JP3616166B2 - Trehalose and its production method and use - Google Patents

Description

【００６１】
精製した酵素標品を７．５ｗ／ｖ％濃度ポリアクリルアミドを含むゲル電気泳動により酵素標品の純度を検定したところ、蛋白バンドは単一で純度の高い標品であった。
【００６２】
【実験３ 酵素の性質】
実験２の方法で得た精製マルトース・トレハロース変換酵素標品をＳＤＳ−ポリアクリルアミドゲル電気泳動法（ゲル濃度１０ｗ／ｖ％）に供し、同時に泳動した分子量マーカー（日本バイオ・ラッド・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５７，０００乃至６７，０００ダルトンであった。
【００６３】
精製マルトース・トレハロース変換酵素標品を２ｗ／ｖ％アンフォライン（ファルマシア・エルケイビー社製）含有等電点ポリアクリルアミドゲル電気泳動法に供し、泳動後、蛋白バンド及びゲルのｐＨを測定して本酵素の等電点を求めたところ、等電点はｐＩ約４．１乃至５．１であった。
【００６４】
本酵素活性に及ぼす温度、ｐＨの影響を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２０℃付近、至適ｐＨは、２５℃、６０分間反応で約７．０乃至８．０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、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２０℃、６０分間保持した後、ｐＨを７．０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３０℃付近までであり、ｐＨ安定性は約６．０乃至９．０であった。なお、本酵素活性は、１ｍＭＣｕ^＋＋、Ｈｇ^＋＋又は５０ｍＭトリス塩酸緩衝液で阻害された。
【００６５】
【実験４ 各種糖質への作用】
各種糖質を用いて、基質になりうるかどうかの試験をした。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、マルトヘプタオース、可溶性澱粉、アミロース（平均重合度１８）、トレハロース、ネオトレハロース、ゲンチオビオース、コージビオース、イソマルトース、セロビオース、マルチトール、シュクロース、マルツロース、ツラノース、パラチノース、トレハルロース、あるいはラクトースの溶液、更に、α−グルコース・１−リン酸と等量のグルコース、又は、β−グルコース・１−リン酸と等量のグルコースとを含む溶液を調製した。
【００６６】
これらの溶液に、実験２の方法で得た精製マルトース・トレハロース変換酵素を基質固形物グラム当たりそれぞれ２単位ずつ加え、基質濃度を５ｗ／ｖ％になるよう調製し、これを２０℃、ｐＨ７．０で２４時間作用させた。酵素反応前後の反応液をメルク社製『キーゼルゲル６０』（アルミプレート、２０×２０ｃｍ）を用いた薄層クロマトグラフィー（以下、ＴＬＣと略称する。）にかけ、それぞれの糖質に対する酵素作用の有無を確認した。ＴＬＣは展開溶媒に１−ブタノール：ピリジン：水＝６：４：１（容積比）を用い、室温で１回展開した。発色は２０％硫酸−メタノール溶液を噴霧し、１１０℃で１０分間加熱しておこなった。結果を表２に示す。
【００６７】
【表２】

【００６８】
表２の結果から明かなように、本発明の酵素は、試験した多種の糖質のうち、マルトースとトレハロースにのみ作用し、その他の糖質、とりわけ、α−グルコース・１リン酸とグルコースとを含む系や、β−グルコース・１−リン酸とグルコースとを含む系に作用しないことから、従来知られているマルトース・ホスホリラーゼやトレハロース・ホスホリラーゼなどのホスホリラーゼとは違い、新規な酵素であることが判明した。
【００６９】
【実験５ マルトース又はトレハロースからの生成物】
最終濃度５％のマルトース水溶液に実験２の方法で得た精製マルトース・トレハロース変換酵素を基質固形物グラム当たり２単位加え、２０℃、ｐＨ７．０で２４時間作用させた。酵素反応液の糖組成は、ガスクロマトグラフィー（以下、ＧＬＣと略称する。）で分析した。酵素反応液の一部を乾固し、ピリジンに溶解した後トリメチルシリル化したものを分析試料とした。ガスクロマトグラフ装置は株式会社島津製作所製『ＧＣ−１６Ａ』、カラムはジー・エル・サイエンス株式会社製『２％シリコンＯＶ−１７／クロモゾルブＷ』を充填したステンレスカラム（３ｍｍφ×２ｍ）、キャリアーガスは窒素ガスを流量４０ｍｌ／分で、カラムオーブン温度は１６０℃から３２０℃まで７．５℃／分の昇温速度で分析した。検出は水素炎イオン検出器を用いた。その結果を表３に示す。
【００７０】
【表３】

【００７１】
表３の結果から明かなように、反応生成物Ｘが多量に生成し、その保持時間が市販トレハロースのそれと一致していることが判明した。反応生成物Ｘを同定するために次の確認試験を行った。すなわち、前述のマルトースを基質とした酵素反応液を糖濃度２％になるよう２０ｍＭ酢酸緩衝液、ｐＨ４．５で希釈し、この０．５ｍｌにグルコアミラーゼ（生化学工業株式会社製）０．１単位を加え４０℃で２０時間反応させた。
【００７２】
また、同様に酵素反応液を糖濃度２％になるよう２０ｍＭリン酸緩衝液、ｐＨ７．０で希釈し、この０．５ｍｌにトレハラーゼ０．５単位を加え４０℃で２０時間反応させた。マルトースを基質とした酵素反応液、そのグルコアミラーゼ処理液及びトレハラーゼ処理液をＧＬＣで分析、比較したところ、グルコアミラーゼ処理によりマルトースは完全にグルコースに分解され、反応生成物Ｘは分解されずに残存していた。
【００７３】
一方、トレハラーゼ処理によりマルトースは残存していたが、反応生成物Ｘは完全にグルコースに分解された。グルコアミラーゼ及びトレハラーゼの反応特性を考慮すると、本発明の新規酵素によって生成するマルトースからのオリゴ糖はトレハロースであると判断される。
【００７４】
更に、トレハロースを基質として、マルトースの場合と同様の条件で精製酵素を作用させ、その反応液も同様にＧＬＣ分析したところ、本発明の酵素によってトレハロースからはマルトースが生成することが判明した。以上のＧＬＣ分析結果をまとめて表４に示す。
【００７５】
【表４】

【００７６】
表４の結果から明かなように、本発明の酵素は、マルトースをトレハロースに変換し、トレハロースをマルトースに変換する。その平衡点は、トレハロース側に片寄っており、マルトースからのトレハロースへの変換率が高く、約７０％以上になることが判明した。
【００７７】
【実験６ トレハロース生成に及ぼすマルトース濃度の影響】
マルトース濃度を２．５％、５％、１０％、２０％あるいは４０％で、温度２０℃、ｐＨ７．０にて、実験２の方法で得た精製マルトース・トレハロース変換酵素をマルトースグラム当たり２単位加えて反応させ、経時的に反応液を採取し、１００℃で１０分間加熱して酵素を失活させた。
【００７８】
この反応液の全糖量をアンスロン硫酸法で、還元糖量をソモギー・ネルソン法でグルコース換算で定量し、全糖量に対する還元糖量の割合を還元力として算出した。
【００７９】
また、この反応液を糖濃度約１％になるよう希釈し、少量限外濾過器、日本ミリポアリミテッド製『モルカットＩＩ ＬＧＣ』にて除蛋白を行い、高速液体クロマトグラフィー（以下、ＨＰＬＣと略称する。）にて糖組成を分析した。ＨＰＬＣの装置は東ソー株式会社製『ＣＣＰＤシステム』、分析カラムは株式会社ワイエムシィー製『ＹＭＣ−ｐａｃｋ ＰＡ−０３』（４．６ｍｍφ×２５０ｍｍ）、溶離液はアセトニトリル：水＝７８：２２（容積比）を流速１．２ｍｌ／ｍｉｎで、検出は示差屈折計で行った。それらの結果を表５に示す。
【００８０】
【表５】

【００８１】
表５の結果から明かなように、基質のマルトース濃度に関係なく、マルトースからのトレハロースへの変換反応はよく進行し、トレハロースへ約８０％変換した。
【００８２】
【実験７ トレハロース生成に及ぼす温度の影響】
マルトース濃度２０％で、ｐＨ７．０にして、実験２の方法で得た精製マルトース・トレハロース変換酵素をマルトースグラム当たり２単位加えて、温度５℃、１０℃、１５℃、２０℃あるいは２５℃で反応させ、経時的に反応液を採取し、１００℃で１０分間加熱して酵素を失活させた。この酵素反応液を実験６と同様にして、ＨＰＬＣにて糖組成を分析した。各温度、各時間でのトレハロース含量を表６に示す。
【００８３】
【表６】

【００８４】
表６の結果から明かなように、反応温度が高いほどトレハロース生成速度は大きくなる傾向にあったが、温度５℃でもマルトースからのトレハロースへの変換反応はよく進行し、トレハロースへ約８２％変換した。
【００８５】
【実験８ マルトースからのトレハロースの調製】
マルトース（株式会社林原生物化学研究所製）１０重量部を水４０重量部に溶解し、温度１５℃、ｐＨ７．０にて、実験２の方法で得た精製マルトース・トレハロース変換酵素をマルトースグラム当たり２単位加えて４８時間反応させ、次いで１００℃で１０分間加熱して酵素を失活させた。本溶液には、トレハロースを固形物当たり約７４％含有していた。本溶液を活性炭で脱色し、イオン交換樹脂（Ｈ型及びＯＨ型）にて脱塩して精製し、濃度約７８％に濃縮して、トレハロース含水結晶を種晶として固形物当たり０．１％添加し、室温に一夜放置したところ、結晶が析出した。得られたマスキットを分蜜し、結晶に少量の水をスプレーして結晶を洗浄し、純度９９．８％の極めて高純度のトレハロース含水結晶約３．０重量部を得た。
【００８６】
【実験９ 酵素の生産】
グルコース２．０ｗ／ｖ％、硫酸アンモニウム１．０ｗ／ｖ％、リン酸二カリウム０．１ｗ／ｖ％、リン酸一ナトリウム０．０６ｗ／ｖ％、硫酸マグネシウム０．０５ｗ／ｖ％、炭酸カルシウム０．３ｗ／ｖ％、及び水からなる液体培地を、５００ｍｌ容三角フラスコに１００ｍｌずつ入れ、オートクレーブで１１５℃で、３０分間滅菌し、冷却して、シュードモナス・プチダ Ｈ２６２（ＦＥＲＭＢＰ−４５７９）を接種し、２７℃、２００ｒｐｍで２４時間回転振とう培養したものを種培養とした。
【００８７】
容量３０ｌのファーメンターに種培養の場合と同組成の培地を約２０ｌ入れて、加熱滅菌、冷却して温度２７℃とした後、種培養液１ｖ／ｖ％を接種し、温度２７℃、ｐＨ６．５乃至８．０に保ちつつ、約２０時間通気攪拌培養した。
【００８８】
培養液のマルトース・トレハロース変換酵素活性は、０．１２単位／ｍｌであった。培養液の一部を採り、遠心分離して菌体と培養液上清とに分離し、更に菌体を５０ｍＭリン酸塩緩衝液（ｐＨ７．０）に懸濁し、元の培養液と同じ液量とした後、菌体懸濁液と培養液上清のマルトース・トレハロース変換酵素活性を測定したところ、菌体懸濁液には、０．１１単位／ｍｌの活性が、培養液上清には、０．０１単位／ｍｌの活性が認められた。なお、本酵素の活性は、反応温度を３５℃にして測定した。
【００８９】
【実験１０ 酵素の精製】
実験９で得た培養液を遠心分離して湿重量約０．４５ｋｇの菌体を回収し、これを１０ｍＭリン酸緩衝液（ｐＨ７．０）に懸濁した。この菌体懸濁液約２ｌを超高圧菌体破砕装置（大日本製薬株式会社販売『ミニラボ』）で処理し、菌体を破砕し、この破砕処理液を遠心分離（１５，０００Ｇ、３０分間）することにより、約１．７ｌの上清を得た。その上清液に飽和度０．７になるように硫安を加え溶解させ、４℃、一夜放置した後、遠心分離することにより硫安塩析物を回収した。
【００９０】
得られた硫安塩析物を１０ｍＭリン酸緩衝液（ｐＨ７．０）に溶解させた後、同じ緩衝液に対して２４時間透析し、遠心分離して不溶物を除いた。その透析液（４００ｍｌ）を２回に分けて、『ＤＥＡＥ−トヨパール』を用いたイオン交換カラムクロマトグラフィー（ゲル量３００ｍｌ）を行った。
【００９１】
本発明のマルトース・トレハロース変換酵素は『ＤＥＡＥ−トヨパール』に吸着し、食塩を含む同緩衝液でカラムから溶出した。溶出した酵素活性画分を回収した後、同緩衝液に対して透析し、再度、『ＤＥＡＥ−トヨパール』を用いたイオン交換カラムクロマトグラフィー（ゲル量８０ｍｌ）を行った。吸着したマルトース・トレハロース変換酵素を食塩０．１Ｍから０．３Ｍのリニアグラジエントによりカラムより溶出させ、酵素活性画分を回収した。
【００９２】
続いて、東ソー株式会社製造『トヨパール ＨＷ−５５Ｓ』を用いたゲル濾過クロマトグラフィー（ゲル量４００ｍｌ）を行い、溶出した酵素活性画分を回収した。精製の各ステップにおける酵素活性量、比活性、収率を表７に示す。
【００９３】
【表７】

【００９４】
精製した酵素標品を７．５ｗ／ｖ％濃度ポリアクリルアミドを含むゲル電気泳動により酵素標品の純度を検定したところ、蛋白バンドは単一で純度の高い標品であった。
【００９５】
【実験１１ 酵素の性質】
実験１０の方法で得た精製マルトース・トレハロース変換酵素標品をＳＤＳ−ポリアクリルアミドゲル電気泳動法（ゲル濃度７．５ｗ／ｖ％）に供し、同時に泳動した分子量マーカー（日本バイオ・ラッド・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１１０，０００乃至１２０，０００ダルトンであった。
【００９６】
精製マルトース・トレハロース変換酵素標品を２ｗ／ｖ％アンフォライン（ファルマシア・エルケイビー社製）含有等電点ポリアクリルアミドゲル電気泳動法に供し、泳動後、蛋白バンド及びゲルのｐＨを測定して本酵素の等電点を求めたところ、等電点はｐＩ約４．１乃至５．１であった。
【００９７】
本酵素活性に及ぼす温度、ｐＨの影響を活性測定方法に準じて調べた。結果を図５（温度の影響）、図６（ｐＨの影響）に示した。酵素の至適温度は、ｐＨ７．０、６０分間反応で３７℃付近、至適ｐＨは、３５℃、６０分間反応で約７．３乃至８．３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、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３５℃、６０分間保持した後、ｐＨを７．０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７（温度安定性）、図８（ｐＨ安定性）に示した。本酵素の温度安定性は４０℃付近までであり、ｐＨ安定性は約６．０乃至９．５であった。なお、本酵素活性は、１ｍＭＣｕ^＋＋、Ｈｇ^＋＋又は５０ｍＭトリス塩酸緩衝液で阻害された。
【００９８】
【実験１２ 各種糖質への作用】
反応温度を３５℃とした以外は、実験４の方法に準じて、実験１０で得たシュードモナス・プチダ Ｈ２６２の精製酵素を各種糖質に作用させて、基質になりうるかどうかの試験をした。その結果、シュードモナス・プチダ Ｈ２６２の酵素は、ピメロバクター・スピーシーズ Ｒ４８の酵素と同様、マルトースとトレハロースにのみ作用しマルトースをトレハロースに変換し、トレハロースをマルトースに変換した。その平衡点は、トレハロース側に片寄っており、マルトースからのトレハロースへの変換率が高く、約７０％になることが判明した。
【００９９】
【実験１３ トレハロース生成に及ぼすマルトース濃度の影響】
マルトース濃度を５％、１０％、２０％あるいは３０％で、温度３５℃、ｐＨ７．０にて、実験１０の方法で得た精製マルトース・トレハロース変換酵素をマルトースグラム当たり２単位加えて反応させ、経時的に反応液を採取し、１００℃で１０分間加熱して酵素を失活させた。
【０１００】
この反応液を用いて、実験６と同様に還元力及び糖組成を測定した。それらの結果を表８に示す。
【０１０１】
【表８】

