、水分０．３％の無水結晶トレハロース粉末を得た。本品は、食品、医薬品、化粧品、その原材料、又は加工中間物などの含水物の脱水剤としてのみならず、上品な甘味を有する白色粉末甘味料としても有利に利用できる。
【００６７】
【実施例Ｂ−１ 甘味料】
実施例Ａ−４の方法で得た非還元性糖質高含有粉末１重量部に、東洋精糖株式会社販売α−グリコシルステビオシド『αＧスイート』０．０１重量部及び味の素株式会社製Ｌ−アスパルチル−Ｌ−フェニルアラニンメチルエステル『アスパルテーム』０．０１重量部を均一に混合し、顆粒成型機にかけて、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、それに配合した高甘味度甘味物の分解もなく、安定性に優れており、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も好適である。
【００６８】
【実施例Ｂ−２ ハードキャンディー】
濃度５５％蔗糖溶液１００重量部に実施例Ａ−３の方法で得た非還元性糖質含有シラップ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、常法に従って成型し、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。
【００６９】
【実施例Ｂ−３ 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、これに蔗糖４重量部及び実施例Ａ−５の方法で得たトレハロース含水結晶粉末３重量部とを加え、更に適量の香料と着色料とを混合し、常法に従って、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。
【００７０】
【実施例Ｂ−４ 加糖練乳】
原乳１００重量部に実施例Ａ−１の方法で得た非還元性糖質含有シラップ３重量部及び蔗糖１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。
【００７１】
【実施例Ｂ−５ 乳酸菌飲料】
脱脂粉乳１７５重量部、実施例Ａ−２の方法で得た非還元性糖質高含有粉末８０重量部及び特開平４−２８１７９５号公報で開示されているラクトスクロース高含有粉末５０重量部を水１，２００重量部に溶解し、６５℃で３０分間殺菌し、４０℃に冷却後、これに、常法に従って、乳酸菌のスターターを３０重量部植菌し、３７℃で８時間培養して乳酸菌飲料を得た。本品は、風味良好な乳酸菌飲料である。また、本品は、オリゴ糖を含有し、乳酸菌を安定に保持するだけでなく、ビフィズス菌増殖促進作用をも有する。
【００７２】
【実施例Ｂ−６ 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重量部に対して、実施例Ａ−２の方法で得た非還元性糖質高含有粉末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℃、風量１５０ｍ^３とし、これに、実施例Ａ−１の方法で得た非還元性糖質含有シラッ プ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、長期に安定であった。
【００７３】
【実施例Ｂ−７ 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の方法で得た非還元性糖質含有シラップ１００重量部、マルトース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。
【００７４】
【実施例Ｂ−８ あん】
原料あずき１０重量部に、常法に従って、水を加えて煮沸し、渋切り、あく抜きして、水溶性夾雑物を除去して、あずきつぶあん約２１重量部を得た。この生あんに、蔗糖１４重量部、実施例Ａ−４の方法で得た非還元性糖質高含有シラップ５重量部及び水４重量部を加えて煮沸し、これに少量のサラダオイルを加えてつぶあんをこわさないように練り上げ、製品のあんを約３５重量部得た。本品は、色焼けもなく、舌ざわりもよく、風味良好で、あんパン、まんじゅう、だんご、もなか、氷菓などのあん材料として好適である。
【００７５】
【実施例Ｂ−９ パン】
小麦粉１００重量部、イースト２重量部、砂糖５重量部、実施例Ａ−２の方法で得た非還元性糖質高含有粉末１重量部及び無機フード０．１重量部を、常法に従って、水でこね、中種を２６℃で２時間発酵させ、その後３０分間熟成し、焼き上げた。本品は、色相、すだちとも良好で適度な弾力、温和な甘味を有する高品質のパンである。
【００７６】
【実施例Ｂ−１０ ハム】
豚もも肉１，０００重量部に食塩１５重量部及び硝酸カリウム３重量部を均一にすり込んで、冷室に１昼夜堆積する。これを水５００重量部、食塩１００重量部、硝酸カリウム３重量部、実施例Ａ−４の方法で得た非還元性糖質高含有粉末４０重量部及び香辛料からなる塩漬液に冷室で７日間漬け込み、次いで、常法に従って、冷水で洗浄し、ひもで巻き締め、燻煙し、クッキングし、冷却包装して製品を得た。本品は、色合いもよく、風味良好な高品質のハムである。
【００７７】
【実施例Ｂ−１１ 粉末ペプチド】
不二製油株式会社製４０％食品用大豆ペプチド溶液『ハイニュートＳ』１重量部に、実施例Ａ−５の方法で得たトレハロース含水結晶粉末２重量部を混合し、プラスチック製バットに入れ、５０℃で減圧乾燥し、粉砕して粉末ペプチドを得た。本品は、風味良好で、プレミックス、冷菓などの製菓用材料としてのみならず、経口流動食、経管流動食などの離乳食、治療用栄養剤などとしても有利に利用できる。
【００７８】
【実施例Ｂ−１２ 粉末卵黄】
生卵から調製した卵黄をプレート式加熱殺菌機で６０乃至６４℃で殺菌し、得られる液状卵黄１重量部に対して、実施例Ａ−６の方法で得た無水結晶トレハロース粉末４重量部の割合で混合した後バットに移し、一夜放置して、トレハロース含水結晶に変換させてブロックを調製した。本ブロックを切削機にかけて粉末化し、粉末卵黄を得た。本品は、プレミックス、冷菓、乳化剤などの製菓用材料としてのみならず、経口流動食、経管流動食などの離乳食、治療用栄養剤などとしても有利に利用できる。また、美肌剤、育毛剤などとしても有利に利用できる。
【００７９】
【実施例Ｂ−１３ 化粧用クリーム】
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、実施例Ａ−２の方法で得た非還元性糖質高含有粉末２重量部、α−グリコシル ルチン１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ン１０重量部及び防腐剤の適量を常法に従って加熱溶解し、これにＬ−乳酸２重量部、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、抗酸化性を有し、安定性が高く、高品質の日焼け止め、美肌剤、色白剤などとして有利に利用できる。
【００８０】
【実施例Ｂ−１４ 固体製剤】
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）を、常法に従って、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−５の方法で得たトレハロース含水結晶粉末を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倍量の株式会社林原商事販売無水結晶マルトース粉末『ファイントース』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、一日当たり、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、トレハロースと共にマルトースが安定剤として作用し、室温でも放置してもその活性を長期間よく維持する。
【００８１】
【実施例Ｂ−１５ 糖衣錠】
重量１５０ｍｇの素錠を芯剤とし、これに実施例Ａ−５の方法で得たトレハロース含水結晶粉末４０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、同じトレハロース含水結晶粉末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在る外観の優れた糖衣錠を得た。本品は、耐衝撃性にも優れており、高品質を長期間維持する。
【００８２】
【実施例Ｂ−１６ 流動食用固体製剤】
実施例Ａ−５の方法で製造したトレハロース含水結晶粉末５００重量部、粉末卵黄２７０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１．８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し、この配合物２５グラムずつ防湿性ラミネート小袋に充填し、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の水に溶解して流動食とし、経口的、又は鼻腔、胃、腸などへ経管的使用方法により利用され、生体へのエネルギー補給用に有利に利用できる。
【００８３】
【実施例Ｂ−１７ 外傷治療用膏薬】
実施例Ａ−５の方法で製造したトレハロース含水結晶粉末２００重量部及びマルトース３００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１０ｗ／ｖ％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、ヨウ素による殺菌作用のみならず、トレハロースによる細胞へのエネルギー補給剤としても作用することから、治癒期間が短縮され、創面もきれいに治る。
【００８４】
【発明の効果】
上記から明らかなように、本発明の新規耐熱性非還元性糖質生成酵素は、５５℃を越える温度で酵素反応が容易に行えるため、微生物汚染を懸念することなく、澱粉部分分解物を同じグルコース重合度の非還元性糖質に高収率で変換する。その非還元性糖質の分離、精製も容易であり、このようにして得られる非還元性糖質、これを含む低還元性糖質及びこれから製造されるトレハロースは安定性に優れ、良質で上品な甘味を有している。非還元性糖質、これを含む低還元性糖質及びトレハロースは甘味料、呈味改良剤、品質改良剤、安定剤、賦形剤などとして、各種飲食物、化粧品、医薬品など各種組成物に有利に利用できる。
【００８５】
従って、本発明の確立は、安価で無限の資源である澱粉に由来する澱粉部分分解物から、従来、望むべくして容易に得られなかった末端にトレハロース構造を有する非還元性糖質、これを含む低還元性糖質、及びこれから容易に製造されるトレハロースを、工業的に大量かつ安価に供給できる全く新しい道を拓くこととなり、それが与える影響の大きさは、食品、化粧品、医薬品分野は勿論のこと、農水畜産業、化学工業にも及び、これら産業界に与える工業的意義は計り知れないものがある。
【図面の簡単な説明】
【図１】本発明の耐熱性非還元性糖質生成酵素の酵素活性に及ぼす温度の影響を示す図である。
【図２】本発明の耐熱性非還元性糖質生成酵素の酵素活性に及ぼすｐＨの影響を示す図である。
【図３】本発明の耐熱性非還元性糖質生成酵素の安定性に及ぼす温度の影響を示す図である。
【図４】本発明の耐熱性非還元性糖質生成酵素の安定性に及ぼすｐＨの影響を示す図である。[0001]
[Industrial applications]
The present invention relates to a thermostable non-reducing saccharide-forming enzyme, a method for producing the same, and a use thereof. More specifically, a trehalose structure is formed at the terminal from one or more kinds of partially degradable reducing starch having a glucose polymerization degree of 3 or more. Novel heat-resistant non-reducing saccharide-forming enzyme capable of producing non-reducing saccharides having the same, a method for producing the same, and a non-reducing saccharide having a trehalose structure at a terminal produced using the novel heat-resistant non-reducing saccharide-forming enzyme The present invention relates to a saccharide, a low-reducing saccharide containing the saccharide, trehalose produced therefrom, and a composition containing these non-reducing saccharides.
