JP3556690B2 - Method for producing sugar carboxylic acid and novel sugar carboxylic acid - Google Patents

Description

で表わされるマルトデキストリンの有するヒドロキシメチル基の少なくとも１つがカルボキシル基に酸化された糖カルボン酸、
式（ＩＩ）
【００１３】
【化５】

で表わされるステビオール配糖体の有するヒドロキシメチル基の少なくとも１つがカルボキシル基に酸化された糖カルボン酸、
式（ＩＩＩ）
【化６】

で表わされるバリダマイシンＡの有するヒドロキシメチル基の少なくとも１つがカルボキシル基に酸化された糖カルボン酸、
モグロシドの有するヒドロキシメチル基の少なくとも１つがカルボキシル基に酸化された糖カルボン酸並びにそれらの塩、
デキストランの有するヒドロキシメチル基がカルボキシル基に酸化された式（ＶＩＩＩ）
【化７】

（但し、式中、ｎ＝１〜５０，好ましくは１０〜１５）で表わされる糖カルボン酸〔すなわち、デキストラニルグルクロン酸（グルクロニルデキストリン、デキストラン−グルクロン酸とも称される）〕、その塩、又はそれらと金属塩との錯体あるいは複合体（以下単に複合体と称する）及び
デキストラニルグルクロン酸（デキストラン−グルクロン酸と称されることもある）が有するヘミアセタール水酸基を有する炭素原子がカルボキシル基に酸化された式（ＩＸ）
【化８】

（式中、ｎ＝１〜５０，好ましくは１０〜１５）で表わされる糖カルボン酸（すなわち、グルクロニルデキストラニルグルコン酸）、その塩又はそれらと金属塩との複合体等は産業上有用な新規化合物である。
【００１４】
上記の出発物質の糖類を、上記したシュードグルコノバクター属に属する菌体もしくはその処理物と接触させることにより酸化反応を行う際、該糖類は、１級水酸基やヘミアセタール水酸基の数やその性質を反映して、位置選択的、かつ段階的に酸化され、対応する糖カルボン酸を特異的に与えることも、このシュードグルコノバクター属に属する菌体あるいは処理物による酸化反応の特徴である。
目的物の分離が容易な場合等には、上記したシュードグルコノバクター属の微生物を、上記糖類含有培地中で培養してもよい。この場合の培養条件としては上記培養液を得る方法と同様な条件で行うことができる。
このようにして生成蓄積された糖カルボン酸は公知の手段又はそれに準じる方法により採取、精製することができる。例えば濾過、遠心分離、活性炭や吸着体処理、溶媒抽出、クロマトグラフィー、沈澱、塩析等の手段を単独で又は適宜組み合わせて適用して、目的物を単離、生成することができる。
上述のように、陰イオン交換樹脂の存在下で酸化を行なう場合、反応液と陰イオン交換樹脂を静地する方法，遠心分離法等により分離し、陰イオン交換樹脂を、溶離剤を用いて溶出処理し、目的物を含む溶出画分を集め、これを上記公知の手段又はそれに準じる方法に付し、目的とする糖カルボン酸を単離、精製する。
【００１５】
デキストランカルボン酸と鉄塩との複合体は通常、デキストランと鉄塩との複合体を製造する公知方法又はそれに準じて製造することができる。例えば、デキストランカルボン酸と水酸化第二鉄等鉄塩のゾルとを反応させることにより製造することもできる。より好ましくは透析により、脱塩、精製した水酸化鉄ゾルにデキストランカルボン酸を加えついでｐＨ８．０〜１０で、加温された温度条件たとえば１００℃〜１２０℃におけるオートクレーブなどで、３０分間加熱処理することにより、コロイド溶液または懸濁液が得られる。
この方法により、鉄は塩形成、キレート形成水和化などのプロセスなどにより、デキストランカルボン酸誘導体鉄塩複合体が形成される。後述するように、この水相中コロイド懸濁鉄塩複合体の元素鉄含有量は溶液状態の約５０〜２５０ｍｇ／ｍｌであり、所望により蒸発、濃縮などの操作により、低鉄分含有品から高鉄分含有鉄塩複合体を調製することもできる。このようにして得られた、高鉄分含有鉄塩複合体はそれ自体長期間極めて安定であり、特に水相中でそのコロイド状態が安定に保持される特性を有している。
このようにして得られたデキストランカルボン酸と水酸化第２鉄等の鉄塩との複合体は、注射用蒸留水、生理食塩水等で希釈して注射剤として非経口的に動物に投与してもよく、又、公知の製剤学的製造法に準じ、所望により製剤学的に許容される希釈剤、賦型剤を用い、錠剤、粉剤、顆粒剤、カプセル剤、乳剤、液剤、プレミックス剤、シロップ剤等として経口的に投与することができる。又、一剤とした後直接又は担体に分散させたものと飼料、飲水等に混ぜて用いることができる。さらにそれぞれの物質を別途所望により製剤原料に許容される希釈剤、賦型剤等を用い、製剤化し用時希釈剤等を用いて一剤とした後、飼料、飲水等の中に混ぜて投与することもできる。さらに上記したようにそれぞれ別途製剤化したものを、別個に同時にまたは時間差をおいて、同一対象に対して同一経路または異なった経路で投与することもできる。
本発明の方法によりカルボン酸を遊離体で得ても、塩で得てもよく、塩で得られた場合は、慣用方法により遊離体に、又、塩で得られた場合は遊離体に変換できることはいうまでもない。又培地に鉄、リチウム、ナトリウム、カリウム等のアルカリ金属、マグネシウム、カルシウム等のアルカリ土類金属を存在せしめることにより糖カルボン酸を生成蓄積しながらその塩を生成することもできる。更に、得られた生成物は、元素分析、融点、比旋光度、赤外線吸収スペクトル、ＮＭＲスペクトル、クロマトグラフィー等の慣用手段により同定できる。
【００１６】
このようにして得られた本発明の糖カルボン酸には、種々の用途、特性が認められる。例えば前記ステビオール配糖体としては例えば次のような化合物があげられ、これらを酸化して得られるグルクロン酸型ステビオール配糖体誘導体はいずれも新規物質である。
【表１】

これらを含む各種ステビオール配糖体の糖カルボン酸誘導体は、甘味質が改善され強甘味とさわやかな味質が認められた。低カロリー性、抗う蝕性、酵素に安定な高甘味食品素材として菓子類、清涼飲料水、漬物、冷菓等に従来の甘味料に準じて用いることができる。さらに易溶解性、低毒性、崩壊性、分解性等の点でもすぐれているので、味の改善が可能で矯味剤として用いることができる。例えば、錠剤、散剤等の製剤に、常法に従い添加、配合して用いることができる。またβ−マルトシル−β−シクロデキストリンの糖カルボン酸誘導体は、シクロデキストリン類での初めてのカルボン酸誘導体で、水に対する溶解性が、著しく改善された（溶解度＞２００ｇ／１００ｍｌ、水、２５℃）。従って、例えば、プロスタグランジン，ステロイド，バルビトール酸等の難溶性医薬品を本発明のシクロデキストリンの糖カルボン酸類で包接化することにより水溶性が向上し、注射剤として適用できる。このように糖カルボン酸類を包接剤として用いる場合、従来知られている包接剤と同様な方法で用いることができる。また、シュークロースを反応させて得られるβ−Ｄ−フラクトシル−（２→１）−α−Ｄ−グルクロン酸や、α−Ｄ−グルクロニル−（１→２）−β−Ｄ−６−フラクチュロン酸は、甘味のない新たな砂糖誘導体で、かつ、グルコシダーゼ，ペクチナーゼ，グルクロニダーゼ，インベルターゼにより分解されないことが判明した。シュークロースより酵素分解しにくい糖誘導体である。従って、難消化性，低カロリー性砂糖誘導体として菓子、ケーキ、冷菓等の食品素材として有用である。またそのカルシウム塩，マグネシウム塩，鉄塩はそれら金属イオンの吸収改善剤として有用である。従って、ビスケット等の菓子類、清涼飲料水等に添加し、骨粗しょう症予防の飲食品を得るのに用いることができる。
【００１７】
また、パラチノースを反応させて得られる β−Ｄ−フラクトシル−（６→１）−α−Ｄ−グルクロン酸，α−Ｄ−グルクロニル−（６→１）−α−Ｄ−フラクチュロン酸は、難消化性，低カロリー性の抗う触剤として用いられる。従って、チューイングガム等の菓子類をはじめとする各種食品に通常の甘味料、調味料と同様にして用いることができる。さらに、Ｄ−トレハロースを反応させて得られる α−Ｄ−グルクロニル−（１→１）−α−Ｄ−グルクロン酸は、難消化性保湿剤，抗体製剤の安定化剤として有用である。またＤ−グルコサミンを反応して得られる β−アミノ−２−デオキシ−Ｄ−グルクロン酸は保湿性のすぐれた化粧品の基材として有用である。
一方、イノシンなどのヌクレオシドを反応して得られる核酸誘導体のうち、特に、５′−カルボキシ−イノシン，５′−カルボキシ−アデノシンは、すぐれた呈味性を有し、かつ、酵素的に安定な呈味剤として有用であり、食品・調味料の成分として、とりわけ保存食品の調味に利用できる。
また２，７−アンヒドロ−β−Ｄ−アルトロ−ヘプチューロースを反応させて得られる １−カルボキシ−２，７−アンヒドロ−β−Ｄ−アルトロ−ヘプチュロースは、弱い甘味を有する鉄、カルシウム，マグネシウムの溶解補助剤としてビスケット等の菓子類、清涼飲料水等に用いることができる。ストレプトゾトシンの糖カルボン酸は、酵素に対して安定な抗菌剤，抗ガン剤として用いられる。又その抗菌作用を利用して、そのまま又は水等溶媒で希釈して病室や、手足の消毒・殺菌に用いることができる。モグロシドの糖カルボン酸誘導体は高甘味剤として有用であり、清涼飲料水、菓子等に利用できる。
【００１８】
マルトデキストリンを酸化して得られる糖カルボン酸は、難消化性、低カロリー食品素材として、菓子、ケーキ、冷菓等に常法により添加、配合して用いられる。
また、デキストランのヒドロキシメチル基をカルボキシル基に酸化して得られるデキストラニルグルクロン酸は、それ自体デキストランと同様なあるいはより安定でより機能的医薬品添加剤として有用であるばかりでなく、水酸化第２鉄ゾルの安定化剤として、優れた特性を発揮する。又、デキストラニルグルコン酸の有するヒドロキシメチル基がカルボキシル基に酸化されるかあるいはデキストラニルグルクロン酸の有するヘミアセタール水酸基をもつ炭素原子がカルボキシル基に酸化されたグルクロニルデキストラニルグルコン酸またはその塩は医薬用製剤素材として有用であり、又食品の粘性保持剤等として、デキストランと同様な使い方で使用できる。又、デキストラニルグルクロン酸及びグルクロニルデキストラニルグルコン酸又はこれらの塩と金属塩との複合体を作る金属塩としては例えば、第１価、第２価又は第３価の金属の塩が含まれ、例えば、カルシウム，マグネシウム，鉄，ナトリウム，リチウム，カリウム等の金属との塩が挙げられる。特に水酸化第二鉄等の鉄化合物との複合体は鉄の補給剤として動物の鉄欠乏性貧血に対し、抗貧血剤としてデキストランと鉄との複合剤と同様な方法で用いることができる。
またバリダマイシンＡは前記式（ＩＩＩ）で表わされる化合物でイネ紋枯病などに対する殺菌活性を有する化合物で本菌体反応による酸化体も、菌のグルコシダーゼに対して抵抗性を有し、作用持続型バリダマイシン誘導体が期待され、改善された持続性を有する農業用殺菌剤として有用である。アスコルビン酸誘導体の糖カルボン酸誘導体も酵素分解に対し安定性が改善されるので、酵素に対して安定な酸化防止剤として用いられる。酸化防止剤としては、アスコルビン酸と同様な方法で鉄食品等に添加、配合して用いることができる。
このように、種々の糖のヒドロキシメチル基及び／又はヘミアセタール水酸基を有する炭素原子を酸化して得られる本発明の糖カルボン酸は、それぞれ原料の糖が有しない特性が付与され従来になかった有用性を有する。
【００１９】
本発明で得られるヒドロキシメチル基がカルボキシル基に酸化された糖カルボン酸誘導体は、一般に、基質であるヒドロキシメチル体と比較し、酵素的に安定でかつ、水に対する溶解性が向上し、かつ、所望により、金属塩にすることができ、食品用途などでは、難消化，カロリーになりにくい、などのダィエット食の素材、金属の吸収改善効果などが期待される。また、ステビオール配糖体（ステビオシド，レバウオシド，ルブソシド等）のカルボン酸は、驚くべきことに、ステビオシドやルブソシドが示す、にがみや後味の悪さがなくなり、清涼感のある甘味質で、砂糖の１００〜２５０倍の甘味を示し、実質的なカロリーのない、甘味剤として有用である。
また本発明のヒドロキシメチル基がカルボキシル基に酸化されたシクロデキストリン誘導体のカルボン酸は、易溶解性，低毒性，崩壊性，分解性などの点ですぐれた特長を有するので、油溶性薬物，脂肪酸，塩基性薬物等の包摂化が可能である。このような包摂化によって活性成分の可溶化、安定化，味の改善が達成できるので矯味、矯臭剤として用いられる。また酵素分解性の特性を利用した製剤基剤としても有用である。より具体的には、例えば錠剤，カプセル剤，丸剤，散剤，顆粒剤，軟膏剤，注射剤，シロップ剤，懸濁剤，点鼻剤などの製剤用のより機能的な基剤としての利用が挙げられる。
このように本発明は、巾広い糖類のヒドロキシメチル基のカルボキシル基への酸化反応を可能にし、種々の新規糖カルボン酸を提供するものである。
又、本発明で得られるヘミアセタール水酸基がカルボキシル基に酸化された糖カルボン酸は基質であるヘミアセタール水酸基をもつ糖類と比較し、酵素的に安定で、かつ水に対する溶解性が向上し、かつ、所望により、金属塩にすることができ、食品用途では難消化、カロリーになりにくいなどのダイエット食の素材、金属の吸収改善効果などが期待される。また医薬品製剤用途では、活性成分の安定化、酵素分解性の特性を利用した腸溶剤などとして有用である。
【００２０】
【発明の効果】
本発明は巾広い糖類からヒドロキシメチル基及び／又はヘミアセタール水酸基をもつ炭素原子がカルボキシル基に特異的に酸化された糖カルボン酸が、高選択的に高収率で製造できる。得られたカルボン酸は、酵素に対する安定性がすぐれ、水に対する溶解性が向上する等のすぐれた特性を有する。
【００２１】
【実施例】
次に、実施例により、本発明をより具体的に説明するが、本発明はこれら実施例に限定されるものではない。
実施例１ β−Ｄ−フラクトシル−（２→１）−α−Ｄ−グルクロン酸ナトリウム塩の製造
シュードグルコノバクター・サッカロケトゲネス（Ｐｓｅｕｄｏｇｌｕｃｏｎｏｂａｃｔｅｒ ｓａｃｃｈａｒｏｋｅｔｏｇｅｎｅｓ）ＴＨ１４−８６菌液の調製；
シュードグルコノバクター・サッカロケトゲネス（Ｐｓｅｕｄｏｇｌｕｃｏｎｏｂａｃｔｅｒ ｓａｃｃｈａｒｏｋｅｔｏｇｅｎｅｓ）ＴＨ１４−８６株スラントを２０ｍｌの下記培地に対して１白金耳加え、これを２００ｍｌのスムースフラスコで３０℃，１日間，回転振盪機を用いて、撹拌下培養する。ついで、２０ｍｌの培地当り前記培養液１ｍｌをとり、これを３０℃，２日間，振盪培養を行い、種培養とした。一方、Ｂａｃｉｌｌｕｓ ｍｅｇａｔｅｒｉｕｍ ＩＦＯ １２１０８株のスラントを２０ｍｌの培地（下記）当り、１白耳，２００ｍｌのスムースフラスコに加え、３０℃，２日間振盪培養を行う。
次に、以下の本培養を行った。上記ＴＨ１４−８６株の種培養（Ｓｅｅｄ 培地）１００ｍｌおよび、Ｂａｃｉｌｌｕｓ ｍｅｇａｔｅｒｉｕｍ のＳｅｅｄ 培地１．５ｍｌを９００ｍｌの下記本培養培地に加えついで、３０℃，約２０時間振盪培養し、Ｐｓｅｕｄｏｇｌｕｃｏｎｏｂａｃｔｅｒ ｓａｃｃｈａｒｏｋｅｔｏｇｅｎｅｓ ＴＨ１４−８６の菌液とした。
菌体反応；
バイオット社製５リットル ジャーファーメンターに、シュークロース３０ｇを滅菌水２００ｍｌにとかした溶液を加え、これに前記のように調製した菌液１リットルを加え、ついで滅菌水８００ｍｌを加え、全量を２リットルとした。３２℃，８００回転（ｒｐｍ）で撹拌しながら、空気を１リットル／分で通気し、１０％ＮａＯＨ溶液でｐＨ＝６．３になるように、反応の進行にともない、自動的に滴加した。２時間で、モノカルボン酸に変換した。
【００２２】
分離，精製；
上記反応液２リットルを８０００回転で冷却遠心器で分離し、菌体を除き、上澄液を活性炭（特製しらさぎクロマト炭，４００ｇ）カラムクロマトに付し、水で洗浄（１．２リットル）後、さらに水３リットルで溶出し、目的物の画分を集め、減圧濃縮し、β−Ｄ−フラクトシル−（２→１）−α−Ｄ−グルクロン酸ナトリウム塩１９．７０ｇの白色粉末を得た。
Ｎａ−塩；ｉｎ Ｄ_２Ｏ（９０ＭＨｚ）δｐｐｍ ６１．３９， ６２．８４， ７１．４８， ７２．４２， ７３．０９， ７３．６５， ７４．６５， ７６．８８， ８２．０１， ９２．７０， １０４．１８， １７６．４９．β−Ｄ−フラクトシル−（２→１）−α−Ｄ−グルクロン酸のマグネシウム塩の製造
β−Ｄ−フラクトシル−（２→１）−α−Ｄ−グルクロン酸のナトリウム塩１．８９ｇを水１０ｍｌにとかし、ついで、１Ｒ−１２０（Ｈ^＋型）ｃｏｌｕｍｎ（５０ｍｌ）に通導し、通過液に酸化マグネシウム０．１０３ｇを加え、１０分間撹拌した。ついでミリポアフィルター（Ｔｙｐｅ ＧＳ ０．２２μｍ）で濾過し、濾液を濃縮し、得られた粘稠なシロップをエタノール・アセトンで白色粉末のβ−Ｄ−フラクトシル−（２→１）−α−Ｄ−グルクロン酸のマグネシウム塩１．４５ｇを得た。
元素分析 Ｃ_２４Ｈ_３８Ｏ_２４Ｍｇ・６Ｈ_２Ｏ
計算値 Ｃ，３４．２０； Ｈ，５．９８
実測値 Ｃ，３４．３３； Ｈ，５．８２
【００２３】
培地組成
〔ＴＨ１４−８６株用〕
Ｓｅｅｄ 培地（１回目、２回目共通）
ラクトース １％
酵母エキス（プロディベル） １
（ＮＨ_４）_２ＳＯ_４ ０．３
コーン・スティープ・リカー ３
ＣａＣＯ_３（スーパー） ２
ＣａＣＯ_３ 投入前
ｐＨ＝７．０
バチリス メガトリウム株用
〔Ｓｅｅｄ 培地〕
シュークロース ４％
プロフロ［Ｎ源，トレイダス・プロテイン（Ｔ＆Ｐ）社］ ４
Ｋ_２ＨＰＯ_４ ０．６５
ＫＨ_２ＰＯ_４ ０．５５
ＮａＣｌ ０．０５
（ＮＨ_４）_２ＳＯ_４ ０．０５
ＭｇＳＯ_４・７Ｈ_２Ｏ ０．００５
パントテン酸カルシウム ０．０２５
滅菌前 ｐＨ＝７．０
〔Ｍａｉｎ 培地〕
シュークロース ０．０５％ 別滅菌
コーン・スティープ・リカー ２ 別滅菌
（ＮＨ_４）_２ＳＯ_４ ０．３ 別滅菌
ＦｅＳＯ_４・７Ｈ_２Ｏ ０．１ 別滅菌
Ｖ． Ｂ_２ １ｍｇ／Ｌ
滅菌前 ｐＨ＝７．０
ソルボース １０％ 別滅菌
ＬａＣｌ_３ ０．０１％ 別滅菌
ＣａＣＯ_３（スーパー） ４％ 別滅菌
【００２４】
実施例２ α−Ｄ−グルクロニル−（１→２）−β−Ｄ−６−フラクチュロン酸の製造
実施例１と同様な方法でα−Ｄ−グルクロニル−（１→２）−β−Ｄ−フラクチュロン酸のナトリウム塩，マグネシウム塩，カルシウム塩を得た。
ナトリウム塩；白色粉末
^１３Ｃ−ＮＭＲ（Ｄ_２Ｏ）ｐｐｍ：６１．９８， ７１．９６， ７３．０２， ７３．３５；７３．６９， ７６．３７， ７７． ０３， ７９．８２， ９３．９７， １０５．８０， １７５．０３， １７５．７０．
マグネシウム塩；白色粉末
元素分析 Ｃ_１２Ｈ_１６Ｏ_１３Ｍｇ・５Ｈ_２Ｏ
計算値 Ｃ，２９．８６； Ｈ，５．４３
実測値 Ｃ，２９．７６； Ｈ，５．５１
カルシウム塩；白色粉末
元素分析 Ｃ_１２Ｈ_１６Ｏ_１３Ｃａ・３Ｈ_２Ｏ
計算値 Ｃ，３１．１７； Ｈ，４．８０
実測値 Ｃ，３１．２６； Ｈ，４．９９
【００２５】
実施例３
実施例１に記載したシュードグルコノバクター・サッカロケトゲネス（Ｐｓｅｕｄｏｇｌｕｃｏｎｏｂａｃｔｅｒ ｓａｃｃｈａｒｏｋｅｔｏｇｅｎｅｓ）菌液１リットルにパラチノース３０ｇ，滅菌水１リットルを加え、３２℃，８００回転で、空気を１．６リットル／分で通気しながら、１時間反応させ、ついでこの反応液を８０００回転で冷却遠心器で分離し、菌体を除き、上澄液を活性炭（特製しらさぎクロマト炭 ４００ｇ）カラムクロマトに付し、水で洗浄（２リットル）ついで、１０％メタノール水溶液（４リットル）で溶出、さらに５０％メタノール水溶液（４リットル）で溶出される画分を集め、濃縮し、凍結乾燥し、β−Ｄ−フラクトシル−（６→１）−α−Ｄ−グルクロン酸のナトリウム塩３５．８ｇを得た。
このナトリウム塩をＩＲ１２０（Ｈ型）カラムに通導し、通過液を濃縮してβ−Ｄ−フラクトシル−（６→１）−α−Ｄ−グルクロン酸を得た。
^１３Ｃ−ＮＭＲ（Ｄ_２Ｏ）ｐｐｍ：６３．３５， ６８．６０， ７１．７８， ７２．５２， ７２．７７， ７３．４５， ７５．１４，７５．９９， ７９．５３， ９８．８１， １０２．２１， １７６．９３．
元素分析 Ｃ_１２Ｈ_２０Ｏ_１２・４．５Ｈ_２Ｏ
計算値 Ｃ，３２．９６； Ｈ，６．６８
実測値 Ｃ，３３．１７； Ｈ，５．８４
【００２６】
実施例４
実施例３と同様な方法で、以下の出発原料〔（ ）内に記載〕を用いて、対応する糖カルボン酸を得た。
【００２７】
【表２】

