JP2004173620A - alpha-MANNOBIOSE-CONTAINING FODDER - Google Patents
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【０００１】
【発明の属する技術分野】
本発明は、α−マンノビオースを含有する飼料、α−マンノビオース含有組成物及びその製造方法、並びに飼料用添加剤に関する。
【０００２】
【従来の技術】
従来より、パーム核ミール、コプラミール、グアーミール等には、マンノースを構成糖とするマンナンが豊富に含有されることが知られており、これらの天然原料に酵素を作用させてマンノース、マンノオリゴ糖、マンノース多糖類を生成させる方法が種々提案されている。また、このようなマンノース類が添加された飼料には、有害細菌であるサルモネラ菌の腸内での定着を抑制して体外へ排出する排菌効果（サルモネラ菌排除効果）を有することが知られており、この効果を利用した技術も種々提案されている（例えば、非特許文献１）。
【０００３】
例えば、グアーミールやコプラミールなどマンナンを含む素材を酵素分解して得られる、マンノビオース及びマンノトリオースを主成分とし単糖類が混合したマンノオリゴ糖類を配合した飼料は、鶏の卵の品質を向上させることができることが報告されており、同時に、マンノオリゴ糖類がサルモネラ菌の家畜腸内での定着防止に有用である可能性があることが報告されている（特許文献１）。また、マンノース、メチル−α−マンノシド、マンノオリゴ糖や、グアーガム、ローカストビーンガム又は酵母から得られるマンナンの酵素及び／又は酸による加水分解物等のマンノース類を配合する飼料が、サルモネラ菌など有害細菌の感染予防に有用であることが報告されている（特許文献２）。さらには、マンノースの繰り返し単位が４０〜１００の多糖を中心（３０〜８０％）とし、オリゴ糖も（５〜３０％）混在するマンノース系多糖体を配合する家畜用飼料が、サルモネラ菌の家畜腸内での定着防止に効果があることが報告されている（特許文献３）。そしてまた、パーム核ミールやグアーミール等のマンナンリッチな原料に酵素を作用させてマンノオリゴ糖に分解したものを配合した飼料や、コプラミールに酵素を作用させたマンノースを含有する飼料が報告されている（特許文献４、特許文献５）。しかし、α−マンノビオースを含有する飼料は知られていなかった。
【０００４】
【非特許文献１】
「Ｐｏｕｌｔｒｙ Ｓｃｉｅｎｃｅ」 １９８９年 第６８号 ｐ．１３５７
【特許文献１】
特開平７−２３６４２９号公報
【特許文献２】
特開平８−３８０６４号公報
【特許文献３】
特開平８−１７３０５５号公報
【特許文献４】
ＷＯ ９５／１７１０３号公報
【特許文献５】
ＷＯ ９９／０８５４４号公報
【０００５】
【発明が解決しようとする課題】
以上のように種々の提案がなされているが、サルモネラ菌の家畜腸内での定着防止効果は十分でないのが現状であり、更なる改良が望まれている。例えば、マンノースにはサルモネラ菌定着抑制効果（サルモネラ菌排除効果）があるものの、それ自体単純な糖であるため、飼料として家畜に供与しても、大部分は腸内細菌類により簡単に資化されることがわかっており、また、家畜自身によっても消化吸収・排泄がなされるであろうと考えられる。また、本発明者らの実験結果によって、家畜の免疫強化のために生菌剤を投与した場合、さらにマンノース分解活性が強くなり、サルモネラ対策として給与したマンノースは大部分が消失するという知見が得られた。従って、実際に十分なサルモネラ菌の定着抑制効果を得るためには極めて多量のマンノースを投与しなければならない。一方、酵母を用いた凝集試験によると、マンノビオースよりも分子量の大きなマンノオリゴ糖やマンノース系多糖体のサルモネラ菌定着抑制効果は小さいという知見が得られた。本発明はかかる実状に鑑みてなされたものであり、本発明の課題は、動物体内でのサルモネラ菌の定着を抑制してサルモネラ菌を体外へ効率的に排出することができる飼料、かかる飼料の製造に有利に用いることができるα−マンノビオース含有組成物及びその製造方法、並びにα−マンノビオース含有組成物を含む飼料用添加剤を提供することにある。
【０００６】
【課題を解決するための手段】
本発明者らは、マンノースにはサルモネラ菌排除効果があるものの、それ自体単純な糖であるため、飼料として家畜に供与しても、大部分は腸内細菌類により簡単に資化され、また、家畜自身によっても消化吸収・排泄がなされるため消失してしまうとの仮説を立て、マンノースに代わる有効成分を探索するなど鋭意研究を重ねた結果、α−マンノビオースが非常に強いサルモネラ認識活性を持ち、かつ、マンノースのように容易に資化されることがないという知見を得、本発明を完成するに至った。
【０００７】
すなわち、本発明は、α−マンノビオースを含有することを特徴とする飼料（請求項１）や、マンノースにα−マンノシダーゼを作用させて得られるα−マンノビオース含有組成物を含むことを特徴とする請求項１に記載の飼料（請求項２）や、マンナンを含有する天然物にβ−マンノシダーゼ及びα−マンノシダーゼを順次又は同時に作用させて得られるα−マンノビオース含有組成物を含むことを特徴とする請求項１に記載の飼料（請求項３）や、マンナンを含有する天然物から抽出したマンナンにβ−マンノシダーゼ及びα−マンノシダーゼを順次又は同時に作用させて得られるα−マンノビオース含有組成物を含むことを特徴とする請求項１に記載の飼料（請求項４）や、マンナンを含有する天然物が、コプラミール及び／又はパーム核ミールであることを特徴とする請求項３又は４に記載の飼料（請求項５）や、α―マンノビオースが、飼料に含まれる全マンノース・マンノオリゴ糖類に対して少なくとも５重量％含有されていることを特徴とする請求項１〜５のいずれかに記載の飼料（請求項６）や、α−マンノビオースが、０．００１〜０．３重量％含有されていることを特徴とする請求項１〜６のいずれかに記載の飼料（請求項７）に関する。
【０００８】