[0002]
[Prior art]
Trehalose (α, α-trehalose) has long been known as a non-reducing carbohydrate having glucose as a constituent sugar, and its existence is described in “Advances in Carbohydrate Chemistry”. 18: 201-225 (1963) Academic Press, Inc. (USA) and Applied and Environmental Microbiology, 56, 3213-3215. Applied and Environmental Microbiology, Applied and Environmental Microbiology. As described on page (1990) and the like, microorganisms, mushrooms, insects, and the like are spread over a wide range in a small amount. Because trehalose is a non-reducing saccharide, it does not cause an aminocarbonyl reaction with a substance having an amino group such as an amino acid or a protein, and does not impair the amino acid-containing substance. Therefore, it is expected that trehalose can be used and processed without worrying about browning and deterioration. Therefore, establishment of an industrial production method is desired.
[0003]
As a method for producing trehalose, for example, a method using a microorganism reported in JP-A-50-154485, or a combination of maltose phosphorylase and trehalose phosphorylase proposed in JP-A-58-216695 is used. A method for converting maltose is known. However, in the method using a microorganism, the cells are used as a starting material, and the content of trehalose contained therein is usually 15 w / w% per solid matter (hereinafter, w / w unless otherwise specified in the present specification). % Is abbreviated as%), and the steps of extracting and purifying it are complicated and unsuitable as an industrial production method. In addition, in the methods using maltose phosphorylase and trehalose phosphorylase, both involve glucose phosphate, it is difficult to increase the substrate concentration. The production rate is low, and it is difficult to keep the reaction system of both enzymes stable and to allow the reaction to proceed smoothly, and it has not yet been realized as an industrial production method.
[0004]
In connection with this, in the “Oligosaccharides” section of “Current Status and Issues of Starch Utilization Development”, “Monthly Food Chemicals”, August issue, pp. 67-72 (1992), Although the range is conceivable, it is said that enzymatic production of the present saccharide directly from starch carbohydrate using a glycosylation and hydrolysis reaction is not scientifically possible at present. " As described above, it has heretofore been considered scientifically impossible to produce trehalose by using an enzyme reaction from starch.
[0005]
On the other hand, starch partially decomposed products produced using starch as a raw material, for example, starch liquefied products, various dextrins, various maltooligosaccharides, etc., are generally known to have a reducing group at the terminal of the molecule and exhibit reducibility. I have. Such a partially hydrolyzed starch is referred to herein as a partially hydrolyzed starch. In general, the reducing starch partially decomposed product indicates the magnitude of the reducing power per solid substance as Dextrose Equivalent (DE). Those with a large value usually have small molecules, low viscosity and strong sweetness, but are highly reactive and easily cause an aminocarbonyl reaction with substances having an amino group such as amino acids and proteins, causing browning and offensive odor. Therefore, it is known that quality tends to deteriorate.
[0006]
The various properties of such a partially decomposed reducible starch depend on the size of DE, and the relationship between the partially decomposed reductant and DE is extremely important. In the past, the industry has even believed that breaking this relationship was impossible.
[0007]
The only way to break the relationship between the partially decomposed reducible starch and DE is to convert the reductive group of the partially decomposed reductive starch into a sugar alcohol by a high-pressure hydrogenation method or the like to form a non-reducible carbohydrate. It is. However, this method requires a high-pressure autoclave, not only consumes a large amount of hydrogen and energy, but also requires sophisticated safety facilities and management for disaster prevention. In addition, the sugar alcohol of the resulting partially decomposed reductive starch is different in that the raw reductive starch partially decomposed product is composed of glucose and sorbitol, whereas the raw material is composed of glucose and sorbitol. Depending on the condition, it may cause transient, but indigestible, slowing symptoms. Therefore, it has been desired to establish a method for reducing or eliminating the reducing power of glucose, which is a constituent sugar of the partially decomposed starch, without changing glucose.
[0008]
In view of such a situation, the present inventor has conducted an intensive search for an enzyme that produces a saccharide having a trehalose structure from starch sugar, and as a result, is disclosed in Japanese Patent Application No. 5-349216. (JP-A-7-143876) Microorganisms such as microorganism M-11 belonging to the genus Rhizobium isolated from soil and microorganism Q36 belonging to the genus Arthrobacter isolated from soil can reduce glucose polymerization degree 3 or more. It has been found that a completely novel non-reducing saccharide-forming enzyme, which is conventionally unknown, that produces a non-reducing saccharide having a trehalose structure at the terminal from a partially degraded starch is produced. It has also been found that trehalose can be easily produced by allowing glucoamylase or α-glucosidase to act on a non-reducing saccharide having a trehalose structure at the terminal obtained using this enzyme.
[0009]
However, the above-mentioned enzymes of the genus Rhizobium or Arthrobacter are poor in heat resistance, and when these enzymes are used to produce non-reducing saccharides or trehalose having a trehalose structure at the terminal, a temperature of about 55 ° C. or less is required. It is necessary to carry out an enzymatic reaction. Related to this, “Knowledge of Enzyme Applications”, First Edition, pp. 80-129 (1986), “Saccharide-related Enzymes and Its Applications” Under saccharification conditions, at 55 ° C. or lower, there is a risk of bacterial contamination, and the pH decreases during the saccharification reaction. ”In the case of a long-term enzymatic reaction using starch as a raw material, 55 ° C. or lower Under the reaction conditions at a temperature of, there is a concern that the pH of the reaction solution is lowered due to bacterial contamination and the enzyme is deactivated during the reaction, and it may be necessary to prevent bacterial contamination by adding lysozyme or the like or adjust the pH of the reaction solution. . In addition, when the hydrolysis rate of the partially degraded starch is low, there is a concern that insolubilized products may be formed due to aging. On the other hand, since the enzyme reaction of the heat-resistant enzyme proceeds even at a high reaction temperature, the reaction using the heat-resistant enzyme is less likely to cause microbial contamination, and it is considered that aging of the partially degraded starch does not easily occur. Therefore, it is desired to establish a novel method for producing non-reducing saccharides having a trehalose structure at the terminal or trehalose using a thermostable non-reducing saccharide-forming enzyme capable of performing an enzyme reaction at a temperature exceeding 55 ° C.
[0010]
[Problems to be solved by the invention]
An object of the present invention is to provide a novel method for producing a non-reducing saccharide from a partially degraded reducing starch using a thermostable non-reducing saccharide-forming enzyme, the non-reducing saccharide and its use. is there.
[0011]
[Means for Solving the Problems]
In order to solve the above-mentioned problems, the present inventors have hopes for the realization of a thermostable non-reducing saccharide-forming enzyme that produces a non-reducing saccharide having a trehalose structure at the terminal from a partially degraded reducing starch. Therefore, microorganisms producing this enzyme have been widely searched. As a result, microorganisms belonging to the genus Sulfolobus Sulfolobus acidocaldarius ATCC33909 and ATCC49426, furthermore, Sulfolobus solfataricus (Sulfolobus solfataric degraded from AT135 to AT350), and ATCC91 from a reduced form of ATCC33 to AT35 of ATCC33, and a partially reduced ATCC of ATCC91 It has been found that a non-reducing saccharide-forming enzyme having a structure is produced and a novel heat-resistant non-reducing saccharide-forming enzyme that is stable up to around 85 ° C. is produced. By acting on the partially degraded product, it was found that a non-reducing saccharide having a trehalose structure at a terminal can be easily produced under the intended operating condition of over 55 ° C. Further, it was found that trehalose can be easily produced by allowing the heat-resistant non-reducing saccharide-forming enzyme to act on the reduced starch partially degraded product, and then allowing glucoamylase or α-glucosidase to act thereon, The present invention has been completed. In addition, the present invention was completed by establishing a composition of the non-reducing saccharide, a low-reducing saccharide containing the non-reducing saccharide and / or trehalose, such as a food and drink, a cosmetic and a pharmaceutical. In the present specification, unless otherwise specified, a non-reducing saccharide having a trehalose structure at its terminal is produced from a partially decomposed reducible starch, and a novel heat-resistant non-reducing saccharide capable of enzymatic reaction at a temperature exceeding 55 ° C. Sex saccharide-forming enzymes are referred to as thermostable non-reducing saccharide-forming enzymes.
[0012]
In the present invention, not only the above-mentioned bacteria, but also other heat-resistant non-reducing saccharide-forming enzymes that belong to the genus Sulfolobus and produce a non-reducing saccharide having a trehalose structure at the terminal from a partially degradable reducing starch Strains, and mutants of those strains, etc. are also used as appropriate.
[0013]
The medium used for culturing the microorganism of the present invention may be any nutrient medium capable of growing the microorganism and producing the thermostable non-reducing saccharide-forming enzyme of the present invention, and may be either a synthetic medium or a natural medium. The carbon source may be any substance that can be assimilated by microorganisms, for example, glucose, fructose, lactose, sucrose, mannitol, sorbitol, molasses, carbohydrates such as starch partially decomposed products, or citric acid, succinic acid, etc. Organic acids or salts thereof can also be used. The concentration of these carbon sources in the medium is appropriately selected depending on the type of the carbon source. For example, in the case of partially degraded starch, the content is usually preferably 20% or less, and more preferably 5% or less in view of the growth and proliferation of bacteria. Examples of the nitrogen source include inorganic nitrogen compounds such as ammonium salts and nitrates, and organic nitrogen-containing substances such as urea, corn steep liquor, casein, peptone, yeast extract, and meat extract. As the inorganic component, for example, calcium salt, magnesium salt, potassium salt, sodium salt, phosphate, manganese salt, zinc salt, iron salt, copper salt, molybdenum salt, cobalt salt and the like are appropriately used.