【０１０２】
表８の結果から明らかなように、基質のマルトース濃度に関係なく、トレハロースを約７０％生成した。
【０１０３】
【実験１４ マルトースからのトレハロースの調製】
マルトース（株式会社林原生物化学研究所販売）１０重量部を水４０重量部に溶解し、温度３５℃、ｐＨ７．０にして、実験例１０の方法で得た本発明の精製マルトース・トレハロース変換酵素をマルトースグラム当たり２単位加えて４８時間反応させ、次いで１００℃で１０分間加熱して酵素を失活させた。本溶液には、トレハロースを固形物当たり約６９％含有していた。本溶液を活性炭で脱色し、イオン交換樹脂（Ｈ型及びＯＨ型）にて脱塩して精製し、濃度約７８％に濃縮して、トレハロース含水結晶を種晶として固形物当たり０．１％添加し、室温に一夜放置したところ、結晶が析出した。得られたマスキットを分蜜し、結晶に少量の水をスプレーして結晶を洗浄し、純度９９．７％の極めて高純度のトレハロース結晶約２．３重量部を得た。
【０１０４】
【実験１５ 酵素の生産】
ポリペプトン０．５ｗ／ｖ％、酵母エキス０．１ｗ／ｖ％、硝酸ナトリウム０．０７ｗ／ｖ％、リン酸二ナトリウム０．０１ｗ／ｖ％、硫酸マグネシウム０．０２ｗ／ｖ％、塩化カルシウム０．０１ｗ／ｖ％及び水からなる液体培地を、ｐＨ７．５に調整した後、５００ｍｌ容三角フラスコに１００ｍｌずつ入れ、オートクレーブで１２０℃で、２０分間滅菌し、冷却して、サーマス・アクアティカス ＡＴＣＣ３３９２３を接種し、６０℃、２００ｒｐｍで２４時間回転振とう培養したものを種培養とした。
【０１０５】
容量３０ｌのファーメンター２基に種培養の場合と同組成の培地をそれぞれ約２０ｌ入れて、加熱滅菌、冷却して温度６０℃とした後、種培養液１ｖ／ｖ％を接種し、温度６０℃、ｐＨ６．５乃至８．０に保ちつつ、約２０時間通気攪拌培養した。
【０１０６】
培養液のマルトース・トレハロース変換酵素活性は０．３５単位／ｍｌであった。培養液の一部を採り、遠心分離して菌体と培養上清液とに分離し、更に菌体を５０ｍＭリン酸緩衝液（ｐＨ７．０）に懸濁し、元の培養液と同じ液量とした後、菌体懸濁液と培養上清液のマルトース・トレハロース変換酵素活性を測定したところ、菌体懸濁液には０．３３単位／ｍｌの酵素活性が、また、培養液上清には０．０２単位／ｍｌの酵素活性が認められた。なお、本酵素の活性は、反応温度を６０℃にして測定した。
【０１０７】
【実験１６ 酵素の精製】
実験１５で得た培養液を遠心分離して湿重量約０．２８ｋｇの菌体を回収し、これを１０ｍＭリン酸緩衝液（ｐＨ７．０）に懸濁した。この菌体懸濁液約１．９ｌを、超音波破砕機（株式会社日本精機製作所製『モデルＵＳ３００』）で処理し、菌体を破砕した。この破砕処理液を遠心分離（１５，０００Ｇ、３０分間）することにより、約１．８ｌの上清を得た。その上清に飽和度０．７になるように硫安を加え溶解させ、４℃、一夜放置した後、遠心分離機にかけ、硫安塩析物を回収した。
【０１０８】
得られた硫安塩析物を１０ｍＭリン酸緩衝液（ｐＨ７．０）に溶解させた後、同じ緩衝液に対して２４時間透析し、遠心分離して不溶物を除いた。その透析液（１５６０ｍｌ）を、東ソー株式会社製『ＤＥＡＥ−トヨパール ６５０』を用いたイオン交換カラムクロマトグラフィー（ゲル量５３０ｍｌ）を３回に分けて行った。
【０１０９】
本発明のマルトース・トレハロース変換酵素は『ＤＥＡＥ−トヨパール』に吸着し、食塩を含む同緩衝液でカラムから溶出した。溶出した酵素活性画分を回収した後、１Ｍ硫安を含む同緩衝液に対して透析し、次に、『ブチルトヨパール ６５０』を用いた疎水カラムクロマトグラフィー（ゲル量３８０ｍｌ）を行った。吸着したマルトース・トレハロース変換酵素を硫安１Ｍから０Ｍのリニアグラジエントによりカラムより溶出させ、酵素活性画分を回収した。
【０１１０】
次に、『トヨパール ＨＷ−５５Ｓ』を用いたゲル濾過クロマトグラフィー（ゲル量３８０ｍｌ）を行い、溶出した酵素活性画分を回収した。
【０１１１】
続いて、ファルマシア・エルケイビー社製『モノＱ ＨＲ５／５』を用いたイオン交換クロマトグラフィー（ゲル量１．０ｍｌ）を行い、食塩０．１Ｍから０．３５Ｍのリニアグラジエントにより溶出した酵素活性画分を回収した。精製の各ステップにおける酵素活性量、比活性、収率を表９に示す。
【０１１２】
【表９】

【０１１３】
精製した酵素標品を５ｗ／ｖ％濃度ポリアクリルアミドを含むゲル電気泳動により酵素標品の純度を検定したところ、蛋白バンドは単一で純度の高い標品であった。
【０１１４】
【実験１７ 酵素の性質】
実験１６の方法で得た精製マルトース・トレハロース変換酵素標品をＳＤＳ−ポリアクリルアミドゲル電気泳動法（ゲル濃度７．５ｗ／ｖ％）に供し、同時に泳動した分子量マーカー（日本バイオ・ラッド・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１００，０００乃至１１０，０００ダルトンであった。
【０１１５】
精製マルトース・トレハロース変換酵素標品を２％ｗ／ｖアンフォライン（ファルマシア・エルケイビー社製）含有等電点ポリアクリルアミドゲル電気泳動法に供し、泳動後、蛋白バンド及びゲルのｐＨを測定して本酵素の等電点を求めたところ、等電点はｐＩ約３．８乃至４．８であった。
【０１１６】
本酵素活性に及ぼす温度、ｐＨの影響を活性測定方法に準じて調べた。結果を図９（温度の影響）、図１０（ｐＨの影響）に示した。酵素の至適温度は、ｐＨ７．０、６０分間反応で６５℃付近、至適ｐＨは、６０℃、６０分間反応で約６．０乃至６．７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６０℃、６０分間保持した後、ｐＨを７．０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１１（温度安定性）、図１２（ｐＨ安定性）に示した。本酵素の温度安定性は約８０℃付近までであり、ｐＨ安定性は約５．５乃至９．５であった。なお、本酵素活性は、１ｍＭＣｕ^＋＋、Ｈｇ^＋＋又は５０ｍＭトリス塩酸緩衝液で阻害された。
【０１１７】
【実験１８ 各種糖質への作用】
反応温度を５０℃とした以外は、実験４の方法に準じて、実験１６で得たサーマス・アクアティカス ＡＴＣＣ３３９２３の精製酵素を各種糖質に作用させて、基質になりうるかどうかの試験をした。その結果、サーマス・アクアティカスＡＴＣＣ３３９２３の酵素はピメロバクター・スピーシーズ Ｒ４８の酵素、あるいは、シュードモナス・プチダ Ｈ２６２の酵素と同様、マルトースとトレハロースにのみ作用しマルトースをトレハロースに変換し、トレハロースをマルトースに変換した。その平衡点は、トレハロース側に片寄っており、マルトースからトレハロースへの変換率が高く、７０％以上になることが判明した。
【０１１８】
【実験１９ トレハロース生成に及ぼすマルトース濃度の影響】
マルトース濃度を２．５％、５％、１０％、２０％あるいは４０％で、温度６０℃、ｐＨ６．５にて、実験１６の方法で得たサーマス・アクアティカス ＡＴＣＣ３３９２３の精製マルトース・トレハロース変換酵素をマルトースグラム当たり２．５単位加えて反応させ、７２時間目に反応液を採取し、１００℃で３０分間加熱して酵素を失活させた。この反応液を用いて、実験６と同様に還元力及び糖組成を測定した。その結果を表１０に示した。
【０１１９】
【表１０】

【０１２０】
表１０の結果から明らかなように、基質のマルトース濃度に関係なく、トレハロースを約７０％生成した。
【０１２１】
【実験２０ トレハロース生成に及ぼす温度の影響】
マルトース濃度２０％で、ｐＨ６．５にして、実験１６の方法で得たサーマス・アクアティカス ＡＴＣＣ３３９２３の精製マルトース・トレハロース変換酵素をマルトースグラム当たり２．５単位加えて、温度４０℃、５０℃、６０℃、あるいは７０℃で反応させ、経時的に反応液を採取し、１００℃で３０分間加熱して酵素を失活させた。この反応液を実験６と同様にして、ＨＰＬＣにて糖組成を分析した。各温度、各時間でのトレハロース含量を表１１に示す。
【０１２２】
【表１１】

【０１２３】
表１１の結果から明らかなように、マルトースからのトレハロースへの変換率は反応温度が低いほど高く、４０℃においてトレハロースへ約８０％変換した。
【０１２４】
【実験２１ 他の微生物からのマルトース・トレハロース変換酵素の生産とその性質】
公知微生物のうち、本発明のマルトース・トレハロース変換酵素産生能の確認された特定の微生物を、実験１５の場合に準じて三角フラスコにて４８時間培養した。培養液の酵素活性を調べた後、実験１６の方法に準じて、培養液を破砕装置にかけ、その上清を透析して、部分精製酵素を得、実験１７の方法に従って、その性質を調べた。結果を表１２に示した。
【０１２５】
【表１２】

【０１２６】
表１２に示すこれらサーマス属に属する公知微生物由来の部分精製酵素を用いて、実験１８の方法に従って、各種糖質への作用を調べたところ、サーマス・アクアティカス ＡＴＣＣ３３９２３由来の酵素の場合と同様に、マルトースとトレハロースにのみ作用し、マルトースからトレハロースを生成することが判明した。
【０１２７】
また、サーマス・ルーバー ＡＴＣＣ３５９４８のマルトース・トレハロース変換酵素は、サーマス・アクアティカス ＡＴＣＣ３３９２３の酵素に比し、その至適温度、安定温度は低かったが、他のサーマス属の酵素は、サーマス・アクアティカス ＡＴＣＣ３３９２３の酵素とほぼ同じ性質を示し、耐熱性の高いことが判明した。
【０１２８】
【実験２２ 調製したトレハロースの理化学的性質】
実験８の方法で調製したトレハロースの高純度標品を用いて理化学的性質を調べた。融点は９７．０℃、比旋光度は［α］^２０ _Ｄ＋１９９゜（ｃ＝５）、融解熱 は５７．８ＫＪ／ｍｏｌｅ、溶解度は２５℃の水に対し、無水物として７７．０ｇであった。これらの物性値は、同時に測定した市販トレハロース含水結晶（和光純薬工業株式会社製）の値とよく一致した。
【０１２９】
【実験２３ 生体内での利用試験】
厚治等が、『臨床栄養』、第４１巻、第２号、第２００乃至２０８頁（１９７２年）で報告している方法に準じて、実験８において調製した高純度トレハロース標品（純度９９．８％）３０ｇを２０ｗ／ｖ％水溶液とし、これをボランティア３名（健康な２６才、２７才、３０才の男性）にそれぞれ経口投与し、経時的に採血して、血糖値及びインスリン値を測定した。対照としては、グルコースを用いた。その結果、トレハロースは、グルコースの場合と同様の挙動を示し、血糖値、インスリン値ともに、投与後、約０．５乃至１時間で最大値を示した。トレハロースは、容易に消化吸収、代謝利用されて、エネルギー源になることが判明した。従って、本発明の方法で得られるトレハロース及びこれを含む糖質は、エネルギー補給用糖源として好適である。
【０１３０】
【実験２４ 急性毒性試験】
マウスを使用して、実施例Ａ−６において調製した高純度トレハロース含水結晶を経口投与して急性毒性試験を行った。その結果、トレハロースは低毒性の物質で、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。従って、正確な値とはいえないが、そのＬＤ_５０値は、５０ｇ／ｋｇ以上であった。
【０１３１】
【実験２５ 培養法によるトレハロース生成に与えるマルトース濃度の影響】
マルトース・トレハロース変換酵素産生能を有する微生物を、マルトースを２乃至２０ｗ／ｖ％含有せしめた栄養培地に培養し、培養物中のトレハロース収量に与えるマルトース濃度の影響を調べた。培養方法は、ピメロバクター・スピーシーズ Ｒ４８（ＦＥＲＭ ＢＰ−４３１５）の場合、栄養培地が、グルコース２．０ｗ／ｖ％の代わりに、別滅菌したマルトースを２乃至２０ｗ／ｖ％を含有せしめた培地とした以外は、実験１と同様にファーメンターで２７℃、７２時間培養し、更に界面活性剤（ポリオキシエチレン・ソルビタン・モノパルミテート、和光純薬工業株式会社販売『Ｔｗｅｅｎ ４０』）を０．１ｖ／ｖ％加えて２４時間培養を続けた。また、サーマス・アクアティカス ＡＴＣＣ３３９２３の場合、栄養培地を別滅菌したマルトースを２乃至２０ｗ／ｖ％を含有せしめた培地とした以外は、実験１５と同様にファーメンターで６０℃、２４時間培養し、更に、界面活性剤『Ｔｗｅｅｎ ４０』を０．１ｖ／ｖ％加えて２４時間培養を続けた。培養物は、遠心分離して、上清に含まれるトレハロース含量（ｍｇ／ｍｌ）をＨＰＬＣで測定した。結果は、表１３に示す。
【０１３２】
【表１３】