【００２８】
【表３】

【００２９】
【表４】

【００３０】
【表５】

【００３１】
【表６】

【００３２】
実施例５
実施例１に記載したシュードグルコノバクター サッカロケトゲネス（Ｐｓｅｕｄｏｇｌｕｃｏｎｏｂａｃｔｅｒ ｓａｃｃｈａｒｏｋｅｔｏｇｅｎｅｓ）菌液１リットルに、ステビア抽出物（ステビオシドを主成分とするステビオール配糖体）３０ｇをバイオット社製５リットルのジャーファーメンターに入れ、ついで滅菌水１リットルを加えて反応液とした。これを３２℃，８００回転で撹拌しながら、空気を１．６リットル／分で通気し、１０％ ＮａＯＨ溶液でｐＨ＝６．３になるように、反応の進行にともない、自動的に滴加した。１．５時間で、基質の消失が認められたので、反応を止め、ついで、反応液２リットルを８０００回転で冷却遠心器で分離し、菌体を除き、上澄液をＨＰ−２０（芳香族系合成吸着剤、三菱化成）カラム（１． ８リットル）に通導し、Ｈ_２Ｏ（８リットル）で洗浄後、５０％ＥｔＯＨ（５リットル）で溶出される画分より、グルクロン酸型ステビオール配糖体混合物が得られた。濃縮後、凍結乾燥により、白色粉末１９．２８ｇを得た。本品は強い甘味とさわやかな味質が認められた。
【００３３】
本品を下記条件でＨＰＬＣに付した。

本品の^１３Ｃ−ＮＭＲ（Ｄ_２Ｏ）ｐｐｍ：１７７．８７（ｅｓｔｅｒ）， １７１．８４（ＣＯＯ⁻）に、カルボニル炭素のシグナルが認められ、カルボキシ基に基づく、炭素の新生を認めた。ＨＰＬＣの知見から、本品は、ステビオシドのＣ_１９位に結合するグルコースと、Ｃ_１３位に結合するグルコースがグルクロン酸型に変換した化合物を主成分とすることが判った。
【００３４】
実施例６
実施例５と同様の方法で、ステビア抽出物（ステビオシドを主成分とするステビオール配糖体）３０ｇに、シュードグルコノバクター サッカロケトゲネス菌液１リットル、３２℃，８００回転、空気を１．６リットル／分で通気する条件で、酸化反応させ、５％ＮａＯＨ溶液５９ｍｌ（約２当量の酸化に相当）を滴下によって加えたところで、反応を停止した。所要酸化反応時間は２１時間であった。反応終了後、反応液を遠心濾過し、菌体を除去したあと、上清液（１８５０ｍｌ）を精製（実施例５と同様の方法）し、白色粉末のグルクロン酸型ステビオール配糖体混合物のＮａ 塩１３．０ｇを得た。本品は強い甘味とさわやかな味質が認められた。
ＨＰＬＣ；カラム ＯＤＰ，温度３５℃
移動層；ＣＨ_３ＣＮ：Ｈ_２Ｏ＝３：７の溶液にトリフロロ酢酸を０．１％添加
流速；１ｍｌ／分
ＨＰＣＬパターン（ＲＩ検出）を〔図２〕に示した。
【００３５】
実施例７
実施例５と同様の方法で、ステビオシド３０ｇを基質として、シュードグルコノバクター サッカロケトゲネス菌による酸化を行った。５％水酸化ナトリウム３０ｍｌを消費し、その反応時間は９０分であった。反応液を、精製し白色粉末２０ｇを得た。本品は高甘味かつ、清涼感のある甘味を呈した。
本品のＨＰＣＬパターン（ＲＩ検出）を〔図３〕に示した。この白色粉末４００ｍｇを逆相系カラムＯＤＰ−５０カラム（φ２１．５×３００ｍｍ 旭化成）と０．１％トリフロロ酢酸を含む水／アセトニトリル（７２：２８）の溶離液系を用い、主要成分を分取し、ついで凍結乾燥により白色粉末１２４ｍｇを得た。本品はメタノール：水＝９：１の混合溶媒から再結晶し、無色針状晶が得られた。
融点 ２２６−２３０℃（分解）．
ＩＲ（ＫＢｒ）ｃｍ−^１：３５００〜３２５０，１７１５，１６０５，１０８０−１０１０，８９０．
【表７】

元素分析 Ｃ_３８Ｈ_５７Ｏ_１９Ｎａ・１．５Ｈ_２Ｏ
計算値 Ｃ，５２．５９； Ｈ，６．９７
実測値 Ｃ，５２．５６； Ｈ，７．１５
以上から、本反応で得られた主要酸化生成物は前記式（ＩＩ）においてＲ_１＝−β−Ｇｌｃ−２−β−Ｇｌｃ，Ｒ_２＝−β−Ｇｌｃ ＵＡの化合物のナトリウム塩つまり下記式（ＩＶ）で表わされる １３−Ｏ−β−ソホロシル，１９−Ｏ−β−Ｄ−グルクロニルステビオール ナトリウム塩であると結論した。
【化９】

【００３６】
実施例８
実施例５と同様の方法で，ステビオシド３０ｇを基質としてシュードグルコノバクター サッカロケトゲネス菌による酸化を行った。８０分間で、５％水酸化ナトリウム溶液を６７ｍｌ消費した、反応液を精製し白色粉末３２ｇを得た。本品は高甘味でかつ清涼感のある甘味を呈した。本品のＨＰＣＬパターン（ＲＩ検出）を〔図４〕に示した。この白色粉末１ｇから、逆相系カラムＯＤＰ−５０（φ２１．５×３００ｍｍ 旭化成）と２％酢酸／アセトニトリルのグラディエント系を用いたＨＰＣＬの手法を用いて、主要酸化生成物を０．２２ｇを分取した。本品の二次イオン質量分析（ｓｅｃｏｎｄａｒｙ ｉｏｎ ｍａｓｓ ｓｐｅｃｔｒｏｍｅｔｒｙ． ＳＩ−ＭＳ）でｍ／ｚ８６９（Ｍ＋２Ｈ_２０・Ｈ^＋）のピークが観察できた。
^１３Ｃ−ＮＭＲ（ｄ_６−ピリジン）ｐｐｍ：１５．１２， １８．９５， ２０．２１， ２１．６９， ２７．７８， ３７．９１， ３７．９６， ３９．３３， ３９．４１， ４０．３５， ４２．１２， ４３．５８， ４３．９１， ４７．２３， ５３．５５， ５６．８６， ６２．５７， ７０．０７， ７１．５４， ７２．４９， ７２．７５， ７５．７５， ７６．１６， ７６．７９， ７６．８３， ７７．５１， ７７．７２， ７７．７８， ８６．００， ８６．９７， ９５．０７， ９７．６０， １０３．８９， １０３．９４， １５３．５０， １７１．２６， １７１．８９， １７６．２３．
以上の知見から本品は前記式（ＩＩ）において、Ｒ_１＝−β−Ｇｌｃ−２−β−Ｇｌｃ ＵＡ，Ｒ_２＝−β−Ｇｌｃ ＵＡの化合物のナトリウム塩つまり下記式（Ｖ）で表される１３−Ｏ−β−Ｄ−グルクロニル−β−Ｄ−グルコシル，１９−Ｏ−β−Ｄ−グルクロニルステビオール ２ナトリウム塩と結論した。
【化１０】

【００３７】
実施例９
実施例５の方法に従って、ステビオシド３０ｇをバイオット社製５リットルのジャーファーメンターに入れ、ついで滅菌水１．６リットルを加えて反応液とした。これを３２℃，８００回転で撹拌しながら、空気を１リットル／分で通気し、炭酸カルシウム２０ｇを加え、４時間反応させた。ついで、反応液２リットルを８０００回転で冷却遠心器で分離し、菌体を除き、上澄液をＨＰ−２０（芳香族系合成吸着剤、三菱化成）カラム（１．８リットル）に通導し、Ｈ_２Ｏ（８リットル）で洗浄後、５０％エタノール（５リットル）で溶出される画分より、ウロン酸型ステビオシドのカルシウム塩が得られた。濃縮後、凍結乾燥により、白色粉末１２．９８ｇを得た。これをメタノールから再結晶し無色粉末状晶を得た。本品は弱い甘味が認められた。
本品の^１３Ｃ−ＮＭＲ（ｄ_６−ピリジン）ｐｐｍ；１５．９３， １９．７４， ２０．９２， ２１．００， ２８．５８， ３８．６６， ３８．７１， ４０．０６， ４１．０４， ４１．８６， ４２．００， ４２．９１， ４４．３３， ４８．１９， ５４．５０， ５７．６３， ７３．１１， ７３．３０， ７３．４５， ７３．９７， ７４．０８， ７６．５３， ７７．７２， ７７．８６， ７７．８９，７８．３４， ７８．３９， ８４．７９， ８６．７９， ９５．８４， ９８．２１， １０５．４６， １０７．０２， １５３．７０， １７１．９３， １７２．１８， １７２．９５， １７６．７８．
以上の知見から、本品は前記式（ＩＩ）において、Ｒ_１＝−β−Ｇｌｃ ＵＡ−２−β−Ｇｌｃ ＵＡ，Ｒ_２＝−β−Ｇｌｃ ＵＡの化合物のカルシウム塩つまり下記式（ＶＩ）で表される１３−Ｏ−β−Ｄ−グルクロニル−β−Ｄ−グルクロニル−１９−Ｏ−β−Ｄ−グルクロニルステビオール カルシウム塩と結論した。
【化１１】