[0014]
The cultivation is usually performed at a temperature of 40 to 95 ° C, preferably 50 to 90 ° C, and a pH of 2 to 7, preferably 2 to 6. The culturing time may be any time as long as the microorganism can grow, and is preferably 10 to 100 hours. The concentration of dissolved oxygen in the culture solution is not particularly limited, but is usually preferably 0.5 to 20 ppm. Therefore, means such as adjusting the amount of ventilation, stirring, adding oxygen to the ventilation, and increasing the pressure in the fermenter are employed. The culture method may be either batch culture or continuous culture.
[0015]
After culturing the microorganism in this way, the enzyme of the present invention is recovered. The present enzyme activity is mainly observed in the cells of the culture, and it is desirable to purify and use it by a known method. For example, a crude enzyme preparation obtained by subjecting a cell extract to ammonium sulfate precipitation and concentration is dialyzed, and then anion exchange column chromatography using a gel “DEAE-Toyopearl” manufactured by Tosoh Corporation and a gel “butyl toyopearl” manufactured by Tosoh Corporation And the like, and a partially purified enzyme preparation from which contaminating enzymes have been almost removed can be obtained. Subsequently, purification is performed by gel filtration chromatography using Sepracor gel "Ultrogel AcA44" or the like, or anion exchange column chromatography using Pharmacia LKB gel "Mono Q" or the like. A single enzyme can also be obtained electrophoretically.
[0016]
The thermostable non-reducing saccharide-forming enzyme of the present invention thus obtained has the following physicochemical properties.
(1) Action
Is glucose polymerization degree 3 or more? Selection A non-reducing saccharide having a trehalose structure at a terminal is produced from one or more kinds of partially decomposed reducing starch.
(2) Molecular weight
Approximately 69000 to 79,000 daltons by SDS-gel electrophoresis.
(3) Isoelectric point
PI of about 5.4 to 6.4 by electrophoresis containing ampholine.
(4) Optimal temperature
pH 5.5, 60 minutes reaction, around 75 ° C.
(5) Optimum pH
The pH is around 5.0 to 5.5 at 60 ° C for 60 minutes.
(6) Temperature stability
Stable up to around 85 ° C by holding pH 7.0 for 60 minutes.
(7) pH stability
PH of about 4.0 to 9.5 at 25 ° C for 16 hours.
[0017]
The activity of the thermostable non-reducing saccharide-forming enzyme of the present invention is measured as follows. 1.0 ml of the enzyme solution was added to 4 ml of maltopentaose 1.25 w / v% (20 mM acetate buffer, pH 5.5) as a substrate and reacted at 60 ° C. for 60 minutes. Stop the reaction, dilute the reaction solution exactly 10 times with deionized water, and measure the reducing power of the diluted solution by the Somogyi Nelson method. As a control, the same measurement is performed using an enzyme solution which has been inactivated by heating at 100 ° C. for 30 minutes in advance. The reaction is stopped by adding a copper solution, and the reducing power is measured by the Somogy-Nelson method. Using the above measurement method, the amount of the enzyme that reduces the reducing power corresponding to 1 μmole maltopentaose per minute was defined as one unit.
[0018]
As a substrate of the present enzyme, a partially decomposed reductive starch obtained by partially hydrolyzing starch such as starch, amylopectin, and amylose with amylase or an acid is used. Examples of the partially degraded reducing starch degraded by amylase include “Handbook of Amylases and Related Enzymes”, (1988) Pergamon Press (Tokyo). The partially degradable reductive starch degraded by the described amylase such as α-amylase, maltotriose-forming amylase, maltotetraose-forming amylase, maltopentaose-forming amylase, and maltohexaose-forming amylase is used. Furthermore, when preparing a partially decomposed product of reducing starch, a debranching enzyme such as pullulanase and isoamylase may optionally be allowed to act. It is also advantageous to use one or more maltooligosaccharides such as maltotriose, maltotetraose, maltopentaose, maltohexaose and maltoheptaose.
[0019]
The substrate concentration is not particularly limited. For example, the reaction of the present enzyme proceeds even when used as a 0.1% substrate solution, but industrially, a high concentration reaction of 2% or more, preferably 5 to 50% is suitable. A non-reducing saccharide having a trehalose structure can be advantageously produced. The reaction may be carried out at a temperature at which the enzyme is stable, that is, up to around 85 ° C, but preferably around 55 to 70 ° C. The reaction pH may be usually adjusted to a range of 3 to 9, but is preferably adjusted to a range of about 4 to 7. The reaction time may be appropriately selected depending on the progress of the enzymatic reaction, and is usually about 0.1 to 100 hours with an amount of the enzyme of about 0.1 to 100 units per gram of the solid substrate.
[0020]
The reaction solution containing the non-reducing saccharide obtained by the above reaction has a remarkably reduced reducing power as compared with the partially decomposed reducible starch used as the substrate. For example, when maltopentaose is used as the substrate, the reducing power of the reaction solution due to the present enzyme reaction is reduced to about 25%, since about 75% of the initial reducing power of the substrate maltopentaose solution disappears.
[0021]
The reaction solution is filtered, centrifuged, and the like to remove insoluble matter, then decolorized with activated carbon, desalted with an H-type or OH-type ion exchange resin, and concentrated to obtain a syrup-like product. Furthermore, it is optional to dry it into a powdery product. If necessary, further purification, for example, ion-exchange column chromatography, activated carbon column chromatography, fractionation by column chromatography such as silica gel column chromatography, fractionation with organic solvents such as alcohol and acetone, membrane with appropriate separation performance By the combination of one or more methods such as fermentation treatment with yeast, decomposition and removal of remaining reducing saccharides by alkali treatment, etc. It is also easy to obtain sex carbohydrate products.
[0022]
In particular, as an industrial mass production method, the use of ion exchange column chromatography is preferable. For example, strongly acidic cations disclosed in JP-A-58-23799, JP-A-58-72598, and the like can be used. Contaminant saccharides can be removed by column chromatography using an exchange resin, and a non-reducing saccharide having an improved content of the target substance can be advantageously produced. At this time, it is optional to adopt any of the fixed bed system, the moving bed system, and the simulated moving bed system.
[0023]
The thus obtained non-reducing saccharide having a trehalose structure at the terminal thereof or a low-reducing saccharide comprising the same, if necessary, may be used as an amylase, for example, α-amylase, β-amylase, glucoamylase, etc. Alternatively, it can be decomposed with α-glucosidase to adjust the sweetness, reducing power, etc., or to reduce the viscosity, or to reduce the remaining reducing sugars by hydrogenation to sugar alcohol to reduce the reducing power. Further processing such as disappearing is optional.
[0024]
In particular, trehalose can be easily produced by allowing glucoamylase or α-glucosidase to act on the non-reducing saccharide of the present invention or the low-reducing saccharide containing the same. That is, glucoamylase or α-glucosidase is allowed to act on these non-reducing or low-reducing carbohydrates to form a mixed solution of trehalose and glucose, which is purified by the above-described purification method, for example, ion exchange column chromatography. Then, glucose is removed and a trehalose-rich fraction is collected. This can be advantageously carried out by purifying and concentrating the product to obtain a syrup-like product, or by further concentrating it to supersaturation and crystallization to obtain trehalose-containing crystals or anhydrous trehalose crystals.
[0025]
In order to produce trehalose-containing crystals, for example, a trehalose-containing solution having a purity of 60% or more and a concentration of 65 to 90% is placed in an auxiliary crystal can, and if necessary, in the presence of a 0.1 to 20% seed crystal, the temperature is increased. The mixture is gradually cooled with stirring at a temperature of 95 ° C. or lower, preferably 10 to 90 ° C., to produce a mass kit containing trehalose hydrate crystals. It is also advantageous to employ a continuous crystallization method in which crystallization is performed while concentrating under reduced pressure. As a method for producing trehalose hydrated crystals or honey-containing crystals containing the same from a mass kit, for example, a known method such as a honey separating method, a block crushing method, a fluid granulation method, and a spray drying method may be employed.
[0026]
In the case of the honey separation method, the mass kit is usually placed in a basket-type centrifugal separator to separate the trehalose hydrated crystals from the nectar (mother liquor), and it is also easy to wash the crystals by spraying a small amount of cold water, if necessary. This method is suitable for producing higher purity trehalose hydrated crystals. In the case of the spray drying method, usually, a mass kit having a concentration of about 60 to 85% and a crystallization rate of about 20 to 60% is sprayed from a nozzle with a high-pressure pump, and hot air at a temperature at which the crystal powder is not dissolved, for example, 60 to 100 ° C. And then aged for about 1 to 20 hours with warm air at 30 to 60 ° C., non-hygroscopic or hardly hygroscopic honey-containing crystals can be easily produced. In the case of the block pulverization method, usually, a mass kit having a water content of about 10 to 20% and a crystallization rate of about 10 to 60% is allowed to stand for several hours to 3 days to crystallize and solidify the whole in a block shape. If powdered and dried by a method such as pulverization or cutting, non-hygroscopic or poorly hygroscopic honey-containing crystals can be easily produced.
[0027]
In order to produce anhydrous crystalline trehalose, hydrated trehalose crystals can be converted by drying. However, in general, a high-concentration trehalose-rich solution having a water content of less than 10% is placed in an auxiliary crystal can and mixed with seed crystals. A mass kit containing anhydrous crystalline trehalose is produced while stirring at a temperature of 50 to 160 ° C., preferably 80 to 140 ° C. under a high temperature drying condition, for example, a block pulverization method, a fluid granulation method. It is produced by crystallization and powdering by a method such as spray drying.