また、本発明は、マンナンを含有する天然物にβ−マンノシダーゼ及びα−マンノシダーゼを順次又は同時に作用させることを特徴とするα−マンノビオース含有組成物の製造方法（請求項８）や、マンナンを含有する天然物から抽出したマンナンにβ−マンノシダーゼ及びα−マンノシダーゼを順次又は同時に作用させることを特徴とするα−マンノビオース含有組成物の製造方法（請求項９）や、マンナンを含有する天然物が、コプラミール及び／又はパーム核ミールであることを特徴とする請求項８又は９に記載のα−マンノビオース含有組成物の製造方法（請求項１０）や、請求項８〜１０のいずれかに記載の製造方法により製造されたことを特徴とするα−マンノビオース含有組成物（請求項１１）や、請求項１１に記載のα−マンノビオース含有組成物を含むことを特徴とする飼料用添加剤（請求項１２）に関する。
【０００９】
【発明の実施の形態】
本発明の飼料としては、マンノース２分子がα−グリコシド結合したα−１，２−マンノビオース（α−１，２−ｍａｎｎｏｂｉｏｓｅ）、α−１，３−マンノビオース（α−１，３−ｍａｎｎｏｂｉｏｓｅ）、α−１，４−マンノビオース（α−１，４−ｍａｎｎｏｂｉｏｓｅ）、α−１，６−マンノビオース（α−１，６−ｍａｎｎｏｂｉｏｓｅ）を１種又は２種以上含有するα−マンノビオース含有飼料であれば特に制限されるものではなく、通常、α−マンノビオースやその含有物を従前用いられている飼料に配合することにより本発明のα−マンノビオース含有飼料を得ることができる。かかる本発明の飼料として、具体的には、精製されたα−マンノビオースを含む飼料や、マンノースにα−マンノシダーゼを作用させて得られるα−マンノビオース含有組成物を含む飼料や、マンナンを含有する天然物にβ−マンノシダーゼ及びα−マンノシダーゼを順次又は同時に作用させて得られるα−マンノビオース含有組成物を含む飼料や、マンナンを含有する天然物から抽出したマンナンにβ−マンノシダーゼ及びα−マンノシダーゼを順次又は同時に作用させて得られるα−マンノビオース含有組成物を含む飼料を例示することができる。かかる飼料の形態は特に制限されず、家畜又は家禽用の固体状の飼料の他、液状、ペースト状等の飼料を例示することができる。
【００１０】
上記精製されたα−マンノビオースを含む飼料は、例えば、マンノースから合成する方法により得られたα−マンノビオースを含み、マンノースや他のマンノオリゴ糖を含まない。また、マンノースからの合成方法としては、例えば、「ＳＹＮＴＨＥＳＩＳ ＯＦ ＭＡＮＮＯＳＥ ＯＬＩＧＯＳＡＣＣＨＡＲＩＤＥＳ ＶＩＡ ＲＥＶＥＲＳＡＬ ＯＦ ＴＨＥ α−ＭＡＮＮＯＳＩＤＡＳＥ ＲＥＡＣＴＩＯＮ」（Ｂｉｏｔｅｃｈｎｏｌｏｇｙ ｌｅｔｔｅｒｓ ｖｏｌ８ ｎｏ６ ｐｐ４２１−４２４，１９８６）に記載の方法を挙げることができる。
【００１１】
上記マンノースにα−マンノシダーゼを作用させて得られるα−マンノビオース含有組成物を含む飼料は、マンノースにα−マンノシダーゼを作用させる方法により得られたα−マンノビオース含有組成物を含み、原料となるマンノースは、精製されたマンノース試薬の他、いかなる起源のものも用いることができ、例えば、コプラミールやパーム核ミールのようなマンナンを含有する天然物にβ−マンノシダーゼを作用させて、遊離してくるマンノースを用いることもできる。また、α−マンノシダーゼとしては、α−マンノシダーゼ活性を持つものであれば特に制限されるものではなく、いかなる起源のものであってもよく、例えば、特開平６−２６９２９０号公報に記載されたＡｓｐｅｒｇｉｌｌｕｓ ｎｉｇｅｒ由来のα−マンノシダーゼや、スミチームＡＣ（新日本化学工業株式会社製）、スミチームＡＧＳ（新日本化学工業株式会社製）、ヘミセルラーゼＧＭ「アマノ」（天野製薬株式会社製）、トランスグルコシダーゼ「アマノ」（天野製薬株式会社製）などの市販の酵素類を例示することができる。
【００１２】
上記マンナンを含有する天然物にβ−マンノシダーゼ及びα−マンノシダーゼを順次又は同時に作用させて得られるα−マンノビオース含有組成物を含む飼料は、マンナンを含有する天然物にβ−マンノシダーゼを作用させてマンノースを遊離させ、かかる遊離したマンノースにα−マンノシダーゼを作用させることにより製造されるα−マンノビオースやマンノースを含有する組成物を含む。このα−マンノビオース含有組成物の製造おいては、マンナンを含有する天然物にβ−マンノシダーゼを作用させマンノースを生成させた後、必要に応じて水分を調整し、α−マンノシダーゼを作用させてもよいし、マンナンを含有する天然物にβ−マンノシダーゼ及びα−マンノシダーゼを同時に作用させてもよい。この反応に用いられるα−マンノシターゼとしては、上述のα―マンノシターゼを用いることができ、β−マンノシターゼとしては、マンナンを分解してマンノースを遊離するものであれば特に制限されるものではなく、いかなる起源のものであってもよく、具体的には、例えば、セルロシンＧＭ５（エイチビィアイ株式会社製）等を例示することができる。そして、前記マンナンを含有する天然物としては、例えば、コプラミール、パーム核ミール、グアーミール等が挙げられ、入手の容易な点、マンナンリッチな点等から、コプラミール、パーム核ミールが好ましい。
【００１３】
上記マンナンを含有する天然物から抽出したマンナンにβ−マンノシダーゼ及びα−マンノシダーゼを順次又は同時に作用させて得られるα−マンノビオースを含む飼料は、マンナンを含有する天然物から抽出したマンナンにβ−マンノシダーゼを作用させてマンノースを遊離させ、かかる遊離したマンノースにα−マンノシダーゼを作用させることにより製造されるα−マンノビオースやマンノースを含有する組成物を含む。このα−マンノビオース含有組成物の製造においては、マンナンを含有する天然物から抽出したマンナンにβ−マンノシダーゼを作用させマンノースを生成させた後、必要に応じて水分を調整し、α−マンノシダーゼを作用させてもよいし、マンナンを含有する天然物から抽出したマンナンにβ−マンノシダーゼ及びα−マンノシダーゼを同時に作用させてもよい。この反応に用いられるα−マンノシターゼとしては、上述のα―マンノシターゼを用いることができ、β−マンノシターゼとしては、マンナンを分解してマンノースを遊離するものであれば特に制限されるものではなく、いかなる起源のものであってもよく、具体的には、例えば、セルロシンＧＭ５（エイチビィアイ株式会社製）等を例示することができる。そして、前記マンナンを含有する天然物としては、例えば、コプラミール、パーム核ミール、グアーミール等が挙げられ、入手の容易な点、マンナンリッチな点等から、コプラミール、パーム核ミールが好ましい。また、マンナンを含有する天然物から、ミセルを作り水不溶のマンナンを抽出する方法としては、アルカリ溶液、例えば５％冷水酸化ナトリウム液や、１０％水酸化ナトリウム液での抽出方法を例示することができる。