【００３８】
実施例１０
実施例５と同様の方法で、レバウディオシドーＡ１０ｇを基質として、シュードグルコノバクター サッカロケトゲネス菌体１５．８ｇ、滅菌水２０００ｍｌを加え３２℃，８００回転、空気１．６リットル／分の条件で撹拌しながら行い、反応は２％ＮａＯＨ溶液を加え、ｐＨ６．３にコントロールした。２％ＮａＯＨ２３ｍｌを消費したところで反応を停止し、遠心分離を行い上清を分離した。反応時間は１３５分を要した。上清液を実施例５と同様な精製法により、処理し、白色粉末１０．１６ｇを得た。
本品のＨＰＬＣパターン（ＲＩ検出）を〔図５〕に示した。
ＩＲ（ＫＢｒ）ｃｍ^−１：３３４０， １７２０， １６１０， １４１０， １０７０ また本品は高甘味で、清涼感のあるすぐれた甘味質である。
【００３９】
実施例１１
実施例１に記載したシュードグルコノバクター サッカロケトゲネス菌液１リットルにステビオシド３０ｇをバイオット社５リットルのジャーファーメンターに入れ、ついで滅菌水１リットルを加え、さらに、アンバーライト ＩＲＡ−６８（以下単にＩＲＡ−６８と略す）２００ミリリットルを加えて反応液とした。これを３２℃，８００回転で撹拌しながら、空気１．６リットル／分で通気した。２時間で、上澄液にステビオシドが認められなくなったので、反応を停止し、遠心分離により、上清とＩＲＡ−６８とを分け、ＩＲＡ−６８は、カラム（φ４×２４ｃｍ）につめ、ついで、２Ｎ−食塩水２リットルで溶出し、溶出画分をＨＰ−２０のカラム（０．５リットル）に通導した。水１リットルで洗浄後、５０％メタノール０．８リットルで溶出し、濃縮乾涸し、白色粉末９．２０ｇを得た。
本品をメタノール：水＝９：１の混合溶媒から再結晶し無色針状晶を得た。
ｍｐ．２２６−２３０℃（分解）
本品は実施例７に示した、１３−Ｏ−β−ソホロシル−１９−Ｏ−β−グルクロニルステビオール ナトリウム塩（ＩＶ）と認定した。
【００４０】
実施例１２
実施例１１に記載した方法に順じて、シュードグルコノバクター サッカロケトゲネス菌液１リットルにステビア抽出物（少なくとも６種のステビオール配糖体混合物）３０ｇをバイオット社５リットルのジャーファーメンターに入れ、ついで滅菌水１リットルを加え、さらにＩＲＡ−６８ ２００ミリリットルを加えて反応液とした。これを３２℃，８００回転で撹拌しながら、空気を１．６リットル／分で通気した。２時間で、上清液にステビオシドが認められなくなったので反応を停止し、遠心分離により、上清とＩＲＡ−６８とを分け、ＩＲＡ−６８はカラム（φ４×２５ｃｍ）につめ、ついで２Ｎ−ＮａＣｌ １リットルで溶出し、溶出画分をＨＰ−２０カラム（０．５リットル）に通導した。水１リットルで洗浄後５０％メタノール１リットルで溶出し濃縮乾涸し白色粉末８．９ｇを得た。
本品のＨＰＬＣパターン（ＲＩ検出）を〔図６〕に示す。
ＩＲ（ＫＢｒ）ｃｍ^−１：３５００〜３２００， １７２０， １７０５， １６１０， １４００ を示した。
【００４１】
実施例１３
糖転移ステビア配糖体（ＳＫスィート、山陽国策パルプ（株）製造）３０ｇを実施例６と同様の方法で、酸化し、精製処理し、白色粉末２８．３ｇを得た。本品の^１３Ｃ−ＮＭＲ（Ｄ_２Ｏ）で、カルボニル炭素の領域に８本のシグナル（１７９．５０，１７９．４６， １７９．４０， １７９．３７， １７９．３２， １７９．２６， １７７．１０， １７７．０７ ｐｐｍ）が観察されたことから、少なくとも糖の１級酸基の１つ以上がカルボン酸に変換したことが認められた。本品は、清涼感のあるさわやかな甘味を呈した。
【００４２】
実施例１４
羅漢果の果実に含まれる高甘味配糖体モグロシドＶ、２００ｍｇを実施例１１に記載した方法に順じて、シュードグルコノバクター サッカロケトゲネス菌１０ｍｌ分（０．３２ｇ）を水３ｍｌに懸濁して加え、ついでＩＲＡ−６８ ５ｍｌを加えた。この混合液を、３０℃，２２９回転で５時間半撹拌した。反応液を静置して、上清を除いたあと、ＩＲＡ−６８をカラムにつめ、水洗（５０ｍｌ）後、０．０１Ｎ ＨＣｌで溶出、溶出画分を集め、凍結乾燥し、白色粉末（３２ｍｇ）を得た。本品はＩＲ（ＫＢｒ）ｃｍ^−１：３３００， １６００， １４１０， １０６０−１０００ であることから、糖鎖がグルクロン酸型に変換されたものと確認した。本品は砂糖のような甘味を呈した。
【００４３】
実施例１５
ルブソシド１．０ｇを滅菌水６０ｍｌにとかし、これに、ＩＲＡ−６８ １３ｍｌを加え、ついで、実施例１に記載したシュードグルコノバクター サッカロケトゲネス（Ｐｓｕｅｄｏｇｌｃｏｎｏｂａｃｔｅｒ ｓａｃｃｈａｒｏｋｅｔｏｇｅｎｅｓ）菌液３０ｍｌを加え、３２℃，６００回転／分で撹拌しながら、空気を６０リットル／分で通気し、２４０分間撹拌しながら反応させた。反応液から、ＩＲＡ−６８をとり出し、カラムに充填し、水１５ｍｌで水洗したあと、２Ｎ−ＮａＣｌ溶液をＳＶ０．５で２００ｍｌを用いて溶出し、溶出液をＨＰ−２０カラム（２０ｍｌ）に吸着し、水１５０ｍｌで洗った後、５０％ＭｅＯＨ溶液（１００ｍｌ）で、ＳＶ０．５で溶出、溶出液を濃縮乾固し、析出物をメタノールから再結晶し、無色粒状晶０．４８ｇを得た。
ｍ．ｐ． １８４−１８８℃（分解）
元素分析 Ｃ_３２Ｈ_４７Ｏ_１４Ｎａ・５Ｈ_２Ｏ
理論値 Ｃ，４９．９９； Ｈ，７．４７
実測値 Ｃ，５０．０５； Ｈ，７．５４
【表８】

以上のデータから、その構造を（ＶＩＩ）と表わされると結論した。
【化１２】

また本品（ＶＩＩ）は、にがみのない、砂糖のような良好な高甘味物質であった。
【００４４】
実施例１６
塩酸フルスルチアミン １０ｍｇ
ビタミンＢ_２ ２ｍｇ
タウリン １０００ｍｇ
安息香酸 ３０ｍｇ
パラオキシ安息香酸ブチル ２．５ｍｇ
グルクロン酸型ステビオシドナトリウム塩（ＩＶ） ５０ｍｇ
クエン酸 １００ｍｇ
グリシン ５００ｍｇ
上記組成に従い、下記方法によりドリンク剤を得た。すなわち約７０℃に加温した水約３０ｍｌにパラオキシ安息香酸ブチル、安息香酸を溶解し、約２５℃まで冷却後、塩酸フルスルチアミン，ビタミンＢ_２，タウリン，甘味料としてのグルクロン酸型ステビオシド，クエン酸，グリシンを溶解し、１Ｎ−かせいソーダを添加し、ｐＨを３．５としたのち、水を加えて全量５０ｍｌとする。
【００４５】
実施例１７
沈降炭酸カルシウム３０ｇ，炭酸マグネシウム５ｇ，乳糖５８．５ｇ，ヒドロキシプロピルセルロース６ｇを均一に混合し、水３００ｍｌを加え、上記の混合物を造粒する。得られた顆粒を乾燥した後粉砕する。得られた粉末と矯味剤としてのグルクロン酸型ステビオシド０．０５ｇ及びステアリン酸マグネシウム０．５ｇを添加して混合し、常法に従って打錠することによって、直径８．５ｍｍで１錠２００ｍｇの錠剤を得た。
【００４６】
実施例１８
実施例１に記載したシュードグルコノバクター サッカロケトゲネス（Ｐｓｅｕｄｏｇｌｕｃｏｎｏｂａｃｔｅｒ ｓａｃｃｈａｒｏｋｅｔｏｇｅｎｅｓ）菌液１．５リットルにデキストラン−４（分子量：４０００〜６０００；エキストラシンテーゼ社（仏国）製）３０ｇをバイオット社製５リットルのジャーファンメンターに入れ、ついで滅菌水１．７リットルを加え反応液とした。これを３２℃，６００回転／分で撹拌しながら、空気を１．６リットル／分で通気し、０．５％ＮａＯＨ溶液でｐＨ＝６．３になるように反応の進行にともない自動的に滴加した。３時間で反応を止めついで、反応液１２．０リットルを８０００回転で冷却遠心器で分離し、菌体を除き、上澄液１．９８リットルを得た。この上澄液をセルロースアセテートメンブランフィルター（φ０．２〜μｍ）で濾過し、再度除菌化した濾液を、アンバーライト ＩＲＡ−６８（ＯＨ型）カラム（５０ｍｌ）に通導し、少量の水（１００ｍｌ）で洗い、通過液２リットルを得た。この通過液をＨＰ−２０カラム（９００ｍｌ）に通導した。ついで、水２リットルで溶離し、初めの通過液７００ｍｌはすて、ついで溶出される液３．３リットルを集め、これに濃塩酸１３．８ｍｌを加え、酸性とし、これを、セルロースアセテートメンブランフィルター（φ０．２μｍ）で濾過し、濾液をセファビーズ（商品名）ＳＰ２０５（三菱化成社製）１０００ｍｌに通導した。ついで、０．０５Ｍ塩酸７００ｍｌ，水２リットルで洗浄後、２０％ＭｅＯＨ溶液２リットルで溶出し、目的物の画分を得た。これを減圧濃縮し、デキストラニル−グルクロン酸の白色粉末１４ｇを得た。本品のＨＰＬＣ（条件：アサヒパックＧＳ３２０（φ７．６ｍｍ×５０ｍｍ；旭化成社製）、移動相；水１ｍｌ／ｍｉｎ，検出；ＲＩ（ウォータース４１０）及び２００ｎｍ（東ソー ＵＶ−８０２０）サンプル０．５％水溶液２０μｌ 注入）でＲｔ＝８．４８に単一のピークを示した。（原料のデキストラン４のＲｔ＝１０．８３であった）
本品の構造確認のため、酵素消化処理実験を行った。本品の１．５％水溶液１００μｌ にグルコアミラーゼ（和光純薬製）溶液（４ｍｇ／ｍｌ）１００μｌ を加え、３０℃，１昼夜インキュベーションを行い、この酵素処理液をＨＰＬＣで検討を行った。対象実験として、デキストラン−４も同様にグルコアミラーゼ処理を行った。その結果、本品はグルコアミラーゼ消化を受けないことが分った。一方デキストラン−４は、グルコアミラーゼ消化を受け、基質は消失し、グルコースが新生した。このことから、本品は、上記式（ＶＩＩＩ）（但し、式中、ｎ＝１５）で示されるデキストラニルグルクロン酸と確認した。
【００４７】
実施例１９
実施例１に準じて、シュードグルコノバクター サッカロケトゲネス（Ｐｓｅｕｄｏｇｌｕｃｏｎｏｂａｃｔｅｒ Ｓａｃｃｈａｒｏｋｅｔｏｇｅｎｅｓ）Ｋ５９１Ｓ菌株をバクトペプトン１％，イーストエキス１％からなるペプトンイースト培地（ＰＹ）培地を用いて、３０℃、２３０回転の振とう下ｐＨ７．５で３日間培養を行った。ついで、メイン培養に移し、ペプトンイースト培地（ＰＹ培地）で、０．１％の塩化カルシウムを添加し、３０℃、２３０ｒｐｍ で振とうしながら、４８時間培養した。ついで、培養液を１００００回転で、１０分間遠心分離し、集菌し、ついで水５００ｍｌで洗菌し、ブロース１リットル当り、７．５９ｇの菌体を得た。ついで、デキストラン−４（分子量４０００〜６０００；エキストラシンテーゼ社（仏国）製）３０ｇを水１リットルにとかし、ついで湿菌体５６ｇを水１リットルに懸濁した液を加え、３２℃、８００ｒｐｍ で撹拌しながら、空気を１分間６０ｍｌで通気し、０．１％ＮａＯＨ溶液でｐＨ６．３に制御しながら６．５時間反応させた。反応液を遠心分離し、上清２リットルを分取し、メンブランフィルターで濾過除菌し、ＨＰ−２０カラム（９００ｍｌ）に通導し、水２０００ｍｌで溶出した。通過液に最終濃度０．０５Ｍになるように、濃塩酸を加え、酸性とし、メンブランフィルターで濾過したあと、濾液をＳＰ−２０５カラム（１００ｍｌ）に通導し、初め、０．０５Ｍ ＨＣｌ（１０００ｍｌ）、ついで水（２０００ｍｌ）で洗浄後、２０％ＭｅＯＨ（２０００ｍｌ）で溶出される画分を集め、濃縮後、凍結乾燥し、１４ｇの白色粉末のデキストラニルグルコン酸を得た。本品のＨＰＬＣ〔条件：アサヒパックＧＳ３２０（φ７．６ｍｍ×５０ｍｍ：旭化成社製）、移動相：水１ｍｌ／ｍｉｎ，ＲＩ（ウォータース４１０）及び２００ｎｍ（東ソーＵＶ−８０２０）、サンプル０．５％％水溶液２０μｌ 注入〕でＲｔ＝８．７０に単一のピークを示した。本品を前記した条件でグルコアミラーゼ消化を行ったところ、容易に消化され、グルコースとグルコシルグルコン酸を検出することができた。従って、本化合物をデキストラニルグルコン酸と確認した。
【００４８】
実施例２０
実施例１９と同様にして、シュードグルコノバクター サッカロケトゲネス（Ｐｓｅｕｄｏｇｌｕｃｏｎｏｂａｃｔｅｒ Ｓａｃｃｈａｒｏｋｅｔｏｇｅｎｅｓ）菌を培養しこの菌体（湿菌体）５０ｇをデキストラニルグルクロン酸（実施例１８で記載の方法で調製した）３０ｇを水１リットルに溶解させた溶液に加え、３２℃、８００ｒｐｍ で撹拌しながら、空気を１分間６０ｍｌで通気し、０．１％ＮａＯＨ溶液でｐＨ６．３に制御しながら、２１時間反応させた。反応液を遠心分離し、上清２リットルを分取し、メンブランフィルターで濾過除菌し、ＨＰ−２０カラム（９００ｍｌ）に通導し、水１．５リットルで溶出し、目的画分を集め、濃縮後凍結乾燥し１５ｇの上記式（ＩＸ）で表わされるグルクロニルデキストラニルグルコン酸のナトリウム塩の白色粉末１２ｇを得た。
ＨＰＬＣ：Ｒｔ＝９．１２ １ピーク
ＨＰＬＣ条件；カラム：ＧＳ−３２０
移動相：Ｈ_２Ｏ，流速１ｍｌ／分
検 出：ＲＩ
^１３Ｃ−ＮＭＲ（Ｄ_２Ｏ）δｐｐｍ： １７９．５５４， １７８．１１９， １７２．４３４ に観察され、その強度が１：０．５：０．５で、その帰属は、グルクロン酸部のカルボニル炭素を１７９．５５４ とし、他はグルコン酸部（１部ラクトン型を想定）のカルボニル炭素とした。
【００４９】
実施例２１
塩化第２鉄・６水和物の５０％水溶液５０ｍｌを３０℃に加温し、ついで２４％Ｎａ_２ＣＯ_３溶液５０ｍｌを１分間０．４ｍｌの速度でよく撹拌しながら滴加する。ついで、実施例１８で得たデキストラニルグルクロン酸を用いた１０％デキストラニルグルクロン酸溶液５０ｍｌを、３ｍｌ／分の速度で滴加し、１６％Ｎａ_２ＣＯ_３溶液を０．４ｍｌ／分の速度で加え、ｐＨ４．３に調整した。次に、エタノール２００ｍｌを加え、強く撹拌しスラリー状にし、遠心分離（５０００ｒｐｍ）し、沈殿を集めついで、水４０ｍｌに溶かし、６０ｍｌのエタノールでよくかくはんし、遠心分離し可溶物を除き、沈殿物に水２０ｍｌを加え、よく分散させ、減圧下、エバポレーターでよくエタノールを除き、１０％ＮａＯＨ溶液で０．２ｍｌ／分で速度でよく撹拌しながら加え、ｐＨ５〜６に調整し、１００℃，２０分間加熱し、ついで、フェノールを４ｍｇ／ｍｌになるように加え、デキストラニルグルクロン酸水酸化鉄ゾル溶液２２ｍｌを得た。本品の１ｍｌの幼若ブタへの筋肉内投与により貧血症状を著しく改善する効果が認められた。デキストラニルグルコン酸水酸化鉄ゾルも同様な方法で製造することができ、また幼若ブタの貧血を著しく改善する効果が認められた。
【００５０】
実施例２２
デキストラニルグルコン酸の水酸化鉄ゾルの製造法 塩化第２鉄（ＦｅＣｌ_３・６Ｈ_２Ｏ）１００ｇを１００ｍｌの水に溶解させ、この溶液をＢｒａｎｓｏｎ Ｂ−２２０で超音波処理を１時間行い、充分溶解させる。ついで、水を加え、２００ｍｌとし、５００ｍｌのビーカーに溶液を移す。ついで２４％炭酸ソーダ溶液２００ｍｌをよく撹拌しながら３０℃，０．８ｍｌ／分の速度で添加し淡黄褐色の水酸化鉄ゾルを調製した。
このようにして調製した水酸化鉄ゾル１００ｍｌを分取し、３００ｍｌのビーカーに移し、３０℃でよく撹拌しながら、１０％デキストラニルグルコン酸溶液５０ｍｌを徐々に加える。ついで、１６％ＮａＣＯ_３溶液を、極めてゆっくり、０．１〜０．２ｍｌ／分の速度で加え、ｐＨを４．３に調整した。ついで、エタノール２００ｍｌを撹拌しながら加え、生じた沈澱を遠心分離し、上清と分け、沈澱を水８０ｍｌを加え、よく懸濁させたあと、エタノール１２０ｍｌを加え、再沈澱させ、沈澱物を遠心分離し、分取する。この操作をさらにもう一回繰り返して得た沈澱物を水２０ｍｌで懸濁、よく撹拌し１０％ＮａＯＨ溶液を０．１〜０．２ｍｌ／分の速度で加え、ｐＨを６．０になるまで加えた。この溶液を１２０℃１０分間、オートクレーブ処理し、防腐剤としてフェノール（１％含となるように）を加え、ついで、エバポレーターで濃縮し、デキストラニルグルコン酸の水酸化鉄ゾル溶液を調整した。本品は、安定した水酸化鉄ゾルを形成した。その性状を分析したところ、以下のとおりであった。
総鉄塩濃度：２００ｍｇ／ｍｌ，粘度；３２ｃＰ，電導度４７ｍＳ／ｃｍであった。
【００５１】
実施例２３
デキストラニルグルクロン酸の透析法による鉄塩化 塩化第２鉄・６水和物の５０％水溶液５０ｍｌを３０℃に加温し、ついで２４％Ｎａ_２ＣＯ_３溶液５０ｍｌを１分間０．３０ｍｌの速度でよく撹拌しながら滴加し、やや黒味がかった黄土色の水酸化鉄ゾル（ｐＨ１．５４）を得た。この水酸化鉄ゾルを、透析膜（ｄｉａｌｙｓｅｓ ｔｕｂｅ）に移し、超純水中で一夜透析した。透析後の水酸鉄ゾルは約１７０ｍｌ、ｐＨ４．４７黒褐色を呈した。ついで、この透析処理した水酸鉄ゾルに、実施例１８で得たデキストラニルグルクロン酸の１０％水溶液５０ｍｌを加え、よく撹拌し、１６％Ｎａ_２ＣＯ_３溶液でｐＨ４．３に調整した。これを１００℃、３０分間オートクレーブ処理した後、１０％ＮａＯＨ溶液を加え、ｐＨ１２に調整し、再度１２１℃、２０分間オートクレーブ処理をし、完全溶解させ、黒褐色のデキストラニルグルクロン酸の水酸化第２鉄塩複合体が得られた。次いでこれを透析膜で一夜透析後濃縮しこれに、フェノールを４ｍｇ／ｍｌになるように加え、デキストラニルグルクロン酸水酸化第２鉄塩複合体の注射剤２２ｍｌを得た。本品は安定したゾル溶液であり、その性状を分析したところ以下のとおりであった。
総鉄塩濃度：２０１ｍｇ／ｍｌ，粘度２３．９ｃＰ，電導度３．７ｍＳ／ｃｍであった。
本品の１ｍｌの幼若ブタへの筋肉内投与により貧血症状を著しく改善する効果が認められた。デキストラニルグルクロン酸水酸化第２鉄塩複合体も同様な方法で製造することができ、また幼若ブタの貧血を著しく改善する効果が認められた。
【００５２】
実施例２４
実施例１１と同様にして、シュードグルコノバクター サッカロケトゲネス菌液１リットルにレバウディオシド−Ａ３０ｇをバイオット社製５リットルのジャーファーメンターに入れ、ついで滅菌水１．５リットルを加え、さらにＩＲＡ−６８ ４００ｍｌを加えて、反応液とした。これを３２℃、６００回転で撹拌しながら、空気１．６リットル／分で通気した。１時間で、上澄液にレバウディオシド−Ａが認められなくなったので、反応を停止し、遠心分離により上清とＩＲＡ−６８とを分け、ＩＲＡ−６８はカラム（φ４×４０ｃｍ）につめ、ついで２Ｎ−食塩水（８．１リットル）で溶出し、溶出画分をＨＰ−２０（１リットル）カラムに通導、吸着せしめ、水洗（８リットル）後、１０％〜５０％エタノールで溶出することにより、目的とするグルクロン酸型レバウディオシド−Ａのナトリウム塩が得られた。濃縮後、凍結乾燥することにより、無色粉末１９．６ｇを得た。これをＭｅＯＨ−Ｈ_２Ｏから再結晶することにより、無色針状晶１０．６ｇを得た。ｍ．ｐ．２２０℃（分解）本品のＨＰＬＣ（ＯＤＰカラム，アセトニトリル：水＝３０：７０）はＲｔ＝９．７０に１本のピークを与えた。本品の^１３Ｃ−ＮＭＲ（Ｄ_２Ｏ）ｐｐｍ：１８．０６， ２１．５２， ２２．９１， ２４．１９， ３０．８９， ３９．５１， ４０．１５， ４２．０２， ４３．０１， ４３．７３， ４４．５８， ４６．６９， ４６．７９， ４９．８９， ５６．１７， ５９．６４， ６３．５１， ６３．７１， ６４．３７， ７１．３６， ７２．４３， ７３．１４， ７４．４０， ７４．６７， ７６．２９， ７６．９９， ７８．１２， ７８．７０， ７８．７２，７８．８１， ７８．９３， ７９．２７， ７９．３８， ８１．４９， ８７．９９， ９０．３１， ９６．６４， ９８．７０， １０４．８７， １０５．０６， １０７．３２， １５５．９９， １７７．７９， １８１．６４
以上の知見から、本品は、前記式（ＩＩ）において、
【化１３】