[0028]
The non-reducing saccharide of the present invention, the low-reducing saccharide containing the non-reducing saccharide, and trehalose produced in this manner have a low reducing property and are stable compared with the raw material partially decomposed by reducing starch. Materials, especially amino acids, oligopeptides, proteins and other amino acids or amino group-containing substances, even when mixed and processed, does not brown, does not generate off-flavors, and can damage other mixed materials Few. Further, unlike the case of the partially decomposed starch, the reducing power is low, despite the low power, and when the average glucose polymerization degree is low, it has good and elegant sweetness. .
[0029]
Furthermore, amylase, for example, decomposed by pancreatic α-amylase to produce low-molecular non-reducing oligosaccharides and low-molecular maltooligosaccharides, and these oligosaccharides are also easily decomposed by α-glucosidase and small intestinal enzymes, It produces glucose and trehalose, and the produced trehalose is easily decomposed into glucose by trehalase, so that it is digested and absorbed by oral ingestion and used as a calorie source. Further, it can be used as a sweetener that is hardly fermented by caries-inducing bacteria or the like and hardly causes dental caries. In addition, it has properties such as osmotic pressure controllability, shaping property, shine imparting property, moisturizing property, viscosity, anti-crystallization property of other sugars, poor fermentation property, and anti-aging property of starch.
[0030]
In addition, the trehalose of the present invention is used parenterally as a tube feeding agent, an infusion solution, and the like, and is well metabolized and used without concern about toxicity and side effects, and can be advantageously used for replenishing energy to a living body. it can. In addition, in the case of a trehalose hydrated crystal product, because it is a stable sweetener, it can be advantageously used as a sugar coating for tablets in combination with a binder such as pullulan, hydroxyethyl starch, or polyvinylpyrrolidone.
[0031]
In addition, in the case of anhydrous crystalline trehalose, it can be advantageously used as a dehydrating agent for hydrates such as foods, pharmaceuticals, cosmetics, raw materials thereof, and processing intermediates, and can be used for solids such as stable, high-quality powders, granules, and tablets. The product can be easily manufactured.
[0032]
Therefore, the non-reducing saccharide of the present invention, or the low-reducing saccharide containing the trehalose and the trehalose produced therefrom are used as sweeteners, taste improvers, quality improvers, stabilizers, excipients, dehydrating agents. For example, it can be advantageously used for various compositions such as food and drink, favorite food, feed, feed, cosmetics, and pharmaceuticals.
[0033]
The non-reducing saccharide of the present invention, the low-reducing saccharide containing the saccharide, and trehalose produced therefrom can be used directly as a seasoning for sweetening. If necessary, for example, powdered candy, glucose, maltose, sucrose, isomerized sugar, honey, maple sugar, isomaltooligosaccharide, galactooligosaccharide, fructooligosaccharide, lactose, sorbitol, maltitol, lactitol, dihydrochalcone, stevioside, α -Glycosyl stevioside, rebaudioside, glycyrrhizin, L-aspartyl-L-phenylalanine methyl ester, saccharin, glycine, alanine, etc., may be used in admixture with one or more appropriate amounts of other sweeteners, If necessary, it can be used in admixture with a bulking agent such as dextrin, starch, lactose and the like.
[0034]
In addition, the non-reducing saccharide of the present invention, a low-reducing saccharide containing the same, and a powder or crystalline product of trehalose produced therefrom, as it is or as necessary, a bulking agent, an excipient, Mixing with a binder or the like and optionally shaping into various shapes such as granules, spheres, short rods, plates, cubes, and tablets may be used.
[0035]
In addition, the non-reducing saccharide of the present invention, the low-reducing saccharide containing the same, and the sweetness of trehalose produced therefrom have other tastes such as acidity, salty taste, astringency, umami, and bitterness. Since it is in harmony with various substances and has high acid resistance and heat resistance, it can be advantageously used for sweetening and taste improvement of general foods and drinks and for quality improvement.
[0036]
For example, amino acids, peptides, soy sauce, powdered soy sauce, miso, powdered miso, moromi, mashed, sprinkled, mayonnaise, dressing, vinegar, three tablespoons vinegar, powdered sushi vinegar, Chinese noodles, tentsuyu, noodle soup, sauce, ketchup, grilled meat It can be advantageously used as various cooking ingredients such as sauce, curry roe, stew ingredients, soup ingredients, dash ingredients, nucleic acid seasonings, complex seasonings, mirin, new mirin, table sugar, coffee sugar and the like.
[0037]
In addition, for example, various types of Japanese confectionery such as rice crackers, hail, rice bran, rice cakes, steamed buns, sea cucumber, sea cucumber, sheep shrimp, mizu-yori, nishikidama, jelly, castella, candy, bread, biscuit, crackers, cookies, pies , Pudding, butter cream, custard cream, cream puff, waffle, sponge cake, donut, chocolate, chewing gum, caramel, candy and other sweets, ice cream, sherbet, etc., ice confectionery, fruit syrup pickles, ice honey and other syrups, Pastes such as flower paste, peanut paste, fruit paste, spread, jams, marmalade, syrup pickles, fruits such as sugar fruits, processed vegetable foods, pickles such as fukujin pickles, betta pickles, senmai pickles, pickled pickles, etc. Pickles, such as Chinese cabbage pickles Raw meat, meat products such as ham, sausage, etc., fish meat products such as fish ham, fish sausage, kamaboko, chikuwa, tempura, various delicacies such as sea urchin, salted squid, vinegar konbu, sasakisume, fugumirin dried, Tsukuda stew made from seaweed, wild vegetables, sardines, small fish, shellfish, etc., prepared foods such as boiled beans, potato salad, konbu-maki, dairy products such as yogurt, cheese, fish meat, animal meat, fruits, vegetable bottled and canned Liquor, such as liquor, sake, synthetic liquor, liqueur, and Western liquor, coffee, tea, cocoa, juice, carbonated drinks, lactic acid drinks, soft drinks such as lactic acid drinks, pudding mix, hot cake mix, instant shiroko, instant soup, etc. Instant foods, sweetening to various foods such as baby foods, therapeutic foods, drinks, peptide foods, frozen foods, taste improvement, and quality It can be advantageously used in such good.
[0038]
In addition, it can be used for raising the preference of feeds, feeds, and the like for livestock, poultry, and other bred animals such as bees, silkworms, and fish. In addition, various solids such as tobacco, toothpaste, lipstick, lip balm, oral liquid, tablets, troches, liver oil drops, mouth fresheners, mouth flavors, gargles, etc. Can be advantageously used as a sweetener for various compositions, or as a taste improver, a flavoring agent, a quality improver, a stabilizer and the like. As a quality improving agent and a stabilizer, it can be advantageously applied to various physiologically active substances which easily lose their active ingredients and activities, or health foods and pharmaceuticals containing the same. For example, interferon-α, interferon-β, interferon-γ, Tsumore necrosis factor-α, Tsumore necrosis factor-β, macrophage migration inhibitory factor, colony stimulating factor, transfer factor, lymphokines such as interleukin II, insulin Hormones such as growth hormone, prolactin, erythropoietin, and egg cell stimulating hormone, BCG vaccine, Japanese encephalitis vaccine, measles vaccine, live polio vaccine, pox seedling, tetanus toxoid, hub antitoxin, biologics such as human immunoglobulin, penicillin, erythromycin, Antibiotics such as chloramphenicol, tetracycline, streptomycin, kanamycin sulfate, thiamine, riboflavin, L-ascorbic acid, liver oil, carotino Vitamin such as id, ergosterol and tocopherol, enzymes such as lipase, elastase, urokinase, protease, β-amylase, isoamylase, glucanase and lactase, ginseng extract, turmeric extract, chlorella extract, aloe extract, propolis extract and other extracts Various biologically active substances such as germs, viruses, lactic acid bacteria and yeast, and various biologically active substances such as royal jelly are also stable and high-quality liquid, paste or solid health foods and pharmaceuticals without losing their active ingredients and activities. It can be easily manufactured.
[0039]
The method for incorporating the above-described various compositions into a non-reducing saccharide having a trehalose structure at a terminal, a low-reducing saccharide containing the trehalose structure, and trehalose produced therefrom is a process until the product is completed. For example, known methods such as mixing, dissolving, melting, dipping, penetrating, spraying, coating, coating, spraying, pouring, crystallization, and solidifying are appropriately selected. The amount is usually 0.1% or more, preferably 1% or more.
[0040]
Next, the present invention will be described more specifically by experiments.
[0041]
[Experiment 1 Preparation of a thermostable non-reducing saccharide-forming enzyme derived from Sulfolobus acidocardarius ATCC 33909]
Peptone 0.1 w / v%, yeast extract 0.1 w / v%, ammonium sulfate 0.2 w / v%, monopotassium phosphate 0.05 w / v%, magnesium sulfate heptahydrate 0.02 w / v, potassium chloride 0 About 100 ml of a liquid medium containing 0.02 w / v% and water was put into a 500 ml Erlenmeyer flask, sterilized in an autoclave at 120 ° C. for 20 minutes, cooled, and adjusted to pH 3.0 with sulfuric acid. This liquid medium was inoculated with Sulfolobus acidocardarius ATCC 33909, and cultured at 75 ° C. and 130 rpm for 24 hours to obtain a primary seed culture.
[0042]
About 5 l of a medium having the same composition as that of the primary seed culture is put into a fermenter having a volume of 10 l, sterilized, cooled to pH 3.0 and a temperature of 75 ° C., and then 1 v / v% of the primary seed culture is added. The inoculum was incubated at a temperature of 75 ° C. and aerated at 500 ml / min for about 48 hours to obtain a secondary seed culture.