【０１３３】
表１３の結果から明らかなように、栄養培地中のマルトースが濃度２０ｗ／ｖ％以下、望ましくは１５ｗ／ｖ％以下、更に望ましくは５乃至１０ｗ／ｖ％付近でトレハロース収量が高く、トレハロース生産に好適であることが判明した。
【０１３４】
以下、本発明のマルトース・トレハロース変換酵素産生能を有する微生物を利用したトレハロース、又はこれを含む糖質の製造方法を実施例Ａで、トレハロース、又は、これを含む糖質を含有せしめた組成物を実施例Ｂで示す。
【０１３５】
【実施例Ａ−１】
濃度１０％馬鈴薯澱粉乳（ｐＨ５．５）にα−アミラーゼ（ナガセ生化学工業株式会社製『スピターゼ ＨＳ』）を澱粉グラム当たり２単位加えて攪拌下、加熱糊化・液化させ、直ちにオートクレーブ（１２０℃）を２０分間行った後、温度５０℃、ｐＨ５．０に調整した。これにβ−アミラーゼ（ナガセ生化学工業株式会社製）を澱粉グラム当たり２０単位及びイソアミラーゼ（株式会社林原生物化学研究所製）を澱粉グラム当たり５００単位の割合になるように加えて２４時間反応させ、次いで、９５℃に加熱して酵素を失活させ、濾過、脱色して、マルトース含量約９２％の糖液を得た。糖源として、グルコース２．０ｗ／ｖ％の代わりに、前述の糖液を別滅菌して固形物当たり１０ｗ／ｖ％を使用した以外は、実験１の方法に準じて調製した栄養培地をファーメンターにとり、これにマルトース・トレハロース変換酵素産生能を有するピメロバクター・スピーシーズ Ｒ４８（ＦＥＲＭ ＢＰ−４３１５）の種培養物を１ｖ／ｖ％植菌し、実験１と同様に２７℃で７２時間通気攪拌培養し、更に、界面活性剤（アルキルフェノール・ポリオキシエチレンエーテル、和光純薬工業株式会社販売『Ｔｒｉｔｏｎ Ｘ−１００』）を０．２ｖ／ｖ％加えて培養を２４時間続けた。この培養物を濾過して不溶物を除去し、得られる濾液を、９５℃に加熱して酵素を失活させた後、濃縮し、常法に従って活性炭で脱色・濾過し、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たり約６５％の収率で得た。本品は、固形物当たりトレハロースを約４４％含有しており、温和な甘味、適度の粘度、保湿性を有し、甘味料、呈味改良剤、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。
【０１３６】
【実施例Ａ−２】
実施例Ａ−１の方法で得た培養物の濾液に、固形物グラム当たり１０単位のグルコアミラーゼ、ナガセ生化学工業株式会社製『グルコチーム』をｐＨ５．０、５０℃で２４時間作用させ、次いで、加熱失活、脱色、脱塩精製して得られる糖液を原糖液とし、トレハロースの含量を高めるため、ナトリウム型強酸性カチオン交換樹脂（東京有機化学工業株式会社製『ＸＴ−１０１６』、架橋度４％）を用いたイオン交換カラムクロマトグラフィーを行った。樹脂を内径５．４ｃｍのジャケット付き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６０℃に維持しつつ、糖液を樹脂に対して、５ｖ／ｖ％加え、これに６０℃の温水をＳＶ０．１５で流して分画し、グルコースを除去し、トレハロース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、トレハロース高含有粉末を固形物当たり、約３５％の収率で得た。本品は、トレハロースを約９７％含有しており、極めて低い還元性、まろやかで上品な甘味を有し、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。
【０１３７】
【実施例Ａ−３】
実施例Ａ−２の方法で得たトレハロース高含有画分を、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て精製した溶液を濃度約７０％に濃縮した後、助晶機にとり、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 より送風して底部に設けた移送金網コンベア上に捕集し、コンベアの下より４５℃の温風を送りつつ、金網コンベア上に捕集した結晶粉末を乾燥塔外に徐々に移動させ取り出した。この取り出した結晶粉末を、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原料のトレハロース高含有糖液に対して固形物当たり約９０％の収率で得た。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、安定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。
【０１３８】
【実施例Ａ−４】
実施例Ａ−２の方法で得たトレハロース高含有画分を、実施例Ａ−３と同様に精製し、次いで、蒸発釜にとり、減圧下で煮詰め、水分約３．０％のシラップとした。次いで、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物当たり１％加え、１２０℃で攪拌助晶し、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、水分約０．３％の無水結晶トレハロース粉末を、原料のトレハロース高含有糖液に対して、固形物当たり約８５％の収率で得た。本品は、食品、化粧品、医薬品、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、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。
【０１３９】
【実施例Ａ−５】
濃度３３％とうもろこし澱粉乳に炭酸カルシウムを０．１％加えた後、ｐＨ６．５に調整し、これにα−アミラーゼ（ノボ社製『ターマミール６０Ｌ』）を澱粉グラム当たり０．２％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３０分間行った後、５５℃に冷却し、これにイソアミラーゼ（株式会社林原生物化学研究所製）を澱粉グラム当たり５００単位及びβ−アミラーゼ（ナガセ生化学工業株式会社製）を澱粉グラム当たり３０単位加え、４８時間反応させ、次いで、９５℃に加熱して酵素を失活させ、濾過、脱色して、マルトース含量約８４％の糖液を得た。糖源として、前述の糖液を別滅菌して固形物当たり１０ｗ／ｖ％を追加した以外は、実験９の方法に準じて調製した栄養培地をファーメンターにとり、これにマルトース・トレハロース変換酵素産生能を有するシュードモナス・プチダ Ｈ２６２（ＦＥＲＭ ＢＰ−４５７９）の種培養物を１ｖ／ｖ％植菌し、実験９と同様に２７℃で４８時間通気撹拌培養し、更に、界面活性剤『Ｔｗｅｅｎ ４０』を０．２ｖ／ｖ％加えて２４時間培養を続けた。この培養物を濾過して不溶物を除去し、得られる濾液を、９５℃に加熱して酵素を失活させた後、濃縮し、常法に従って活性炭で脱色・濾過し、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たり約５０％の収率で得た。本品は、固形物当たりトレハロースを約６４％を含有しており、低還元性、温和な甘味、適度の粘度、保湿性を有し、各種飲食物、化粧品、医薬品などの各種組成物に有利に利用できる。
【０１４０】
【実施例Ａ−６】
実施例Ａ−５の方法で得たシラップを濃度約８０％に濃縮して助晶機にとり、種晶としてトレハロース含水結晶粉末約１％を加え、攪拌しつつ徐冷、晶出させた。次いで、バスケット型遠心分離機で分蜜し、結晶を少量の水でスプレーし、洗浄して高純度トレハロース含水結晶を固形物当たり約２０％の収率で得た。本品は、実験２２と同様の理化学的性質を示し、甘味料、呈味改良剤、品質改良剤、安定剤などとして、各種飲食物、化粧品、医薬品などの各種組成物に有利に利用できる。更には、工業試薬、化学原料などに利用することも有利に実施できる。
【０１４１】
【実施例Ａ−７】
濃度１０％とうもろこし澱粉乳（ｐＨ５．５）にα−アミラーゼ『スピターゼＨＳ』を澱粉グラム当たり２単位加えて撹拌下、加熱糊化・液化させ、直ちにオートクレイブ（１２０℃）を２０分間行った後、温度５５℃、ｐＨ５．０に調整した。これにイソアミラーゼ（株式会社林原生物化学研究所製）を澱粉グラム当たり３００単位及びβ−アミラーゼ（ナガセ生化学工業株式会社製）を澱粉グラム当たり２０単位の割合になるように加えて２４時間反応させ、次いで、９５℃に加熱して酵素を失活させ、濾過、脱色して、マルトース含量約９２％の糖液を得た。糖源として、前述の糖液を別滅菌して固形物当たり１０ｗ／ｖ％を追加した以外は、実験１５の方法に準じて調製した栄養培地をファーメンターにとり、これにマルトース・トレハロース変換酵素産生能を有するサーマス・アクアティカスＡＴＣＣ ３３９２３の種培養物を１ｖ／ｖ％植菌し、実験１５と同様に６０℃で４０時間通気撹拌培養し、更に、界面活性剤『Ｔｒｉｔｏｎ Ｘ−１００』を０．１ｖ／ｖ％及び培養液１ｌ当たり卵白リゾチーム５０ｍｇを加えて１６時間培養を続けた。この培養物を濾過して不溶物を除去し、得られる濾液を、９５℃に加熱して酵素を失活させた後、濃縮し、常法に従って活性炭で脱色・濾過し、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たり約５５％の収率で得た。本品は、固形物当たりトレハロースを約６８％含有しており、温和な甘味、適度の粘度、保湿性を有し、甘味料、呈味改良剤、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。
【０１４２】
【実施例Ａ−８】
実施例Ａ−７の方法で得たシラップを濃度約８５％に濃縮して助晶機にとり、種晶約１％を混合した後、バットにとり、２０℃で４日間静置して晶出固化させ、次いで切削機にて粉砕し、乾燥して含蜜型トレハロース含水結晶粉末を固形物当たり約９５％の収率で得た。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、安定剤などとして、各種飲食物、化粧品、医薬品などの各種組成物に有利に利用できる。
【０１４３】
【実施例Ａ−９】
実施例Ａ−７の方法で得たシラップを濃度約８０％に濃縮して助晶機にとり、実施例Ａ−６と同様に晶出、分蜜して高純度のトレハロース含水結晶を固形物当たり約２０％の収率で得た。本品は、実験２２と同様の理化学的性質を示し、実施例Ａ−６と同様に、各種飲食物、化粧品、医薬品などの各種組成物、更には、工業試薬、工業原料、化学原料などに有利に利用できる。
【０１４４】
【実施例Ｂ−１ 甘味料】
実施例Ａ−３の方法で得たトレハロース含水結晶粉末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『αＧスイート』）０．０１重量部及びＬ−アスパルチル−Ｌ−フェニルアラニンメチルエステル（味の素株式会社販売『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。
【０１４５】
【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−１の方法で得たトレハロース含有シラップ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、変形も起こらない高品質のハードキャンディーである。
【０１４６】
【実施例Ｂ−３ 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３０重量部、実施例Ａ−６の方法で得た高純度トレハロース含水結晶２０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。
【０１４７】
【実施例Ｂ−４ 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−３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。
【０１４８】
【実施例Ｂ−５ 加糖練乳】
原乳１００重量部に実施例Ａ−５の方法で得たトレハロース含有シラップ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。
【０１４９】
【実施例Ｂ−６ 乳酸菌飲料】
脱脂粉乳１７５重量部、実施例Ａ−２の方法で得たトレハロース高含有粉末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。
【０１５０】
【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重量部に対して、実施例Ａ−６の方法で得た高純度トレハロース含水結晶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−５の方法で得たトレハロース高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。
【０１５１】
【実施例Ｂ−８ カスタードクリーム】
コーンスターチ１００重量部、実施例Ａ−１の方法で得たトレハロース含有シラップ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。
【０１５２】
【実施例Ｂ−９ ういろうの素】
米粉９０重量部に、コーンスターチ２０重量部、蔗糖４０重量部、実施例Ａ−３の方法で得たトレハロース含水結晶粉末８０重量部及びプルラン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、日持ちも良い。
【０１５３】
【実施例Ｂ−１０ 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−７の方法で得たトレハロース含有シラップ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。
【０１５４】
【実施例Ｂ−１１ 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の方法で得たトレハロース高含有粉末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、すだちともに良好で適度な弾力、温和な甘味を有する高品質のパンである。
【０１５５】
【実施例Ｂ−１２ 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−２の方法で得たトレハロース高含有粉末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、常法に従い、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。
【０１５６】
【実施例Ｂ−１３ 粉末ペプチド】
濃度４０％食品用大豆ペプチド溶液（不二製油株式会社製『ハイニュートＳ』）１重量部に、実施例Ａ−６の方法で得た高純度トレハロース含水結晶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。
【０１５７】
【実施例Ｂ−１４ 粉末味噌】
赤味噌１重量部に実施例Ａ−４の方法で得た無水結晶トレハロース粉末３重量部を混合し、多数の半球状凹部を設けた金属板に流し込み、これを室温下で一夜静置して固化し、離型して１個当たり約４ｇの固形味噌を得、これを粉砕機にかけて粉末味噌を得た。本品は、即席ラーメン、即席吸物などの調味料として有利に利用できる。また、固形味噌は、固形調味料としてだけでなく味噌菓子などとしても利用できる。
【０１５８】
【実施例Ｂ−１５ 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−４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。
【０１５９】
【実施例Ｂ−１６ 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の方法で得たトレハロース高含有粉末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ル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。
【０１６０】
【実施例Ｂ−１７ 粉末薬用人参エキス】
薬用人参エキス０．５重量部に実施例Ａ−４の方法で得た無水結晶トレハロース粉末１．５重量部を混捏した後、バットに移し、２日間放置してトレハロース含水結晶に変換させ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、分級して粉末薬用人参エキスを得た。本品を適量のビタミンＢ１及びビタミンＢ２粉末とともに顆粒成型機にかけ、ビタミン含有顆粒状薬用人参エキスとした。本品は、疲労回復剤、強壮、強精剤などとして有利に利用できる。また、育毛剤などとしても利用できる。
【０１６１】
【実施例Ｂ−１８ 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、安定剤であるウシ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−６の方法で得た高純度トレハロース含水結晶を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『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、本発明の非還元性糖質と無水結晶マルトースが共に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。
【０１６２】
【実施例Ｂ−１９ 糖衣錠】
重量１５０ｍｇの素錠を芯剤とし、これに実施例Ａ−９の方法で得た高純度トレハロース含水結晶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。
【０１６３】
【実施例Ｂ−２０ 練歯磨】
配合
第２リン酸カルシウム ４５．０重量部
プルラン ２．９５重量部
ラウリル硫酸ナトリウム １．５重量部
グリセリン ２０．０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
防腐剤 ０．０５重量部
実施例Ａ−８の方法で得たトレハロース含水結晶粉末 １２．０重量部
マルチトール ５．０重量部
水 １３．０重量部
上記の材料を常法に従って混合し、練歯磨を得た。本品は、適度の甘味を有しており、特に子供用練歯磨として好適である。
【０１６４】
【実施例Ｂ−２１ 流動食用固体製剤】
実施例Ａ−６の方法で製造した高純度トレハロース含水結晶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。
【０１６５】
【実施例Ｂ−２２ 輸液剤】
実施例Ａ−６の方法で製造した高純度トレハロース含水結晶を水に濃度約１０ｗ／ｖ％に溶解し、次いで、常法に従って、精密濾過してパイロジェンフリーとし、プラスチック製ボトルに無菌的に充填し施栓して製品を得た。本品は、経日変化もなく安定な輸液剤で、静脈内、腹腔内などに投与するのに好適である。本品は濃度１０ｗ／ｖ％で血液と等張で、グルコースの場合の２倍濃度でエネルギー補給できる。
【０１６６】
【実施例Ｂ−２３ 輸液剤】
実施例Ａ−９の方法で製造した高純度トレハロース含水結晶と下記の組成のアミノ酸配合物とがそれぞれ５ｗ／ｖ％、３０ｗ／ｖ％になるように水に混合溶解し、次いで実施例Ｂ−２２と同様に精製してパイロジェンフリーとし、更に、プラスチック製バックに充填し施栓して製品を得た。
アミノ酸配合物の組成（ｍｇ／１００ｍｌ）
Ｌ−イソロイシン １８０
Ｌ−ロイシン ４１０
Ｌ−リジン塩酸塩 ６２０
Ｌ−メチオニン ２４０
Ｌ−フェニルアラニン ２９０
Ｌ−スレオニン １８０
Ｌ−トリプトファン ６０
Ｌ−バリン ２００
Ｌ−アルギニン塩酸塩 ２７０
Ｌ−ヒスチジン塩酸塩 １３０
グリシン ３４０
本品は、糖質とアミノ酸との複合輸液剤にもかかわらず、トレハロースが還元性を示さないため、経日変化もなく安定な輸液剤で、静脈内、腹腔内などへ投与するのに好適である。本品は、生体へのエネルギー補給のみならず、アミノ酸補給のためにも有利に利用できる。
【０１６７】
【実施例Ｂ−２４ 外傷治療用膏薬】
実施例Ａ−８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。
【０１６８】
【発明の効果】
上記から明らかなように、マルトースを含有せしめた栄養培地に、マルトース・トレハロース変換酵素産生能を有する微生物を培養することにより、培養物から非還元性糖質を極めて簡便、短時間に製造することができることとなり、トレハロース、又は、これを含む糖質の工業的な製造を容易にする。また、マルトースとして、澱粉を液化した溶液にβ−アミラーゼ又はβ−アミラーゼとともに澱粉枝切酵素を作用させて得られるマルトースを利用すれば、澱粉からのトレハロース収量を著しく高め、その工業的な製造を容易にする。このようにして得られるトレハロース又はこれを含む糖質は、安定性に優れ、良質で上品な甘味を有している。また、経口摂取により消化吸収され、カロリー源となる。とりわけ、トレハロースは非経口的にも使用され、よく代謝利用される。従って、本発明のトレハロース、又は、これを含む糖質は、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。
【０１６９】
本発明の確立は、安価で無限の資源である澱粉から、従来望むべくして容易に得られなかったトレハロース、又は、これを含む糖質を工業的に大量かつ安価に提供できる全く新しい道を拓くこととなり、それが与える影響は、食品、化粧品、医薬品業界は言うに及ばず、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】ピメロバクター・スピーシーズ Ｒ４８のマルトース・トレハロース変換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】ピメロバクター・スピーシーズ Ｒ４８のマルトース・トレハロース変換酵素の酵素活性に及ぼすｐＨの影響を示す図である。
【図３】ピメロバクター・スピーシーズ Ｒ４８のマルトース・トレハロース変換酵素の安定性に及ぼす温度の影響を示す図である。
【図４】ピメロバクター・スピーシーズ Ｒ４８のマルトース・トレハロース変換酵素の安定性に及ぼすｐＨの影響を示す図である。
【図５】シュードモナス・プチダ Ｈ２６２のマルトース・トレハロース変換酵素の酵素活性に及ぼす温度の影響を示す図である。
【図６】シュードモナス・プチダ Ｈ２６２のマルトース・トレハロース変換酵素の酵素活性に及ぼすｐＨの影響を示す図である。
【図７】シュードモナス・プチダ Ｈ２６２のマルトース・トレハロース変換酵素の安定性に及ぼす温度の影響を示す図である。
【図８】シュードモナス・プチダ Ｈ２６２のマルトース・トレハロース変換酵素の安定性に及ぼすｐＨの影響を示す図である。
【図９】サーマス・アクアティカス ＡＴＣＣ３３９２３のマルトース・トレハロース変換酵素の酵素活性に及ぼす温度の影響を示す図である。
【図１０】サーマス・アクアティカス ＡＴＣＣ３３９２３のマルトース・トレハロース変換酵素の酵素活性に及ぼすｐＨの影響を示す図である。
【図１１】サーマス・アクアティカス ＡＴＣＣ３３９２３のマルトース・トレハロース変換酵素の安定性に及ぼす温度の影響を示す図である。
【図１２】サーマス・アクアティカス ＡＴＣＣ３３９２３のマルトース・トレハロース変換酵素の安定性に及ぼすｐＨの影響を示す図である。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to trehalose, a method for producing the same, and uses, and more specifically, trehalose obtained by culturing a microorganism capable of producing maltose / trehalose converting enzyme in a nutrient medium containing maltose, or a sugar containing the same. And a method for producing the sugar and its use.
[0002]
[Prior art]
Trehalose (α, α-trehalose) has long been known as a non-reducing saccharide with glucose as a constituent sugar, and its existence is “Advanceds in Carbohydrate Chemistry”. 18: 201-225 (1963) Academic Press (USA) and “Applied and Environmental Microbiology”, 56: 3213-3215 As described in the page (1990), it covers a wide range of microorganisms, mushrooms, insects, etc. in small quantities. Non-reducing carbohydrates such as trehalose do not cause an aminocarbonyl reaction with substances having amino groups such as amino acids and proteins, and do not damage amino acid-containing substances, so that they can be used and processed without concern about browning and deterioration. Therefore, establishment of an industrial production method is desired.
[0003]
Examples of the method for producing trehalose include a method using a microbial cell reported in Japanese Patent Laid-Open No. 50-154485, and a method using maltose phosphorylase and trehalose phosphorylase proposed in Japanese Patent Laid-Open No. 58-216695. A method of converting maltose in combination is known. However, in the method using a microbial cell, the content of trehalose contained in the microbial cell is usually 15 w / w% per solid (hereinafter, unless otherwise specified, w / w% is simply abbreviated as%), and the process of extracting and purifying this is complicated and unsuitable for an industrial production method. In addition, the methods using maltose phosphorylase and trehalose phosphorylase are both via glucose-1 phosphate, and it is difficult to increase the substrate concentration, and the reaction system of both enzymes is a reversible reaction. The production rate of the product is low, and furthermore, it is difficult to keep the reaction system of both enzymes stable and allow the reaction to proceed smoothly, and it has not yet been realized as an industrial production method.
[0004]
In this connection, “Monthly Food Chemical”, August issue, pages 67 to 72 (1992), “Current status and issues of starch utilization development”, “Oligosaccharide”, Although the range of application is conceivable, it is said that enzymatic production using the direct sugar transfer and hydrolysis reaction of the present sugar from starch sugar is currently impossible scientifically. " As described above, it has been conventionally considered that it is impossible to produce trehalose by starch using starch as a raw material.
[0005]
On the other hand, it is known that partially decomposed starch produced from starch as a raw material, for example, starch liquefaction product, various dextrins, various maltooligosaccharides, etc., usually has a reducing group at the end of the molecule and exhibits reducibility. Yes. Such a starch partial decomposition product is referred to as a reducing starch partial decomposition product in the present specification. In general, a reducing starch partially decomposed product expresses the magnitude of reducing power per solid as Dextrose Equivalent (DE). Those with large values usually have small molecules, low viscosity, and strong sweetness, but are highly reactive and easily cause aminocarbonyl reactions with substances having amino groups such as amino acids and proteins. Thus, it is known that the quality is easily deteriorated.
[0006]
Various properties of such a reduced starch partial decomposition product depend on the size of DE, and the relationship between the reduced starch partial decomposition product and DE is extremely important. Traditionally, the industry has even believed that it was impossible to break this relationship.
[0007]
The only way to break the relationship between the degradable starch partially decomposed product and the DE is to convert the reducing starch partially decomposed product into sugar alcohol by a high-pressure hydrogenation method or the like to make a non-reducing carbohydrate. It is. However, this method requires a high-pressure autoclave, which not only consumes a large amount of hydrogen and energy, but also requires advanced safety facilities and management from the viewpoint of disaster prevention. In addition, the sugar alcohol of the resulting reduced starch partial decomposition product differs in that it is composed of glucose and sorbitol, whereas the raw reduced starch partial decomposition product is composed of glucose only and must be ingested. Depending on the condition, there is a concern that the symptoms of indigestion and laxity may occur, although it is transient. Therefore, it is desired to establish a method for reducing or eliminating the reducing power without changing glucose, which is a constituent sugar of the reduced starch partial decomposition product.
[0008]
In order to solve this problem, the present inventors previously described in Japanese Patent Application No. 6-144092 a novel maltose / trehalose converting enzyme that converts maltose to trehalose (this enzyme is referred to as maltose throughout the present specification). (Hereinafter referred to as “trehalose converting enzyme”), and a method for producing trehalose and a sugar containing the same from maltose was established using the present maltose / trehalose converting enzyme.
[0009]
However, in addition to the culture time for culturing microorganisms to produce the enzyme, the enzyme recovery time and the enzyme reaction time from maltose to trehalose are required, and this requires a considerable amount of time for industrial implementation. It turns out that there is. When producing trehalose from maltose, it is desired to establish a method for producing trehalose that can be carried out more easily and easily, including the production of the enzyme.
[0010]
[Problems to be solved by the invention]
The present invention establishes a new method capable of producing trehalose from maltose easily and in a short time, and also provides its use.
[0011]
[Means for Solving the Problems]
In order to solve the above-mentioned problems, the present inventors have continued intensive studies on the culture state of microorganisms capable of producing maltose / trehalose converting enzyme and the production state of the enzyme. As a result, it has been found that the microorganism produces maltose / trehalose converting enzyme from a very early stage, and easily produces and accumulates trehalose by allowing maltose to coexist in the nutrient medium during cultivation. The present invention has been completed.
[0012]
That is, the present invention comprises culturing a microorganism having the ability to produce maltose / trehalose converting enzyme in a nutrient medium containing maltose, the resulting trehalose, or a saccharide containing the same, a method for producing the saccharide, and The present invention establishes a composition containing the carbohydrate. In the present invention, maltose is particularly preferably obtained by reacting starch liquefaction with β-amylase or β-amylase together with starch debranching enzyme, and as microorganisms, maltose is particularly preferred. -The use of microorganisms capable of producing trehalose converting enzyme is extremely advantageous for trehalose production. The trehalose and saccharides containing the same thus obtained have high stability, are easy to handle, and can be used in a wide range of applications. For example, they can be advantageously used in various compositions such as foods and drinks, cosmetics, and pharmaceuticals.
[0013]
The microorganism having the ability to produce maltose / trehalose converting enzyme used in the present invention may be any microorganism that produces an enzyme that converts maltose into trehalose. For example, the genus Pimelobacter disclosed in Japanese Patent Application No. 6-144092 Microorganisms belonging to the genus Pseudomonas and Thermus are preferred.
[0014]
The medium used for culturing the microorganism is a nutrient medium that can grow microorganisms and can produce maltose / trehalose converting enzyme, and contains maltose that is a substrate for producing trehalose. If necessary, other carbon sources that can be assimilated by the microorganism, for example, sugars such as glucose, fructose, lactose, sucrose, mannitol, sorbitol, molasses, organic acids such as citric acid, succinic acid, or It is also optional to use the salt together. The maltose concentration contained in the medium is preferably 20 w / v% or less, desirably 15 w / v% or less, and more desirably 5 to 10 w / v%. As the nitrogen source, for example, inorganic nitrogen compounds such as ammonium salts and nitrates, and organic nitrogen-containing materials such as urea, corn steep liquor, casein, peptone, yeast extract and meat extract are appropriately used. Moreover, as an inorganic component, calcium salt, magnesium salt, potassium salt, sodium salt, phosphate, manganese salt, zinc salt, iron salt, copper salt, molybdenum salt, cobalt salt etc. are used suitably, for example. Furthermore, amino acids, vitamins and the like are also used as necessary.
[0015]
The culture conditions may be as long as microorganisms grow and produce maltose / trehalose converting enzyme. Usually, the temperature is 4 to 80 ° C., preferably 20 to 75 ° C., preferably pH 5 to 9, and preferably 6 to 8.5. Done aerobically. The culture time may be a time longer than the microorganisms can grow, and is preferably 10 to 100 hours. Moreover, although there is no restriction | limiting in particular in the dissolved oxygen concentration of a culture solution, Usually, 0.5 thru | or 20 ppm is preferable. For this purpose, means such as adjusting the aeration amount, stirring, adding oxygen to the aeration, and increasing the pressure in the fermenter are adopted. The culture method may be any of batch culture, continuous culture, or semi-continuous culture.
[0016]
In this way, a microorganism having the ability to produce maltose / trehalose converting enzyme can be cultured in a nutrient medium containing maltose, and trehalose can be produced and accumulated in the culture. If necessary, the maltose / trehalose converting enzyme prepared separately may be supplemented to the nutrient medium during cultivation to increase the production rate of trehalose. It is also advantageous to increase the rate of trehalose production by adding a lytic enzyme such as an activator and / or egg white lysozyme to the nutrient medium in culture. The trehalose produced and accumulated in this manner is contained in the liquid portion obtained by separating insoluble matters in the culture.
[0017]
The culture containing trehalose is first made into a sterilized solution by a known solid-liquid separation method such as filtration or centrifugation, concentrated according to a conventional method, decolorized with activated carbon, H-type, OH-type ions. It is desalted with an exchange resin, purified and concentrated to a syrup-like product. It is optional to further dry it into a powdered product. If necessary, the culture is directly filtered through a membrane such as a flat membrane or a hollow fiber membrane to remove insoluble matter such as bacterial cells and high-molecular substances such as soluble proteins and nucleic acids, or in advance by centrifugation or the like. After removing the insoluble matter and then removing the soluble polymer by membrane filtration, it is purified by concentrating, decolorizing and desalting according to conventional methods to advantageously produce trehalose or a carbohydrate product containing the same. be able to.
[0018]
Next, the maltose / trehalose converting enzyme contributing to the production of trehalose according to the present invention will be described. Enzymatic activity is observed in both the microbial cells and the sterilization solution of the culture, and the microbial cells and the sterilization solution can be collected as a crude enzyme solution, or the entire culture can be used as a crude enzyme solution. A known solid-liquid separation method is employed to remove the cells from the culture. For example, a method of centrifuging the culture itself as it is, a method of separating by filtration using a precoat filter or the like, a method of separating by membrane filtration such as a flat membrane or a hollow fiber membrane, etc. are appropriately employed. The sterilizing solution can be used as an enzyme solution as it is, but generally it is used after being concentrated. As the concentration method, for example, an ammonium sulfate salting-out method, an acetone and alcohol precipitation method, a membrane concentration method such as a flat membrane or a hollow fiber membrane, or the like is employed.
[0019]
Furthermore, the disinfectant solution and the concentrate thereof can be immobilized by a known method. For example, a binding method to an ion exchanger, a covalent bond / adsorption method with a resin and a membrane, a comprehensive method using a polymer substance, and the like are employed. Moreover, although the microbial cell isolate | separated from the culture can be used as it is as a crude enzyme, you may fix | immobilize and use this. As an example, this is mixed with sodium alginate and dropped into a calcium chloride solution to be gelled and fixed in a granular form. The granulated product may be further fixed with polyethyleneimine or glutaraldehyde. It is also possible to extract an enzyme from the cells and use the extract as a crude enzyme solution. For example, an enzyme can be extracted from the cells by ultrasonic crushing, glass beads and alumina mechanical crushing, French press crushing, etc., and a clear crude enzyme solution can be obtained by centrifugation or membrane filtration. .
[0020]
This enzyme solution can be used as it is, but can be further purified and used by a known method. As an example, after dialysis of a crude enzyme preparation concentrated by subjecting the treated solution of the culture solution to salting out with ammonium sulfate, anion exchange column chromatography using “DEAE-Toyopearl” manufactured by Tosoh Corporation, followed by “ A single enzyme can be obtained electrophoretically by purifying it using hydrophobic column chromatography using "Butyl Toyopearl" etc. or gel filtration chromatography using "Toyopearl HW-55" etc. .
[0021]
The maltose / trehalose converting enzyme thus obtained generally has the following physicochemical properties, for example.
(1) Action
Convert maltose to trehalose and convert trehalose to maltose.
(2) Molecular weight
About 57,000 to 120,000 daltons by SDS-gel electrophoresis.
(3) Isoelectric point
PI of about 3.8 to 5.1 by an ampholine-containing electrophoresis.
(4) Activity inhibition
1 mM Cu⁺⁺, Hg⁺⁺Alternatively, it is inhibited with 50 mM Tris-HCl buffer.
(5) Origin
It is an enzyme produced by a microorganism.
[0022]
The following is a specific example based on the difference in the source microorganism.
[Mitrose trehalose converting enzyme derived from Pimerobacter sp. R48]
(1) Action
Convert maltose to trehalose and convert trehalose to maltose.
About 1 mole of trehalose or maltose is produced from 1 mole of maltose or trehalose, respectively.
(2) Molecular weight
About 57,000 to 67,000 daltons by SDS-gel electrophoresis.
(3) Isoelectric point
PI of about 4.1 to 5.1 as determined by ampholine-containing electrophoresis.
(4) Activity inhibition
1 mM Cu⁺⁺, Hg⁺⁺Alternatively, it is inhibited with 50 mM Tris-HCl buffer.
(5) Optimal temperature
pH 7.0, reaction for 60 minutes, around 20 ° C.
(6) Optimum pH
PH of about 7.0 to 8.0 after reaction at 25 ° C. for 60 minutes.
(7) Temperature stability
Stable to around 30 ° C with pH 7.0 and 60 minutes holding.
(8) pH stability
PH of about 6.0 to 9.0 after holding at 20 ° C. for 60 minutes.
[0023]
[Maltose trehalose converting enzyme derived from Pseudomonas putida H262]
(1) Action
Convert maltose to trehalose and convert trehalose to maltose.
About 1 mole of trehalose or maltose is produced from 1 mole of maltose or trehalose, respectively.
(2) Molecular weight
About 110,000 to 120,000 daltons by SDS-gel electrophoresis.
(3) Isoelectric point
PI of about 4.1 to 5.1 as determined by ampholine-containing electrophoresis.
(4) Activity inhibition
1 mM Cu⁺⁺, Hg⁺⁺Alternatively, it is inhibited with 50 mM Tris-HCl buffer.
(5) Optimal temperature
pH 7.0, reaction for 60 minutes, around 37 ° C.
(6) Optimum pH
PH of about 7.3 to 8.3 after reaction at 35 ° C. for 60 minutes.
(7) Temperature stability
Stable to around 40 ° C with pH 7.0 and 60 minutes holding.
(8) pH stability
Holding at 35 ° C. for 60 minutes, pH is about 6.0 to 9.5.
[0024]
[Maltose / trehalose converting enzyme derived from Thermus aquaticus ATCC 33923]
(1) Action
Convert maltose to trehalose and convert trehalose to maltose.
About 1 mole of trehalose or maltose is produced from 1 mole of maltose or trehalose, respectively.
(2) Molecular weight
About 100,000 to 110,000 daltons by SDS-gel electrophoresis.
(3) Isoelectric point
PI of about 3.8 to 4.8 as determined by ampholine-containing electrophoresis.
(4) Activity inhibition
1 mM Cu⁺⁺, Hg⁺⁺Alternatively, it is inhibited with 50 mM Tris-HCl buffer.
(5) Optimal temperature
pH 7.0, reaction for 60 minutes, around 65 ° C.
(6) Optimum pH
The pH is about 6.0 to 6.7 after reaction at 60 ° C. for 60 minutes.
(7) Temperature stability
Stable up to around 80 ° C at pH 7.0, held for 60 minutes.
(8) pH stability
Holding at 60 ° C. for 60 minutes, pH is about 5.5 to 9.5.
[0025]
The activity of maltose / trehalose converting enzyme is measured as follows. 1 ml of enzyme solution is added to 1 ml of maltose 20 w / v% (10 mM phosphate buffer, pH 7.0) as a substrate, the reaction temperature is 25 ° C., 35 ° C. or 60 ° C., and the reaction is performed for 60 minutes. The reaction is stopped by heating for 10 minutes. This reaction solution was accurately diluted 11 times with 50 mM phosphate buffer pH 7.5, and 0.1 ml of a trehalase-containing solution (1 unit / ml) was added to 0.4 ml of the diluted solution at 45 ° C. and 120 ° C. After incubating for minutes, the amount of glucose in the reaction solution is quantified by the glucose oxidase method. As a control, the same measurement is performed using an enzyme solution and trehalase previously inactivated by heating at 100 ° C. for 10 minutes. Using the above measurement method, the amount of trehalose produced by maltose / trehalose converting enzyme is determined from the increasing amount of glucose, and one unit of activity is defined as the amount of enzyme that produces 1 μmole of trehalose per minute.
[0026]
The reaction temperature is 25 ° C. when the maltose / trehalose converting enzyme is derived from a microorganism belonging to the genus Pymelobacter, 35 ° C. when it is derived from a microorganism belonging to the genus Pseudomonas, and 60 ° C. when derived from a microorganism belonging to the genus Thermus. It was.
[0027]
Next, the starch used in the present invention may be ground starch such as corn starch, rice starch and wheat starch, or underground starch such as potato starch, sweet potato starch and tapioca starch. In order to liquefy the starch, usually starch milk in which the starch is suspended in water, preferably at a concentration of 10% or more, more preferably about 20 to 50%, can be heated and mechanically liquefied. Or you may liquefy with an enzyme. The degree of liquefaction is suitably low, preferably less than DE15, more preferably less than DE10. When liquefying with an acid, for example, it may be liquefied with hydrochloric acid, phosphoric acid, oxalic acid, etc., and then neutralized to the required pH with calcium carbonate, calcium oxide, sodium carbonate or the like. In the case of liquefying with an enzyme, α-amylase, in particular, heat-resistant liquefied α-amylase is suitable.
[0028]
Β-amylase used for producing maltose from a starch liquefaction solution in the present invention may be prepared from a plant such as sweet potato, soybean, wheat straw, etc., a culture of a microorganism belonging to the genus Bacillus, etc. by a known method. It is also optional to use commercially available enzyme agents. Further, the starch debranching enzyme used in the present invention is an enzyme that acts on a liquefied solution of starch having a relatively low DE, preferably hydrolyzing less than DE 15, and hydrolyzes the branching bonds of starch. Pullulanase, isoamylase and the like can be used advantageously, and a commercially available enzyme agent can also be used advantageously.
[0029]
The amount of the enzyme used may be appropriately selected depending on the operating conditions and reaction time, but is usually selected from about 1 to 100 units in the case of β-amylase per gram of solid relative to the starch liquefaction solution as a substrate, In the case of starch debranching enzyme, it is selected from about 1 to 2,000 units.
[0030]
The maltose thus obtained can be advantageously used as trehalose by the culture method of the present invention or as a sugar source for producing a carbohydrate containing the same. Also, it is possible to advantageously use a commercially available maltose. Maltose may be added to the nutrient medium as long as trehalose can be produced, and it is optional to add maltose from the beginning of the culture or from the middle of the culture. In the case of continuous or semi-continuous culture, for example, a part of the culture medium in which trehalose is produced may be extracted, and a nutrient medium containing the same volume of maltose may be added and cultured. In addition, it is optional to carry out the culture in a nutrient medium in which starch liquefaction solution coexists with β-amylase or β-amylase together with starch debranching enzyme.
[0031]
The culture containing trehalose thus obtained is filtered, centrifuged, etc. to remove insoluble matter such as bacterial cells, and then concentrated, decolorized with activated carbon, and H-type and OH-type ion exchange resins. It is desalted, purified and concentrated to a syrup-like product. Furthermore, it is optional to dry it into a powdered product. If necessary, further purification, for example, fractionation by column chromatography such as ion exchange column chromatography, activated carbon column chromatography, silica gel column chromatography, fractionation by organic solvent such as alcohol and acetone, membrane having appropriate separation performance Trehalose products of the highest purity by purifying by one or a combination of two or more methods, such as separation by fermentation, and further, such as fermentation treatment with yeast and decomposition removal of remaining reducing sugars by alkali treatment, etc. It is also easy to obtain.
[0032]
In particular, as an industrial mass production method, it is preferable to employ ion exchange column chromatography. For example, strongly acidic cations disclosed in JP-A-58-23799 and JP-A-58-72598 are disclosed. A low-reducing carbohydrate having an improved target trehalose content can be advantageously produced by removing contaminating saccharides by column chromatography using an exchange resin. At this time, it is optional to adopt any of a fixed floor method, a moving floor method, and a simulated moving floor method.
[0033]
The sugar containing the trehalose of the present invention thus obtained can be decomposed with glucoamylase or α-glucosidase, if necessary, to adjust sweetness, reducing power, etc., or to reduce the viscosity. Further, it is optional to carry out further processing such as hydrogenation to make the reducing sugar remaining as sugar alcohol to extinguish the reducing power.
[0034]
In particular, a glucoamylase or α-glucosidase is allowed to act on the carbohydrate containing trehalose of the present invention to form a mixed solution of trehalose and glucose, which is purified by the above-described purification method, for example, ion exchange column chromatography. If glucose is removed, a fraction containing high trehalose can be collected. This can be purified and concentrated to obtain a syrup-like product, or further concentrated to be supersaturated and crystallized to obtain trehalose hydrous crystals or anhydrous crystalline trehalose.
[0035]
To produce trehalose hydrous crystals, for example, a trehalose high liquid with a purity of about 60% or more and a concentration of about 65 to 90% is placed in an auxiliary crystal can and, if necessary, in the presence of 0.1 to 20% seed crystals. Then, the mixture is gradually cooled with stirring at a temperature of 95 ° C. or lower, desirably 10 to 90 ° C., to produce a mass kit containing trehalose hydrated crystals. It is also possible to advantageously employ a continuous crystallization method in which crystallization is performed while concentrating under reduced pressure. As a method for producing a trehalose hydrated crystal or a honey-containing crystal containing the same from a mass kit, a known method such as a honey mash method, a block pulverization method, a fluid granulation method, or a spray drying method may be employed.
[0036]
In the case of the honey method, the mass kit is usually applied to a basket-type centrifuge to separate the trehalose-containing crystals and the honey (mother liquor), and if necessary, it can be easily washed by spraying a small amount of cold water. And is suitable for producing higher-purity water-containing trehalose crystals. In the case of the spray drying method, a mass kit having a concentration of 70 to 85% and a crystallization rate of 20 to 60% is usually sprayed from a nozzle with a high-pressure pump, and hot air at a temperature at which the crystal powder does not dissolve, for example, 60 to 100 ° C. And then aged for about 1 to 20 hours with hot air of 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 10 to 20% and a crystallization rate of 10 to 60% is allowed to stand for about 0.1 to 3 days to crystallize and solidify the whole into a block shape. If this is pulverized and dried by a method such as grinding or cutting, a non-hygroscopic or hardly hygroscopic honey-containing crystal can be easily produced.
[0037]
In order to produce anhydrous crystalline trehalose, it is possible to dry and convert the trehalose hydrated crystal, but generally, a high concentration trehalose-rich solution with a moisture content of less than 10% is placed in an auxiliary crystal can and coexists with seed crystals. A mass kit containing anhydrous crystalline trehalose is produced under stirring at a temperature of 50 to 160 ° C., preferably 80 to 140 ° C., and this is subjected to relatively high temperature drying conditions such as block grinding, fluid granulation, It is produced by crystallization and pulverization by a method such as drying.
[0038]
The trehalose of the present invention produced in this way, or a sugar containing the same, is stable with low reducibility and is mixed and processed with other materials, particularly substances having amino acids such as amino acids, oligopeptides and proteins. However, it does not turn brown, does not generate a strange odor, and does not damage other mixed materials. In addition, it has a low viscosity despite its low reducing power, and has a good quality and elegant sweetness.
[0039]
Furthermore, since trehalose of the present invention is easily decomposed into glucose by trehalase, it is digested and absorbed by ingestion and used as a calorie source. It can also be used as a sweetener that is not easily fermented by caries-causing bacteria and that is unlikely to cause caries. In addition, it has properties such as osmotic pressure controllability, shaping, irradiating properties, moisture retention, viscosity, anti-crystallization of other sugars, difficult fermentation, and anti-aging properties of gelatinized starch.
[0040]
Further, the trehalose of the present invention is used parenterally as a tube feeding agent, an infusion solution, etc., has no concern about toxicity and side effects, is frequently metabolized and utilized, and can be advantageously used for energy supply to the living body. it can. Further, since it is a stable sweetener, trehalose-containing crystals can be advantageously used as a sugar coating for tablets in combination with binders such as pullulan, hydroxyethyl starch, and polyvinylpyrrolidone.
[0041]
In the case of anhydrous crystalline trehalose, it can also be advantageously used as a dehydrating agent for water-containing products such as foods, pharmaceuticals, cosmetics, raw materials, and processed intermediates, and it can be used as solid, stable, high-quality powders, granules, tablets, etc. Things can be easily manufactured.
[0042]
Therefore, the trehalose of the present invention or a saccharide containing the same is used as a sweetener, a taste improver, a quality improver, a stabilizer, an excipient, a dehydrator, and the like as food, drink, food, feed, and cosmetics. It can be advantageously used in various compositions such as pharmaceuticals.
[0043]
The trehalose of the present invention or a saccharide containing the same can be used as it is as a seasoning for sweetening. If necessary, for example, flour, glucose, maltose, sucrose, isomerized sugar, honey, maple sugar, isomaltoligosaccharide, galactooligosaccharide, fructooligosaccharide, lactosucrose, sorbitol, maltitol, lactitol, dihydrochalcone, stevioside, α -May be used in admixture with one or more suitable amounts of other sweeteners such as glycosyl stevioside, rebaudioside, glycyrrhizin, L-aspartyl-L-phenylalanine methyl ester, saccharin, glycine, alanine, If necessary, it can be used by mixing with a bulking agent such as dextrin, starch, lactose and the like.
[0044]
Further, the trehalose of the present invention, or a saccharide powder or crystalline product containing the same, or as necessary, mixed with a filler, excipient, binder, etc. It is also optional to use it in various shapes such as short rod, plate, cube and tablet.
[0045]
The sweetness of the trehalose of the present invention or a saccharide containing the same is well harmonized with various substances having other tastes such as acidity, salt to taste, astringency, umami, bitterness, acid resistance, heat resistance Therefore, it can be advantageously used for sweetening and taste improvement of general foods and drinks and quality improvement.
[0046]
For example, amino acids, peptides, soy sauce, powdered soy sauce, miso, powdered miso, moromi, hishio, sprinkle, mayonnaise, dressing, vinegar, three cups of vinegar, sushi vinegar, Chinese food, tempura soup, noodle soup, sauce, ketchup, yakiniku It can be advantageously used as various seasonings such as sauce, curry roux, stew, soup, dashi, nucleic acid-based seasoning, compound seasoning, mirin, new mirin, table sugar, coffee sugar.
[0047]
In addition, for example, Japanese rice cakes such as rice crackers, hail, rice cakes, rice cakes, manju, oirou, chanterelles, sheep mushrooms, water sheep rice cakes, brocade balls, jelly, castella, rice cakes, bread, biscuits, crackers, cookies, pie , Pudding, butter cream, custard cream, cream puff, waffle, sponge cake, donut, chocolate, chewing gum, caramel, candy, etc. Paste such as paste, peanut paste, fruit paste, spread, fruits such as jam, marmalade, syrup pickles, sugar cane, processed foods of vegetables, pickles such as Fukujin pickles, bedara pickles, thousand pickles, pickled pickles, takan pickles, Pickles such as Chinese cabbage pickles Meat products such as fish, ham and sausage, fish meat ham, fish sausage, fish products such as kamaboko, chikuwa, tempura, sea urchin, squid salt, vinegar kombu, sakisume, fugurin dried and other delicacies, Tsukudani, made with seaweed, wild vegetables, shrimp, small fish, shellfish, boiled beans, potato salad, side dish foods such as kombu rolls, dairy products such as yogurt and cheese, fish meat, livestock meat, fruits, bottled canned vegetables, canned foods Alcohol, Sake, Synthetic Sake, Liqueur, Western Sake, Coffee, Tea, Cocoa, Juice, Carbonated Beverages, Lactic Beverages, Lactic Bacteria Beverages, Soft Drinks, Pudding Mix, Hot Cake Mix, Instant Shiko, Instant Soup, etc. Improving taste for instant foods, sweetening foods such as baby foods, therapeutic foods, drinks, peptide foods, frozen foods, and dried foods. It was, can be advantageously used in such as quality improvement.
[0048]
Moreover, it can also be used for the purpose of improving the palatability of feed, feed, etc. for domestic animals, poultry, and other domestic animals such as bees, cormorants and fish. In addition, tobacco, toothpaste, lipstick, lip balm, oral solution, tablets, troches, liver oil drop, mouth freshener, mouth fragrance, mouthwash, etc. It can be advantageously used as a sweetener for various compositions, as a taste improver, a corrigent, and as a quality improver, stabilizer and the like.
[0049]
The quality improver and stabilizer can be advantageously applied to various physiologically active substances that easily lose active ingredients, activity, etc., health foods containing them, and pharmaceuticals. 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, lymphokine such as interleukin II, insulin , Hormones such as growth hormone, prolactin, erythropoietin, egg cell stimulating hormone, BCG vaccine, Japanese encephalitis vaccine, measles vaccine, polio vaccine, seedling, tetanus toxoid, hub antitoxin, human immunoglobulin and other biologics, penicillin, erythromycin, Chloramphenicol, tetracycline, suGAntibiotics such as leptomycin, kanamycin sulfate, thiamine, riboflavin, L-ascorbic acid, liver oil, carotenoid, ergosterol, tocopherol and other vitamins, lipase, elastase, urokinase, protease, β-amylase, isoamylase, glucanase, lactase, etc. Enzymes, ginseng extract, suppon extract, chlorella extract, aloe extract, propolis extract and other extracts, viruses, lactic acid bacteria, live bacteria such as yeast, and various physiologically active substances such as royal jelly also lose their active ingredients and activities Therefore, it can be easily manufactured into a stable, high-quality liquid, pasty or solid health food or medicine.
[0050]
The method of adding trehalose or a saccharide containing the same to various compositions as described above may be included in the process until the product is completed, for example, mixing, dissolving, melting, soaking, and penetrating. Well-known methods such as spraying, coating, coating, spraying, pouring, crystallization, and solidification are appropriately selected. The amount is usually 0.1% or more, preferably 1% or more. Next, the present invention will be described more specifically by experiments.
[0051]
First, maltose trehalose converting enzyme from novel microorganisms Pimerobacter sp. R48, Pseudomonas putida H262 and Thermus aquaticus ATCC 33923 will be described, and then maltose trehalose converting enzyme from other known microorganisms will be described.
[0052]
[Experiment 1 Enzyme production]
Glucose 2.0 w / v%, polypeptone 0.5 w / v%, yeast extract 0.1 w / v%, dipotassium phosphate 0.1 w / v%, monosodium phosphate 0.06 w / v%, magnesium sulfate Liquid medium consisting of heptahydrate 0.05 w / v%, calcium carbonate 0.5 w / v%, and water is placed in 100 ml portions in a 500 ml Erlenmeyer flask, sterilized in an autoclave at 115 ° C. for 30 minutes, cooled, Pimelobacter sp. R48 (FERM BP-4315) was inoculated and cultured under rotation at 27 ° C. and 200 rpm for 24 hours as a seed culture.
[0053]
About 20 liters of medium having the same composition as in seed culture is placed in a 30 liter fermenter, heat-sterilized and cooled to a temperature of 27 ° C., then inoculated with 1 v / v% of the seed culture solution, temperature 27 ° C., pH 6 The culture was aerated and stirred for about 40 hours while maintaining the temperature at 0.0 to 8.0.
[0054]
The maltose / trehalose converting enzyme activity of the culture broth was 0.55 units / ml. A part of the culture solution is taken and centrifuged to separate the cells and the culture supernatant, and the cells are suspended in 50 mM phosphate buffer (pH 7.0), and the same solution as the original culture solution. Then, when the maltose / trehalose converting enzyme activity of the cell suspension and the culture supernatant was measured, the cell suspension had an activity of 0.5 units / ml in the culture supernatant. Activity of 0.05 units / ml was observed. The activity of this enzyme was measured at a reaction temperature of 25 ° C.
[0055]
[Experiment 2 Enzyme Purification]
The culture solution obtained in Experiment 1 was centrifuged to collect cells having a wet weight of about 0.5 kg and suspended in 10 mM phosphate buffer (pH 7.0). About 5 l of this bacterial cell suspension was applied to “Vibrogen Cell Mill” manufactured by Edmund Bühler, the bacterial cells were crushed, and the crushed solution was centrifuged (15,000 G, 30 minutes) to obtain about 4.5 l. A supernatant was obtained. The supernatant was dissolved by adding ammonium sulfate to a saturation degree of 0.3, allowed to stand at 4 ° C. for 4 hours, and then centrifuged to collect the supernatant.
[0056]
Further, ammonium sulfate was added to the solution so that the degree of saturation was 0.8, and the mixture was allowed to stand at 4 ° C. overnight, and then centrifuged to collect ammonium sulfate salted-out.
[0057]
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 to remove insoluble matters. The dialysate (400 ml) was divided into two portions and subjected to ion exchange column chromatography (gel amount: 300 ml) using “DEAE-Toyopearl”.
[0058]
The maltose / trehalose converting enzyme of the present invention was adsorbed on “DEAE-Toyopearl” and eluted from the column with the same buffer containing sodium chloride. The eluted enzyme active fraction is collected, dialyzed against the same buffer containing 1M ammonium sulfate, the dialyzed solution is centrifuged to remove insoluble matters, and then “Butyl Toyopearl 650” manufactured by Tosoh Corporation. Hydrophobic column chromatography (gel amount: 300 ml) was performed. The adsorbed maltose / trehalose converting enzyme was eluted from the column with an ammonium sulfate 1M to 0M linear gradient, and the enzyme activity fraction was recovered.
[0059]
Subsequently, ion exchange chromatography (gel amount: 10 ml) using “Mono Q HR5 / 5” manufactured by Pharmacia El-Cabi was performed, and the eluted enzyme activity fraction was collected. Table 1 shows the amount of enzyme activity, specific activity, and yield in each step of purification.
[0060]
[Table 1]