【００１４】
飼料中におけるα−マンノビオースの含有量としては、０．００１〜０．３重量％であることが好ましく、０．００１〜０．０３重量％であることがより好ましい。α−マンノビオースの含有量が上記範囲であれば本発明の効果を有効に奏することができると共にマンノース類の含有量を大幅に少量化することができる。また、飼料中にα−マンノビオース以外の他のマンノース・マンノオリゴ糖類が含有される場合、α−マンノビオースは、飼料に含まれる全マンノース・マンノオリゴ糖類に対して少なくとも５重量％（５〜１００重量％）含有されていることが好ましく、１０重量％以上含有されていることがより好ましい。なお、ここでマンノオリゴ糖類とは、２〜８個のマンノース単位から構成されるオリゴ糖をいう。
【００１５】
本発明の飼料は、α−マンノビオースを含有するので、動物体内でのサルモネラ菌の定着を抑制してサルモネラ菌を体外へ効率的に排出することができる。即ち、α−マンノビオースが非常に強いサルモネラ認識活性を持ち、かつ、体内で容易に資化されることがないので、効率的にサルモネラ菌を体外へ排出することができ、飼料中のマンノース類の含有量を少量化することが可能となって、経済的負担も大幅に軽減される。具体的には、後述するサルモネラ菌凝集抑制試験及び代謝試験の結果から明らかなように、α−マンノビオースは、マンノースの４倍のサルモネラ認識活性があり、代謝試験によると、腸内細菌類によってほとんど資化されないことから、マンノースを使用する場合と比較して少なくとも４分の１以下の量で十分にサルモネラ菌排除効果を発揮することができる。また、本発明の飼料は、家畜の免疫強化のために成鶏盲腸内容菌などの生菌剤を併用する場合において特に有用である。
【００１６】
本発明のα−マンノビオース含有組成物の製造方法としては、例えばコプラミールやパーム核ミール等のマンナンを含有する天然物にβ−マンノシダーゼ及びα−マンノシダーゼを順次又は同時に作用させる製造方法や、マンナンを含有する天然物から抽出したマンナンにβ−マンノシダーゼ及びα−マンノシダーゼを順次又は同時に作用させる製造方法であれば特に制限されるものではなく、その形態としては液状、ペースト状、スラリー状、固体状のものを挙げることができる。天然物に直接酵素を作用させて製造するα−マンノビオース含有組成物は、α−マンノビオースを天然物から分離抽出することなくそのまま飼料に添加し、本発明の飼料を製造することができ、これにより簡便かつ低コストで本発明の飼料を提供することが可能となる。かかるマンノビオース含有組成物を飼料添加用としてそのまま流通、使用する場合には、黴、菌類の発生が危惧されることから、流動層乾燥などの方法により、水分が１０重量％以下となるように乾燥することもできる。また、液状のα−マンノビオース含有組成物をそのまま添加する場合は、乾燥処理を省略できることからより低コストで本発明の飼料を提供することが可能となる。さらに、本発明のα−マンノビオース含有組成物としては、上記本発明のα−マンノビオース含有組成物の製造方法により得られるものであればどのようなものでもよく、本発明の飼料用添加剤としては、かかるα−マンノビオース含有組成物からなるものや、該α−マンノビオース含有組成物に保存料等の他の添加成分が配合されたものを例示することができる。
【００１７】
【実施例】
以下、本発明の実施例を示し、本発明を詳細に説明するが、本発明は以下の実施例に限定されるものではない。なお、実施例中の「部」は、特に断りがない限り「重量部」を表すものとする。
試験例１（サルモネラ菌凝集抑制試験）
各種糖類のサルモネラ菌凝集抑制試験を以下のように行った。サルモネラ菌サルモネラ・エンテリティディス（伊藤忠飼料株式会社にて分離したＳａｌｍｏｎｅｌｌａ ｅｎｔｅｒｉｔｉｄｉｓ ＫＴＥ−６１株 菌数１×１０８ｃｆｕ／ｍｌ）の懸濁液０．１ｍｌ及び蒸留水０．１ｍｌを十分混合させ、これに酵母キャンディダ・アルビカンス（社団法人動物用生物学的製剤協会より購入したＣａｎｄｉｄａ ａｌｂｉｃａｎｓ ＫＩ−１０２００１ ）懸濁液０．１ｍｌを加え混合した。この混合液に凝集が生じることを実体顕微鏡（オリンパス社製）で確認する予備試験を行った。
【００１８】
次に、予備試験と同様の方法で、蒸留水に代えて各種糖水溶液を用い、それぞれ濃度を変化させて混合液の凝集が阻害される最低濃度（凝集抑制最低濃度）を求めた。被験糖類は以下に示す（１）〜（９）であり、いずれも試薬特級（マンノビオース２種はＳｉｇｍａ社製、その他は和光純薬社製）を使用した。
（１）マンノース（Ｄ−ｍａｎｎｏｓｅ）
（２）α−１，４−マンノビオース（α−１，４−ｍａｎｎｏｂｉｏｓｅ）
（３）α−１，６−マンノビオース（α−１，６−ｍａｎｎｏｂｉｏｓｅ）
（４）α−メチル−Ｄ−マンノピラノシド（α−ｍｅｔｈｙｌ−Ｄ−ｍａｎｎｏｐｙｒａｎｏｓｉｄｅ）
（５）Ｄ−フルクトース（Ｄ−ｆｒｕｃｔｏｓｅ）
（６）Ｌ−アラビノース（Ｌ−ａｒａｂｉｎｏｓｅ）
（７）Ｄ−ガラクトース（Ｄ−ｇａｌａｃｔｏｓｅ）
（８）Ｄ−グルコース（Ｄ−ｇｌｕｃｏｓｅ）
（９）スクロース（ｓｕｃｒｏｓｅ）
【００１９】