[0043]
About 250 liters of a medium having the same composition as in the case of the primary seed culture is put into a fermenter having a capacity of 300 liters, sterilized, cooled to pH 3.0 and a temperature of 75 ° C., and then 1 v / v% of the secondary seed culture is added. After inoculation, aeration culture was performed at a temperature of 75 ° C. and an aeration rate of 100 l / min for about 42 hours. Approximately 170 l of the resulting culture was centrifuged with an SF membrane to collect 258 g of wet cells as a wet weight. After 300 ml of 10 mM phosphate buffer (pH 7.0) was added to the cells and suspended, the cells were crushed using an ultrasonic crusher model “US300” manufactured by Nippon Seiki Seisaku-sho, Ltd. The crushed liquid was centrifuged (10,000 rpm, 30 minutes) to obtain about 300 ml of a centrifuged supernatant. Ammonium sulfate was added to and dissolved in the solution to a saturation degree of 0.7, left at 4 ° C. for 24 hours, and then centrifuged to collect a salted-out product. The obtained salted-out product was dissolved in 10 mM Tris / hydrochloric acid buffer (pH 8.5), dialyzed against the same buffer for 24 hours, and centrifuged to remove insolubles. The dialysate (about 600 ml) was divided into two portions and subjected to ion exchange column chromatography using DEAE-Toyopearl (gel amount: about 350 ml). The adsorbed present enzyme was eluted from the column with a linear gradient of 0 M to 0.3 M sodium chloride concentration, and an enzyme activity fraction eluted at around 0.1 M sodium chloride concentration was collected. The obtained enzyme-active fraction was dialyzed against 10 mM Tris / hydrochloric acid buffer (pH 8.5) containing 1 M ammonium sulfate, and the dialysate was centrifuged to remove insolubles. The product was subjected to hydrophobic column chromatography (gel amount: 350 ml) using a company-made gel “butyl toyopearl 650”. The adsorbed present enzyme was eluted from the column with a linear gradient of 1M to 0M ammonium sulfate concentration, and about 440 units of the enzyme active fraction eluted at around 0.8M ammonium sulfate concentration were recovered. The obtained partially purified preparation showed a specific activity of about 20 units / mg protein.
[0044]
The partially purified sample was dialyzed against 10 mM Tris / hydrochloric acid buffer (pH 8.5) containing 0.2 M sodium chloride, and the dialysate was centrifuged to remove insolubles, and the resulting supernatant was subjected to Ultrogel AcA44. The fraction was subjected to gel filtration chromatography (gel amount: 350 ml) using, and the enzyme-active fraction was collected. After that, the fraction was dialyzed against 10 mM Tris / hydrochloric acid buffer (pH 8.5), and the dialysate was centrifuged and insoluble. The resulting supernatant was subjected to ion exchange column chromatography using Mono Q (gel amount: 10 ml), and the adsorbed enzyme was eluted from the column with a linear gradient of 0 M to 0.2 M sodium chloride concentration. About 40 units of a heat-resistant non-reducing saccharide-forming enzyme-active fraction eluted at around 0.1 M sodium chloride concentration were collected.
[0045]
The obtained purified heat-resistant non-reducing saccharide-forming enzyme preparation exhibited a specific activity of about 81 units / mg protein, and its purity was assayed by electrophoresis using an SDS-polyacrylamide gel (gel concentration: 10%). However, it was shown that the protein band was single, and it was a single sample with high purity by electrophoresis.
[0046]
[Experiment 2 Physicochemical properties of thermostable non-reducing saccharide-forming enzyme]
[Experiment 2-1 action]
As a substrate, a 10% aqueous solution of glucose, maltose, maltotriose, maltotetraose, maltopentaose, maltohexaose, and maltoheptaose was prepared, and the purified heat-resistant non-reduced solution obtained by the method of Experiment 1 was prepared for each. A saccharide-forming enzyme was added at a ratio of 2 units per gram of solid substrate, allowed to act at 60 ° C. and pH 5.5 for 48 hours, desalted, and then subjected to column “Wakobeads WB-T” manufactured by Wako Pure Chemical Industries, Ltd. The reaction product was analyzed by high performance liquid chromatography using "-330". The high performance liquid chromatography was performed at room temperature, and water was flowed at a flow rate of 0.5 ml / min as an eluent, and analyzed by a differential refractometer “RI-8012” manufactured by Tosoh Corporation. Table 1 shows the results.
[0047]
[Table 1]

[0048]
As is evident from the results in Table 1, the purified enzyme is a non-reducing saccharide having a trehalose structure at its terminal from maltotriose to maltoheptaose, which is a partially degraded starch having a degree of polymerization of glucose of 3 or more. -Glucosyltrehalose to α-maltopentaosyltrehalose. In addition to non-reducing carbohydrates produced without changing each substrate and glucose polymerization degree remaining in the reaction product, relatively small amounts of substrate hydrolysates such as glucose and low-molecular-weight maltooligosaccharides and formed from them It has been found that non-reducing saccharides are present and have a weak but hydrolytic action in addition to the non-reducing saccharide-forming action. The rates of production of non-reducing saccharides and reducing saccharides produced by hydrolysates from the respective substrates by the purified enzyme were 30.2% and 27.6% from maltotriose, and maltotetraose. From 65.4% and 18.4%, from maltopentaose to maltoheptaose from about 74 to 75% and from about 2 to 3%, and from maltooligosaccharides having a degree of glucose polymerization of 5 or more, non-reducing saccharides Was produced at a high production rate, and the production of hydrolyzate was found to be slight. In addition, it turned out that glucose and maltose do not produce new carbohydrates.
[0049]
[Experiment 2-2 Molecular weight]
When SDS-polyacrylamide gel electrophoresis was carried out according to the method reported by U.K.Laemli in Nature, Vol. 227, pp. 680-685 (1970), the enzyme was found to have a molecular weight of A single band was shown at a position corresponding to about 69,000 to 79,000 daltons. The molecular weight markers at this time include myosin (200,000 dalton), β-galactosidase (116,250 dalton), phosphorylase B (97,400 dalton), serum albumin (66,200 dalton) and ovalbumin. (45,000 daltons) was used.
[0050]
[Experiment 2-3 Isoelectric point]
The purified heat-resistant non-reducing saccharide-forming enzyme is subjected to isoelectric focusing using a polyacrylamide gel (containing 2% ampholine, manufactured by Pharmacia LKB), and after electrophoresis, the gel pH is measured. When the isoelectric point was determined, the isoelectric point was about 5.4 to 6.4.
[0051]
[Experiment 2-4 optimal temperature]
When the test was carried out by a conventional method under the condition of incubating in a 20 mM acetate buffer (pH 5.5) for 60 minutes, as shown in FIG. 1, the enzyme showed an optimum temperature around 75 ° C.
[0052]
[Experiment 2-5 Optimum pH]
According to a conventional method, the enzyme was incubated at 60 ° C. for 60 minutes in McKilvain's buffer having a different pH. As shown in FIG. 2, the enzyme reached a pH of about 5.0 to 5.5. Appropriate pH was shown.
[0053]
[Experiment 2-6 Temperature stability]
According to a conventional method, a test was performed under conditions of incubation for 60 minutes in a 10 mM phosphate buffer (pH 7.0). As shown in FIG. 3, the enzyme was stable up to around 85 ° C.
[0054]
[Experiment 2-7 pH stability]
According to a conventional method, the test was carried out under conditions of incubation at 25 ° C. for 16 hours in a McKilvain buffer solution having a different pH or a sodium carbonate-sodium hydrogen carbonate buffer solution. As shown in FIG. The pH was stable up to around 4.5 to 9.5.
[0055]
[Experiment 2-8 N-terminal amino acid sequence]
After a portion of the purified heat-resistant non-reducing saccharide-forming enzyme preparation obtained by the method of Experiment 1 was dialyzed against distilled water, about 80 μg of protein was used as a sample for N-terminal amino acid sequence analysis. . The N-terminal amino acid sequence was analyzed from the N-terminal to 10 residues using Applied Biosystems Japan Gas Phase Protein Sequencer “473A”. The N-terminal sequence obtained is shown below.
[0056]

[0057]
[Experiment 3 Preparation of thermostable non-reducing saccharide-forming enzyme derived from other Sulfolobus microorganism]
The fermenter was cultured for 42 hours in the same manner as in Experiment 1, except that Sulfolobus acidocardarius ATCC49909, Sulfolobus solfataricus ATCC35091 and Sulfolobus solfataricus ATCC35092 were used instead of Sulfolobus acidocardarius ATCC33909. Cells were collected from about 170 l of each culture, sonicated, and the supernatant was subjected to ammonium sulfate precipitation, dialyzed, and further subjected to ion exchange column chromatography and hydrophobic column chromatography to obtain a partially purified enzyme preparation. Its properties were investigated. The results are summarized in Table 2.
[0058]
[Table 2]

[0059]
When the production of non-reducing saccharides was examined using these partially purified enzymes according to the method of Experiment 2-1, all enzymes were found to be heat-resistant non-reducing saccharides derived from Sulfolobus acidocardarius ATCC 33909. As in the case of the synthase, it was found that non-reducing saccharides having a trehalose structure at the terminal and having a degree of glucose polymerization of 3 or more were produced from partially degradable reducing starches having a glucose polymerization degree of 3 or more.
[0060]
Hereinafter, the method for producing a non-reducing saccharide of the present invention, a low-reducing saccharide containing the same, and a method for producing trehalose will be described in Example A. The impregnated composition is shown in Example B.