[0061]
When the purity of the enzyme preparation was assayed by gel electrophoresis containing 7.5 w / v% polyacrylamide in the purified enzyme preparation, the protein band was a single and high purity preparation.
[0062]
[Experiment 3 Properties of enzyme]
A purified molecular weight marker obtained by the method of Experiment 2 was subjected to SDS-polyacrylamide gel electrophoresis (gel concentration: 10 w / v%), and simultaneously migrated molecular weight marker (manufactured by Nippon Bio-Rad Laboratories, Inc.) ) And the molecular weight of the enzyme was measured to be about 57,000 to 67,000 daltons.
[0063]
The purified maltose / trehalose converting enzyme preparation is subjected to isoelectric focusing polyacrylamide gel electrophoresis containing 2 w / v% ampholine (Pharmacia LC), and after electrophoresis, the protein band and gel pH are measured to measure the enzyme. The isoelectric point was found to be about 4.1 to 5.1.
[0064]
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. for reaction at pH 7.0 for 60 minutes, and the optimum pH was about 7.0 to 8.0 for reaction at 25 ° C. for 60 minutes. The temperature stability of the enzyme was determined by measuring the remaining enzyme activity after holding the enzyme solution (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 50 mM buffer at each pH at 20 ° C. for 60 minutes, adjusting the pH to 7.0, 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 up to about 30 ° C., and the pH stability was about 6.0 to 9.0. The enzyme activity is 1 mM Cu⁺⁺, Hg⁺⁺Alternatively, it was inhibited with 50 mM Tris-HCl buffer.
[0065]
[Experiment 4 Effects on various carbohydrates]
Various sugars were used to test whether they could become substrates. Glucose, maltose, maltotriose, maltotetraose, maltopentaose, maltohexaose, maltoheptaose, soluble starch, amylose (average polymerization degree 18), trehalose, neotrehalose, gentiobiose, cordobiose, isomaltose, cellobiose, multi A solution of tall, sucrose, maltulose, turanose, palatinose, trehalulose, or lactose, and an equivalent amount of glucose to α-glucose · 1-phosphate, or an equivalent amount of glucose to β-glucose · 1-phosphate A solution containing was prepared.
[0066]
To these solutions, add 2 units each of purified maltose / trehalose converting enzyme obtained by the method of Experiment 2 per gram of substrate solids so that the substrate concentration is 5 w / v%.MadeThis was allowed to act at 20 ° C. and pH 7.0 for 24 hours. The reaction solution before and after the enzyme reaction was subjected to thin layer chromatography (hereinafter abbreviated as TLC) using “Kieselgel 60” (aluminum plate, 20 × 20 cm) manufactured by Merck & Co. confirmed. TLC was developed once at room temperature using 1-butanol: pyridine: water = 6: 4: 1 (volume ratio) as a developing solvent. Coloring was performed by spraying a 20% sulfuric acid-methanol solution and heating at 110 ° C. for 10 minutes. The results are shown in Table 2.
[0067]
[Table 2]