なお、本方法においては、サルモネラ菌懸濁液の濃度、サルモネラ菌の感受性、酵母懸濁液濃度などの試験条件により凝集阻害糖濃度が異なるので、（１）マンノースを陽性対照として相対比較した。表１に、混合液の凝集を阻害するまでの実際の凝集抑制最低濃度（μｇ／ｍｌ）、及びマンノースの凝集抑制最低濃度を１とした場合の各種糖類の相対濃度（濃度比）を示す。相対濃度が１以下の場合、マンノースよりも低い濃度で同じ凝集抑制効果が得られることを示している。
【００２０】
【表１】
表１に示すように、（２）α−１，４−マンノビオース、（３）α−１，６−マンノビオース及び（４）α−メチル−Ｄ−マンノピラノシドは、マンノースよりも低濃度でサルモネラ菌による凝集を抑制できることが判明した。中でも、本発明の飼料が含有する（２）α−１，４−マンノビオース及び（３）α−１，６−マンノビオースは非常に高いサルモネラ菌による酵母凝集抑制効果を有することが判明した。なお、Ｄ−フルクトースはサルモネラ菌の凝集を抑制するためにはマンノースの４倍程度の量が必要であり、Ｌ−アラビノース、Ｄ−ガラクトース、Ｄ−グルコース及びスクロースは、サルモネラ菌による凝集抑制能を全く示さなかった。
【００２２】
実施例１（マンノースからのα−１，６−マンノビオースの製造）
Ｄ−マンノース（和光純薬社製「試薬特級」）１００部に、酵素溶液トランスグルコシダーゼ「アマノ」（天野製薬株式会社製）１００部を加え、５０℃で３６時間反応させた。この反応物中のα−１，６−マンノビオース含有量をイオン交換クロマトグラフィー法で測定したところ２５．４部であった。この反応物から高速液体クロマトグラフィー（日本分光社製、ｃｏｌｕｍｎ Ｓｈｏｄｅｘ ＤＣ−６１３）により、α−１，６−マンノビオースを分取し、得られたα−１，６−マンノビオースについて、試験例１と同様にしてサルモネラ菌による酵母凝集抑制能を調べた。その結果、マンノースに対する相対濃度は０．２５６であり、マンノースにトランスグルコシダーゼを作用させて生成させたα−１，６−マンノビオースについても非常に高いサルモネラ菌・酵母凝集阻害効果を有することが判明した。
【００２３】
実施例２（α−マンノビオース含有パーム核ミールの製造；酵素順次添加）
パーム核ミール（不二製油株式会社製）１００部に、酵素セルロシンＧＭ５（エイチビィアイ株式会社製）０．６部を溶解した酵素溶液１２５部を加え、６０℃で７２時間作用させてマンノース１７．５０部を生成させた。これを水分５％程度まで乾燥させ、トランスグルコシダーゼ「アマノ」（天野製薬株式会社製）１０部を加えよく混合し、さらに５５℃で６９時間作用させて、α−マンノビオース含有パーム核ミールを得た。このα−マンノビオース含有パーム核ミールを再度水分５％まで乾燥させた。この乾燥α−マンノビオース含有パーム核ミールについて、イオン交換クロマトグラフィー法でα−マンノビオース及びマンノースの含有量を測定したところ、α−１，６−マンノビオースが３．３５部、マンノースが１４．０５部であった。なお、乾燥は６０℃のインキュベータ内で静置することにより行った。
【００２４】
実施例３（α−マンノビオース含有パーム核ミールの製造；酵素同時添加）
パーム核ミール１００部に、酵素セルロシンＧＭ５（エイチビィアイ株式会社製）０．６部及び酵素溶液トランスグルコシダーゼ「アマノ」（天野製薬株式会社製）１０部を溶解した酵素液１２５部を加えて、６０℃で３３６時間作用させて、α−マンノビオース含有パーム核ミールを得た。このα−マンノビオース含有パーム核ミールを水分５％まで乾燥させた。この乾燥α−マンノビオース含有パーム核ミールについて、イオン交換クロマトグラフィー法でα−マンノビオース及びマンノースの含有量を測定したところ、α−１，６−マンノビオースが３．５部、マンノースが２１．６部であった。
【００２５】
比較例１
パーム核ミール１００部に、酵素セルロシンＧＭ５（エイチビィアイ株式会社製）０．６部を溶解した酵素液１２５部を加え、６０℃で７２時間作用させてマンノース含有パーム核ミールを製造した。含有されるマンノース量を前記方法で測定したところ１７．５０部であった。
【００２６】
実施例４（サルモネラ菌・酵母凝集抑制試験）
実施例２において得られた乾燥済みのα−マンノビオース含有パーム核ミールを１０倍量の水に混合懸濁した後、１００００ｒｐｍで１０分間遠心分離し、α−マンノビオースをはじめ各種水溶性成分が溶解している上清を得た。一方、比較例１において得られたマンノース含有パーム核ミールも同様に処理してマンノースをはじめ各種水溶性成分を含む溶液を得た。これら２つの抽出液について、試験例１と同様にサルモネラ菌・酵母凝集抑制能を調べた。その結果、実施例２に係るα−マンノビオース含有パーム核ミールからの抽出液は、比較例１に係るマンノース含有パーム核ミールからの抽出液に比して１．５倍のサルモネラ菌・酵母凝集抑制活性を示した。
【００２７】
実施例５（代謝試験）
実施例２で製造した乾燥済みのα−マンノビオース含有パーム核ミールを１０％（ｗ／ｖ）となるようにＢＨＩ培地（日水製薬社製）に添加し、オートクレーブ滅菌後、鶏盲腸内容構成菌であるインテクリーン（伊藤忠飼料株式会社製）を接種し、３７℃で培養した。培養液を４時間ごとに取り出し、それぞれ１００００ｒｐｍで１０分間遠心分離後、その上澄のマンノース及びα−マンノビオース濃度（ｍｇ／ｍｌ）を分析した。その結果を表２に示す。表２から明らかなように、マンノースは資化されるのに対し、α−マンノビオースはほとんど資化されないことが判明した。
【００２８】
【表２】
【発明の効果】
本発明によれば、動物体内でのサルモネラ菌の定着を抑制してサルモネラ菌を体外へ効率的に排出することができる飼料、かかる飼料の製造に有利に用いることができるα−マンノビオース含有組成物及びその製造方法、並びにα−マンノビオース含有組成物を含む飼料用添加剤を提供することができる。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a feed containing α-mannobiose, an α-mannobiose-containing composition, a method for producing the same, and a feed additive.