の化合物のナトリウム塩つまり下記式（Ｘ）で表わされると結論した。
【化１４】

【００５３】
試験例
実施例４で得た６−Ｏ−α−Ｄ−グルクロニル（１→４）−α−Ｄ−グルコシル−β−シクロデキストリンＮａ 塩（１）の各種酵素に対する安定性試験を下記方法により行った。比較対照として、６−Ｏ−α−マルトシル−β−シクロデキストリンを用いた。
試験方法
１０ｍＭの供試シクロデキストリン水溶液に、下記酵素の所定量をそれぞれ加えた後、３７℃の温浴中で静置する。５００μｌ ずつサンプルを分取し、１００℃で１５分間加熱することにより酵素を失活させた後、遠心分離（１５，０００ｒ．ｐ．ｍ． ５分）する。さらにミリポアＵＳＹ−１（分画分子量１０，０００）で濾過する。１０倍に希釈して下記条件によりＨＰＬＣ分析に付した。
ＨＰＬＣ 分析条件；
カラム；ＮＨ２Ｐ−５０（Ａｓａｈｉｐａｋ）
移動相；ＣＨ_３ＣＮ：Ｈ_２Ｏ＝４８：５２にＰＩＣ試薬０．００５Ｍを添加した。
流 速；０．８ｍｌ／分
検 出；ＲＩ
上記操作で得られた各試料のＨＰＩＣから、１２０分までの時間ごとのシクロデキストリンの残存率を求めた。
【００５４】
使用酵素と酵素濃度；
【表９】

結果
６−Ｏ−α−Ｄ−グルクロニル（１→４）−α−Ｄ−グルコシル−β−シクロデキストリンＮａ 塩（１）のおよび６−Ｏ−α−マルトシル−β−シクロデキストリンの各酵素による残存率の経時変化を〔図１１〕〜〔図１４〕に示す。
（１）は、α−アミラーゼ，グルコアミラーゼ，プルラナーゼの酵素処理に対して、対照とした６−Ｏ−α−マルトシル−β−シクロデキストリンに比し、安定であることが判明した。
一方プルラナーゼの酵素処理に対する検討では、対照とした６−Ｏ−α−マルトシル−β−シクロデキストリンが約６０％分解されるのに対して、（１）は、安定であった。
【図面の簡単な説明】
【図１】は実施例５で得られたグルクロン酸型ステビオール配糖体混合物のＨＰＬＣパターン（ＲＩ検出）を示す。
【図２】は実施例６で得られたグルクロン酸型ステビオール配糖体混合物のＨＰＬＣパターン（ＲＩ検出）を示す。
【図３】は実施例７で得られたグルクロン酸型ステビオール配糖体ナトリウム塩のＨＰＬＣパターン（ＲＩ検出）を示す。
【図４】は実施例８で得られたグルクロン酸型ステビオール配糖体２ナトリウム塩のＨＰＬＣパターン（ＲＩ検出）を示す。
【図５】は実施例１０で得られたレバウディオシド−Ａの１当量酸化体のＨＰＬＣパターン（ＲＩ検出）を示す。
【図６】は実施例１２で得られたグルクロン酸型ステビオール配糖体混合物のＨＰＬＣパターン（ＲＩ検出）を示す。
【図７】は実施例４で得られた６−Ｏ−（２−カルボキシエチル）−β−シクロデキストリンＮａ 塩と６，６−ジ−Ｏ−（２−カルボキシエチル）−β−シクロデキストリンの^１３Ｃ−ＮＭＲ（２７０ＭＨｚ，Ｄ_２Ｏ）δｐｐｍ のチャートを示す。
【図８】は実施例４で得られた６−Ｏ−α−Ｄ−グルクロニル−（１→４）−α−Ｄ−グルコシル−α−シクロデキストリンの^１３Ｃ−ＮＭＲ（２７０ＭＨｚ，Ｄ_２Ｏ）δｐｐｍ のチャートを示す。
【図９】は実施例４で得られた６−Ｏ−α−Ｄ−グルクロニル−β−シクロデキストリンの^１３Ｃ−ＮＭＲ（２７０ＭＨｚ，Ｄ_２Ｏ）δｐｐｍ のチャートを示す。
【図１０】は実施例４で得られた６−Ｏ−（２−カルボキシ２−ハイドロキシエチル）−β−シクロデキストリンＮａ 塩と６，６−ジ−Ｏ−（２−カルボキシ２−ハイドロキシエチル）−β−シクロデキストリンＮａ 塩の^１３Ｃ−ＮＭＲ（２７０ＭＨｚ，Ｄ_２Ｏ）δｐｐｍ のチャートを示す。
【図１１】は試験例で得られた６−Ｏ−α−Ｄ−グルクロニル（１→４）−α−Ｄ−グルコシル−β−シクロデキストリンＮａ塩及び対照の６−Ｏ−α−マルトシル−β−シクロデキストリンのα−アミラーゼに対する安定性試験の結果を示し、両化合物をα−アミラーゼ処理した場合の残存率の経時変化を示す。
【図１２】は試験例で得られた６−Ｏ−α−Ｄ−グルクロニル（１→４）−α−Ｄ−グルコシル−β−シクロデキストリンＮａ塩及び対照の６−Ｏ−α−マルトシル−β−シクロデキストリンのグルコアミラーゼに対する安定性試験の結果を示し、両化合物をグルコアミラーゼ処理した場合の残存率の経時変化を示す。
【図１３】は試験例で得られた６−Ｏ−α−Ｄ−グルクロニル（１→４）−α−Ｄ−グルコシル−β−シクロデキストリンＮａ塩及び対照の６−Ｏ−α−マルトシル−β−シクロデキストリンのプルラナーゼに対する安定性試験の結果を示し、両化合物をプルラナーゼ処理した場合の残存率の経時変化を示す。
【図１４】は試験例で得られた６−Ｏ−α−Ｄ−グルクロニル（１→４）−α−Ｄ−グルコシル−β−シクロデキストリンＮａ塩及び対照の６−Ｏ−α−マルトシル−β−シクロデキストリンのβ−グルクロニダーゼに対する安定性試験の結果を示し、両化合物をβ−グルクロニダーゼ処理した場合の残存率の経時変化を示す。
【符号の説明】
〔図１１〕〜〔図１４〕において、●は６−Ｏ−α−マルトシル−シクロデキストリンを、▲は６−Ｏ−α−Ｄ−グルクロニル（１→４）−α−Ｄ−シクロデキストリンＮａ塩を示す。[0001]
[Industrial applications]
The present invention relates to a novel sugar carboxylic acid or a salt thereof, and a method for producing the novel sugar carboxylic acid. Specifically, a method for efficiently producing a corresponding carboxylic acid from a saccharide having a primary hydroxyl group (hydroxymethyl group) using a bacterium of the genus Pseudogluconobacter or a processed product thereof, hydroxymethyl contained in the saccharide A novel sugar carboxylic acid in which the group is oxidized to a carboxyl group, the hemiacetal hydroxyl group of a saccharide having a hemiacetal hydroxyl group is converted to a carboxyl group using a bacterium of the genus Pseudogluconobacter or a treated product thereof. The present invention relates to a method for producing a corresponding carboxylic acid by oxidation and a novel sugar carboxylic acid in which a carbon atom having a hemiacetal hydroxyl group is oxidized to a carboxyl group.
[0002]
[Prior art]
Examples of microorganisms that catalyze the oxidation of a hydroxymethyl group to a carboxyl group include, for example, bacteria of the genus Acetobacter [Agricultural Biological Chemistry (Agric. Biol. Chem.), Vol. 42, 2331 (1978)], Alcohol dehydrogenases such as bacteria of the genus Novobacter [Agricultural Biological Chemistry (Agric. Biol. Chem.), Vol. 42, 2045 (1978)] or bacterium of the genus Pseudomonas [for example, Biochemical Journal (Biochem)]. J.), Vol. 223, Item 921 (1984)], methanol dehydrogenase of methanol bacteria [Agricultural Biological Chemistry, Agric. Biol. Chem., Vol. 54, Item 3123 (1). 990)] are known, but none of them has a limited substrate specificity, and there is no report that they act on other than alcohols such as methanol and ethanol.
[0003]
Gluconobacterium is a microorganism that acts on saccharides such as D-sorbitol and L-sorbose (hereinafter sometimes simply referred to as sorbose) to catalyze the oxidation of hydroxymethyl groups to carboxyl groups. D-sorbitol dehydrogenase [Agricultural Biological Chemistry (Agric. Biol. Chem.), Vol. 46, paragraph 135 (1982)], sorbose dehydrogenase [Agricultural Biological Chemistry (Agric. Biol. Chem.), 55, 363 (1991)], glucose dehydrogenase [Agricultural Biological Chemistry, 44, 1505 (1980)] and the like. As is known, these Also, its substrate specificity are limited, which is insufficient from the viewpoint of versatility. In addition, a soil-separating bacterium, already named Pseudogluconobacter saccharoketogenes, produced a remarkable amount of 2-keto-L-gulonic acid from L-sorbose (see JP-A-62-228288 and JP-A-64-85088). It has also been found that 2-keto-D-glucaric acid is produced from D-glucose or the like (see Japanese Patent Application Laid-Open No. 62-228287).
[0004]
On the other hand, among polysaccharides, for example, dextran is a generic name for high molecular glucans mainly composed of α1 → 6 bonds generated from sucrose by leuconostoc mesenteroides belonging to lactic acid bacteria and the like. Various methods have been tried, but it is difficult to obtain a product with a regioselectively high reaction rate in a commonly used chemical reaction, and a large number of by-products are given. As described above, many polysaccharides such as dextran are composed of homologues of various polymers having different molecular weights.Thus, in chemical modification, various side reactions occur to complicate the product and the product There are many cases in which the substance cannot be clarified. [See "Biotransformations in Preparative Organic Chemistry HG Davis et al, Academic Press."] [Biotransformations in Preparative Organic Chemistry HG Davis et al, Academic Press]
In particular, attempts have been made to chemically oxidize dextran with oxidants such as sodium hypochlorite, sodium hypobromite, chlorine, bromine, and iodine, but the structure of the resulting product is not always clear. Or, there is not enough support. For example, chemical oxidation of dextran is described in, for example, a Japanese application by Tosuni, Coelho and Patel (Patent Publication No. 61-23001).
[0005]
[Problems to be solved by the invention]
As described above, conventionally, oxidation of a compound such as a saccharide having a hydroxymethyl group to a carboxylic acid using a microorganism has been extremely limited to monosaccharides and the like due to its substrate specificity. In addition, the chemical oxidation of the hemiacetal hydroxyl group and the hydroxymethyl group of saccharides, particularly oligosaccharides and polysaccharides, has many side reactions as described above, and the purification process is inevitably complicated. Technology was required. Thus, it is an object of the present invention to use a microorganism having a wide substrate specificity to produce industrially and industrially useful substances, to have a hydroxymethyl group and / or a hemiacetal hydroxyl group with high yield and high selectivity. An object of the present invention is to provide a method for producing a sugar carboxylic acid by oxidizing a carbon atom.
[0006]
[Means for Solving the Problems]
The present inventors have conducted intensive studies on various microorganisms in order to achieve the above-mentioned object. As a result, Pseudogluconobacter saccharoketogenes has a hydroxymethyl group (-CH ₂ OH) and / or a wide range of sugars having carbon atoms with hemiacetal OH and their derivatives have been found to specifically oxidize these groups to readily oxidize sugar carboxylic acids in high yields and selectivity. Also, surprisingly, they found that they are extremely versatile and have very wide substrate specificity, and further studied to complete the present invention.
That is, the present invention
1) Oxidizing a carbon atom having a hydroxymethyl group and / or a hemiacetal hydroxyl group to a carboxyl group in a monosaccharide derivative, oligosaccharide or a derivative thereof, a polysaccharide or a derivative thereof having a hydroxymethyl group and / or a hemiacetal hydroxyl group A method of producing a sugar carboxylic acid or a salt thereof characterized by acting a microorganism belonging to the genus Pseudogluconobacter having the ability or a processed product thereof, producing and accumulating the corresponding carboxylic acid, and collecting the same.
2) A novel sugar carboxylic acid in which at least one of carbon atoms having a hydroxymethyl group and / or a hemiacetal hydroxyl group of a saccharide or a derivative thereof is oxidized to a carboxyl group.
[0007]
The microorganism belonging to the genus Pseudogluconobacter which can be used in the present invention is a microorganism belonging to the genus Pseudogluconobacter having the ability to oxidize a carbon atom having a hydroxymethyl group and / or a hemiacetal hydroxyl group of a saccharide to a carboxyl group. Any mutant may be used, and examples thereof include mutant strains obtained by ordinary mutagenesis procedures, for example, treatment with a mutagen such as nitrosoguanidine, ultraviolet irradiation, or gene recombination. In particular, bacteria of Pseudogluconobacter saccharoketogenes are preferred. More specifically, for example, the following strains described in European Patent Publication No. 221 707 are mentioned as typical examples.
Pseudogluconobacter saccharoketogenes K591s strain: FERM BP-1130, IFO 14464
Pseudogluconobacter saccharoketogenes 12-5 strain: FERM BP-1129, IFO 14465
Pseudogluconobacter saccharoketogenes TH 14-86 strain: FERM BP-1128, IFO 14466
Pseudogluconobacter saccharoketogenes 12-15 strain: FERM BP-1132, IFO 14482
Pseudogluconobacter saccharoketogenes 12-4 strain: FERM BP-1131, IFO 14483
Pseudogluconobacter saccharoketogenes 22-3 strain: FERM BP-1133, IFO 11484.
[0008]
In the method of the present invention, the cells of the microorganism of the genus Pseudogluconobacter may be allowed to act, or a processed product thereof may be used. As a processed product, for example, a culture solution of these microorganisms can be used. Further, enzymes produced by these microorganisms may be used. However, in the case of the microorganism of the genus Pseudogluconobacter according to the present invention, the enzyme is usually accumulated in the cells. Usually, it is convenient to use the cells themselves and contact and act on the starting saccharides to produce carboxylic acids. In particular, it is preferable to use resting cells. The cells or a culture solution thereof can be produced, for example, according to the method described in JP-A-64-85088.
That is, seed culture is performed from the slant, and then main culture is performed to obtain a fermentation broth. If necessary, the fermentation broth is centrifuged, and the precipitate is collected. The resulting precipitate can be subjected to a bacterial reaction. Under aerobic conditions, the microorganism may be a source of nutrients that can be used by the strain, ie, a carbon source (a carbohydrate such as glucose, sucrose, starch or an organic substance such as peptone or yeast extract), a nitrogen source (ammonium salts, urea, etc.). Inorganic and organic nitrogen compounds such as corn steep liquor and peptone; inorganic salts (salts such as potassium, sodium, calcium, magnesium, iron, manganese, cobalt, copper phosphate and thiosulfate); and CoA and pantothene as micronutrients It can be cultured in a liquid medium containing vitamins and coenzymes such as acids, biotin, thiamine, riboflavin, and FMN (flavin mononucleoside) or amino acids such as L-cysteine and L-glutamic acid or natural products containing them. The culture solution thus obtained may be used in the method of the present invention. The cultivation can be performed at pH 4 to 9, preferably pH 6 to 8.
[0009]
The culture time varies depending on the microorganism used, the composition of the medium, and the like, but is preferably 10 to 100 hours. A suitable temperature range for culturing is 10 to 40 ° C, preferably 25 to 35 ° C. At the time of culturing, the desired product can be produced more efficiently by adding a rare earth element to the medium. Rare earth elements added to the medium include scandium (Sc), yttrium (Y), lanthanum (La), cerium (Ce), praseodymium (Pr), neodymium (Nd), samarium (Sm), europium (Eu), Examples include gadolinium (Gd), terbium (Tb), dysprosium (Dy), holmium (Ho), erbium (Er), thulium (Tm), ytterbium (Yb), and lutetium (Lu). These rare earth elements may be added as metal powder or metal pieces, or may be used as compounds such as chlorides, carbonates, sulfates, nitrates, oxides or oxalates. They may be used alone or two or more rare earth elements such as cerium carbonate and lanthanum chloride may be used simultaneously. Furthermore, a crude product obtained in the process of separating and purifying various elements can also be used. The amount of the rare earth element added to the medium may be selected within a range that does not suppress the growth of the microorganism used, and is usually 0.000001 to 0.1% (W / V), preferably 0.0001 to 0.05%. The range of (W / V) is effective. As a method of adding to the medium, the medium may be added in advance to the medium, but may be added intermittently or continuously during the culture.
[0010]
In practicing the present invention, the microorganisms may be brought into contact with the saccharide to be subjected to the reaction using water or a solvent miscible with water, such as methanol, acetone or polyethylene glycol. The amount of the solvent to be used may be selected within a range that does not delay the reaction, and the substrate concentration is usually 0.1 to 20% (W / V), preferably 1 to 5%. A suitable temperature range for carrying out the oxidation reaction by the microorganism of the present invention is 10 to 40 ° C, preferably 25 to 35 ° C. The reaction is preferably carried out under aerobic conditions. For example, the mixture may be stirred at 50 to 2,000 revolutions as necessary while passing air at 0.1 to 5 liter / min. The reaction time varies depending on the nature of the primary hydroxyl group and / or hemiacetal hydroxyl group substituted with the saccharide to be subjected to the reaction, but is usually 5 minutes to 3 days, usually 1 hour to 24 hours. The reaction is preferably adjusted for pH. Usually, it is effective to carry out in the range of pH 4 to 9, preferably pH 6 to 8. The base used for pH adjustment can be used as long as it does not inhibit the reaction. For example, inorganic salts such as sodium hydroxide, potassium hydroxide, calcium carbonate, magnesium hydroxide and ferrous hydroxide, and organic salts such as sodium morpholinoethanesulfonate and calcium morpholinoethanesulfonate can be used. In addition, by adding an anion exchange resin if desired, it is not necessary to add the above-described neutralizing agent such as an alkali metal salt for adjusting the pH, and the reaction can be selectively controlled. The method of adding this anion exchange resin is particularly preferable when the reaction is carried out selectively to obtain one equivalent oxidized product. As the anion exchange resin used here, any anion exchange resin may be used as long as it can adsorb the generated carboxylic acid. In particular, styrene and acrylic anion exchange resins are preferred. Specifically, for example, Amberlite (trade name, Organo Corporation) IRA-400, IRA-401, IRA-402, IRA-410, IRA-900, IRA-910, IRA-35, IRA-68, IRA-94S Diaion (trade name, Mitsubishi Kasei) SA-10A, SA-20A, PA-306, PA-308, PA-406, WA-10, WA-11, WA-20, WA-30, and the like. .
These anion exchange resins are stirred when the sugars added as a substrate (monosaccharide derivatives having a hydroxymethyl group and / or hemiacetal hydroxyl group, oligosaccharides or derivatives thereof, polysaccharides or derivatives thereof) disappear in the reaction solution. Is stopped, the reaction solution is separated from the anion exchange resin, and the anion exchange resin is eluted by adding an appropriate eluent to obtain the desired product. Examples of the eluent include an aqueous solution of sodium chloride, an alkali metal salt, or the like, or an acidic aqueous solution of hydrochloric acid, sulfuric acid, phosphoric acid, citric acid, or the like. The sugar carboxylic acid thus eluted and accumulated can be collected and purified by known means or a method analogous thereto.
[0011]
According to the method of the present invention, D-glucose, D-fructol, D-galactose, D-ribose, D-mannose can be used as a saccharide to specifically oxidize a primary hydroxyl group (hydroxymethyl group) to give a corresponding sugar carboxylic acid. , Derivatives of monosaccharides such as L-sorbose [eg, amino sugars such as D-glucosamine, N-acetyl-D-glucosamine, N-acetyl-chitobiose, tri-N-acetyl-chitotriose, glucosyl-L-ascorbic acid] , L-ascorbic acid and other ascorbic acid-related compounds, inosine, adenosine, uridine, guanosine, cytidine, thymidine, 2-deoxyinosine, 2-deoxyadenosine, 2-deoxyuridine, 2-deoxyguanosine, 2-deoxycytidine, 2 -Nucleic acid-related compounds such as deoxythymidine And streptozotocin (streptozocin)], oligosaccharides such as sucrose, lactose, palatinose, raffinose, lactosucrose, glucosyl sucrose, galactosyl sucrose, xylobiose, maltotriose, maltotetraose, isomalttriose, panose, and the like. Starch-based sugars such as maltitol, amino sugars such as validamycin A, cellooligosaccharides such as cellobiose, cellotriose, cellohexaose, stevioside, rebaudioside-A, rebaudioside-C, rebaudioside-D, rebaudioside-E, rubusoside-A, and rubusoside-A. [In the following formula (II), R ₁ = Β-Glc, R ₂ = -Β-Glc), oligosaccharides such as mogroside or derivatives thereof, and polysaccharides such as cyclodextrin, soluble starch, dextrin, dextran, β-1,3-glucan, and derivatives thereof. .
The derivatives of oligosaccharides and polysaccharides also include sugar carboxylic acids obtained by oxidizing carbon atoms having a hemiacetal hydroxyl group of oligosaccharides and polysaccharides to carboxyl groups by the method of the present invention described below.
[0012]
Dextran, cellulose, chitin, amylose, amylopectin, maltotriose, panose, isomaltose, cellobiose, lactose, maltose, etc., as saccharides which give a sugar carboxylic acid by the specific oxidation of the hemiacetal hydroxyl group by the method of the present invention. No. Examples of oligosaccharide and polysaccharide derivatives include sugar carboxylic acids in which the hydroxymethyl group of the primary hydroxyl group of oligosaccharides and polysaccharides is oxidized to a carboxyl group by the method of the present invention, such as dextranyl glucuronic acid. Is also included.
Among the sugar carboxylic acids obtained from these sugars,
For example, a sugar carboxylic acid in which at least one of the hydroxymethyl groups of palatinose has been oxidized to a carboxyl group,
A sugar carboxylic acid in which at least one of the hydroxymethyl groups of sucrose has been oxidized to a carboxyl group,
A sugar carboxylic acid in which at least one of the hydroxymethyl groups of D-trehalose is oxidized to a carboxyl group,
A sugar carboxylic acid in which at least one of the hydroxymethyl groups of maltosyl-β-cyclodextrin has been oxidized to a carboxyl group,
A sugar carboxylic acid in which at least one hydroxymethyl group of 2-O-α-D-glucopyranosyl-L-ascorbic acid is oxidized to a carboxyl group,
A sugar carboxylic acid in which at least one of the hydroxymethyl groups of streptozotocin is oxidized to a carboxyl group,
Sugar carboxylic acid in which the hydroxymethyl group of heptulose has been oxidized to a carboxyl group,
Formula (I)
Embedded image