[0068]
As is clear from the results in Table 2, the enzyme of the present invention acts only on maltose and trehalose among the various carbohydrates tested, and other carbohydrates, particularly α-glucose monophosphate and glucose. It is a novel enzyme unlike conventional phosphorylases such as maltose phosphorylase and trehalose phosphorylase because it does not act on a system containing glucose or a system containing β-glucose / 1-phosphate and glucose. There was found.
[0069]
[Experiment 5: Product from maltose or trehalose]
Two units of the purified maltose / trehalose converting enzyme obtained by the method of Experiment 2 was added to a final 5% maltose aqueous solution per gram of substrate solids and allowed to act at 20 ° C. and pH 7.0 for 24 hours. The sugar composition of the enzyme reaction solution was analyzed by gas chromatography (hereinafter abbreviated as GLC). A part of the enzyme reaction solution was dried, dissolved in pyridine and then trimethylsilylated, and used as an analytical sample. The gas chromatograph is “GC-16A” manufactured by Shimadzu Corporation, the column is a stainless steel column (3 mmφ × 2 m) packed with “2% silicon OV-17 / chromosolve W” manufactured by GL Sciences Inc., and the carrier gas is Nitrogen gas was analyzed at a flow rate of 40 ml / min, and the column oven temperature was 160 ° C. to 320 ° C. at a heating rate of 7.5 ° C./min. For detection, a flame ion detector was used. The results are shown in Table 3.
[0070]
[Table 3]

[0071]
As is clear from the results in Table 3, it was found that a large amount of reaction product X was formed, and the retention time was consistent with that of commercial trehalose. In order to identify the reaction product X, the following confirmation test was conducted. That is, the enzyme reaction solution using maltose as a substrate is diluted with 20 mM acetate buffer, pH 4.5 so that the sugar concentration becomes 2%, and 0.5 ml of glucoamylase (Seikagaku Corporation) 0.1 is added. The unit was added and reacted at 40 ° C. for 20 hours.
[0072]
Similarly, the enzyme reaction solution was diluted with 20 mM phosphate buffer, pH 7.0 so that the sugar concentration was 2%, 0.5 units of trehalase was added to 0.5 ml, and the mixture was reacted at 40 ° C. for 20 hours. Analyzing and comparing the enzyme reaction solution with maltose as a substrate, its glucoamylase treatment solution and trehalase treatment solution by GLC, maltose is completely decomposed into glucose by the glucoamylase treatment, and the reaction product X remains without being decomposed. Was.
[0073]
On the other hand, maltose remained due to the trehalase treatment, but the reaction product X was completely decomposed into glucose. Considering the reaction characteristics of glucoamylase and trehalase, it is determined that the oligosaccharide from maltose produced by the novel enzyme of the present invention is trehalose.
[0074]
Furthermore, when purified enzyme was allowed to act on trehalose as a substrate under the same conditions as in maltose and the reaction solution was similarly subjected to GLC analysis, it was found that maltose was produced from trehalose by the enzyme of the present invention. The above GLC analysis results are summarized in Table 4.
[0075]
[Table 4]