[0002]
[Prior art]
Conventionally, palm kernel meal, copra meal, guar meal, and the like are known to contain abundant mannan having mannose as a constituent sugar, and mannose, manno-oligosaccharide, mannose are produced by reacting an enzyme with these natural materials. Various methods for producing polysaccharides have been proposed. In addition, it is known that feed containing such mannoses has a bactericidal effect (salmonella elimination effect) that suppresses colonization of the harmful bacterium Salmonella in the intestine and discharges it out of the body. Various techniques using this effect have been proposed (for example, Non-Patent Document 1).
[0003]
For example, a feed that is obtained by enzymatically decomposing a material containing mannan such as guar meal or copra meal and that contains manno-oligosaccharides containing mannobiose and mannotriose as main components and a mixture of monosaccharides can improve the quality of chicken eggs. At the same time, it is reported that mannooligosaccharides may be useful for preventing the colonization of Salmonella in livestock intestines (Patent Document 1). In addition, feed containing mannose, such as mannose, methyl-α-mannoside, manno-oligosaccharide, and a hydrolyzate of mannan obtained from guar gum, locust bean gum or yeast and / or an acid is used as a feed for harmful bacteria such as Salmonella. It is reported that it is useful for preventing infection (Patent Document 2). Furthermore, livestock feeds containing mannose-based polysaccharides mainly composed of polysaccharides having a repeating unit of mannose of 40 to 100 (30 to 80%) and also containing oligosaccharides (5 to 30%) are used in livestock intestines of Salmonella. It has been reported that it is effective in preventing fixation in a paper (Patent Document 3). In addition, there have been reported feeds in which a mannan-rich raw material such as palm kernel meal or guar meal is decomposed into mannooligosaccharides by the action of an enzyme, or feed containing mannose in which the enzyme is acted on copra meal ( Patent Document 4, Patent Document 5). However, no feed containing α-mannobiose was known.
[0004]
[Non-patent document 1]
"Poultry Science" 1989, No. 68, p. 1357
[Patent Document 1]
JP-A-7-236429 [Patent Document 2]
JP-A-8-38064 [Patent Document 3]
Japanese Patent Application Laid-Open No. 8-173,055 [Patent Document 4]
WO 95/17103 [Patent Document 5]
WO 99/08544 [0005]
[Problems to be solved by the invention]
Although various proposals have been made as described above, the effect of preventing the colonization of Salmonella in the livestock intestine is not sufficient at present, and further improvement is desired. For example, although mannose has a salmonella colonization inhibitory effect (salmonella elimination effect), since it is a simple sugar itself, even if it is provided to livestock as feed, most of it is easily assimilated by intestinal bacteria. It is known that digestion, absorption and excretion will be achieved by livestock themselves. In addition, according to the experimental results of the present inventors, it has been found that when a probiotic agent is administered to enhance the immunity of livestock, the activity of degrading mannose is further increased, and most of the mannose supplied as a measure against Salmonella disappears. Was done. Therefore, an extremely large amount of mannose must be administered in order to actually obtain a sufficient effect of inhibiting the colonization of Salmonella. On the other hand, according to the agglutination test using yeast, it was found that a manno-oligosaccharide or a mannose-based polysaccharide having a higher molecular weight than mannobiose had a small effect on inhibiting Salmonella colonization. The present invention has been made in view of such circumstances, and an object of the present invention is to provide a feed capable of suppressing the establishment of Salmonella in an animal body and efficiently discharging Salmonella out of the body, and a method for producing such a feed. An object of the present invention is to provide an α-mannobiose-containing composition which can be advantageously used, a method for producing the composition, and a feed additive containing the α-mannobiose-containing composition.
[0006]
[Means for Solving the Problems]
The present inventors have found that although mannose has an effect of eliminating Salmonella, it is a simple sugar itself, so even if it is provided to livestock as feed, most of it is easily assimilated by intestinal bacteria, Based on the hypothesis that digestion, absorption and excretion are also carried out by the livestock themselves, they disappear, and as a result of intensive studies such as searching for an active ingredient that replaces mannose, α-mannobiose has a very strong salmonella recognition activity. Further, they have found that they are not easily assimilated unlike mannose, and have completed the present invention.
[0007]
That is, the present invention provides a feed characterized by containing α-mannobiose (claim 1) and an α-mannobiose-containing composition obtained by reacting α-mannosidase on mannose. The feed according to claim 1 (claim 2) or an α-mannobiose-containing composition obtained by allowing β-mannosidase and α-mannosidase to act on natural products containing mannan sequentially or simultaneously. The feed according to claim 1 (Claim 3) or an α-mannobiose-containing composition obtained by allowing β-mannosidase and α-mannosidase to act on mannan extracted from a natural product containing mannan sequentially or simultaneously. The feed according to claim 1 (claim 4) or the natural product containing mannan is copra meal and / or palm kernel powder. 5. The feed according to claim 3 or 4, wherein α-mannobiose is contained in the feed at least 5% by weight based on the total amount of mannose mannooligosaccharides contained in the feed. The feed according to any one of claims 1 to 5 (claim 6), and 0.001 to 0.3% by weight of α-mannobiose. 6. The feed according to any one of claims 6 to 7 (claim 7).
[0008]
Also, the present invention provides a method for producing an α-mannobiose-containing composition (claim 8), wherein β-mannosidase and α-mannosidase are allowed to act sequentially or simultaneously on a natural product containing mannan. The method for producing an α-mannobiose-containing composition, wherein β-mannosidase and α-mannosidase are allowed to act sequentially or simultaneously on mannan extracted from a natural product, wherein the natural product containing mannan is: The method for producing an α-mannobiose-containing composition according to claim 8 or 9, which is copra meal and / or palm kernel meal (claim 10), and the production according to any one of claims 8 to 10. An α-mannobiose-containing composition (claim 11) produced by the method, or the α-mannobiose according to claim 11. Feed additive, which comprises a chromatography scan-containing compositions relating (claim 12).