A sugar carboxylic acid in which at least one of the hydroxymethyl groups of maltodextrin represented by is oxidized to a carboxyl group,
Formula (II)
[0013]
Embedded image

A sugar carboxylic acid in which at least one of the hydroxymethyl groups of the steviol glycoside represented by is oxidized to a carboxyl group,
Formula (III)
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A sugar carboxylic acid in which at least one of the hydroxymethyl groups of validamycin A represented by is oxidized to a carboxyl group,
Sugar carboxylic acids in which at least one of the hydroxymethyl groups of the mogroside has been oxidized to a carboxyl group, and salts thereof,
Formula (VIII) in which the hydroxymethyl group of dextran is oxidized to a carboxyl group
Embedded image

(Wherein, n = 1 to 50, preferably 10 to 15) sugar carboxylic acid [that is, dextranyl glucuronic acid (also referred to as glucuronyl dextrin or dextran-glucuronic acid)], Salts or complexes or complexes of these with metal salts (hereinafter simply referred to as complexes); and
Formula (IX) wherein a carbon atom having a hemiacetal hydroxyl group of dextranyl glucuronic acid (sometimes referred to as dextran-glucuronic acid) is oxidized to a carboxyl group
Embedded image

(Wherein n = 1 to 50, preferably 10 to 15), sugar carboxylic acids (that is, glucuronyldextranyl gluconic acid), salts thereof, and complexes of these with metal salts are industrially available. It is a useful new compound.
[0014]
When performing the oxidation reaction by contacting the saccharide of the starting material with the cells belonging to the genus Pseudogluconobacter or a processed product thereof, the saccharide has the number and properties of primary hydroxyl groups and hemiacetal hydroxyl groups. It is also a feature of the oxidation reaction by the cells or the processed products belonging to the genus Pseudogluconobacter that they are regioselectively and stepwise oxidized in consideration of the above and specifically give the corresponding sugar carboxylic acid.
When the target product can be easily separated, the microorganism of the genus Pseudogluconobacter may be cultured in the saccharide-containing medium. In this case, the culture can be performed under the same conditions as in the method for obtaining the culture solution.
The sugar carboxylic acid thus produced and accumulated can be collected and purified by known means or a method analogous thereto. For example, the target product can be isolated and produced by applying means such as filtration, centrifugation, treatment with activated carbon or an adsorbent, solvent extraction, chromatography, precipitation, salting-out or the like, alone or in an appropriate combination.
As described above, when oxidation is performed in the presence of an anion exchange resin, the reaction solution and the anion exchange resin are separated by a static method, centrifugation, or the like, and the anion exchange resin is separated using an eluent. An elution treatment is carried out, and the eluted fraction containing the target substance is collected and subjected to the above-mentioned known means or a method analogous thereto, thereby isolating and purifying the target sugar carboxylic acid.
[0015]
The complex of dextran carboxylic acid and an iron salt can be usually produced by a known method for producing a complex of dextran and an iron salt or according to the method. For example, it can be produced by reacting dextran carboxylic acid with a sol of an iron salt such as ferric hydroxide. More preferably, dextran carboxylic acid is added to the desalted and purified iron hydroxide sol by dialysis and then heated at pH 8.0 to 10 in a heated temperature condition, for example, in an autoclave at 100 ° C to 120 ° C for 30 minutes. By doing so, a colloid solution or suspension is obtained.
By this method, dextran carboxylic acid derivative iron salt complex is formed by processes such as salt formation and chelate formation hydration of iron. As described below, the elemental iron content of the iron salt complex suspended in the aqueous phase is about 50 to 250 mg / ml in the form of a solution. An iron-containing iron salt complex can also be prepared. The iron salt complex containing high iron content thus obtained is itself extremely stable for a long period of time, and in particular, has a property that its colloidal state is stably maintained in the aqueous phase.
The complex of dextran carboxylic acid thus obtained and an iron salt such as ferric hydroxide is diluted with distilled water for injection, physiological saline or the like, and administered parenterally to an animal as an injection. Tablets, powders, granules, capsules, emulsions, liquids, premixes, if necessary, using pharmaceutically acceptable diluents and excipients according to known pharmaceutical manufacturing methods. It can be orally administered as a preparation, syrup or the like. It can also be used as a single agent, mixed directly or dispersed in a carrier with a feed, drinking water or the like. Furthermore, if necessary, each substance is separately formulated into a single preparation using a diluent, excipient, etc., which is acceptable to the drug substance, and then mixed with feed, drinking water, etc. for administration. You can also. Furthermore, as described above, those separately formulated can be separately administered to the same subject at the same time or at different times at the same time or at different times.
The carboxylic acid may be obtained as a free form or as a salt according to the method of the present invention.If the carboxylic acid is obtained as a salt, the carboxylic acid is converted to the free form by a conventional method. It goes without saying that you can do it. In addition, the presence of an alkali metal such as iron, lithium, sodium and potassium, and an alkaline earth metal such as magnesium and calcium in a medium can produce a salt thereof while producing and accumulating a sugar carboxylic acid. Further, the obtained product can be identified by conventional means such as elemental analysis, melting point, specific rotation, infrared absorption spectrum, NMR spectrum, chromatography and the like.
[0016]
The thus obtained sugar carboxylic acid of the present invention has various uses and properties. For example, the steviol glycosides include, for example, the following compounds, and the glucuronic acid-type steviol glycoside derivatives obtained by oxidizing them are all novel substances.
[Table 1]