[0061]
Example A-1
Sulfolobus acidocardarius ATCC33909 was cultured in a fermenter for about 42 hours according to the method of Experiment 1. After the culture, the cells were concentrated using an SF membrane, and the cells were collected by centrifugation. According to the method of Experiment 3, the cells were sonicated, and the supernatant was subjected to ammonium sulfate precipitation and dialysis, and further subjected to ion exchange column chromatography and hydrophobic column chromatography to obtain a portion having a specific activity of about 20 units / mg. A purified enzyme solution (18.0 units / ml) was obtained. After heating and gelatinizing potato starch milk having a concentration of 6%, the pH was adjusted to 4.5 and the temperature was adjusted to 50 ° C., and isoamylase (manufactured by Hayashibara Biochemical Laboratory Co., Ltd.) was added thereto at a rate of 2500 units per gram of starch. And reacted for 20 hours. The reaction solution was adjusted to pH 6.5, autoclaved (120 ° C.) for 10 minutes, and then cooled to 60 ° C., to which α-amylase “Termamyl 60L” manufactured by Novo Co. was added at a ratio of 30 units per gram of starch. And reacted for 20 hours. The reaction solution was autoclaved (120 ° C.) for 20 minutes, cooled to 65 ° C. and adjusted to pH 5.5, and the heat-resistant non-reducing saccharide-forming enzyme prepared above was added to the starch in an amount of 1 / gram of starch. The mixture was added so as to have a unit ratio and reacted for 96 hours. The reaction solution was kept at 97 ° C. for 30 minutes, cooled, filtered, and the filtrate obtained was decolorized with activated carbon and desalted with H-type and OH-type ion exchange resins in accordance with a conventional method. And further concentrated to give a syrup with a concentration of about 70% at about 90% per solid. This product has a DE of 24.6 and contains a non-reducing saccharide at 12.0% of α-glucosyltrehalose, 5.5% of α-maltosyltrehalose and 29.9% of α-maltotriosyltrehalose per solid content. Contains 1.5% of α-maltotetraosyltrehalose and 2.2% of α-maltopentaosyltrehalose, and has a mild and elegant sweetness, a moderate viscosity, a moisturizing property, a sweetener, As a taste improver, a quality improver, a stabilizer, an excipient, etc., it can be advantageously used in various compositions such as various foods and drinks, cosmetics and pharmaceuticals.
[0062]
Example A-2
The sugar solution obtained by the method of Example A-1 was used as a raw sugar solution, and in order to increase the content of non-reducing sugars, a sodium-type strongly acidic cation exchange resin “XT-1016” manufactured by Tokyo Organic Chemical Industry Co., Ltd. ( Column fractionation using a crosslink degree of 4%) was performed. The resin was packed in four jacketed stainless steel columns having an inner diameter of 5.4 cm and connected in series to make the total length of the resin layer 20 m. While maintaining the temperature in the column at 55 ° C., a sugar solution was added to the resin at 5 v / v%, and hot water at 55 ° C. was flowed at SV 0.13 to fractionate the fraction, thereby removing glucose and maltose-rich fractions. Then, a non-reducing sugar-rich fraction was collected. Further, purification, concentration, vacuum drying and pulverization gave a non-reducing sugar-rich powder at about 64% per solid. This product has a DE of 4.8 and contains non-reducing carbohydrates at 18.2% of α-glucosyltrehalose, 7.9% of α-maltosyltrehalose and 46.6% of α-maltotriosyltrehalose per solid. , 2.3% of α-maltotetraosyltrehalose and 3.4% of α-maltopentaosyltrehalose. As in Example A-1, mild sweet taste, moderate viscosity, It has a moisturizing property, and can be advantageously used as a sweetener, a taste improver, a quality improver, a stabilizer, an excipient, and the like in various compositions such as various foods and drinks, cosmetics, and pharmaceuticals.
[0063]
Example A-3
After adding calcium carbonate to 33% corn starch milk to a final concentration of 0.1%, the pH was adjusted to 6.5, and 60 L of termamyl was added to 0.2% per gram of starch. The reaction was performed for 15 minutes. The reaction mixture was subjected to autoclave (120 ° C.) for 10 minutes, cooled to 55 ° C., and then reacted with maltotheolaose-forming amylase manufactured by Hayashibara Biochemical Laboratory Co., Ltd. disclosed in JP-A-63-240783. Was added at a rate of 5 units per gram of starch, and reacted for 6 hours. To this, 30 units per gram of starch were added with α-amylase “α-amylase 2A” manufactured by Ueda Chemical Co., Ltd., and further reacted at 65 ° C. for 4 hours. I let it. The reaction solution was autoclaved (120 ° C.) for 10 minutes, and then cooled to 65 ° C. to adjust the pH to 5.5, and then added to the heat-resistant non-reducing saccharide prepared by the method of Example A-1. The resulting enzyme was added at a ratio of 2 units per gram of starch and allowed to react for 48 hours. The reaction solution was kept at 97 ° C. for 30 minutes, cooled, filtered, and the filtrate obtained was decolorized with activated carbon and desalted with H-type and OH-type ion exchange resins in accordance with a conventional method. And concentrated to give a syrup having a concentration of about 70% in a yield of about 90% per solid. This product has a DE of 17.1 and has a non-reducing sugar content of 8.9% of α-glucosyltrehalose, 29.3% of α-maltosyltrehalose and 0.8% of α-maltotriosyltrehalose per solid. Containing 0.7% of α-maltotetraosyltrehalose and 0.7% of α-maltopentaosyltrehalose, having mild and elegant sweetness, moderate viscosity and moisturizing property, As a taste improver, a quality improver, a stabilizer, an excipient, etc., it can be advantageously used in various compositions such as various foods and drinks, cosmetics and pharmaceuticals.
[0064]
Example A-4
The sugar solution obtained by the method of Example A-3 was used as a raw sugar solution, and in order to increase the content of α-maltosyltrehalose in the solution, a magnesium type strong acid cation exchange sold by Dow Chemical Company as a fractionation resin. Column chromatography was performed according to the method of Example A-2 except that the resin “Dowex 50W × 4” was used, and a α-maltosyltrehalose-rich fraction was collected. Furthermore, purification, concentration, and spray drying were performed to obtain a non-reducing saccharide-rich powder at a yield of about 41% per solid. This product contains 10.9% of α-glucosyl trehalose, 61.3% of α-maltosyl trehalose, and 1.0% of α-maltotriosyl trehalose per solid, and its DE Represents 2.5, has a very low reducibility, and has a mild and elegant sweetness, a suitable viscosity and a moisturizing property as in Example A-3, and is a sweetener, a taste improver, and a quality improver. It can be advantageously used as a stabilizer, an excipient, etc. in various compositions such as various foods and drinks, cosmetics, and pharmaceuticals.
[0065]
Example A-5
40 parts by weight of partially degraded starch “Paindex # 4” manufactured by Matsutani Chemical Industry Co., Ltd. was dissolved by heating in 60 parts by weight of water, and this solution was adjusted to 65 ° C. and pH 5.5, followed by the method of Example A-1. The heat-resistant non-reducing saccharide-forming enzyme prepared in the above was added at a ratio of 1 unit per gram of partially degraded starch and reacted for 96 hours, and then heated to 97 ° C. for 30 minutes to inactivate the enzyme. Was. The reaction solution was diluted to a concentration of about 20%, and glucoamylase "glucozyme" manufactured by Nagase Seikagaku Co., Ltd., was added in an amount of 10 units per gram of partially degraded starch and reacted for 10 hours, followed by heating to inactivate the enzyme. Was. 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. This sugar solution contained 30.1% trehalose per solid. Column chromatography was performed according to the method of Example A-2, except that a sodium-type strongly acidic cation exchange resin “CG6000” sold by Organo Corporation was used as the fractionation resin, and a trehalose-rich fraction was collected. The high content liquid contained about 97% trehalose per solid. After concentrating this solution to a concentration of about 75%, the solution was placed in an auxiliary crystal can, and about 2% of trehalose hydrated crystals were added as seed crystals, followed by slow cooling to obtain a mass kit having a crystallization rate of about 45%. 150kg / cm from the nozzle on the drying tower ² At high pressure. At the same time, the powder was gradually moved out of the drying tower while hot air at 85 ° C was being sent from the upper part of the drying tower at a temperature of 45 ° C to take out the powder. The crystal powder was filled in a maturation tower, aged for 10 hours while sending hot air, and crystallization and drying were completed to obtain a trehalose hydrate crystal powder. This product has practically no hygroscopicity, is easy to handle, and has various compositions such as sweeteners, taste improvers, quality improvers, stabilizers, excipients, etc. It can be used to advantage for things.
[0066]
Example A-6
According to the method of Experiment 3, Sulfolobus solfataricus ATCC 35091 was cultured in a fermenter for about 42 hours. After culturing, the cells are concentrated using an SF membrane, and the cells are collected by centrifugation, sonicated, and the supernatant is subjected to salting out with ammonium sulfate, dialyzed, and further subjected to ion exchange column chromatography and hydrophobic column. Chromatography was performed to obtain a partially purified enzyme solution (19.0 units / ml) having a specific activity of about 18 units / mg. Using 30% corn starch milk, according to the method of Example A-3, 60 L of tamamar, then maltotetraose-forming amylase (manufactured by Hayashibara Biochemical Laboratory Co., Ltd.) and α-amylase 2A are allowed to act, and an autoclave ( (120 ° C.), and then cooled to 65 ° C., and the heat-resistant non-reducing saccharide-forming enzyme prepared by the above-mentioned method was added at 2 units per gram of starch, and reacted for 64 hours. The enzyme was then inactivated by heating to 97 ° C. for 30 minutes. This reaction solution was treated with glucozyme, decolorized and desalted according to the method of Example A-5, and concentrated to a concentration of about 60%. The sugar solution contained about 23% trehalose per solid. This saccharide solution was subjected to column chromatography using a salt type strongly acidic cation exchange resin according to the method of Example A-5, and a trehalose-rich fraction was collected. The high content liquid contained about 95% trehalose per solid. This solution is taken in an evaporator and boiled down under reduced pressure to form a syrup having a water content of 4.0%. The syrup is transferred to an auxiliary crystallizer, and 1% of anhydrous trehalose is added as a seed crystal to the syrup solid content, followed by stirring at 95 ° C. for 5 minutes. The crystal was assisted and then taken out into an aluminum vat and crystallized and aged at 100 ° C. for 6 hours to prepare a block. Next, this block was pulverized with a cutting machine and fluidized and dried to obtain anhydrous crystalline trehalose powder having a water content of 0.3%. The product can be advantageously used not only as a dehydrating agent for hydrates such as foods, pharmaceuticals, cosmetics, raw materials thereof, or processed intermediates, but also as a white powder sweetener having an elegant sweetness.