[0009]
BEST MODE FOR CARRYING OUT THE INVENTION
As the feed of the present invention, α-1,2-mannobiose (α-1,2-mannobiose), α-1,3-mannobiose (α-1,3-mannobiose) having two α-glycosidic bonds of mannose, α-mannobiose-containing feed containing one or more kinds of α-1,4-mannobiose (α-1,4-mannobiose) and α-1,6-mannobiose (α-1,6-mannobiose) There is no particular limitation, and the α-mannobiose-containing feed of the present invention can be usually obtained by blending α-mannobiose and its contents with the feed conventionally used. Specific examples of the feed of the present invention include a feed containing purified α-mannobiose, a feed containing an α-mannobiose-containing composition obtained by allowing α-mannosidase to act on mannose, and a natural feed containing mannan. Or a feed containing an α-mannobiose-containing composition obtained by allowing β-mannosidase and α-mannosidase to act sequentially or simultaneously on a product, or β-mannosidase and α-mannosidase sequentially or selectively on mannan extracted from a natural product containing mannan. A feed containing the α-mannobiose-containing composition obtained by acting simultaneously can be exemplified. The form of the feed is not particularly limited, and examples thereof include liquid feeds and pasty feeds in addition to solid feeds for livestock and poultry.
[0010]
The feed containing the purified α-mannobiose contains, for example, α-mannobiose obtained by a method of synthesizing mannose, and does not contain mannose or other mannooligosaccharides. As a method for synthesizing from mannose, for example, “SYNTHESIS OF MANNOSE OLIGOSACCHARIDES VIA REVERSAL OF THE α-MANNOSIDASE REACTION” (Biotechnology Letters vol8 no.
[0011]
The feed containing the α-mannobiose-containing composition obtained by allowing α-mannosidase to act on the mannose contains the α-mannobiose-containing composition obtained by the method of causing α-mannobiose to act on mannose, and the mannose used as a raw material is Other than the purified mannose reagent, any source can be used.For example, β-mannosidase is allowed to act on a natural product containing mannan such as copra meal or palm kernel meal to release mannose. It can also be used. The α-mannosidase is not particularly limited as long as it has α-mannosidase activity, and may be of any origin, for example, Aspergillus described in JP-A-6-269290. niger-derived α-mannosidase, Sumiteam AC (manufactured by Nippon Chemical Industry Co., Ltd.), Sumiteam AGS (manufactured by Nippon Chemical Industry Co., Ltd.), hemicellulase GM “Amano” (manufactured by Amano Pharmaceutical Co., Ltd.), transglucosidase “Amano (Manufactured by Amano Pharmaceutical Co., Ltd.).
[0012]
The feed containing the α-mannobiose-containing composition obtained by allowing β-mannosidase and α-mannosidase to act on the natural product containing mannan sequentially or simultaneously is obtained by reacting β-mannosidase on a natural product containing mannan to produce mannose. And a composition containing α-mannobiose and mannose produced by allowing α-mannosidase to act on the released mannose. In the production of this α-mannobiose-containing composition, β-mannosidase is allowed to act on a natural product containing mannan to produce mannose, and then, if necessary, the water content is adjusted to allow α-mannosidase to act. Alternatively, β-mannosidase and α-mannosidase may be allowed to act simultaneously on a natural product containing mannan. As the α-mannosidase used in this reaction, the above-mentioned α-mannosidase can be used, and β-mannosidase is not particularly limited as long as it decomposes mannan and releases mannose, and any It may be of origin, and specific examples thereof include Cellulosin GM5 (manufactured by HBI Co., Ltd.). Examples of the natural product containing mannan include copra meal, palm kernel meal, guar meal and the like, and copra meal and palm kernel meal are preferred from the viewpoint of easy availability and rich mannan.
[0013]
The feed containing α-mannobiose obtained by allowing β-mannosidase and α-mannosidase to act on the mannan extracted from the natural product containing mannan sequentially or simultaneously is obtained by adding β-mannosidase to mannan extracted from the natural product containing mannan. And a composition containing α-mannobiose or mannose produced by reacting α-mannosidase on the released mannose. In the production of the α-mannobiose-containing composition, β-mannosidase is applied to mannan extracted from a mannan-containing natural product to produce mannose, and then, if necessary, the water content is adjusted to allow α-mannosidase to act. Alternatively, β-mannosidase and α-mannosidase may simultaneously act on mannan extracted from a natural product containing mannan. As the α-mannosidase used in this reaction, the above-mentioned α-mannosidase can be used, and β-mannosidase is not particularly limited as long as it decomposes mannan and releases mannose, and any It may be of origin, and specific examples thereof include Cellulosin GM5 (manufactured by HBI Co., Ltd.). Examples of the natural product containing mannan include copra meal, palm kernel meal, guar meal and the like, and copra meal and palm kernel meal are preferred from the viewpoint of easy availability and rich mannan. In addition, as a method for extracting micelles insoluble in water by forming micelles from a natural product containing mannan, an extraction method using an alkali solution, for example, a 5% cold sodium hydroxide solution or a 10% sodium hydroxide solution is exemplified. Can be.
[0014]
The content of α-mannobiose in the feed is preferably from 0.001 to 0.3% by weight, more preferably from 0.001 to 0.03% by weight. When the content of α-mannobiose is within the above range, the effects of the present invention can be exhibited effectively, and the content of mannoses can be significantly reduced. When the feed contains other mannose manno-oligosaccharides other than α-mannobiose, α-mannobiose is at least 5% by weight (5 to 100% by weight) based on the total mannose manno-oligosaccharides contained in the feed. It is preferably contained, more preferably at least 10% by weight. Here, the manno-oligosaccharide means an oligosaccharide composed of 2 to 8 mannose units.