The sugar carboxylic acid derivatives of various steviol glycosides including these had improved sweetness and strong sweetness and refreshing taste were recognized. As a low-calorie, anti-cariogenic, enzyme-stable, high-sweet food material, it can be used in confectionery, soft drinks, pickles, frozen desserts, etc. according to conventional sweeteners. Furthermore, it is excellent in terms of easy solubility, low toxicity, disintegration, decomposability, etc., so that the taste can be improved and it can be used as a flavoring agent. For example, it can be added to and blended with tablets, powders and other preparations according to conventional methods. The sugar carboxylic acid derivative of β-maltosyl-β-cyclodextrin is the first carboxylic acid derivative among cyclodextrins, and has significantly improved solubility in water (solubility> 200 g / 100 ml, water, 25 ° C.). . Therefore, for example, by incorporating a poorly soluble drug such as prostaglandin, steroid or barbituric acid with the sugar carboxylic acid of cyclodextrin of the present invention, the water solubility is improved, and the drug can be applied as an injection. When sugar carboxylic acids are used as clathrates in this manner, they can be used in the same manner as conventionally known clathrates. Further, β-D-fructosyl- (2 → 1) -α-D-glucuronic acid or α-D-glucuronyl- (1 → 2) -β-D-6-fracturonic acid obtained by reacting sucrose. Was found to be a new sugar derivative without sweetness, and was not decomposed by glucosidase, pectinase, glucuronidase or invertase. It is a sugar derivative that is less prone to enzymatic degradation than sucrose. Therefore, it is useful as an indigestible, low-calorie sugar derivative as a food material for confectionery, cakes, frozen desserts and the like. The calcium, magnesium, and iron salts are useful as an agent for improving the absorption of these metal ions. Therefore, it can be added to confectionery such as biscuits, soft drinks, etc., and used to obtain foods and drinks for osteoporosis prevention.
[0017]
Β-D-fructosyl- (6 → 1) -α-D-glucuronic acid and α-D-glucuronyl- (6 → 1) -α-D-fructuronic acid obtained by reacting palatinose are hardly digested. It is used as a neutral, low-calorie antidepressant. Therefore, it can be used for various foods such as confectionery such as chewing gum in the same manner as ordinary sweeteners and seasonings. Further, α-D-glucuronyl- (1 → 1) -α-D-glucuronic acid obtained by reacting D-trehalose is useful as an indigestible humectant and a stabilizer for antibody preparations. Further, β-amino-2-deoxy-D-glucuronic acid obtained by reacting D-glucosamine is useful as a base material for cosmetics having excellent moisture retention.
On the other hand, among nucleic acid derivatives obtained by reacting nucleosides such as inosine, 5'-carboxy-inosine and 5'-carboxy-adenosine have excellent taste and are enzymatically stable. It is useful as a flavoring agent and can be used as a component of foods and seasonings, especially for flavoring preserved foods.
1-carboxy-2,7-anhydro-β-D-alto-heptulose obtained by reacting 2,7-anhydro-β-D-alto-heptulose is iron, calcium, magnesium having a weak sweet taste. Can be used as a solubilizing agent for confectionery such as biscuits, soft drinks, and the like. The sugar carboxylic acid of streptozotocin is used as an antibacterial agent and an anticancer agent which are stable to the enzyme. By utilizing its antibacterial action, it can be used as it is or diluted with a solvent such as water for disinfection and sterilization of hospital rooms and limbs. The sugar carboxylic acid derivative of mogroside is useful as a sweetener and can be used for soft drinks, confectionery and the like.
[0018]
The sugar carboxylic acid obtained by oxidizing maltodextrin is used as an indigestible, low-calorie food material by adding and blending it to confectionery, cakes, frozen desserts, etc. by a conventional method.
Also, dextranyl glucuronic acid obtained by oxidizing the hydroxymethyl group of dextran to a carboxyl group is not only useful as a dextran itself or a more stable and more functional pharmaceutical excipient, but It exhibits excellent properties as a stabilizer for iron sol. Also, glucuronyl dextranyl gluconic acid in which a hydroxymethyl group of dextranyl gluconic acid is oxidized to a carboxyl group or a carbon atom having a hemiacetal hydroxyl group of dextranyl glucuronic acid is oxidized to a carboxyl group Alternatively, a salt thereof is useful as a pharmaceutical preparation material, and can be used in the same manner as dextran as a food viscosity preservative. Examples of dextranyl glucuronic acid and glucuronyl dextranyl gluconic acid or metal salts forming complexes of these salts with metal salts include, for example, monovalent, divalent or trivalent metal salts. And, for example, salts with metals such as calcium, magnesium, iron, sodium, lithium and potassium. In particular, a complex with an iron compound such as ferric hydroxide can be used as an iron supplement against iron deficiency anemia in an animal in the same manner as a dextran / iron complex as an anti-anemic agent.
Validamycin A is a compound represented by the above formula (III), which has a bactericidal activity against rice sheath blight and the like. An oxidized product of this bacterial reaction also has resistance to bacterial glucosidase, Validamycin derivatives are expected and are useful as agricultural fungicides with improved persistence. A sugar carboxylic acid derivative of an ascorbic acid derivative is also used as an enzyme-stable antioxidant because its stability against enzyme degradation is improved. As an antioxidant, it can be added to and blended with iron foods and the like in the same manner as for ascorbic acid.
As described above, the sugar carboxylic acids of the present invention obtained by oxidizing the carbon atom having the hydroxymethyl group and / or the hemiacetal hydroxyl group of various sugars have characteristics that the raw material sugar does not have, and have not been available in the past. Has utility.
[0019]
The sugar carboxylic acid derivative in which the hydroxymethyl group obtained by the present invention is oxidized to a carboxyl group is generally enzymatically stable and has improved solubility in water, as compared with the hydroxymethyl form as a substrate, and If desired, it can be converted into a metal salt, and in food applications, it is expected to have an effect of improving the absorption of metals, such as diet food materials, such as indigestibility and low calorie. The carboxylic acid of steviol glycosides (stevioside, rebauside, rubusoside, etc.) is, surprisingly, free of bitterness and aftertaste exhibited by stevioside and rubusoside, and has a refreshing sweetness and a sugar content of 100%. It is ~ 250 times sweeter and has no substantial calories and is useful as a sweetener.
Further, the carboxylic acid of the cyclodextrin derivative in which the hydroxymethyl group is oxidized to a carboxyl group according to the present invention has excellent characteristics in terms of easy solubility, low toxicity, disintegration, decomposability, and the like. , Basic drugs and the like can be included. Such inclusion allows the solubilization, stabilization and improvement of taste of the active ingredient to be achieved, so that it is used as a flavoring and flavoring agent. It is also useful as a formulation base utilizing enzymatic degradation properties. More specifically, use as a more functional base for pharmaceuticals such as tablets, capsules, pills, powders, granules, ointments, injections, syrups, suspensions, nasal drops, etc. Is mentioned.
As described above, the present invention enables the oxidation reaction of a hydroxymethyl group of a wide variety of saccharides to a carboxyl group, and provides various novel sugar carboxylic acids.
Further, the sugar carboxylic acid obtained by oxidizing a hemiacetal hydroxyl group to a carboxyl group obtained in the present invention is enzymatically stable and has improved solubility in water as compared with a saccharide having a hemiacetal hydroxyl group as a substrate, and If desired, it can be converted to a metal salt, which is expected to be a material for diet foods such as hardly digestible and less calorie in food applications, and an effect of improving metal absorption. Further, in pharmaceutical preparation use, it is useful as an enteric agent utilizing the properties of stabilizing the active ingredient and enzymatic degradation.
[0020]
【The invention's effect】
According to the present invention, a sugar carboxylic acid in which a carbon atom having a hydroxymethyl group and / or a hemiacetal hydroxyl group is specifically oxidized to a carboxyl group can be produced from a wide variety of saccharides with high selectivity and high yield. The obtained carboxylic acid has excellent properties such as excellent stability to enzymes and improved solubility in water.
[0021]
【Example】
Next, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples.
Example 1 Production of β-D-fructosyl- (2 → 1) -α-D-glucuronic acid sodium salt
Preparation of Pseudogluconobacter saccharoketogenes TH14-86 bacterial solution;
Pseudogluconobacter saccharoketogenes TH14-86 strain slant was added to 20 ml of the following medium with one platinum loop, and this was added to a 200 ml smooth flask at 30 ° C. for 1 day using a rotary shaker. Incubate with stirring. Then, 1 ml of the above-mentioned culture solution was taken per 20 ml of the medium, and this was subjected to shaking culture at 30 ° C. for 2 days to obtain seed culture. On the other hand, a slant of Bacillus megaterium IFO 12108 strain is added to a 200 ml smooth flask of 1 white ear per 20 ml of the medium (described below), followed by shaking culture at 30 ° C. for 2 days.
Next, the following main culture was performed. 100 ml of the seed culture (Seed medium) of the above TH14-86 strain and 1.5 ml of Seed medium of Bacillus megaterium were added to 900 ml of the following main culture medium, followed by shaking culture at 30 ° C. for about 20 hours. A bacterial solution was used.
Bacterial reaction;
A solution prepared by dissolving 30 g of sucrose in 200 ml of sterilized water was added to a 5-liter jar fermenter manufactured by Biot, 1 liter of the bacterial solution prepared as described above was added, and 800 ml of sterilized water was added. And While stirring at 32 ° C. and 800 rotations (rpm), air was bubbled at 1 liter / minute, and was automatically added dropwise as the reaction progressed to a pH of 6.3 with a 10% NaOH solution. . In 2 hours, it was converted to the monocarboxylic acid.
[0022]
Separation, purification;
The reaction solution (2 liters) was separated by a cooling centrifuge at 8000 rpm, the cells were removed, and the supernatant was applied to activated carbon (400 g of specially made shirasagi chromatograph) column chromatography, and washed with water (1.2 liters). Further, the fraction was eluted with 3 liters of water, and the desired fraction was collected and concentrated under reduced pressure to obtain 19.70 g of β-D-fructosyl- (2 → 1) -α-D-glucuronic acid sodium salt (19.70 g). .
Na-salt; in D ₂ O (90 MHz) δ ppm 61.39, 62.84, 71.48, 72.42, 73.09, 73.65, 74.65, 76.88, 82.01, 92.70, 104.18, 176 .49. Production of magnesium salt of β-D-fructosyl- (2 → 1) -α-D-glucuronic acid
Dissolve 1.89 g of the sodium salt of β-D-fructosyl- (2 → 1) -α-D-glucuronic acid in 10 ml of water and then add 1R-120 (H ⁺ (Type) column (50 ml), and 0.103 g of magnesium oxide was added to the passing solution, followed by stirring for 10 minutes. Then, the mixture was filtered through a Millipore filter (Type GS 0.22 μm), the filtrate was concentrated, and the obtained viscous syrup was treated with ethanol / acetone to obtain β-D-fructosyl- (2 → 1) -α-D- as a white powder. 1.45 g of magnesium salt of glucuronic acid was obtained.
Elemental analysis C ₂₄ H ₃₈ O ₂₄ Mg 6H ₂ O
Calculated value C, 34.20; H, 5.98
Found C, 34.33; H, 5.82.
[0023]
Medium composition
[For TH14-86 strain]
Seed medium (1st, 2nd common)
Lactose 1%
Yeast extract (Prodivel) 1
(NH ₄ ) ₂ SO ₄ 0.3
Corn Steep Liquor 3
CaCO ₃ (Super) 2
CaCO ₃ Before launch
pH = 7.0
For Bacillus Megathorium strain
[Seed medium]
4% sucrose
PROFLO [N source, Trades Protein (T & P)] 4
K ₂ HPO ₄ 0.65
KH ₂ PO ₄ 0.55
NaCl 0.05
(NH ₄ ) ₂ SO ₄ 0.05
MgSO ₄ ・ 7H ₂ O 0.005
Calcium pantothenate 0.025
Before sterilization pH = 7.0
[Main medium]
Sucrose 0.05% Separate sterilization
Corn Steep Liquor 2 Separate Sterilization
(NH ₄ ) ₂ SO ₄ 0.3 Separate sterilization
FeSO ₄ ・ 7H ₂ O 0.1 Separate sterilization
V. B ₂ 1mg / L
Before sterilization pH = 7.0
Sorbose 10% Separate sterilization
LaCl ₃ 0.01% Separate sterilization
CaCO ₃ (Super) 4% Separate sterilization
[0024]
Example 2 Production of α-D-glucuronyl- (1 → 2) -β-D-6-fracturonic acid
The sodium, magnesium and calcium salts of α-D-glucuronyl- (1 → 2) -β-D-fracturonic acid were obtained in the same manner as in Example 1.
Sodium salt; white powder
^Thirteen C-NMR (D ₂ O) ppm: 61.98, 71.96, 73.02, 73.35; 73.69, 76.37, 77. 03, 79.82, 93.97, 105.80, 175.03, 175.70.
Magnesium salt; white powder
Elemental analysis C ₁₂ H ₁₆ O _Thirteen Mg ・ 5H ₂ O
Calculated value C, 29.86; H, 5.43
Found, C, 29.76; H, 5.51.
Calcium salt; white powder
Elemental analysis C ₁₂ H ₁₆ O _Thirteen Ca ・ 3H ₂ O
Calculated C, 31.17; H, 4.80.
Found C, 31.26; H, 4.99.
[0025]
Example 3
To 1 liter of Pseudogluconobacter saccharoketogenes described in Example 1, 30 g of palatinose and 1 liter of sterilized water were added to 1 liter of the bacterial solution, and air was supplied at 32 ° C., 800 rpm and air at 1.6 liter / min. While reacting for 1 hour, the reaction solution was separated by a cooling centrifuge at 8000 rpm to remove the cells, the supernatant was applied to activated carbon (400 g of specially made shirasagi chromato charcoal) column chromatography, and washed with water ( The fraction eluted with a 10% aqueous methanol solution (4 liters) and further with a 50% aqueous methanol solution (4 liters) was collected, concentrated, lyophilized, and treated with β-D-fructosyl- (6 → 1) 35.8 g of sodium salt of -α-D-glucuronic acid was obtained.
This sodium salt was passed through an IR120 (H type) column, and the passing solution was concentrated to obtain β-D-fructosyl- (6 → 1) -α-D-glucuronic acid.
^Thirteen C-NMR (D ₂ O) ppm: 63.35, 68.60, 71.78, 72.52, 72.77, 73.45, 75.14, 75.99, 79.53, 98.81, 102.21, 176. 93.
Elemental analysis C ₁₂ H ₂₀ O ₁₂ ・ 4.5H ₂ O
Calculated C, 32.96; H, 6.68
Found, C, 33.17; H, 5.84.
[0026]
Example 4
In the same manner as in Example 3, using the following starting materials (described in parentheses), the corresponding sugar carboxylic acids were obtained.
[0027]
[Table 2]

[0028]
[Table 3]

[0029]
[Table 4]

[0030]
[Table 5]

[0031]
[Table 6]

[0032]
Example 5
To 1 liter of Pseudogluconobacter saccharoketogenes bacterial solution described in Example 1, 30 g of stevia extract (a steviol glycoside containing stevioside as a main component) was added to 30 g of a steviol glycoside in a 5-liter jar fermenter manufactured by Biot. Then, 1 liter of sterilized water was added to obtain a reaction solution. While stirring the mixture at 32 ° C. and 800 rotations, air was bubbled in at 1.6 liters / minute, and was automatically added dropwise as the reaction progressed so that the pH became 6.3 with a 10% NaOH solution. did. After 1.5 hours, the disappearance of the substrate was observed. The reaction was stopped. Then, 2 liters of the reaction solution was separated by a cooling centrifuge at 8000 rpm to remove the cells, and the supernatant was separated from HP-20 (fragrance). Group adsorbent, Mitsubishi Kasei) column (1.8 liter) ₂ After washing with O (8 L), a glucuronic acid-type steviol glycoside mixture was obtained from the fraction eluted with 50% EtOH (5 L). After concentration, freeze drying gave 19.28 g of a white powder. This product had strong sweetness and refreshing taste.
[0033]
This product was subjected to HPLC under the following conditions.

This product ^Thirteen C-NMR (D ₂ O) ppm: 177.87 (ester), 171.84 (COO ⁻ ), A signal of a carbonyl carbon was observed, and a new carbon was recognized based on the carboxy group. From the HPLC findings, it was found that stevioside C ₁₉ Glucose that binds to the _Thirteen It was found that the main component was a compound in which glucose bound to the position was converted to a glucuronic acid type.
[0034]
Example 6
In the same manner as in Example 5, 30 g of stevia extract (steviol glycoside containing stevioside as a main component), 1 liter of Pseudogluconobacter saccharoketogenes bacterium, 800 rpm at 32 ° C, and 1.6 times of air were added. The reaction was oxidized under aeration at a rate of 1 liter / minute, and the reaction was stopped when 59 ml of a 5% NaOH solution (corresponding to about 2 equivalents of oxidation) was added dropwise. The required oxidation reaction time was 21 hours. After completion of the reaction, the reaction solution was subjected to centrifugal filtration to remove the cells, and the supernatant (1850 ml) was purified (the same method as in Example 5), and the white powder of glucuronic acid-type steviol glycoside mixture Na 13.0 g of salt were obtained. This product had strong sweetness and refreshing taste.
HPLC; column ODP, temperature 35 ° C
Moving layer; CH ₃ CN: H ₂ 0.1% trifluoroacetic acid added to a solution of O = 3: 7
Flow rate: 1 ml / min
The HPCL pattern (RI detection) is shown in FIG.
[0035]
Example 7
In the same manner as in Example 5, oxidation with Pseudogluconobacter saccharoketogenes was performed using 30 g of stevioside as a substrate. 30 ml of 5% sodium hydroxide was consumed and the reaction time was 90 minutes. The reaction solution was purified to obtain 20 g of a white powder. This product had a high sweetness and refreshing sweetness.
The HPCL pattern (RI detection) of this product is shown in FIG. 400 mg of this white powder was fractionated using a reverse phase column ODP-50 column (φ21.5 × 300 mm Asahi Kasei) and an eluent system of water / acetonitrile (72:28) containing 0.1% trifluoroacetic acid to separate major components. Then, freeze-drying was performed to obtain 124 mg of a white powder. This product was recrystallized from a mixed solvent of methanol: water = 9: 1 to give colorless needles.
226-230 ° C (decomposition).
IR (KBr) cm- ¹ : 3500-3250, 1715, 1605, 1080-1010, 890.
[Table 7]

Elemental analysis C ₃₈ H ₅₇ O ₁₉ Na 1.5H ₂ O
Calculated value C, 52.59; H, 6.97
Found C, 52.56; H, 7.15.
From the above, the main oxidation product obtained by this reaction is represented by R in the above formula (II). ₁ = -Β-Glc-2-β-Glc, R ₂ = -Β-Glc UA was concluded to be a sodium salt of a compound of UA, that is, a sodium salt of 13-O-β-sophorosyl and 19-O-β-D-glucuronyl steviol represented by the following formula (IV).
Embedded image

[0036]
Example 8
In the same manner as in Example 5, oxidation was performed with Pseudogluconobacter saccharoketogenes using 30 g of stevioside as a substrate. In 80 minutes, 67 ml of the 5% sodium hydroxide solution was consumed, and the reaction solution was purified to obtain 32 g of a white powder. This product had a high sweetness and refreshing sweetness. The HPCL pattern (RI detection) of this product is shown in FIG. From 1 g of this white powder, 0.22 g of a main oxidation product was separated using a reverse phase column ODP-50 (φ21.5 × 300 mm Asahi Kasei) and a HPCL technique using a gradient system of 2% acetic acid / acetonitrile. I took it. The product was found to have m / z 869 (M + 2H) by secondary ion mass spectrometry (SI-MS) by secondary ion mass spectrometry. ₂ 0 · H ⁺ ) Peak was observed.
^Thirteen C-NMR (d ₆ -Pyridine) ppm: 15.12, 18.95, 20.21, 21.69, 27.78, 37.91, 37.96, 39.33, 39.41, 40.35, 42.12, 43 .58, 43.91, 47.23, 53.55, 56.86, 62.57, 70.07, 71.54, 72.49, 72.75, 75.75, 76.16, 76.79 , 76.83, 77.51, 77.72, 77.78, 86.00, 86.97, 95.07, 97.60, 103.89, 103.94, 153.50, 171.26, 171 89, 176.23.
Based on the above findings, the product is represented by the formula (II) ₁ = -Β-Glc-2-β-Glc UA, R ₂ = -Β-Glc Sodium salt of UA compound, ie, 13-O-β-D-glucuronyl-β-D-glucosyl, 19-O-β-D-glucuronyl steviol represented by the following formula (V) 2 Sodium salt was concluded.
Embedded image

[0037]
Example 9
According to the method of Example 5, 30 g of stevioside was placed in a 5 liter jar fermenter manufactured by Biot, and 1.6 liter of sterilized water was added to obtain a reaction solution. While stirring the mixture at 32 ° C. and 800 rotations, air was bubbled at 1 liter / minute, and 20 g of calcium carbonate was added, followed by a reaction for 4 hours. Then, 2 liters of the reaction solution was separated by a cooling centrifuge at 8000 rpm to remove the cells, and the supernatant was passed through an HP-20 (aromatic synthetic adsorbent, Mitsubishi Kasei) column (1.8 liter). And H ₂ After washing with O (8 l), the uronic acid-type stevioside calcium salt was obtained from the fraction eluted with 50% ethanol (5 l). After concentration, freeze drying gave 12.98 g of a white powder. This was recrystallized from methanol to obtain colorless powdery crystals. This product had a weak sweetness.
This product ^Thirteen C-NMR (d ₆ -Pyridine) ppm; 15.93, 19.74, 20.92, 21.00, 28.58, 38.66, 38.71, 40.06, 41.04, 41.86, 42.00, 42 .91, 44.33, 48.19, 54.50, 57.63, 73.11, 73.30, 73.45, 73.97, 74.08, 76.53, 77.72, 77.86. , 77.89, 78.34, 78.39, 84.79, 86.79, 95.84, 98.21, 105.46, 107.02, 153.70, 171.93, 172.18, 172. .95, 176.78.
Based on the above findings, the product is represented by the formula (II) ₁ = -Β-Glc UA-2-β-Glc UA, R ₂ = -Β-Glc Calcium salt of UA compound, ie, 13-O-β-D-glucuronyl-β-D-glucuronyl-19-O-β-D-glucuronyl steviol calcium represented by the following formula (VI) Concluded with salt.
Embedded image