[0067]
Example B-1 Sweetener
1 part by weight of the non-reducing carbohydrate-rich powder obtained by the method of Example A-4 was added to 0.01 part by weight of α-glycosyl stevioside “αG suite” sold by Toyo Seikagaku Co., Ltd. and L-aspartyl manufactured by Ajinomoto Co., Ltd. 0.01 part by weight of L-phenylalanine methyl ester "aspartame" was uniformly mixed and subjected to a granulator to obtain a granular sweetener. The product has excellent sweetness, has about twice the sweetness of sucrose, and has reduced calories per sweetness to about 1/2 that of sucrose. This sweetener does not decompose high-intensity sweeteners contained in it and has excellent stability.As a low-calorie sweetener, low-calorie foods for obese and diabetics who have restricted caloric intake It is suitable for sweetening products and the like. In addition, the present sweetener is suitable for sweetening foods and the like that suppress tooth decay because it produces less acid by caries-inducing bacteria and produces less insoluble glucan.
[0068]
[Example B-2 Hard candy]
30 parts by weight of the non-reducing saccharide-containing syrup obtained by the method of Example A-3 was mixed with 100 parts by weight of a 55% strength sucrose solution by heating, and then concentrated by heating under reduced pressure until the water content was less than 2%. Was mixed with 1 part by weight of citric acid and an appropriate amount of a lemon flavor and a coloring agent, 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 crystallization of sucrose.
[0069]
[Example B-3 Chewing gum]
3 parts by weight of the gum base is heated and melted to a degree that it is softened, 4 parts by weight of sucrose and 3 parts by weight of the trehalose hydrated crystal powder obtained by the method of Example A-5 are added, and an appropriate amount of a flavor and a coloring agent are further added. The mixture was mixed, kneaded with 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.
[0070]
Example B-4 Sweetened Condensed Milk
3 parts by weight of the non-reducing carbohydrate-containing syrup and 1 part by weight of sucrose obtained by the method of Example A-1 were dissolved in 100 parts by weight of raw milk, sterilized by heating with a plate heater, and then concentrated to a concentration of 70%. The product was obtained by canning under aseptic conditions. This product has a mild sweetness and good flavor, and can be advantageously used for seasoning infant foods, fruits, coffee, cocoa, black tea and the like.
[0071]
[Example B-5 Lactic acid bacteria drink]
175 parts by weight of skim milk powder, 80 parts by weight of a non-reducing carbohydrate-rich powder obtained by the method of Example A-2, and 50 parts by weight of a lactosucrose-rich powder disclosed in JP-A-4-281795 Dissolved in 1,200 parts by weight, sterilized at 65 ° C. for 30 minutes, cooled to 40 ° C., inoculated with 30 parts by weight of a lactic acid bacterium starter, and cultured at 37 ° C. for 8 hours. I got a drink. This product is a lactic acid bacteria beverage having a good flavor. In addition, the product contains oligosaccharides and not only stably retains lactic acid bacteria, but also has a bifidobacterium growth promoting effect.
[0072]
Example B-6 Powdered Juice
33 parts by weight of orange juice powder produced by spray drying, 50 parts by weight of the non-reducing carbohydrate-rich powder obtained by the method of Example A-2, 10 parts by weight of sucrose, 0.65 parts by weight of citric anhydride 0.1 part by weight of malic 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 This was charged into a fluidized bed granulator, and the exhaust air temperature was 40 ° C and the air volume was 150m. ³ Then, the non-reducing sugar-containing syrup obtained by the method of Example A-1 was sprayed as a binder, granulated for 30 minutes, weighed and packaged to obtain a product. This product is a powdered juice having a fruit juice content of about 30%. This product had no off-flavor or off-flavor and was stable for a long period of time.
[0073]
[Example B-7 Custard cream]
100 parts by weight of corn starch, 100 parts by weight of the non-reducing sugar-containing syrup obtained by the method of Example A-3, 80 parts by weight of maltose, 20 parts by weight of sucrose and 1 part by weight of sodium chloride are thoroughly mixed, and 280 parts by weight of chicken egg Add and stir, 1,000 parts by weight of boiled milk is gradually added, and further, it is heated and kept stirring, and when the corn starch is completely gelatinized and the whole becomes translucent, the fire is stopped, After cooling, an appropriate amount of vanilla flavor was added, and the product was weighed, filled and packaged to obtain a product. This product has a smooth luster and is mildly sweet and delicious.
[0074]
[Example B-8 Ann]
Water was added to 10 parts by weight of the raw material Azuki according to a conventional method, and the mixture was boiled, astringently cut out, and air-extracted to remove water-soluble impurities, thereby obtaining about 21 parts by weight of Azuki tsubuan. To this raw bean paste, 14 parts by weight of sucrose, 5 parts by weight of a syrup containing a high content of non-reducing sugar obtained by the method of Example A-4 and 4 parts by weight of water are added and boiled, and a small amount of salad oil is added thereto. The bean paste was kneaded so as not to be broken, and about 35 parts by weight of the product was obtained. This product has no color burning, has a good texture, and has a good flavor, and is suitable as an ingredient for anpan bread, steamed bun, dumpling, monaka, frozen dessert and the like.
[0075]
Example B-9 Bread
100 parts by weight of flour, 2 parts by weight of yeast, 5 parts by weight of sugar, 1 part by weight of the non-reducing carbohydrate-rich powder obtained by the method of Example A-2 and 0.1 part by weight of an inorganic food were prepared according to a conventional method. The kneaded seeds were kneaded with water and fermented at 26 ° C. for 2 hours, then aged for 30 minutes and baked. This product is a high-quality bread having good hue, good elasticity, moderate elasticity and mild sweetness.
[0076]
Example B-10 Ham
To 1000 parts by weight of pork leg, 15 parts by weight of salt and 3 parts by weight of potassium nitrate are evenly rubbed, and piled up in a cold room all day and night. This was mixed with 500 parts by weight of water, 100 parts by weight of salt, 3 parts by weight of potassium nitrate, 40 parts by weight of the non-reducing carbohydrate-rich powder obtained by the method of Example A-4, and spices in a cold room for 7 days in a cold room. The product was immersed, washed with cold water, wound with a string, smoked, cooked, and cooled and packaged according to a conventional method to obtain a product. This product is a high quality ham with good color and good flavor.
[0077]
Example B-11 Powder Peptide
2 parts by weight of the trehalose hydrate crystal powder obtained by the method of Example A-5 were mixed with 1 part by weight of a 40% soybean peptide solution for food "Hinute S" manufactured by Fuji Oil Co., Ltd., and put in a plastic vat. It was dried under reduced pressure at 50 ° C. and crushed to obtain a powdered peptide. This product has a good flavor and can be advantageously used not only as a confectionery material such as premix and frozen dessert, but also as a baby food such as an oral liquid food and a tube food liquid, a therapeutic nutrient and the like.
[0078]
[Example B-12 Powdered egg yolk]
The yolk prepared from raw eggs is sterilized at 60 to 64 ° C. with a plate-type heat sterilizer, and 4 parts by weight of the anhydrous crystalline trehalose powder obtained by the method of Example A-6 is used for 1 part by weight of the obtained liquid yolk. After mixing at a ratio, the mixture was transferred to a vat and left overnight to be converted into trehalose hydrated crystals to prepare a block. The block was pulverized by a cutting machine to obtain powdered egg yolk. The product can be advantageously used not only as a confectionary material such as a premix, a frozen dessert, an emulsifier, but also as a baby food such as an oral liquid food and a tube food, a therapeutic nutrient, and the like. In addition, it can also be advantageously used as a beautifying agent, a hair restorer and the like.
[0079]
Example B-13 Cosmetic Cream
2 parts by weight of polyoxyethylene glycol monostearate, 5 parts by weight of self-emulsifying glyceryl monostearate, 2 parts by weight of a non-reducing saccharide-rich powder obtained by the method of Example A-2, 1 part by weight of α-glycosyl rutin Parts, 1 part by weight of liquid paraffin, 10 parts by weight of glycerin trioctanoate and an appropriate amount of a preservative are dissolved by heating according to a conventional method, and 2 parts by weight of L-lactic acid, 5 parts by weight of 1,3-butylene glycol and 66 parts by weight of purified water are dissolved therein. Then, the mixture was added to a homogenizer, emulsified, and then an appropriate amount of a flavor was added, followed by stirring and mixing to produce a cream. The product has antioxidant properties, high stability, and can be advantageously used as a high-quality sunscreen, skin-beautifying agent, fair-skinning agent, and the like.
[0080]
Example B-14 Solid preparation
A human natural interferon-α sample (manufactured by Hayashibara Biochemical Laboratory Co., Ltd.) is applied to an immobilized anti-human interferon-α antibody column according to a conventional method to adsorb human natural interferon-α contained in the sample. The bovine serum albumin, which is a stabilizer, is removed by passing through, and then the pH is changed to obtain a physiological saline containing 5% of hydrated trehalose hydrous crystal powder obtained by the method of Example A-5. Elution was carried out using saline. This liquid is subjected to precision filtration, added to about 20 times the amount of anhydrous crystalline maltose powder “Fine Tose” sold by Hayashibara Shoji Co., Ltd., dehydrated and pulverized, and then tableted with a tableting machine to give 1 tablet (about 200 mg). A tablet containing about 150 units of human natural interferon-α per unit was obtained. The product is orally administered as sublingual tablets, for example, about 1 to 10 tablets per day, and can be advantageously used for the treatment of viral diseases, allergic diseases, rheumatism, diabetes, malignant tumors and the like. In particular, it can be advantageously used as a therapeutic agent for AIDS, hepatitis, etc., in which the number of patients has rapidly increased in recent years. In this product, maltose acts as a stabilizer together with trehalose, and its activity is well maintained for a long time even at room temperature.