[0015]
Since the feed of the present invention contains α-mannobiose, it is possible to suppress the colonization of Salmonella in the animal body and to efficiently discharge Salmonella out of the body. That is, since α-mannobiose has a very strong Salmonella recognition activity and is not easily assimilated in the body, it is possible to efficiently discharge Salmonella bacteria out of the body, and to contain mannoses in feed. The amount can be reduced, and the economic burden is greatly reduced. Specifically, as evident from the results of the Salmonella agglutination inhibition test and the metabolism test described below, α-mannobiose has a salmonella recognition activity four times that of mannose, and according to the metabolism test, almost no intestinal bacteria contribute to it. Since mannose is not used, the effect of eliminating Salmonella can be sufficiently exerted with at least a quarter or less of the amount when mannose is used. In addition, the feed of the present invention is particularly useful in the case of using a probiotic agent such as adult cecal bacterium for enhancing immunity of livestock.
[0016]
As a method for producing the α-mannobiose-containing composition of the present invention, for example, a production method in which β-mannosidase and α-mannosidase act sequentially or simultaneously on a natural product containing mannan such as copra meal or palm kernel meal, or containing mannan It is not particularly limited as long as it is a production method in which β-mannosidase and α-mannosidase act on mannan extracted from a natural product to be used sequentially or simultaneously, and the form is liquid, paste, slurry, or solid. Can be mentioned. The α-mannobiose-containing composition produced by directly acting an enzyme on a natural product can be added directly to the feed without separating and extracting α-mannobiose from the natural product to produce the feed of the present invention. The feed of the present invention can be provided simply and at low cost. When such a mannobiose-containing composition is distributed and used as it is for feed addition, it is dried by a method such as fluidized bed drying so that the water content becomes 10% by weight or less, since generation of fungi and fungi is feared. You can also. When the liquid α-mannobiose-containing composition is directly added, the feed of the present invention can be provided at a lower cost because the drying treatment can be omitted. Further, the α-mannobiose-containing composition of the present invention may be any composition obtained by the method for producing the α-mannobiose-containing composition of the present invention, and as the feed additive of the present invention, Examples thereof include a composition comprising the α-mannobiose-containing composition and a composition in which other additive components such as a preservative are added to the α-mannobiose-containing composition.
[0017]
【Example】
Hereinafter, the present invention will be described in detail with reference to Examples of the present invention, but the present invention is not limited to the following Examples. In the examples, "parts" means "parts by weight" unless otherwise specified.
Test Example 1 (Salmonella Agglutination Inhibition Test)
Salmonella agglutination inhibition tests of various saccharides were performed as follows. 0.1 ml of a suspension of Salmonella enteritidis (Salmonella enteritidis KTE-61 strain 1 × 10 8 cfu / ml isolated by ITOCHU FOOD CO., LTD.) And 0.1 ml of distilled water were thoroughly mixed, and 0.1 ml of a suspension of yeast Candida albicans (Candida albicans KI-102001 purchased from the Association of Animal Biologicals) was added and mixed. Preliminary tests were conducted to confirm that agglomeration occurred in the mixed liquid with a stereoscopic microscope (manufactured by Olympus Corporation).
[0018]
Next, in the same manner as in the preliminary test, various types of aqueous saccharide solutions were used in place of distilled water, and the respective concentrations were varied to determine the minimum concentration at which aggregation of the mixture was inhibited (the minimum concentration at which aggregation was inhibited). The test saccharides are (1) to (9) shown below, all of which used reagents of special grade (mannobiose of two types manufactured by Sigma and others manufactured by Wako Pure Chemical Industries).
(1) Mannose (D-mannose)
(2) α-1,4-mannobiose (α-1,4-mannobiose)
(3) α-1,6-mannobiose (α-1,6-mannobiose)
(4) α-methyl-D-mannopyranoside
(5) D-fructose
(6) L-arabinose
(7) D-galactose
(8) D-glucose
(9) Sucrose
[0019]
In this method, the concentration of the aggregation-inhibiting sugar was different depending on the test conditions such as the concentration of the Salmonella suspension, the sensitivity of the Salmonella, and the concentration of the yeast suspension. Therefore, (1) Mannose was used as a positive control for relative comparison. Table 1 shows the actual minimum concentration for inhibiting aggregation (μg / ml) before inhibiting the aggregation of the mixture and the relative concentration (concentration ratio) of various saccharides when the minimum concentration for inhibiting aggregation of mannose is set to 1. The case where the relative concentration is 1 or less indicates that the same aggregation suppressing effect can be obtained at a concentration lower than that of mannose.
[0020]
[Table 1]
As shown in Table 1, (2) α-1,4-mannobiose, (3) α-1,6-mannobiose and (4) α-methyl-D-mannopyranoside aggregated by Salmonella at a lower concentration than mannose. It has been found that can be suppressed. Among them, (2) α-1,4-mannobiose and (3) α-1,6-mannobiose contained in the feed of the present invention were found to have a very high effect of inhibiting yeast agglutination by Salmonella. In addition, D-fructose requires about four times the amount of mannose to suppress the aggregation of Salmonella, and L-arabinose, D-galactose, D-glucose and sucrose show no ability to inhibit aggregation by Salmonella. Did not.
[0022]
Example 1 (Production of α-1,6-mannobiose from mannose)
To 100 parts of D-mannose ("reagent special grade" manufactured by Wako Pure Chemical Industries, Ltd.), 100 parts of enzyme solution transglucosidase "Amano" (manufactured by Amano Pharmaceutical Co., Ltd.) was added and reacted at 50 ° C for 36 hours. The content of α-1,6-mannobiose in this reaction product was measured by ion exchange chromatography and found to be 25.4 parts. From this reaction product, α-1,6-mannobiose was fractionated by high performance liquid chromatography (column spectroscopy DC-613, manufactured by JASCO Corporation), and the obtained α-1,6-mannobiose was tested according to Test Example 1. Similarly, the ability of Salmonella to inhibit yeast aggregation was examined. As a result, the relative concentration with respect to mannose was 0.256, and it was found that α-1,6-mannobiose produced by allowing transglucosidase to act on mannose also has a very high salmonella / yeast aggregation inhibitory effect.