[0076]
As is clear from the results in Table 4, the enzyme of the present invention converts maltose to trehalose and converts trehalose to maltose. The equilibrium point is shifted to the trehalose side, and it has been found that the conversion rate from maltose to trehalose is high and is about 70% or more.
[0077]
[Experiment 6: Effect of maltose concentration on trehalose production]
Purified maltose / trehalose converting enzyme obtained by the method of Experiment 2 at a maltose concentration of 2.5%, 5%, 10%, 20% or 40% at a temperature of 20 ° C. and pH 7.0, 2 units per maltose gram In addition, the reaction solution was collected over time, and heated at 100 ° C. for 10 minutes to inactivate the enzyme.
[0078]
The total amount of sugar in this reaction solution was quantified in terms of glucose by the anthrone sulfate method and the amount of reducing sugar by glucose conversion by the Somogy-Nelson method, and the ratio of the amount of reducing sugar to the total amount of sugar was calculated as the reducing power.
[0079]
The reaction solution was diluted to a sugar concentration of about 1%, deproteinized with a small amount of ultrafilter, “Morkat II LGC” manufactured by Nihon Millipore Limited, and then subjected to high performance liquid chromatography (hereinafter abbreviated as HPLC). )) To analyze the sugar composition. The HPLC apparatus is “CCPD system” manufactured by Tosoh Corporation, the analytical column is “YMC-pack PA-03” (4.6 mmφ × 250 mm) manufactured by YMC Co., Ltd., and the eluent is acetonitrile: water = 78: 22 (volume ratio). ) At a flow rate of 1.2 ml / min, and detection was performed with a differential refractometer. The results are shown in Table 5.
[0080]
[Table 5]

[0081]
As is clear from the results in Table 5, the conversion reaction from maltose to trehalose progressed well regardless of the maltose concentration of the substrate, and the conversion to trehalose was about 80%.
[0082]
[Experiment 7: Effect of temperature on trehalose production]
At a maltose concentration of 20%, the pH is 7.0, and 2 units of the purified maltose / trehalose converting enzyme obtained by the method of Experiment 2 is added per maltose gram, and the temperature is 5 ° C, 10 ° C, 15 ° C, 20 ° C or 25 ° C. The reaction was collected over time and heated at 100 ° C. for 10 minutes to inactivate the enzyme. The sugar composition of this enzyme reaction solution was analyzed by HPLC in the same manner as in Experiment 6. Table 6 shows the trehalose content at each temperature and time.
[0083]
[Table 6]

[0084]
As can be seen from the results in Table 6, the higher the reaction temperature, the higher the trehalose production rate, but the conversion reaction from maltose to trehalose progressed well even at a temperature of 5 ° C, and the conversion to trehalose was about 82%. did.
[0085]
[Experiment 8: Preparation of trehalose from maltose]
Maltose (manufactured by Hayashibara Biochemical Laboratories, Inc.) 10 parts by weight is dissolved in 40 parts by weight of water, and the purified maltose / trehalose converting enzyme obtained by the method of Experiment 2 is used at a temperature of 15 ° C. and a pH of 7.0. Two units were added and reacted for 48 hours, and then heated at 100 ° C. for 10 minutes to inactivate the enzyme. This solution contained about 74% trehalose per solid. This solution is decolorized with activated carbon, purified by desalting with an ion exchange resin (H type and OH type), concentrated to a concentration of about 78%, and trehalose hydrous crystals as seed crystals, 0.1% per solid matter. When it was added and allowed to stand at room temperature overnight, crystals were precipitated. The obtained mass kit was honeyed and the crystal was washed by spraying a small amount of water to obtain about 3.0 parts by weight of extremely high purity trehalose hydrous crystal having a purity of 99.8%.
[0086]
[Experiment 9 Enzyme production]
Glucose 2.0 w / v%, ammonium sulfate 1.0 w / v%, dipotassium phosphate 0.1 w / v%, monosodium phosphate 0.06 w / v%, magnesium sulfate 0.05 w / v%, calcium carbonate 0 Liquid medium consisting of 3 w / v% and water was put into a 500 ml Erlenmeyer flask, 100 ml each, sterilized by autoclaving at 115 ° C. for 30 minutes, cooled, and inoculated with Pseudomonas putida H262 (FERMBP-4579). The seed culture was cultured at 27 ° C. and 200 rpm for 24 hours with shaking.
[0087]
About 20 liters of medium having the same composition as in seed culture is put into a 30 liter fermenter, heat-sterilized and cooled to a temperature of 27 ° C., then inoculated with 1 v / v% of the seed culture solution, temperature 27 ° C., pH 6 The culture was aerated and agitated for about 20 hours while maintaining at .5 to 8.0.
[0088]
The maltose / trehalose converting enzyme activity of the culture broth was 0.12 units / ml. A part of the culture solution is taken and centrifuged to separate the cells and the culture supernatant, and the cells are suspended in 50 mM phosphate buffer (pH 7.0), and the same solution as the original culture solution. When the maltose / trehalose converting enzyme activity of the cell suspension and the culture supernatant was measured, the cell suspension had an activity of 0.11 unit / ml in the culture supernatant. Activity of 0.01 units / ml was observed. The activity of this enzyme was measured at a reaction temperature of 35 ° C.
[0089]
[Experiment 10 Enzyme Purification]
The culture solution obtained in Experiment 9 was centrifuged to recover a wet cell weight of about 0.45 kg, which was suspended in 10 mM phosphate buffer (pH 7.0). About 2 liters of this bacterial cell suspension is treated with an ultra-high pressure bacterial cell disruption device (Dainippon Pharmaceutical Co., Ltd. “Minilab”) to disrupt the bacterial cells, and this disrupted solution is centrifuged (15,000 G, 30 minutes) ) To obtain about 1.7 l of supernatant. Ammonium sulfate was recovered by adding ammonium sulfate to the supernatant so as to have a saturation degree of 0.7, leaving it to stand at 4 ° C. overnight, and then centrifuging it.
[0090]
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 to remove insoluble matters. The dialysate (400 ml) was divided into two portions and subjected to ion exchange column chromatography (gel amount: 300 ml) using “DEAE-Toyopearl”.
[0091]
The maltose / trehalose converting enzyme of the present invention was adsorbed on “DEAE-Toyopearl” and eluted from the column with the same buffer containing sodium chloride. The eluted enzyme active fraction was collected, dialyzed against the same buffer, and again subjected to ion exchange column chromatography (gel amount 80 ml) using “DEAE-Toyopearl”. The adsorbed maltose / trehalose converting enzyme was eluted from the column with a linear gradient of 0.1 M to 0.3 M of sodium chloride, and the enzyme activity fraction was recovered.
[0092]
Subsequently, gel filtration chromatography (gel amount: 400 ml) using “Toyopearl HW-55S” manufactured by Tosoh Corporation was performed, and the eluted enzyme activity fraction was collected. Table 7 shows the amount of enzyme activity, specific activity, and yield in each step of purification.
[0093]
[Table 7]

[0094]
When the purity of the enzyme preparation was assayed by gel electrophoresis containing 7.5 w / v% polyacrylamide in the purified enzyme preparation, the protein band was a single and high purity preparation.
[0095]
[Experiment 11: Properties of enzyme]
A purified molecular weight marker obtained by the method of Experiment 10 was subjected to SDS-polyacrylamide gel electrophoresis (gel concentration 7.5 w / v%) and simultaneously migrated molecular weight marker (Nippon Bio-Rad Laboratories Co., Ltd.) The molecular weight of this enzyme was measured in comparison with that of the company), and the molecular weight was about 110,000 to 120,000 daltons.
[0096]
The purified maltose / trehalose converting enzyme preparation was subjected to isoelectric focusing polyacrylamide gel electrophoresis containing 2 w / v% ampholine (Pharmacia LC). After electrophoresis, the pH of the protein band and gel was measured to measure the enzyme. The isoelectric point was found to be about 4.1 to 5.1.
[0097]
The effects of temperature and pH on the enzyme activity were examined according to the activity measurement method. The results are shown in FIG. 5 (effect of temperature) and FIG. 6 (effect of pH). The optimum temperature of the enzyme was about 37 ° C. for a reaction of pH 7.0 for 60 minutes, and the optimum pH was about 7.3 to 8.3 for a reaction of 35 ° C. for 60 minutes. The temperature stability of the enzyme was determined by measuring the remaining enzyme activity after holding the enzyme solution (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 35 ° C. for 60 minutes, adjusting the pH to 7.0, and measuring the remaining enzyme activity. The respective results are shown in FIG. 7 (temperature stability) and FIG. 8 (pH stability). The temperature stability of this enzyme was up to around 40 ° C., and the pH stability was about 6.0 to 9.5. The enzyme activity is 1 mM Cu⁺⁺, Hg⁺⁺Alternatively, it was inhibited with 50 mM Tris-HCl buffer.
[0098]
[Experiment 12 Action on Various Sugars]
Except that the reaction temperature was 35 ° C., the purified enzyme of Pseudomonas putida H262 obtained in Experiment 10 was allowed to act on various carbohydrates in accordance with the method of Experiment 4 to test whether it could be a substrate. As a result, the Pseudomonas putida H262 enzyme acted only on maltose and trehalose to convert maltose to trehalose and trehalose converted to maltose, as did the enzyme Pimelobacter species R48. The equilibrium point is shifted to the trehalose side, and it was found that the conversion rate from maltose to trehalose is high, which is about 70%.
[0099]
[Experiment 13: Effect of maltose concentration on trehalose production]
At a maltose concentration of 5%, 10%, 20% or 30%, at a temperature of 35 ° C. and a pH of 7.0, the purified maltose / trehalose converting enzyme obtained by the method of Experiment 10 was added and reacted for 2 units per maltose gram. The reaction solution was collected over time and heated at 100 ° C. for 10 minutes to deactivate the enzyme.
[0100]
Using this reaction solution, the reducing power and sugar composition were measured in the same manner as in Experiment 6. The results are shown in Table 8.
[0101]
[Table 8]

[0102]
As is apparent from the results in Table 8, about 70% of trehalose was produced regardless of the maltose concentration of the substrate.
[0103]
[Experiment 14 Preparation of trehalose from maltose]
Purified maltose / trehalose converting enzyme of the present invention obtained by the method of Experimental Example 10 by dissolving 10 parts by weight of maltose (sales by Hayashibara Biochemical Laboratories, Inc.) in 40 parts by weight of water and adjusting the temperature to 35 ° C. and pH 7.0. Was added for 2 hours per maltose and allowed to react for 48 hours, followed by heating at 100 ° C. for 10 minutes to inactivate the enzyme. This solution contained about 69% trehalose per solid. This solution is decolorized with activated carbon, purified by desalting with an ion exchange resin (H type and OH type), concentrated to a concentration of about 78%, and trehalose hydrous crystals as seed crystals, 0.1% per solid matter. When it was added and allowed to stand at room temperature overnight, crystals were precipitated. The obtained mass kit was honeyed and the crystal was washed by spraying a small amount of water to obtain about 2.3 parts by weight of extremely high purity trehalose crystal having a purity of 99.7%.
[0104]
[Experiment 15 Enzyme production]
Polypeptone 0.5 w / v%, yeast extract 0.1 w / v%, sodium nitrate 0.07 w / v%, disodium phosphate 0.01 w / v%, magnesium sulfate 0.02 w / v%, calcium chloride 0. A liquid medium consisting of 01 w / v% and water was adjusted to pH 7.5, and then 100 ml each was placed in a 500 ml Erlenmeyer flask, sterilized in an autoclave at 120 ° C. for 20 minutes, cooled, and thermus aquaticus ATCC 33923 was added. The seed culture was inoculated and cultured with shaking at 60 ° C. and 200 rpm for 24 hours.
[0105]
About 20 liters of each medium having the same composition as in the case of seed culture was added to two 30 liter fermenters, heat-sterilized and cooled to a temperature of 60 ° C., and then seeded with 1 v / v% of the seed culture solution. The culture was aerated and stirred for about 20 hours while maintaining the temperature at 6.5 ° C. and 8.0 to 8.0.
[0106]
The maltose / trehalose converting enzyme activity of the culture broth was 0.35 units / ml. A portion of the culture solution is taken and centrifuged to separate the cells and culture supernatant, and the cells are further suspended in 50 mM phosphate buffer (pH 7.0), and the same volume as the original culture solution. Then, when the maltose / trehalose converting enzyme activity of the cell suspension and the culture supernatant was measured, the cell suspension had an enzyme activity of 0.33 units / ml, and the culture supernatant Was found to have an enzyme activity of 0.02 units / ml. The activity of this enzyme was measured at a reaction temperature of 60 ° C.
[0107]
[Experiment 16 Purification of enzyme]
The culture solution obtained in Experiment 15 was centrifuged to collect cells having a wet weight of about 0.28 kg and suspended in 10 mM phosphate buffer (pH 7.0). About 1.9 l of this bacterial cell suspension was treated with an ultrasonic crusher (“Model US300” manufactured by Nippon Seiki Seisakusho Co., Ltd.) to crush the bacterial cells. The crushing solution was centrifuged (15,000 G, 30 minutes) to obtain about 1.8 l of supernatant. Ammonium sulfate was added to the supernatant so as to have a saturation degree of 0.7, dissolved, and allowed to stand at 4 ° C. overnight, and then centrifuged to recover ammonium sulfate salted-out.
[0108]
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 to remove insoluble matters. The dialysate (1560 ml) was subjected to ion exchange column chromatography (gel amount 530 ml) using “DEAE-Toyopearl 650” manufactured by Tosoh Corporation in three portions.
[0109]
The maltose / trehalose converting enzyme of the present invention was adsorbed on “DEAE-Toyopearl” and eluted from the column with the same buffer containing sodium chloride. The eluted enzyme activity fraction was collected, dialyzed against the same buffer containing 1M ammonium sulfate, and then subjected to hydrophobic column chromatography (gel amount 380 ml) using “Butyl Toyopearl 650”. The adsorbed maltose / trehalose converting enzyme was eluted from the column with a linear gradient of 1M to 0M ammonium sulfate, and the enzyme activity fraction was recovered.
[0110]
Next, gel filtration chromatography (gel amount: 380 ml) using “Toyopearl HW-55S” was performed, and the eluted enzyme activity fraction was recovered.
[0111]
Subsequently, ion-exchange chromatography (gel amount: 1.0 ml) using “Mono Q HR5 / 5” manufactured by Pharmacia El-Cabi Co., Ltd., and the enzyme activity fraction eluted with a linear gradient from 0.1 M to 0.35 M of sodium chloride Was recovered. Table 9 shows the amount of enzyme activity, specific activity, and yield in each step of purification.
[0112]
[Table 9]

[0113]
When the purity of the enzyme preparation was assayed by gel electrophoresis containing 5% w / v concentration polyacrylamide, the protein band was a single and high purity preparation.
[0114]
[Experiment 17: Properties of enzyme]
A purified molecular weight marker obtained by the method of Experiment 16 was subjected to SDS-polyacrylamide gel electrophoresis (gel concentration 7.5 w / v%), and simultaneously migrated molecular weight marker (Nippon Bio-Rad Laboratories Co., Ltd.) The molecular weight of this enzyme was measured in comparison with that of the company, and the molecular weight was about 100,000 to 110,000 daltons.
[0115]
The purified maltose / trehalose converting enzyme preparation is subjected to isoelectric focusing polyacrylamide gel electrophoresis containing 2% w / v ampholine (Pharmacia LK), and after electrophoresis, the pH of the protein band and gel is measured. When the isoelectric point of the enzyme was determined, the isoelectric point was about 3.8 to 4.8.
[0116]
The effects of temperature and pH on the enzyme activity were examined according to the activity measurement method. The results are shown in FIG. 9 (effect of temperature) and FIG. 10 (effect of pH). The optimum temperature of the enzyme was around 65 ° C. for a reaction of pH 7.0 for 60 minutes, and the optimum pH was about 6.0 to 6.7 for a reaction of 60 ° 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 60 ° C. for 60 minutes, adjusting the pH to 7.0, and measuring the remaining enzyme activity. The respective results are shown in FIG. 11 (temperature stability) and FIG. 12 (pH stability). The temperature stability of this enzyme was up to about 80 ° C., and the pH stability was about 5.5 to 9.5. The enzyme activity is 1 mM Cu⁺⁺, Hg⁺⁺Alternatively, it was inhibited with 50 mM Tris-HCl buffer.
[0117]
[Experiment 18 action on various carbohydrates]
Except that the reaction temperature was 50 ° C., the purified enzyme of Thermus aquaticus ATCC 33923 obtained in Experiment 16 was allowed to act on various carbohydrates according to the method of Experiment 4 to test whether it could be a substrate. As a result, the enzyme of Thermus aquaticus ATCC 33923 acted only on maltose and trehalose to convert maltose to trehalose, and trehalose was converted to maltose, like the enzyme of Pimerobacter species R48 or Pseudomonas putida H262. The equilibrium point is shifted to the trehalose side, and it has been found that the conversion rate from maltose to trehalose is high and is 70% or more.
[0118]
[Experiment 19: Effect of maltose concentration on trehalose production]
Purified maltose trehalose converting enzyme of Thermus aquaticus ATCC 33923 obtained by the method of Experiment 16 at a maltose concentration of 2.5%, 5%, 10%, 20% or 40% at a temperature of 60 ° C. and a pH of 6.5 The reaction solution was collected at 72 hours and heated at 100 ° C. for 30 minutes to inactivate the enzyme. Using this reaction solution, the reducing power and sugar composition were measured in the same manner as in Experiment 6. The results are shown in Table 10.
[0119]
[Table 10]