[0038]
Example 10
In the same manner as in Example 5, 10 g of rebaudioside A was used as a substrate, and 15.8 g of Pseudogluconobacter saccharoketogenes cells and 2000 ml of sterilized water were added. The reaction was carried out while stirring under the conditions, and the reaction was adjusted to pH 6.3 by adding a 2% NaOH solution. When 23 ml of 2% NaOH was consumed, the reaction was stopped and centrifuged to separate the supernatant. The reaction time required 135 minutes. The supernatant was treated by the same purification method as in Example 5 to obtain 10.16 g of a white powder.
The HPLC pattern (RI detection) of this product is shown in FIG.
IR (KBr) cm ^-1 : 3340, 1720, 1610, 1410, 1070 This product has a high sweetness and a refreshing sweet taste.
[0039]
Example 11
To 1 liter of Pseudogluconobacter saccharoketogenes described in Example 1, 30 g of stevioside was placed in a 5 liter jar fermenter of Biot, and then 1 liter of sterilized water was added. (Abbreviated as IRA-68) 200 ml was added to prepare a reaction solution. This was aerated at 1.6 l / min while stirring at 800 rpm at 32 ° C. After 2 hours, no stevioside was observed in the supernatant, the reaction was stopped, the supernatant was separated from the IRA-68 by centrifugation, and the IRA-68 was packed in a column (φ4 × 24 cm). The column was eluted with 2 L of 2N-brine, and the eluted fraction was passed through a column of HP-20 (0.5 L). After washing with 1 liter of water, the residue was eluted with 0.8 liter of 50% methanol and concentrated to dryness to obtain 9.20 g of a white powder.
This product was recrystallized from a mixed solvent of methanol: water = 9: 1 to obtain colorless needles.
mp. 226-230 ° C (decomposition)
This product was identified as 13-O-β-sophorosyl-19-O-β-glucuronyl steviol sodium salt (IV) shown in Example 7.
[0040]
Example 12
According to the method described in Example 11, 30 g of Stevia extract (a mixture of at least 6 types of steviol glycosides) was added to 1 liter of Pseudogluconobacter saccharoketogenes in a 5-liter jar fermenter of Biot. Then, 1 liter of sterilized water was added, and 200 ml of IRA-68 was further added to obtain a reaction solution. While stirring the mixture at 32 ° C. and 800 rpm, air was bubbled at 1.6 liter / minute. After 2 hours, stevioside was not observed in the supernatant, and the reaction was stopped. The supernatant was separated from IRA-68 by centrifugation, and the IRA-68 was packed in a column (φ4 × 25 cm). Elution was performed with 1 liter of NaCl, and the eluted fraction was passed through a HP-20 column (0.5 liter). After washing with 1 liter of water, the residue was eluted with 1 liter of 50% methanol and concentrated to dryness to obtain 8.9 g of a white powder.
The HPLC pattern (RI detection) of this product is shown in FIG.
IR (KBr) cm ^-1 : 3500 to 3200, 1720, 1705, 1610, 1400.
[0041]
Example 13
In a manner similar to that of Example 6, 30 g of glycosyltransferred stevia glycoside (SK suite, manufactured by Sanyo Kokusaku Pulp Co., Ltd.) was oxidized and purified to obtain 28.3 g of a white powder. This product ^Thirteen C-NMR (D ₂ O), eight signals (179.50, 179.46, 179.40, 179.37, 179.32, 179.26, 177.10, 177.07 ppm) were observed in the carbonyl carbon region. Thus, it was confirmed that at least one or more of the primary acid groups of the saccharide was converted to a carboxylic acid. This product exhibited a refreshing sweetness with a refreshing feeling.
[0042]
Example 14
In accordance with the method described in Example 11, 10 mg (0.32 g) of Pseudogluconobacter saccharoketogenes was suspended in 3 ml of water according to the method described in Example 11. In addition, IRA-685 ml was added. The mixture was stirred at 229 rotations at 30 ° C. for 5 半 hours. The reaction solution was allowed to stand, and after removing the supernatant, IRA-68 was packed in a column, washed with water (50 ml), eluted with 0.01 N HCl, and the eluted fractions were collected, lyophilized, and dried with a white powder (32 mg). ) Got. This product is IR (KBr) cm ^-1 : 3300, 1600, 1410, 1060-1000, it was confirmed that the sugar chain was converted to the glucuronic acid type. This product exhibited a sugar-like sweetness.
[0043]
Example 15
Dissolve 1.0 g of rubusoside in 60 ml of sterile water, add 13 ml of IRA-68, then add 30 ml of Pseudogluconobacter saccharoketogenes described in Example 1, and add 32 ml of the solution at 32 ° C and 600 ° C. While stirring at a rotation / minute, air was bubbled at a rate of 60 liters / minute, and the reaction was performed with stirring for 240 minutes. The IRA-68 was taken out of the reaction solution, packed in a column, washed with 15 ml of water, and then eluted with 2N-NaCl solution using SV 0.5 with 200 ml. The eluate was applied to an HP-20 column (20 ml). After adsorption and washing with 150 ml of water, elution was carried out with 50% MeOH solution (100 ml) at SV 0.5, the eluate was concentrated to dryness, and the precipitate was recrystallized from methanol to obtain 0.48 g of colorless granular crystals. Was.
m. p. 184-188 ° C (decomposition)
Elemental analysis C ₃₂ H ₄₇ O ₁₄ Na ・ 5H ₂ O
Theory C, 49.99; H, 7.47
Found, C, 50.05; H, 7.54.
[Table 8]

From the above data, it was concluded that the structure was represented as (VII).
Embedded image

This product (VII) was a good high-sweetness substance such as sugar without any bite.
[0044]
Example 16
Fursultiamine hydrochloride 10mg
Vitamin B ₂ 2mg
Taurine 1000mg
Benzoic acid 30mg
2.5 mg of butyl paraoxybenzoate
Glucuronic acid type stevioside sodium salt (IV) 50mg
Citric acid 100mg
Glycine 500mg
According to the above composition, a drink was obtained by the following method. That is, butyl parahydroxybenzoate and benzoic acid are dissolved in about 30 ml of water heated to about 70 ° C., cooled to about 25 ° C., and fursultiamine hydrochloride, vitamin B ₂ , Taurine, glucuronic acid-type stevioside as a sweetener, citric acid, and glycine are dissolved, and 1N-caustic soda is added to adjust the pH to 3.5, and then water is added to make a total volume of 50 ml.
[0045]
Example 17
30 g of precipitated calcium carbonate, 5 g of magnesium carbonate, 58.5 g of lactose and 6 g of hydroxypropylcellulose are uniformly mixed, and 300 ml of water is added to granulate the above mixture. The obtained granules are dried and pulverized. 0.05 g of glucuronic acid-type stevioside and 0.5 g of magnesium stearate as a flavoring agent were added to the obtained powder, mixed, and tableted according to a conventional method to give a tablet of 8.5 mg in diameter and 200 mg per tablet. Obtained.
[0046]
Example 18
5 liters of 30 g of dextran-4 (molecular weight: 4000-6000; manufactured by Extrasynthesis (France)) in 1.5 liters of the pseudogluconobacter saccharoketogenes described in Example 1 And then added 1.7 liters of sterilized water to obtain a reaction solution. While stirring the mixture at 32 ° C. and 600 rpm, air was bubbled in at 1.6 liters / min, and the pH was automatically adjusted to 0.5 with a 0.5% NaOH solution as the reaction progressed. It was added dropwise. After 3 hours, the reaction was stopped, and 12.0 liters of the reaction solution was separated by a cooling centrifuge at 8000 rpm to remove the cells, thereby obtaining 1.98 liters of a supernatant. This supernatant was filtered through a cellulose acetate membrane filter (φ0.2 to μm), and the filtrate, which had been sterilized again, was passed through an Amberlite IRA-68 (OH type) column (50 ml), and a small amount of water ( 100 ml) to obtain 2 liters of the passing solution. This passing solution was passed through an HP-20 column (900 ml). Then, elution was carried out with 2 liters of water, and the first 700 ml of the eluate was collected. Then, 3.3 liters of the eluted solution was collected, and 13.8 ml of concentrated hydrochloric acid was added thereto to make the solution acidic. (Φ0.2 μm), and the filtrate was passed through 1000 ml of Sephabeads (trade name) SP205 (manufactured by Mitsubishi Kasei Corporation). Then, after washing with 700 ml of 0.05M hydrochloric acid and 2 liters of water, elution was performed with 2 liters of a 20% MeOH solution to obtain a fraction of the desired product. This was concentrated under reduced pressure to obtain 14 g of dextranyl-glucuronic acid white powder. HPLC of this product (conditions: Asahi Pack GS320 (φ7.6 mm × 50 mm; manufactured by Asahi Kasei Corporation), mobile phase; water 1 ml / min, detection; RI (Waters 410) and 200 nm (Tosoh UV-8020) sample 0.5 % Aqueous solution (20 μl injection) showed a single peak at Rt = 8.48. (Rt of raw material dextran 4 was 10.83)
To confirm the structure of this product, an enzyme digestion treatment experiment was performed. To 100 μl of a 1.5% aqueous solution of this product was added 100 μl of a glucoamylase (manufactured by Wako Pure Chemical Industries, Ltd.) solution (4 mg / ml), and the mixture was incubated at 30 ° C. for 24 hours. The enzyme-treated solution was examined by HPLC. As a target experiment, dextran-4 was similarly treated with glucoamylase. As a result, it was found that this product did not undergo glucoamylase digestion. On the other hand, dextran-4 underwent glucoamylase digestion, the substrate disappeared, and glucose was regenerated. From this, the product was identified as dextranyl glucuronic acid represented by the above formula (VIII) (where n = 15).
[0047]
Example 19
According to Example 1, Pseudogluconobacter saccharoketogenes K591S strain was shaken at 30 ° C. and 230 rpm using a peptone yeast medium (PY) medium consisting of 1% bactopeptone and 1% yeast extract. The culture was carried out at pH 7.5 under shaking for 3 days. Then, the mixture was transferred to the main culture, and cultured in a peptone yeast medium (PY medium) for 48 hours while adding 0.1% calcium chloride and shaking at 30 ° C. and 230 rpm. Then, the culture solution was centrifuged at 10,000 rpm for 10 minutes, and the cells were collected. The cells were then washed with 500 ml of water to obtain 7.59 g of cells per liter of broth. Then, 30 g of dextran-4 (molecular weight: 4,000 to 6000; manufactured by Extrasynthesis Co., Ltd., France) was dissolved in 1 liter of water, and a suspension of 56 g of wet cells suspended in 1 liter of water was added. While stirring, air was bubbled at 60 ml for 1 minute, and the reaction was carried out for 6.5 hours while controlling the pH to 6.3 with a 0.1% NaOH solution. The reaction solution was centrifuged, 2 liters of the supernatant was collected, sterilized by filtration through a membrane filter, passed through an HP-20 column (900 ml), and eluted with 2000 ml of water. The passing solution was acidified by adding concentrated hydrochloric acid to a final concentration of 0.05 M, filtered through a membrane filter, and then passed through an SP-205 column (100 ml). ) Then, after washing with water (2000 ml), the fraction eluted with 20% MeOH (2000 ml) was collected, concentrated and freeze-dried to obtain 14 g of dextranyl gluconic acid as a white powder. HPLC of this product [conditions: Asahi Pack GS320 (φ7.6 mm × 50 mm: manufactured by Asahi Kasei Corporation), mobile phase: water 1 ml / min, RI (Waters 410) and 200 nm (Tosoh UV-8020), sample 0.5% % Aqueous solution injected) showed a single peak at Rt = 8.70. When this product was digested with glucoamylase under the conditions described above, it was easily digested, and glucose and glucosylgluconic acid could be detected. Therefore, this compound was identified as dextranyl gluconic acid.
[0048]
Example 20
Pseudogluconobacter saccharoketogenes was cultured in the same manner as in Example 19, and 50 g of the cells (wet cells) were dextranyl glucuronic acid (prepared by the method described in Example 18). 30 g was added to a solution prepared by dissolving 30 g in 1 liter of water, and the mixture was allowed to react for 21 hours while stirring at 32 ° C. and 800 rpm for 1 minute while passing air at 60 ml and controlling the pH to 6.3 with a 0.1% NaOH solution. Was. The reaction solution was centrifuged, 2 liters of the supernatant was collected, sterilized by filtration through a membrane filter, passed through an HP-20 column (900 ml), eluted with 1.5 liters of water, and the target fraction was collected. After concentration and freeze-drying, 15 g of white powder of sodium salt of glucuronyldextranylgluconic acid represented by the above formula (IX) was obtained.
HPLC: Rt = 9.12 1 peak
HPLC conditions; Column: GS-320
Mobile phase: H ₂ O, flow rate 1 ml / min
Detection: RI
^Thirteen C-NMR (D ₂ O) δ ppm: observed at 179.554, 178.119, 172.434, the intensity was 1: 0.5: 0.5, and the assignment was that the carbonyl carbon in the glucuronic acid portion was 179.554. Is the carbonyl carbon of the gluconic acid part (1 part lactone type is assumed).
[0049]
Example 21
50 ml of a 50% aqueous solution of ferric chloride hexahydrate was heated to 30 ° C. ₂ CO ₃ 50 ml of the solution are added dropwise at a rate of 0.4 ml for 1 minute with good stirring. Then, 50 ml of a 10% dextranyl glucuronic acid solution using the dextranyl glucuronic acid obtained in Example 18 was added dropwise at a rate of 3 ml / min, and 16% Na was added. ₂ CO ₃ The solution was added at a rate of 0.4 ml / min and adjusted to pH 4.3. Next, 200 ml of ethanol was added, the mixture was vigorously stirred to form a slurry, centrifuged (5000 rpm), the precipitate was collected, dissolved in 40 ml of water, stirred well with 60 ml of ethanol, centrifuged to remove soluble matter, and precipitated. 20 ml of water was added to the mixture, and the mixture was dispersed well. The ethanol was removed with an evaporator under reduced pressure, and the mixture was added with a 10% NaOH solution at a rate of 0.2 ml / min while stirring well. After heating for 20 minutes, phenol was added to a concentration of 4 mg / ml to obtain 22 ml of a dextranyl glucuronic acid iron hydroxide sol solution. Intramuscular administration of this product to 1 ml of young pigs showed an effect of remarkably improving anemia symptoms. Iron dextranyl gluconate hydroxide sol can be produced in the same manner, and the effect of remarkably improving anemia in young pigs was confirmed.
[0050]
Example 22
Method for producing iron hydroxide sol of dextranyl gluconic acid ferric chloride (FeCl ₃ ・ 6H ₂ O) Dissolve 100 g in 100 ml of water and sonicate this solution with Branson B-220 for 1 hour to fully dissolve. Then, add water to make 200 ml and transfer the solution to a 500 ml beaker. Then, 200 ml of a 24% sodium carbonate solution was added at 30 ° C. at a rate of 0.8 ml / min with good stirring to prepare a pale yellow-brown iron hydroxide sol.
100 ml of the iron hydroxide sol thus prepared is taken, transferred to a 300 ml beaker, and 50 ml of a 10% dextranyl gluconic acid solution is gradually added while stirring well at 30 ° C. Then, 16% NaCO ₃ The solution was added very slowly at a rate of 0.1-0.2 ml / min and the pH was adjusted to 4.3. Then, 200 ml of ethanol was added with stirring, the resulting precipitate was centrifuged, separated from the supernatant, and the precipitate was added with 80 ml of water and suspended well. Then, 120 ml of ethanol was added and reprecipitated, and the precipitate was centrifuged. Separate and separate. This operation was repeated once more, and the resulting precipitate was suspended in 20 ml of water, stirred well, and a 10% NaOH solution was added at a rate of 0.1 to 0.2 ml / min until the pH reached 6.0. added. This solution was autoclaved at 120 ° C. for 10 minutes, phenol (so as to contain 1%) was added as a preservative, and then concentrated by an evaporator to prepare a dextranylgluconic acid iron hydroxide sol solution. The product formed a stable iron hydroxide sol. Its properties were analyzed and the results were as follows.
Total iron salt concentration: 200 mg / ml, viscosity: 32 cP, conductivity 47 mS / cm.
[0051]
Example 23
Iron chloride by dialysis of dextranyl glucuronic acid 50 ml of a 50% aqueous solution of ferric chloride hexahydrate was heated to 30 ° C., and then 24% Na ₂ CO ₃ 50 ml of the solution was added dropwise with good stirring at a rate of 0.30 ml for 1 minute to obtain a slightly blackish ocher iron hydroxide sol (pH 1.54). The iron hydroxide sol was transferred to a dialysis tube and dialyzed overnight in ultrapure water. The iron hydroxide sol after the dialysis showed about 170 ml and pH 4.47 blackish brown. Next, 50 ml of a 10% aqueous solution of dextranyl glucuronic acid obtained in Example 18 was added to the dialyzed iron hydroxide sol, and the mixture was stirred well and mixed with 16% Na. ₂ CO ₃ The solution was adjusted to pH 4.3. After autoclaving the mixture at 100 ° C. for 30 minutes, a 10% NaOH solution was added to adjust the pH to 12. The autoclave treatment was again performed at 121 ° C. for 20 minutes to completely dissolve, and the hydration of dextranyl glucuronic acid in blackish brown was completed. A ferrous salt complex was obtained. Next, this was dialyzed overnight with a dialysis membrane and then concentrated. To this, phenol was added to a concentration of 4 mg / ml to obtain 22 ml of an injection of dextranyl glucuronic acid ferric hydroxide complex. This product was a stable sol solution, and its properties were analyzed as follows.
Total iron salt concentration: 201 mg / ml, viscosity 23.9 cP, conductivity 3.7 mS / cm.
Intramuscular administration of this product to 1 ml of young pigs showed an effect of remarkably improving anemia symptoms. The ferric dextranyl glucuronic acid hydroxide salt complex can be produced in the same manner, and the effect of remarkably improving anemia in young pigs was recognized.
[0052]
Example 24
In the same manner as in Example 11, 30 g of rebaudioside-A was placed in 1 liter of Pseudogluconobacter saccharoketogenes in a 5 liter jar fermenter manufactured by Biot, and then 1.5 liter of sterilized water was added. 400 ml was added to obtain a reaction solution. This was aerated at 1.6 L / min while stirring at 32 ° C. and 600 rotations. After 1 hour, no rebaudioside-A was found in the supernatant. The reaction was stopped, the supernatant was separated from the IRA-68 by centrifugation, and the IRA-68 was packed in a column (φ4 × 40 cm). Elution with 2N-saline (8.1 liter), elution fraction was passed through an HP-20 (1 liter) column, adsorbed, washed with water (8 liter), and eluted with 10% to 50% ethanol. As a result, the desired sodium salt of glucuronic acid rebaudioside-A was obtained. After concentration, the residue was freeze-dried to obtain 19.6 g of a colorless powder. This is MeOH-H ₂ By recrystallizing from O, 10.6 g of colorless needles were obtained. m. p. 220 ° C (decomposition) HPLC of this product (ODP column, acetonitrile: water = 30: 70) gave one peak at Rt = 9.70. This product ^Thirteen C-NMR (D ₂ O) ppm: 18.06, 21.52, 22.91, 24.19, 30.89, 39.51, 40.15, 42.02, 43.01, 43.73, 44.58, 46. 69, 46.79, 49.89, 56.17, 59.64, 63.51, 63.71, 64.37, 71.36, 72.43, 73.14, 74.40, 74.67, 76.29, 76.99, 78.12, 78.70, 78.72, 78.81, 78.93, 79.27, 79.38, 81.49, 87.99, 90.31, 96. 64, 98.70, 104.87, 105.06, 107.32, 155.99, 177.79, 181.64.
From the above findings, this product is represented by the formula (II):
Embedded image