[0023]
Example 2 (Production of palm kernel meal containing α-mannobiose; sequential addition of enzyme)
To 100 parts of palm kernel meal (manufactured by Fuji Oil Co., Ltd.), 125 parts of an enzyme solution obtained by dissolving 0.6 part of the enzyme cellulosin GM5 (manufactured by HBI Co., Ltd.) was added. Part was generated. This was dried to a water content of about 5%, 10 parts of transglucosidase "Amano" (manufactured by Amano Pharmaceutical Co., Ltd.) was added, mixed well, and allowed to act at 55 ° C. for 69 hours to obtain α-mannobiose-containing palm kernel meal. . The α-mannobiose-containing palm kernel meal was dried again to a moisture content of 5%. When the content of α-mannobiose and mannose was measured by ion exchange chromatography on the dried α-mannobiose-containing palm kernel meal, 3.35 parts of α-1,6-mannobiose and 14.05 parts of mannose were measured. there were. In addition, drying was performed by leaving still in 60 degreeC incubator.
[0024]
Example 3 (Production of α-mannobiose-containing palm kernel meal; simultaneous addition of enzyme)
To 100 parts of palm kernel meal, 125 parts of an enzyme solution obtained by dissolving 0.6 part of an enzyme cellulosin GM5 (manufactured by HBI Co., Ltd.) and 10 parts of an enzyme solution transglucosidase “Amano” (manufactured by Amano Pharmaceutical Co., Ltd.) were added. For 336 hours to obtain α-mannobiose-containing palm kernel meal. The α-mannobiose-containing palm kernel meal was dried to a moisture content of 5%. The content of α-mannobiose and mannose was measured by ion-exchange chromatography on the dried α-mannobiose-containing palm kernel meal. As a result, 3.5 parts of α-1,6-mannobiose and 21.6 parts of mannose were obtained. there were.
[0025]
Comparative Example 1
To 100 parts of palm kernel meal, 125 parts of an enzyme solution obtained by dissolving 0.6 part of enzyme cellulosin GM5 (manufactured by HBI Co., Ltd.) was added, and the mixture was allowed to act at 60 ° C. for 72 hours to produce a mannose-containing palm kernel meal. It was 17.50 parts when the amount of mannose contained was measured by the said method.
[0026]
Example 4 (Salmonella / Yeast Agglutination Inhibition Test)
The dried α-mannobiose-containing palm kernel meal obtained in Example 2 was mixed and suspended in 10 times the volume of water, and then centrifuged at 10,000 rpm for 10 minutes to dissolve various water-soluble components including α-mannobiose. Was obtained. On the other hand, the mannose-containing palm kernel meal obtained in Comparative Example 1 was similarly treated to obtain a solution containing mannose and various water-soluble components. For these two extracts, the ability to inhibit the aggregation of Salmonella and yeast was examined in the same manner as in Test Example 1. As a result, the extract from the α-mannobiose-containing palm kernel meal according to Example 2 had 1.5 times the activity of inhibiting Salmonella / yeast aggregation than the extract from the mannose-containing palm kernel meal according to Comparative Example 1. showed that.
[0027]
Example 5 (metabolism test)
The dried α-mannobiose-containing palm kernel meal produced in Example 2 was added to a BHI medium (manufactured by Nissui Pharmaceutical Co., Ltd.) to a concentration of 10% (w / v), sterilized in an autoclave, and then subjected to autoclaving, followed by the constituent bacteria of chicken cecum Was inoculated and cultured at 37 ° C. The culture was taken out every 4 hours, centrifuged at 10,000 rpm for 10 minutes, and the supernatant was analyzed for mannose and α-mannobiose concentrations (mg / ml). Table 2 shows the results. As is clear from Table 2, it was found that mannose was assimilated, whereas α-mannobiose was hardly assimilated.
[0028]
[Table 2]
【The invention's effect】
ADVANTAGE OF THE INVENTION According to this invention, the feed which can suppress the colonization of Salmonella in an animal body and can efficiently discharge Salmonella out of a body, the alpha-mannobiose-containing composition which can be used advantageously for manufacture of such a feed, and its It is possible to provide a production method and an additive for feed containing the α-mannobiose-containing composition.
Claims (12)
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| JP2002345012A JP4294302B2 (en) | 2002-11-28 | 2002-11-28 | Alpha-mannobiose-containing feed |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2006325587A (en) * | 2005-04-27 | 2006-12-07 | Fuji Oil Co Ltd | COMPOSITION, FEED ADDITIVE AND FEED EACH DESIGNED TO CONTROL CAMPYLOBACTER, AND USING METHOD OF beta-1,4-MANNOBIOSE-CONTAINING COMPOSITION |
| JP2009538872A (en) * | 2006-05-30 | 2009-11-12 | ニュートリション サイエンシス エン．ヴェー．／エス．アー． | Tri- and tetra-oligosaccharides suitable as flocculants against enteric pathogens |
| JP6342531B1 (en) * | 2017-02-06 | 2018-06-13 | 不二製油株式会社 | Biological oil and fat composition for bait, method for producing bait oil and fat composition for bait, and method for producing bait organism |
| CN111885922A (en) * | 2018-03-16 | 2020-11-03 | 味之素株式会社 | Additive for feed and feed |
-
2002
- 2002-11-28 JP JP2002345012A patent/JP4294302B2/en not_active Expired - Lifetime
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2006325587A (en) * | 2005-04-27 | 2006-12-07 | Fuji Oil Co Ltd | COMPOSITION, FEED ADDITIVE AND FEED EACH DESIGNED TO CONTROL CAMPYLOBACTER, AND USING METHOD OF beta-1,4-MANNOBIOSE-CONTAINING COMPOSITION |
| JP2009538872A (en) * | 2006-05-30 | 2009-11-12 | ニュートリション サイエンシス エン．ヴェー．／エス．アー． | Tri- and tetra-oligosaccharides suitable as flocculants against enteric pathogens |
| JP6342531B1 (en) * | 2017-02-06 | 2018-06-13 | 不二製油株式会社 | Biological oil and fat composition for bait, method for producing bait oil and fat composition for bait, and method for producing bait organism |
| WO2018143083A1 (en) * | 2017-02-06 | 2018-08-09 | 不二製油グループ本社株式会社 | Oil-and-fat composition for feed organism, production method for oil-and-fat composition for feed organism, and production method for feed organism |
| JP2018126066A (en) * | 2017-02-06 | 2018-08-16 | 不二製油株式会社 | Fat composition for bait organism, method for manufacturing fat composition for bait organism, and method for manufacturing bait organism |
| CN111885922A (en) * | 2018-03-16 | 2020-11-03 | 味之素株式会社 | Additive for feed and feed |
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