[0120]
As is apparent from the results in Table 10, about 70% trehalose was produced regardless of the maltose concentration of the substrate.
[0121]
[Experiment 20: Effect of temperature on trehalose production]
Purified maltose-trehalose converting enzyme of Thermus aquaticus ATCC 33923 obtained by the method of Experiment 16 at a maltose concentration of 20% was added at 2.5 units per maltose gram, and the temperature was 40 ° C, 50 ° C, 60 ° C. The reaction solution was collected over time and heated at 100 ° C. for 30 minutes to inactivate the enzyme. This reaction solution was analyzed for sugar composition by HPLC in the same manner as in Experiment 6. Table 11 shows the trehalose content at each temperature and time.
[0122]
[Table 11]

[0123]
As is clear from the results in Table 11, the conversion rate from maltose to trehalose was higher as the reaction temperature was lower, and about 80% was converted to trehalose at 40 ° C.
[0124]
[Experiment 21 Production and properties of maltose / trehalose converting enzyme from other microorganisms]
Among known microorganisms, a specific microorganism having the ability to produce maltose / trehalose converting enzyme of the present invention was cultured in an Erlenmeyer flask for 48 hours according to Experiment 15. After examining the enzyme activity of the culture solution, according to the method of Experiment 16, the culture solution was applied to a crushing apparatus, and the supernatant was dialyzed to obtain a partially purified enzyme. The property was examined according to the method of Experiment 17. . The results are shown in Table 12.
[0125]
[Table 12]

[0126]
Using the partially purified enzymes derived from known microorganisms belonging to the genus Thermus shown in Table 12, the effects on various carbohydrates were examined according to the method of Experiment 18, as in the case of the enzyme derived from Thermus aquaticus ATCC 33923. It was found that it acts only on maltose and trehalose and produces trehalose from maltose.
[0127]
Further, the maltose trehalose converting enzyme of Thermus louver ATCC 35948 was lower in the optimum temperature and the stable temperature than the enzyme of Thermus aquaticus ATCC 33923, but other enzymes of the genus Thermus were thermus aquaticus ATCC 33923. It was found to have almost the same properties as the enzyme of, and to have high heat resistance.
[0128]
[Experiment 22 Physicochemical properties of prepared trehalose]
The physicochemical properties were examined using a high-purity sample of trehalose prepared by the method of Experiment 8. Melting point is 97.0 ° C, specific rotation is [α]²⁰ _DIt was + 7.0 ° (c = 5), the heat of fusion was 57.8 KJ / mole, and the solubility was 77.0 g as an anhydride with respect to water at 25 ° C. These physical property values agreed well with the values of commercially available trehalose hydrated crystals (manufactured by Wako Pure Chemical Industries, Ltd.) measured simultaneously.
[0129]
[Experiment 23 In vivo use test]
According to the method reported by Koji et al., “Clinical Nutrition”, Vol. 41, No. 2, pages 200 to 208 (1972), a high-purity trehalose preparation (purity 99. 8%) 30 g of 20 w / v% aqueous solution, and this was orally administered to 3 volunteers (healthy 26-year-old, 27-year-old, 30-year-old male), and blood was collected over time to determine blood sugar level and insulin level. It was measured. As a control, glucose was used. As a result, trehalose behaved in the same manner as glucose, and both the blood glucose level and the insulin level showed a maximum value after about 0.5 to 1 hour after administration. Trehalose has been found to be easily digested, absorbed, and metabolized to become an energy source. Therefore, trehalose obtained by the method of the present invention and a saccharide containing the same are suitable as a sugar source for energy supply.
[0130]
[Experiment 24: Acute toxicity test]
Using mice, an acute toxicity test was conducted by orally administering the high-purity trehalose hydrous crystals prepared in Example A-6. As a result, trehalose is a low-toxic substance, and no death was observed even at the maximum dose. Therefore, although it is not an accurate value, its LD₅₀The value was 50 g / kg or more.
[0131]
[Experiment 25: Effect of maltose concentration on trehalose production by culture method]
Microorganisms capable of producing maltose / trehalose converting enzyme were cultured in a nutrient medium containing 2 to 20 w / v% maltose, and the influence of maltose concentration on trehalose yield in the culture was examined. In the case of Pimerobacter species R48 (FERM BP-4315), the culture method is a medium containing 2 to 20 w / v% of maltose separately sterilized instead of 2.0 w / v% of glucose. Except for the above, incubate with fermenter at 27 ° C. for 72 hours in the same manner as in Experiment 1, and further add 0.1v of surfactant (polyoxyethylene sorbitan monopalmitate, “Tween 40” sold by Wako Pure Chemical Industries, Ltd.) / V% was added and the culture was continued for 24 hours. Further, in the case of Thermus aquaticus ATCC 33923, the culture medium was cultured at 60 ° C. for 24 hours with a fermenter in the same manner as in Experiment 15 except that maltose separately sterilized from nutrient medium was changed to a medium containing 2 to 20 w / v%. Furthermore, the surfactant “Tween 40” was added at 0.1 v / v%, and the culture was continued for 24 hours. The culture was centrifuged, and the trehalose content (mg / ml) contained in the supernatant was measured by HPLC. The results are shown in Table 13.
[0132]
[Table 13]