[0081]
Example B-15 Dragee
A plain tablet weighing 150 mg was used as a core, and 40 parts by weight of the trehalose hydrated crystal powder obtained by the method of Example A-5, 2 parts by weight of pullulan (average molecular weight: 200,000), 30 parts by weight of water, and 25 parts by weight of talc And sugar coating using a undercoating solution consisting of 3 parts by weight of titanium oxide until the tablet weight becomes about 230 mg, and then an overhanging solution consisting of 65 parts by weight of the same trehalose hydrated crystal powder, 1 part by weight of pullulan and 34 parts by weight of water. And coated with a wax solution to obtain a sugar-coated tablet excellent in gloss and appearance. This product has excellent impact resistance and maintains high quality for a long time.
[0082]
[Example B-16 Liquid edible solid preparation]
500 parts by weight of hydrated trehalose crystalline powder produced by the method of Example A-5, 270 parts by weight of powdered egg yolk, 209 parts by weight of skim milk powder, 4.4 parts by weight of sodium chloride, 1.8 parts by weight of potassium chloride, 4 parts by weight of magnesium sulfate Of thiamine, 0.01 part by weight of thiamine, 0.1 part by weight of sodium ascorbate, 0.6 part by weight of vitamin E acetate and 0.04 part by weight of nicotinamide, and moisture-proofing 25 g of the mixture at a time. The product was filled in a heat-resistant laminated pouch and heat-sealed to obtain a product. This product is obtained by dissolving one bag in about 150 to 300 ml of water to make a liquid diet, and is used orally or by tube application to the nasal cavity, stomach, intestine, etc., and is advantageous for replenishing energy to living organisms. Available to
[0083]
Example B-17 Trauma for Treatment of Trauma
To 200 parts by weight of the hydrated trehalose crystalline powder and 300 parts by weight of maltose produced by the method of Example A-5, 50 parts by weight of methanol in which 3 parts by weight of iodine was dissolved were added and mixed, and 200 parts by weight of a 10 w / v% aqueous pullulan solution were further added. Was added and mixed to obtain a wound treatment plaster exhibiting moderate elongation and adhesiveness. Since this product acts not only as a bactericidal action by iodine but also as an energy supplement to cells by trehalose, the healing period is shortened and the wound surface is healed neatly.
[0084]
【The invention's effect】
As is clear from the above, the novel thermostable non-reducing saccharide-forming enzyme of the present invention can easily carry out the enzymatic reaction at a temperature exceeding 55 ° C. It is converted into non-reducing saccharides with a high degree of glucose polymerization in high yield. It is easy to separate and purify the non-reducing saccharide. The non-reducing saccharide, low-reducing saccharide containing the non-reducing saccharide and trehalose produced therefrom are excellent in stability, good in quality and elegant. Has a sweet taste. Non-reducing saccharides, low-reducing saccharides containing the same and trehalose are used as sweeteners, taste improvers, quality improvers, stabilizers, excipients, etc. in various compositions such as foods, beverages, cosmetics, and pharmaceuticals. It can be used advantageously.
[0085]
Therefore, the establishment of the present invention is based on inexpensive and unlimited resources from starch partially degraded products derived from starch, a non-reducing saccharide having a trehalose structure at the end, which was not easily obtained conventionally as desired. Will open up an entirely new way of industrially supplying low-reducing sugars containing trehalose and trehalose, which is easily produced therefrom, in large quantities and at low cost. The impact of this will be in the food, cosmetics, and pharmaceutical fields. Needless to say, it extends to the agriculture, fisheries and livestock industries, and the chemical industry, and the industrial significance to these industries is immense.
[Brief description of the drawings]
FIG. 1 is a diagram showing the effect of temperature on the enzymatic activity of the thermostable non-reducing saccharide-forming enzyme of the present invention.
FIG. 2 is a graph showing the effect of pH on the enzyme activity of the thermostable non-reducing saccharide-forming enzyme of the present invention.
FIG. 3 is a graph showing the effect of temperature on the stability of the thermostable non-reducing saccharide-forming enzyme of the present invention.
FIG. 4 is a graph showing the effect of pH on the stability of the thermostable non-reducing saccharide-forming enzyme of the present invention.

Claims (20)

A non-reducing saccharide having a trehalose structure at a terminal is produced from a partially decomposed product of a reducing starch having a glucose polymerization degree of 3 or more, and can be obtained from a microorganism belonging to the genus Sulfolobus and has the following physicochemical properties. Reducing sugar-forming enzyme;
(1) Action A non-reducing saccharide having a trehalose structure at a terminal is produced from one or more kinds of partially decomposed starches having a degree of polymerization of glucose of 3 or more.
(2) Molecular weight: about 69000 to 79,000 daltons by SDS-gel electrophoresis.
(3) pI of about 5.4 to 6.4 by an isophoretic ampholine-containing electrophoresis method.
(4) Approximately 75 ° C. at the optimum temperature of pH 5.5 for 60 minutes.
(5) Optimum pH
The pH is around 5.0 to 5.5 at 60 ° C for 60 minutes.
(6) Temperature stability Stable up to around 85 ° C by holding at pH 7.0 for 60 minutes.
(7) pH stability A pH of about 4.0 to 9.5 at 25 ° C. for 16 hours. A microorganism belonging to the genus Sulfolobus having the ability to produce a heat-resistant non-reducing saccharide-forming enzyme according to claim 1 is cultured in a nutrient medium, and the heat-resistant non-reducing saccharide-forming enzyme is collected from the obtained culture. wherein the method for producing a thermostable non-reducing saccharide-forming enzyme according to claim 1, wherein. The microorganism belonging to the genus Sulfolobus is Sulfolobus acidocardarius (ATCC 33909), Sulfolobus acidocardarius (ATCC 49426), Sulfolobus solfataricus (ATCC 35091), or Sulfolobus solfataricus (ATCC 35092). A method for producing a thermostable non-reducing saccharide enzyme. 2. A non-reducing saccharide having a trehalose structure at a terminal, or a non-reducing saccharide having a trehalose structure at the terminal, wherein the heat-resistant non-reducing saccharide-forming enzyme according to claim 1 is allowed to act on a partially degraded reducing starch having a degree of polymerization of glucose of 3 or more. A method for producing a low-reducing saccharide comprising: 5. The terminal trehalose structure according to claim 4, wherein the reductive starch partially decomposed product is one or more reductive starch partially decomposed products selected from a glucose polymerization degree of 3 or more, a starch liquefaction, a dextrin or a maltooligosaccharide. A method for producing a non-reducing saccharide or a low-reducing saccharide comprising the same. The non-reducing carbohydrate having a trehalose structure at a terminal according to claim 4 or 5, wherein the reducing starch partially decomposed product is a partially degraded reducing starch product obtained by partially hydrolyzing starch with an enzyme or an acid. Or a method for producing a low-reducing saccharide comprising the same. The method for producing a non-reducing saccharide having a trehalose structure at a terminal or a low-reducing saccharide comprising the same according to claim 6, wherein the enzyme is an amylase or a debranching enzyme. 8. The non-reducing saccharide having a trehalose structure at a terminal or a low-reducing saccharide comprising the same according to claim 4, wherein the reducing starch partially decomposed product is a solution containing a reducing starch partially decomposed product. Manufacturing method. A solution containing the non-reducing saccharide having a trehalose structure at the terminal or the low-reducing saccharide containing the same according to any one of claims 4 to 8 is subjected to column chromatography using a strongly acidic cation exchange resin. A method for producing a non-reducing saccharide having a trehalose structure at a terminal or a low-reducing saccharide containing the same, the content of which is improved. 10. A non-reducing saccharide having a trehalose structure at a terminal or a low-reducing saccharide containing the non-reducing saccharide by the method according to any one of claims 4 to 9, and eating and drinking the same. Method of manufacturing a product. Cosmetics characterized by producing a non-reducing saccharide having a trehalose structure at a terminal or a low-reducing saccharide containing the same by the production method according to any one of claims 4 to 9, and incorporating the same. Manufacturing method. A pharmaceutical product characterized by producing a non-reducing saccharide having a trehalose structure at a terminal or a low-reducing saccharide containing the same by the production method according to any one of claims 4 to 9, and incorporating the same. Manufacturing method. The heat-resistant non-reducing saccharide-forming enzyme according to claim 1 is allowed to act on a partially degraded reducing starch having a degree of polymerization of glucose of 3 or more, and then glucoamylase or α-glucosidase is acted on to produce trehalose. A method for producing trehalose, which comprises collecting. The method for producing trehalose according to claim 13, wherein column chromatography using a strongly acidic cation exchange resin is used. The method for producing trehalose according to claim 13 or 14, wherein the trehalose is a hydrous crystal or an anhydrous crystal. 14. The method for producing trehalose according to claim 13, wherein the partially decomposed reducing starch is one or more kinds of partially decomposed reducing starch selected from a glucose polymerization degree of 3 or more, a liquefied starch, a dextrin or a maltooligosaccharide. 17. The method for producing trehalose according to claim 13 or 16, wherein the partially decomposed reducing starch is a solution containing a partially decomposing starch. A method for producing food and drink, comprising producing trehalose by the method for producing trehalose according to any one of claims 13 to 17, and incorporating the same. A method for producing cosmetics, comprising producing trehalose by the method for producing trehalose according to any one of claims 13 to 17, and incorporating trehalose. A method for producing a medicament, comprising producing trehalose by the method for producing trehalose according to any one of claims 13 to 17, and incorporating the trehalose.

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