It was concluded that the compound was represented by the following formula (X):
Embedded image

[0053]
Test example
The stability test for various enzymes of 6-O-α-D-glucuronyl (1 → 4) -α-D-glucosyl-β-cyclodextrin Na salt (1) obtained in Example 4 was performed by the following method. As a control, 6-O-α-maltosyl-β-cyclodextrin was used.
Test method
A predetermined amount of each of the following enzymes is added to a 10 mM test cyclodextrin aqueous solution, and the mixture is allowed to stand in a 37 ° C warm bath. A sample is taken in 500 μl portions, heated at 100 ° C. for 15 minutes to inactivate the enzyme, and then centrifuged (15,000 rpm, 5 minutes). Further, the solution is filtered through Millipore USY-1 (fraction molecular weight: 10,000). It was diluted 10-fold and subjected to HPLC analysis under the following conditions.
HPLC analysis conditions;
Column; NH2P-50 (Asahipak)
Mobile phase; CH ₃ CN: H ₂ At O = 48: 52, PIC reagent 0.005M was added.
Flow rate: 0.8ml / min
Detection; RI
From the HPIC of each sample obtained by the above operation, the residual ratio of cyclodextrin every hour up to 120 minutes was determined.
[0054]
Enzyme used and enzyme concentration;
[Table 9]

result
Residual rate of 6-O-α-D-glucuronyl (1 → 4) -α-D-glucosyl-β-cyclodextrin Na salt (1) and 6-O-α-maltosyl-β-cyclodextrin by each enzyme The changes over time are shown in FIGS. 11 to 14.
(1) was found to be more stable to the enzyme treatment of α-amylase, glucoamylase and pullulanase than 6-O-α-maltosyl-β-cyclodextrin as a control.
On the other hand, in the examination of the enzyme treatment with pullulanase, (1) was stable, while the control 6-O-α-maltosyl-β-cyclodextrin was degraded by about 60%.
[Brief description of the drawings]
1 shows the HPLC pattern (RI detection) of the glucuronic acid-type steviol glycoside mixture obtained in Example 5. FIG.
FIG. 2 shows the HPLC pattern (RI detection) of the glucuronic acid-type steviol glycoside mixture obtained in Example 6.
3 shows an HPLC pattern (RI detection) of glucuronic acid-type steviol glycoside sodium salt obtained in Example 7. FIG.
4 shows the HPLC pattern (RI detection) of the glucuronic acid-type steviol glycoside disodium salt obtained in Example 8. FIG.
FIG. 5 shows the HPLC pattern (RI detection) of one equivalent oxidized form of rebaudioside-A obtained in Example 10.
6 shows the HPLC pattern (RI detection) of the glucuronic acid-type steviol glycoside mixture obtained in Example 12. FIG.
FIG. 7 shows the relationship between 6-O- (2-carboxyethyl) -β-cyclodextrin Na salt obtained in Example 4 and 6,6-di-O- (2-carboxyethyl) -β-cyclodextrin. ^Thirteen C-NMR (270 MHz, D ₂ O) The chart of δ ppm is shown.
FIG. 8 shows the relationship between 6-O-α-D-glucuronyl- (1 → 4) -α-D-glucosyl-α-cyclodextrin obtained in Example 4. ^Thirteen C-NMR (270 MHz, D ₂ O) The chart of δ ppm is shown.
FIG. 9 shows the relationship between 6-O-α-D-glucuronyl-β-cyclodextrin obtained in Example 4. ^Thirteen C-NMR (270 MHz, D ₂ O) The chart of δ ppm is shown.
FIG. 10 shows 6-O- (2-carboxy2-hydroxyethyl) -β-cyclodextrin Na salt obtained in Example 4 and 6,6-di-O- (2-carboxy2-hydroxyethyl). Of β-cyclodextrin Na salt ^Thirteen C-NMR (270 MHz, D ₂ O) The chart of δ ppm is shown.
FIG. 11 shows 6-O-α-D-glucuronyl (1 → 4) -α-D-glucosyl-β-cyclodextrin Na salt obtained in the test example and 6-O-α-maltosyl-β as a control. 2 shows the results of a stability test of cyclodextrin against α-amylase, and shows the change over time in the residual ratio when both compounds were treated with α-amylase.
FIG. 12 shows 6-O-α-D-glucuronyl (1 → 4) -α-D-glucosyl-β-cyclodextrin Na salt obtained in the test example and 6-O-α-maltosyl-β as a control. -Shows the results of a stability test of cyclodextrin against glucoamylase, and shows the change over time in the residual ratio when both compounds are treated with glucoamylase.
FIG. 13 shows 6-O-α-D-glucuronyl (1 → 4) -α-D-glucosyl-β-cyclodextrin Na salt obtained in the test example and 6-O-α-maltosyl-β as a control. -Shows the results of a stability test of cyclodextrin against pullulanase, and shows the change over time in the residual ratio when both compounds are treated with pullulanase.
FIG. 14 shows 6-O-α-D-glucuronyl (1 → 4) -α-D-glucosyl-β-cyclodextrin Na salt obtained in the test example and 6-O-α-maltosyl-β as a control. 2 shows the results of a stability test of cyclodextrin against β-glucuronidase, and shows the change over time in the residual ratio when both compounds were treated with β-glucuronidase.
[Explanation of symbols]
In FIGS. 11 to 14, ● represents 6-O-α-maltosyl-cyclodextrin, and ▲ represents 6-O-α-D-glucuronyl (1 → 4) -α-D-cyclodextrin Na salt. Is shown.

Claims (23)

D-glucosamine having a hydroxymethyl group and / or a hemiacetal hydroxyl group , N-acetyl-D-glucosamine, N-acetyl-chitobiose, tri-N-acetyl-chitotriose, glucosyl-L-ascorbic acid, L-ascorbic acid, Inosine, adenosine, uridine, guanosine, cytidine, thymidine, 2-deoxyinosine, 2-deoxyadenosine, 2-deoxyuridine, 2-deoxyguanosine, 2-deoxycytidine, 2-deoxythymidine, streptozotocin, sucrose, lactose, palatinose , Raffinose, lactosucrose, glucosyl sucrose, galactosyl sucrose, xylobiose, maltotriose, maltotetraose, isomalttriose, panose, mal Tall, validamycin A, cellobiose, cellotriose, cellohexaose, stevioside, rebaudioside -A, rebaudioside -C, rebaudioside -D, rebaudioside -E, dulcoside -A, rubusoside, mogroside, cyclodextrin, soluble starch, dextrin, dextran, beta - 1,3-glucan, cellulose, chitin, amylose, amylopectin, isomaltose, maltose, dextranylglucuronic acid, 6-O-alpha-maltosyl -β- cyclodextrin, 6-O- (3- hydroxypropyl) - β-cyclodextrin, 6,6-di-O- (3-hydroxypropyl) -β-cyclodextrin, 6-O-α-maltosyl-α-cyclodextrin, 6-O-α-D-glucosyl-β- Cyclode String, 6-O-maltosyl-α-cyclodextrin, 6-O- (2,3-di-hydroxypropyl) -β-cyclodextrin or 6,6-di-O- (2,3-di-hydroxypropyl ) -Β -cyclodextrin , Pseudogluconobacter saccharoketogenes strain K591s (FERM BP-1130, IFO 14464), Pseudogluconobacter saccharoketogenes strain 12-5 (FERM BP-1129, IFO 14465), Pseudogluconobacter saccharoketogenes TH 14-86 strain (FERM BP-1128, IFO 14466), Pseudogluconobacter saccharoketogenes strain 12-15 (FERM BP-1132, IFO 14482), Pseudogluconobacter Saccharoketogenes 1 2-4 strain (FERM BP-1131, IFO 14483) and Pseudogluconobacter saccharoketogenes 22-3 strain (FERM BP-1133, IFO 11484), or a culture solution of the microorganism or a microorganism produced therefrom. A method for producing a sugar carboxylic acid or a salt thereof, comprising reacting an enzyme to produce and accumulate a corresponding carboxylic acid, and collecting the carboxylic acid. D-glucosamine having a hydroxymethyl group , N-acetyl-D-glucosamine, N-acetyl-chitobiose, tri-N-acetyl-chitotriose, glucosyl-L-ascorbic acid, L-ascorbic acid, inosine, adenosine, uridine, Guanosine, cytidine, thymidine, 2-deoxyinosine, 2-deoxyadenosine, 2-deoxyuridine, 2-deoxyguanosine, 2-deoxycytidine, 2-deoxythymidine, streptozotocin, sucrose, lactose, palatinose, raffinose, lactose sucrose , Glucosyl sucrose, galactosyl sucrose, xylobiose, maltotriose, maltotetraose, isomalttriose, panose, maltitol, validamycin A, Biose, cellotriose, cellohexaose, stevioside, rebaudioside -A, rebaudioside -C, rebaudioside -D, rebaudioside -E, dulcoside -A, rubusoside, mogroside, cyclodextrin, soluble starch, dextrin, dextran, beta - 1,3 Glucan, 6-O-α-maltosyl-β-cyclodextrin, 6-O- (3-hydroxypropyl) -β-cyclodextrin, 6,6-di-O- (3-hydroxypropyl) -β-cyclodextrin , 6-O-α-maltosyl-α-cyclodextrin, 6-O-α-D-glucosyl-β-cyclodextrin, 6-O-maltosyl-α-cyclodextrin, 6-O- (2,3-di -Hydroxypropyl) -β-cyclodextrin or 6,6- -O- - a (2,3-di-hydroxypropyl)-.beta.-cyclodextrin, pseudoephedrine saccharoketogenes K591s strain (FERM BP-1130, I FO 14464), Pseudogluconobacter saccharoketogenes 12 -5 strains (FERM BP-1129, IFO 14465), Pseudogluconobacter saccharoketogenes TH 14-86 strain (FERM BP-1128, IFO 14466), and Pseudogluconobacter saccharoketogenes 12-15 strain (FERM) BP-1132, IFO 14482), Pseudogluconobacter saccharoketogenes strain 12-4 (FERM BP-1131, IFO 14483) and Pseudogluconobacter saccharoketogenes strain 22-3 (FERM BP-11). 3, IFO 11484) by the action of microorganisms or enzymes culture or the microorganism of the microorganism produced selected from, produce the corresponding carboxylic acid, allowed to accumulate, it saccharide carboxylic acid or a, characterized in that collecting the Method for producing salt. Pseudogluconobacter saccharoketogenes strain K591s (FERM BP-1130, IFO 14464), Pseudogluconobacter saccharoketogenes strain 12-5 (FERM BP-1129, IFO 14465), Pseudogluconobacter saccharoketogenes TH 14-86 strain (FERM BP-1128, IFO 14466), Pseudogluconobacter saccharoketogenes 12-15 (FERM BP-1132, IFO 14482), Pseudogluconobacter saccharoketogenes 12-4 ( FERM BP-1131, IFO 14483) and Pseudogluconobacter saccharoketogenes 22-3 strain (FERM BP-1133, IFO 11484 ) according to claim 1 or 2, wherein the action of microorganisms selected from Manufacturing method. A sugar carboxylic acid or a salt thereof in which at least one of the hydroxymethyl groups of maltosyl-β-cyclodextrin is oxidized to a carboxyl group. A sugar carboxylic acid or a salt thereof in which at least one hydroxymethyl group of 2-O-α-D-glucopyranosyl-L-ascorbic acid is oxidized to a carboxyl group. A sugar carboxylic acid or a salt thereof in which at least one of the hydroxymethyl groups of streptozotocin has been oxidized to a carboxyl group. A sugar carboxylic acid in which the hydroxymethyl group of heptulose has been oxidized to a carboxyl group or a salt thereof. Formula (I)

A sugar carboxylic acid or a salt thereof, wherein at least one of the hydroxymethyl groups of maltodextrin represented by the formula (1) is oxidized to a carboxyl group. Formula (II)

A sugar carboxylic acid or a salt thereof, wherein at least one of the hydroxymethyl groups of the steviol glycoside represented by the formula is oxidized to a carboxyl group. The sugar carboxylic acid or a salt thereof according to claim 9 , wherein the steviol glycoside is stevioside in which R ₁ = -β-Glc-2-β-Glc and R ₂ = -β-Glc in formula (II). Steviol glycoside in formula (II)

The sugar carboxylic acid or a salt thereof according to claim 9 , which is rebaudioside-A. A sugar carboxylic acid or a salt thereof in which at least one of the hydroxymethyl groups of validamycin A is oxidized to a carboxyl group. A sugar carboxylic acid or a salt thereof in which at least one of the hydroxymethyl groups of mogroside is oxidized to a carboxyl group. Small Do Kutomo one of the sugar carboxylic acid or a salt thereof is oxidized to the carboxyl group of hydroxymethyl groups of the dextran. 15. A complex of the sugar carboxylic acid according to claim 14 or a salt thereof and a metal salt selected from iron salts, alkali metal salts and alkaline earth metal salts . Dextran, cellulose, chitin, amylose, amylopectin, maltotriose, panose, isomaltose, cellobiose, lactose, maltose or dextranyl glucuronic acid having a hemiacetal hydroxyl group, and pseudogluconobacter saccharoketogenes strain K591s (FERM BP) -1130, IFO 14464), Pseudogluconobacter saccharoketogenes 12-5 strain (FERM BP-1129, IFO 14465), Pseudogluconobacter saccharoketogenes strain TH14-86 (FERM BP-1128, IFO 14466) ), Pseudogluconobacter saccharoketogenes strain 12-15 (FERM BP-1132, IFO 14482), Pseudogluconobacter saccharoketogenes 1 -4 strain (FERM BP-1131, IFO 14483 ) and Pseudogluconobacter saccharoketogenes 22-3 strain (FERM BP-1133, IFO 11484 ) microorganism or culture or the microorganism of the microorganism selected from the produced A method for producing a sugar carboxylic acid or a salt thereof, comprising reacting an enzyme to produce and accumulate a corresponding carboxylic acid, and collecting the carboxylic acid. Pseudogluconobacter saccharoketogenes strain K591s (FERM BP-1130, IFO 14464), Pseudogluconobacter saccharoketogenes strain 12-5 (FERM BP-1129, IFO 14465), Pseudogluconobacter saccharoketogenes TH 14-86 strain (FERM BP-1128, IFO 14466), Pseudogluconobacter saccharoketogenes 12-15 (FERM BP-1132, IFO 14482), Pseudogluconobacter saccharoketogenes 12-4 ( FERM BP-1131, IFO 14483) and Pseudogluconobacter saccharoketogenes 22-3 strain (FERM BP-1133, IFO 11484 ) manufactured according to claim 16, wherein the action of microorganisms selected from Construction method. 17. The method according to claim 16 , wherein the polysaccharide having a hemiacetal hydroxyl group is dextran. The method according to claim 16 , wherein the derivative of the polysaccharide having a hemiacetal hydroxyl group is dextranyl glucuronic acid. A sugar carboxylic acid or a salt thereof in which a carbon atom having at least one hemiacetal hydroxyl group of dextranyl glucuronic acid is oxidized to a carboxyl group. A complex of the sugar carboxylic acid or a salt thereof according to claim 20 , and a metal salt selected from iron salts, alkali metal salts and alkaline earth metal salts . A method for producing a complex of dextranyl glucuronic acid and ferric hydroxide, which comprises reacting dextranyl glucuronic acid with ferric hydroxide sol. To Cie Yukurosu that having a hydroxymethyl group, pseudoephedrine saccharoketogenes K591s strain (FERM BP-1130, IFO 14464 ), shoe de saccharoketogenes 12-5 strain (FERM BP-1129, IFO 14465), Pseudogluconobacter saccharoketogenes TH 14-86 strain (FERM BP-1128, IFO 14466), Pseudogluconobacter saccharoketogenes 12-15 strain (FERM BP-1132, IFO 14482), pseudoglucose From the strains of Novobacter saccharoketogenes 12-4 (FERM BP-1131, IFO 14483) and Pseudogluconobacter saccharoketogenes 22-3 (FERM BP-1133, IFO 11484) A method for producing a sugar carboxylic acid or a salt thereof , which comprises causing a selected microorganism or a culture solution of the microorganism or an enzyme produced by the microorganism to act to produce and accumulate a corresponding carboxylic acid, and collecting the carboxylic acid .

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