[0133]
As is apparent from the results in Table 13, the trehalose yield is high at a concentration of maltose in the nutrient medium of 20 w / v% or less, desirably 15 w / v% or less, more desirably 5 to 10 w / v%, which is useful for trehalose production. It has been found suitable.
[0134]
Hereinafter, a method for producing trehalose using a microorganism having the ability to produce maltose / trehalose converting enzyme of the present invention or a saccharide containing the same in Example A, a composition containing trehalose or a saccharide containing the same in Example A Is shown in Example B.
[0135]
Example A-1
2 units of α-amylase (“Spitase HS” manufactured by Nagase Seikagaku Corporation) was added to 10% potato starch milk (pH 5.5) per gram of starch, gelatinized and liquefied with stirring, and immediately autoclaved (120 C.) for 20 minutes and then adjusted to a temperature of 50 ° C. and a pH of 5.0. To this, β-amylase (manufactured by Nagase Seikagaku Corporation) was added at a rate of 20 units per gram of starch and isoamylase (produced by Hayashibara Biochemical Laboratories Co., Ltd.) was added at a rate of 500 units per gram of starch. The mixture was then heated to 95 ° C. to deactivate the enzyme, filtered and decolorized to obtain a sugar solution having a maltose content of about 92%. As a sugar source, instead of glucose 2.0 w / v%, the aforementioned sugar solution is eliminated.FungusThen, except that 10 w / v% per solid was used, a nutrient medium prepared according to the method of Experiment 1 was used as a fermenter, and Pyrolobacter sp. R48 (FERM BP- 4315) is inoculated with 1% v / v and cultured with aeration and agitation at 27 ° C. for 72 hours in the same manner as in Experiment 1. Further, a surfactant (alkylphenol polyoxyethylene ether, sold by Wako Pure Chemical Industries, Ltd.) “Triton X-100”) was added at 0.2 v / v%, and the culture was continued for 24 hours. The culture is filtered to remove insoluble matter, and the resulting filtrate is heated to 95 ° C. to inactivate the enzyme, then concentrated, decolorized and filtered with activated carbon according to a conventional method, and H-type and OH-type. Purification by desalting with an ion exchange resin and further concentration gave syrup having a concentration of about 70% in a yield of about 65% per solid. This product contains about 44% trehalose per solid, has mild sweetness, moderate viscosity, and moisturizing properties. Various foods and drinks as sweeteners, taste improvers, stabilizers, excipients, etc. It can be advantageously used in various compositions such as cosmetics and pharmaceuticals.
[0136]
Example A-2
In the culture filtrate obtained by the method of Example A-1, 10 units of glucoamylase per gram of solid matter, “Glucozyme” manufactured by Nagase Seikagaku Corporation was allowed to act at pH 5.0, 50 ° C. for 24 hours, Next, in order to increase the trehalose content by using the sugar solution obtained by heat deactivation, decolorization, and desalting and purification as a raw sugar solution, sodium-type strongly acidic cation exchange resin (“XT-1016” manufactured by Tokyo Organic Chemical Industry Co., Ltd.) , Ion exchange column chromatography using a degree of cross-linking of 4%). The resin was packed into four jacketed stainless steel columns with an inner diameter of 5.4 cm and connected in series to a total resin layer length of 20 m. Column temperatureTheWhile maintaining the temperature at 60 ° C., the sugar solution is added to the resin at 5 v / v%, and then the water is fractionated by flowing 60 ° C. warm water at SV 0.15, glucose is removed, and the trehalose-rich fraction is collected. did. Further purification, concentration, vacuum drying and pulverization gave a powder containing high trehalose in a yield of about 35% per solid. This product contains about 97% trehalose, has extremely low reducibility, mellow and elegant sweetness. Various sweeteners, taste improvers, quality improvers, stabilizers, excipients, etc. It can be advantageously used for various compositions such as foods, drinks, cosmetics and pharmaceuticals.
[0137]
Example A-3
The trehalose-rich fraction obtained by the method of Example A-2 was decolorized with activated carbon according to a conventional method, and the solution purified by desalting with an ion exchange resin was concentrated to a concentration of about 70%. In addition, about 2% of a water-containing trehalose crystal was added as a seed crystal and gradually 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, 85 ° C hot air is blown from the top of the drying tower and collected on a transfer wire mesh conveyor provided at the bottom, and 45 ° C hot air is sent from the bottom of the conveyor while being collected on the wire mesh conveyor. The powder was gradually moved out of the drying tower and taken out. The taken-out crystal powder is filled into an aging tower and aged for 10 hours while sending warm air, crystallization and drying are completed, and the trehalose-containing crystal powder is about Obtained in 90% yield. This product does not substantially exhibit hygroscopicity, is easy to handle, and can be advantageously used in various compositions such as various foods, cosmetics, and pharmaceuticals as sweeteners, taste improvers, stabilizers, and the like.
[0138]
Example A-4
The trehalose-rich fraction obtained by the method of Example A-2 was purified in the same manner as in Example A-3, then taken into an evaporation kettle and boiled under reduced pressure to obtain syrup having a water content of about 3.0%. Next, it was transferred to an auxiliary crystallizer, to which 1% of anhydrous crystalline trehalose was added as a seed crystal per syrup solid, stirred at 120 ° C., then taken out into an aluminum vat, and crystallized and matured at 100 ° C. for 6 hours. Block was prepared. Next, this block is pulverized with a cutting machine, fluidized and dried, and an anhydrous crystalline trehalose powder having a water content of about 0.3% is obtained at a yield of about 85% per solid relative to the raw trehalose-rich sugar solution. Got in. This product is not only used as a dehydrating agent for water-containing products such as foods, cosmetics, pharmaceuticals, raw materials, or processed intermediates, but also as a white powder sweetener with elegant sweetness. It can be advantageously used in the composition.
[0139]
Example A-5
After adding 0.1% of calcium carbonate to corn starch milk having a concentration of 33%, the pH was adjusted to 6.5, and α-amylase (“Termameal 60L” manufactured by Novo) was added to the starch starch in an amount of 0.2% per gram of starch. The reaction was allowed to proceed at 15 ° C. for 15 minutes. The reaction solution was autoclaved (120 ° C.) for 30 minutes, then cooled to 55 ° C., and isoamylase (produced by Hayashibara Biochemical Laboratories Co., Ltd.) with 500 units per gram starch and β-amylase (Nagase Biochemistry). Kogyo Co., Ltd.) was added at 30 units per gram of starch, reacted for 48 hours, then heated to 95 ° C. to deactivate the enzyme, filtered and decolorized to obtain a sugar solution having a maltose content of about 84%. As a sugar source, a nutrient medium prepared according to the method of Experiment 9 is used as a fermenter, except that the above-mentioned sugar solution is separately sterilized and added to solids at 10 w / v%, and maltose / trehalose converting enzyme is produced. 1 v / v% of a seed culture of Pseudomonas putida H262 (FERM BP-4579) having the ability to inoculate and cultured by aeration and agitation at 27 ° C. for 48 hours in the same manner as in Experiment 9, and further the surfactant “Tween 40” Was added at 0.2 v / v%, and the culture was continued for 24 hours. The culture is filtered to remove insoluble matter, and the resulting filtrate is heated to 95 ° C. to inactivate the enzyme, then concentrated, decolorized and filtered with activated carbon according to a conventional method, and H-type and OH-type. Purification by desalting with an ion exchange resin and further concentration gave syrup having a concentration of about 70% in a yield of about 50% per solid. This product contains about 64% trehalose per solid, has low reducing properties, mild sweetness, moderate viscosity, and moisturizing properties, and is advantageous for various foods, cosmetics, pharmaceuticals and other various compositions. Available to:
[0140]
Example A-6
The syrup obtained by the method of Example A-5 was concentrated to a concentration of about 80% and taken in an auxiliary crystal machine. About 1% of a trehalose hydrous crystal powder was added as a seed crystal, and the mixture was slowly cooled and crystallized with stirring. Then, it was honeyed in a basket type centrifuge, and the crystals were sprayed with a small amount of water and washed to obtain high-purity trehalose-containing crystals in a yield of about 20% per solid. This product exhibits physicochemical properties similar to those in Experiment 22, and can be advantageously used in various compositions such as various foods, cosmetics, and pharmaceuticals as a sweetener, a taste improver, a quality improver, a stabilizer, and the like. Further, it can be advantageously used for industrial reagents and chemical raw materials.
[0141]
Example A-7
After adding 2 units of α-amylase “Spitase HS” per gram of starch to corn starch milk (pH 5.5) at a concentration of 10%, the mixture was heated and gelatinized and liquefied with stirring, and immediately autoclaved (120 ° C.) for 20 minutes. The temperature was adjusted to 55 ° C. and pH 5.0. To this was added isoamylase (produced by Hayashibara Biochemical Laboratories Co., Ltd.) at a rate of 300 units per gram starch and β-amylase (produced by Nagase Seikagaku Co., Ltd.) at a rate of 20 units per gram starch and reacted for 24 hours. Then, the mixture was heated to 95 ° C. to deactivate the enzyme, filtered and decolorized to obtain a sugar solution having a maltose content of about 92%. As a sugar source, a nutrient medium prepared in accordance with the method of Experiment 15 is used as a fermenter, except that the above-mentioned sugar solution is separately sterilized and added to solids, and maltose / trehalose converting enzyme is produced. 1% / v% of the seed culture of thermus aquaticus ATCC 33923 having the ability was cultured at 60 ° C. for 40 hours under aeration and agitation in the same manner as in Experiment 15, and the surfactant “Triton X-100” was added to 0%. The culture was continued for 16 hours by adding 50 mg of egg white lysozyme per 1 v / v% and 1 l of the culture solution. The culture is filtered to remove insoluble matter, and the resulting filtrate is heated to 95 ° C. to inactivate the enzyme, then concentrated, decolorized and filtered with activated carbon according to a conventional method, and H-type and OH-type. Purification by desalting with an ion exchange resin and further concentration gave syrup having a concentration of about 70% in a yield of about 55% per solid. This product contains about 68% trehalose per solid, has mild sweetness, moderate viscosity, and moisturizing properties. Various foods and drinks as sweeteners, taste improvers, stabilizers, excipients, etc. It can be advantageously used in various compositions such as cosmetics and pharmaceuticals.
[0142]
[Example A-8]
The syrup obtained by the method of Example A-7 was concentrated to a concentration of about 85% and placed in an auxiliary crystal machine. After mixing about 1% of seed crystals, the seed crystal was placed in a vat and left at 20 ° C. for 4 days to crystallize and solidify. Then, it was pulverized by a cutting machine and dried to obtain a honey-containing trehalose hydrated crystal powder in a yield of about 95% per solid. This product does not substantially exhibit hygroscopicity, is easy to handle, and is useful as a sweetener, taste improver, quality improver, stabilizer, etc., for various foods, cosmetics, pharmaceuticals, and other various compositions. Available to:
[0143]
Example A-9
The syrup obtained by the method of Example A-7 was concentrated to a concentration of about 80% and taken in an auxiliary crystallizer, and crystallized and mashed in the same manner as in Example A-6 to give high-purity trehalose-containing crystals per solid. Obtained in a yield of about 20%. This product exhibits physicochemical properties similar to those in Experiment 22, and in the same manner as in Example A-6, various compositions such as various foods, cosmetics, and pharmaceuticals, as well as industrial reagents, industrial raw materials, and chemical raw materials. It can be used advantageously.
[0144]
Example B-1 Sweetener
To 1 part by weight of the trehalose hydrous crystal powder obtained by the method of Example A-3, 0.01 part by weight of α-glycosyl stevioside (“αG Sweet” sold by Toyo Seika Co., Ltd.) and L-aspartyl-L-phenylalanine methyl ester ( AJI-NO-MOTO Co., Ltd. “Aspartame”) 0.01 parts by weight was uniformly mixed and subjected to a granulating machine to obtain a granular sweetener. This product has excellent sweetness quality, has about twice the sweetness of sucrose, and the calorie per sweetness is reduced to about 1/2 that of sucrose. This sweetener has no degradation of high-sweetness sweetener blended in it, has excellent stability, and as a low-calorie sweetener, low-calorie food and drink for obese people, diabetics, etc. who restrict caloric intake Suitable for sweetening products. In addition, 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.
[0145]
[Example B-2 Hard Candy]
30 parts by weight of trehalose-containing syrup obtained by the method of Example A-1 was heated and mixed with 100 parts by weight of a 55% sucrose solution, and then heated and concentrated under reduced pressure until the water content was less than 2%. Part by weight and an appropriate amount of lemon flavor and color 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 crystallize or deform.
[0146]
[Example B-3 Chocolate]
After mixing 40 parts by weight of cocoa paste, 10 parts by weight of cocoa butter, 30 parts by weight of sucrose, and 20 parts by weight of high-purity trehalose hydrous crystals obtained by the method of Example A-6, the mixture was passed through a refiner to reduce the particle size. And knead for 2 days at 50 ° C. In the meantime, 0.5 part 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.
[0147]
Example B-4 Chewing gum
3 parts by weight of gum base is heated and melted to a degree of softening, and 4 parts by weight of sucrose and 3 parts by weight of trehalose-containing hydrated crystal powder obtained by the method of Example A-3 are added thereto. They were 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.
[0148]
[Example B-5 Sweetened condensed milk]
3 parts by weight of trehalose-containing syrup and 1 part by weight of sucrose obtained by the method of Example A-5 are dissolved in 100 parts by weight of raw milk, sterilized by heating with a plate heater, then concentrated to a concentration of 70%, and canned under 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.
[0149]
[Example B-6 Lactic acid bacteria beverage]
175 parts by weight of skim milk powder, 80 parts by weight of powder containing high trehalose obtained by the method of Example A-2, and 50 parts by weight of powder containing high lactosucrose disclosed in Japanese Patent Laid-Open No. 4-281795 are added to 1,200 parts by weight of water. And then sterilized at 65 ° C. for 30 minutes, cooled to 40 ° C., inoculated with 30 parts by weight of a lactic acid bacteria starter according to a conventional method, and cultured at 37 ° C. for 8 hours to obtain a lactic acid bacteria beverage. . This product is a lactic acid bacteria beverage with good flavor. In addition, this product contains oligosaccharides and not only stably retains lactic acid bacteria, but also has a bifidobacteria growth promoting action.
[0150]
[Example B-7 powdered juice]
50 parts by weight of high-purity trehalose hydrous crystals obtained by the method of Example A-6, 10 parts by weight of sucrose, 0.65 parts by weight of anhydrous citric acid, and malic acid with respect to 33 parts by weight of orange juice powder produced by spray drying 0.1 parts by weight, 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 and fluidized The product is charged into a layer granulator, the exhaust air temperature is 40 ° C., and the trehalose-rich syrup obtained by the method of Example A-5 is sprayed as a binder, granulated for 30 minutes, weighed and packaged. Obtained. 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 was stable for a long time.
[0151]
[Example B-8 Custard cream]
100 parts by weight of corn starch, 100 parts by weight of trehalose-containing syrup obtained by the method of Example A-1, 80 parts by weight of maltose, 20 parts by weight of sucrose and 1 part by weight of sodium chloride were mixed well, and 280 parts by weight of chicken egg was added and stirred. Gradually add 1,000 parts by weight of boiled milk to this, and continue to stir it over fire. When the corn starch is completely gelatinized and the whole becomes translucent, stop the fire and cool it. Appropriate amount of vanilla fragrance was added and weighed, filled and packed to obtain a product. This product has a smooth luster, mild sweetness and deliciousness.
[0152]
[Example B-9 Uiro no Moto]
Rice bran was prepared by uniformly mixing 90 parts by weight of rice flour with 20 parts by weight of corn starch, 40 parts by weight of sucrose, 80 parts by weight of the hydrous crystal powder of trehalose obtained by the method of Example A-3 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.
[0153]
[Example B-10 An]
In accordance with a conventional method, water was added to 10 parts by weight of azuki bean, boiled, astringent, drained, and water-soluble impurities were removed to obtain about 21 parts by weight of azuki bean paste. To this raw bean paste, 14 parts by weight of sucrose, 5 parts by weight of trehalose-containing syrup obtained by the method of Example A-7 and 4 parts by weight of water are boiled, and a small amount of salad oil is added to this so as not to break the crumbs. As a result, about 35 parts by weight of the product was obtained. This product has no color burn, has a good texture, has a good flavor, and is suitable as an ingredient for anpan, bun, dumpling, peach and ice confectionery.
[0154]
Example B-11 Bread
Knead 100 parts by weight of wheat flour, 2 parts by weight of yeast, 5 parts by weight of sugar, 1 part by weight of a powder containing high trehalose obtained by the method of Example A-2 and 0.1 part by weight of an inorganic food according to a conventional method. Medium seeds were fermented at 26 ° C. for 2 hours, then aged for 30 minutes and baked. This product is a high quality bread with good hue and sudoku, moderate elasticity and mild sweetness.
[0155]
[Example B-12 Ham]
The pork thigh is uniformly rubbed with 1,000 parts by weight of meat and 15 parts by weight of sodium chloride and 3 parts by weight of potassium nitrate, and deposited in a cold room day and night. This was soaked in a cold room for 7 days in a salted solution consisting of 500 parts by weight of water, 100 parts by weight of sodium chloride, 3 parts by weight of potassium nitrate, 40 parts by weight of a high trehalose powder obtained by the method of Example A-2 and spices, According to a conventional method, the product was washed with cold water, wound with a string, smoked, cooked, and cooled and packaged to obtain a product. This product is a high quality ham with good color and good flavor.
[0156]
[Example B-13 powdered peptide]
Concentrated 40% soy peptide solution for food (Fuji Oil Co., Ltd. “Hi-Nut S”) is mixed with 2 parts by weight of high-purity trehalose hydrous crystals obtained by the method of Example A-6, and made of plastic. It put into the vat, dried under reduced pressure at 50 ° C., and pulverized 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 confectionery but also as a baby food such as oral liquid food and tube liquid food, and a therapeutic nutrient.
[0157]
[Example B-14 Powdered Miso]
3 parts by weight of anhydrous crystalline trehalose powder obtained by the method of Example A-4 was mixed with 1 part by weight of red miso, poured into a metal plate provided with a number of hemispherical recesses, and allowed to stand overnight at room temperature. Solidified and released from mold to obtain about 4 g of solid miso per piece, which was then pulverized to obtain powdered miso. This product can be advantageously used as a seasoning for instant ramen and instant soup. The solid miso can be used not only as a solid seasoning but also as a miso confectionery.
[0158]
[Example B-15 powdered egg yolk]
Egg yolk prepared from raw eggs is sterilized with a plate-type heat sterilizer at 60 to 64 ° C., and 1 part by weight of the liquid egg yolk obtained is 4 parts by weight of anhydrous crystalline trehalose powder obtained by the method of Example A-4. After mixing at a ratio, the mixture was transferred to a vat and allowed to stand overnight to convert into trehalose hydrous crystals to prepare a block. This block was pulverized with a cutting machine to obtain powdered egg yolk. This product can be advantageously used not only as a confectionery material such as a premix, a frozen dessert, and an emulsifier, but also as a baby food such as an oral liquid food and a tube liquid food, and a therapeutic nutrient. Moreover, it can be advantageously used as a skin beautifying agent, hair restorer and the like.
[0159]
[Example B-16 Cosmetic cream]
2 parts by weight of polyoxyethylene glycol monostearate, 5 parts by weight of glyceryl monostearate, 2 parts by weight of a high trehalose powder obtained by the method of Example A-2, 1 part by weight of α-glycosyl rutin, liquid paraffin 1 part by weight, 10 parts by weight of glyceryl trioctanoate and an appropriate amount of 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 added thereto. Then, the mixture was emulsified with a homogenizer, and an appropriate amount of a fragrance was added, followed by stirring and mixing to produce a cream. This product has antioxidant properties, high stability, and can be advantageously used as a high-quality sunscreen, skin beautifying agent, whitening agent, and the like.
[0160]
[Example B-17 Powdered Ginseng Extract]
After mixing 1.5 parts by weight of anhydrous crystalline trehalose powder obtained by the method of Example A-4 with 0.5 parts by weight of ginseng extract, it is transferred to a vat and left to stand for 2 days to convert into trehalose hydrated crystals. Prepared. The block was pulverized with a cutting machine and classified to obtain a powdered ginseng extract. This product was applied to a granule molding machine together with appropriate amounts of vitamin B1 and vitamin B2 powders to obtain a vitamin-containing granular medicinal carrot extract. This product can be advantageously used as a fatigue recovery agent, tonic, toughening agent and the like. It can also be used as a hair restorer.
[0161]
[Example B-18 Solid preparation]
A human natural interferon-α preparation (manufactured by Hayashibara Biochemical Laboratories, Inc.) is applied to an immobilized anti-human interferon-α antibody column according to a conventional method to adsorb the human natural interferon-α contained in the preparation. The bovine serum albumin, which is a stabilizer, is removed by passage, then the pH is changed, and human natural interferon-α contains 5% of high-purity trehalose hydrous crystals obtained by the method of Example A-6. Elution was performed using physiological saline. This liquid is microfiltered, added to approximately 20 times the amount of anhydrous crystalline maltose powder, “Fine Tose” sold by Hayashibara Shoji Co., Ltd., dehydrated and powdered, and then tableted with a tableting machine to give 1 tablet (approximately 200 mg) ) To obtain tablets containing about 150 units of human natural interferon-α. About 1 to 10 adults per day are administered orally as sublingual tablets, etc., and this product 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., whose number of patients is rapidly increasing in recent years. In this product, both the non-reducing saccharide of the present invention and anhydrous crystalline maltose act as a stabilizer, and maintain its activity well for a long time even when left at room temperature.
[0162]
[Example B-19 Dragee]
An uncoated tablet having a weight of 150 mg is used as a core, and 40 parts by weight of high-purity trehalose-containing crystals obtained by the method of Example A-9, 2 parts by weight of pullulan (average molecular weight 200,000), 30 parts by weight of water, and 25% by weight of talc. Sugar solution using an undercoat liquid consisting of 3 parts by weight of titanium oxide and 3 parts by weight of titanium oxide until the tablet weight is about 230 mg, and then an overcoat consisting of 65 parts by weight of the same trehalose-containing crystal powder, 1 part by weight of pullulan and 34 parts by weight of water. The liquid was used for sugar coating, and it was further polished with wax liquid to obtain a sugar coated tablet with a glossy appearance and excellent appearance. This product has excellent impact resistance and maintains high quality for a long time.
[0163]
[Example B-20 toothpaste]
Formulation
Dicalcium phosphate 45.0 parts by weight
Pullulan 2.95 parts by weight
Sodium lauryl sulfate 1.5 parts by weight
Glycerin 20.0 parts by weight
0.5 parts by weight of polyoxyethylene sorbitan laurate
Preservative 0.05 parts by weight
12.0 parts by weight of water-containing trehalose crystal powder obtained by the method of Example A-8
Maltitol 5.0 parts by weight
13.0 parts by weight of water
The above materials were mixed according to a conventional method to obtain a toothpaste. This product has moderate sweetness and is particularly suitable as a toothpaste for children.
[0164]
[Example B-21 Liquid food solid preparation]
500 parts by weight of high-purity trehalose hydrous crystals produced by the method of Example A-6, 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 of magnesium sulfate A blend consisting of parts by weight, 0.01 parts by weight thiamine, 0.1 parts by weight sodium ascorbate, 0.6 parts by weight vitamin E acetate and 0.04 parts by weight nicotinamide is prepared, and each 25 grams of this blend is prepared. A moisture-proof laminated sachet was filled and heat sealed to obtain a product. This product is dissolved in about 150 to 300 ml of water to make a liquid food, and it is used orally or by tube use to the nasal cavity, stomach, intestine, etc., and is advantageous for supplementing energy to the living body. Available to:
[0165]
[Example B-22 infusion]
The high-purity trehalose-containing crystals produced by the method of Example A-6 are dissolved in water to a concentration of about 10 w / v%, then microfiltered to pyrogen-free according to a conventional method, and aseptically filled into plastic bottles The product was obtained by plugging. This product is a stable infusion that does not change over time and is suitable for administration intravenously, intraperitoneally, and the like. This product is isotonic with blood at a concentration of 10 w / v%, and can be replenished with energy at twice the concentration of glucose.
[0166]
[Example B-23 infusion]
The high-purity trehalose hydrated crystals produced by the method of Example A-9 and the amino acid blend having the following composition were mixed and dissolved in water so that the amounts were 5 w / v% and 30 w / v%, respectively, and then Example B- The product was purified to be pyrogen-free in the same manner as in No. 22, and filled into a plastic bag and plugged to obtain a product.
Composition of amino acid compound (mg / 100ml)
L-isoleucine 180
L-leucine 410
L-lysine hydrochloride 620
L-methionine 240
L-phenylalanine 290
L-threonine 180
L-tryptophan 60
L-Valine 200
L-arginine hydrochloride 270
L-histidine hydrochloride 130
Glycine 340
This product is a stable infusion with no changes over time because trehalose does not exhibit reducing properties despite the complex infusion of carbohydrates and amino acids, and is suitable for administration intravenously, intraperitoneally, etc. It is. This product can be advantageously used not only for replenishing energy to the living body but also for supplementing amino acids.
[0167]
[Example B-24 Ointment for trauma treatment]
To 200 parts by weight of the water-containing trehalose crystal powder and 300 parts by weight of maltose produced by the method of Example A-8, 50 parts by weight of methanol in which 3 parts by weight of iodine were dissolved was added and mixed, and further 200 parts by weight of a 10 w / v% pullulan aqueous solution. Was added and mixed to obtain a plaster for treatment of trauma showing moderate elongation and adhesion. This product not only acts as a bactericidal action with iodine, but also acts as an energy replenisher to cells with trehalose, shortening the healing period and healing the wound surface cleanly.
[0168]
【The invention's effect】
As is clear from the above, non-reducing carbohydrates can be produced from a culture in a very simple and short time by culturing a microorganism capable of producing maltose / trehalose converting enzyme in a nutrient medium containing maltose. This facilitates the industrial production of trehalose or a sugar containing the same. As maltose, if maltose obtained by allowing starch debranching enzyme to act on β-amylase or β-amylase in a solution obtained by liquefying starch, trehalose yield from starch is remarkably increased, and its industrial production make it easier. The trehalose thus obtained or a saccharide containing the same has excellent stability, high quality and elegant sweetness. Moreover, it is digested and absorbed by ingestion and becomes a calorie source. In particular, trehalose is also used parenterally and is frequently metabolized. Therefore, the trehalose of the present invention or a saccharide containing the same is advantageous as a sweetener, a taste improver, a quality improver, a stabilizer, an excipient, and the like for various compositions such as various foods, cosmetics, and pharmaceuticals. Available to:
[0169]
The establishment of the present invention provides a completely new way for industrially providing trehalose that is not easily obtained as desired from starch, which is an endless resource, or a sugar containing the same industrially in large quantities and at low cost. The impact of this will not be limited to the food, cosmetics, and pharmaceutical industries, but also the agricultural, aquatic and livestock industries, and the chemical industry.
[Brief description of the drawings]
FIG. 1 is a graph showing the effect of temperature on the enzyme activity of Pyrolobacter species R48 maltose / trehalose converting enzyme.
FIG. 2 is a graph showing the influence of pH on the enzyme activity of Pyrolobacter species R48 maltose / trehalose converting enzyme.
FIG. 3 is a graph showing the effect of temperature on the stability of Pyrolobacter species R48 maltose trehalose converting enzyme.
FIG. 4 is a graph showing the effect of pH on the stability of Pyrolobacter sp. R48 maltose trehalose converting enzyme.
FIG. 5 is a graph showing the influence of temperature on the enzyme activity of Pseudomonas putida H262 maltose / trehalose converting enzyme.
FIG. 6 is a graph showing the influence of pH on Pseudomonas putida H262 maltose / trehalose converting enzyme activity.
FIG. 7 is a graph showing the effect of temperature on the stability of Pseudomonas putida H262 maltose trehalose converting enzyme.
FIG. 8 is a graph showing the influence of pH on the stability of Pseudomonas putida H262 maltose / trehalose converting enzyme.
FIG. 9 is a graph showing the influence of temperature on the enzyme activity of maltose trehalose converting enzyme of Thermus aquaticus ATCC 33923.
FIG. 10 is a graph showing the influence of pH on the enzymatic activity of maltose / trehalose converting enzyme of Thermus aquaticus ATCC 33923.
FIG. 11 is a graph showing the effect of temperature on the stability of the maltose trehalose converting enzyme of Thermus aquaticus ATCC 33923.
FIG. 12 is a graph showing the influence of pH on the stability of the maltose trehalose converting enzyme of Thermus aquaticus ATCC 33923.

Claims (5)

A microorganism selected from the genera Pimerobacter, Pseudomonas and Thermus having the ability to produce maltose / trehalose converting enzyme is cultured in a nutrient medium containing maltose, and trehalose or a carbohydrate containing the same is collected from the resulting culture. A method for producing trehalose or a carbohydrate containing the same. The method for producing trehalose or a carbohydrate containing the same according to claim 1 , wherein maltose is contained in the nutrient medium in an amount of 20 w / v% or less. A method for producing trehalose or a carbohydrate containing the same according to claim 1 or 2 , wherein the nutrient medium contains a surfactant together with maltose. The maltose is maltose obtained by allowing a starch debranching enzyme to act on β-amylase or β-amylase together with starch liquefied, or trehalose according to any one of claims 1 to 3 Quality manufacturing method. The production of trehalose or a carbohydrate containing the same according to any one of claims 1 to 4, wherein glucoamylase or α-glucosidase is allowed to act on a culture or a carbohydrate containing trehalose obtained therefrom. Method.

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