JP4127864B2 - Gram-negative bacterial growth inhibitor - Google Patents
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Description
【０００１】
【産業上の利用分野】
本発明は、哺乳動物及び鳥類およびそれらから得られる畜産物のグラム陰性菌増殖抑制剤に関する。
【０００２】
【従来の技術】
細菌は菌の表層構造の差異によるグラム染色性によりグラム陽性菌とグラム陰性菌に分別される。グラム陰性菌には、赤痢菌、チフス菌、パラチフス菌、サルモネラ菌、大腸菌、クレブシエラ、セラチア、プロテウス、ペスト菌、エルシニア、コレラ菌、腸炎ビブリオ、緑濃菌、インフルエンザ菌、ブルセラ、レジオネラ、カンピロバクター等があり、病原性の細菌が多い。なかでも大腸菌やサルモネラ菌は代表的な食中毒菌であり、人畜共通の感染症でもある。サルモネラ感染症の場合、動物では牛、馬、めん羊、山羊、豚、犬、猫等の哺乳動物や鶏、あひる、うずら等の鳥類に見られ、牛では下痢や関節炎、豚では敗血症や腸炎、鶏の場合はブロイラーのひな白痢や１０日齢以内の幼ひなの下痢・敗血症を起こし、重度の場合は死に至る。サルモネラ感染症は発生率が高く、損害も大きいと同時に肉用牛や鶏では、人への感染源ともなりうるため公衆衛生上重要な問題である。過去にサルモネラ・エンテリティディスによるサルモネラ食中毒の発生が世界的に増加し、その原因が鶏卵の汚染であることが明らかにされて大きな社会問題となっている。また、大腸菌の場合は体内や食品中で繁殖し、毒素を生成して下痢症の原因となっている。
【０００３】
このようなグラム陰性菌が動物・ヒトの体内へ侵入、増殖したりすることは衛生上また健康上好ましくない。これらグラム陰性菌に対する対策としては消毒剤、抗菌剤、洗浄剤、ワクチン等が知られている。例えば、家畜や鶏のサルモネラ感染症の予防・治療方法としては、畜舎や養鶏場の清掃・消毒の励行はもちろんのこと、抗生物質や他の合成抗菌剤を飼料とともに投与する方法が行われている（特開昭６２−２９４６２３号）。しかし、このような薬剤による方法は薬剤耐性菌の出現や畜産物への薬剤の残留等の問題があり、人の健康上好ましい方法ではない。また特公昭６４−７０４９号には、盲腸内容物の嫌気培養したものを経口投与することにより、腸管内のサルモネラ菌を排除しようとする試みが開示されているが、効果が明確ではなく嫌気培養等に特殊な設備が必要であり、実用的でない。さらに欧米ではワクチンによる感染防除の方法が実施されているが、ワクチン接種にかなりの手間がかかり、また鶏の場合断餌等のストレスにより以後の発育、産卵に影響を及ぼす等の問題があり有効な方法ではない。
【０００４】
最近、鶏のサルモネラ感染症の場合、フラクトオリゴ糖やガラクトオリゴ糖を用いてサルモネラ菌の増殖を阻害する方法が開示されている（特開平３−５０１９７１号、特開平５−２０８９１２号）。しかしながら、これらのオリゴ糖は飼料化するときの加熱、酸に対して不安定であったり、菌に対する増殖阻害効果が試験管レベルでの結果にとどまっており効果が弱い等の欠点を有する。
【０００５】
【発明が解決しようとする課題】
従って、このような状況下、哺乳動物や鳥類およびそれらから得られる畜産物などからグラム陰性菌を除く、または増殖を抑制する安全な物質が強く求められていた。
【０００６】
【課題を解決するための手段】
本発明者らは上記課題を解決すべく鋭意研究した結果、多糖類の分解物を哺乳動物や鳥類などが経口的に摂取することによる、体内のグラム陰性菌の増殖抑制効果を見出した。また、これら哺乳動物や鳥類などから得られる畜産物についても抑制効果の有ることを見出した。さらに、これら多糖類の分解物とタンニン類を組み合わせたもの、多糖類の分解物とグリセリン脂肪酸エステルまたはポリグリセリン脂肪酸エステルを組み合わせたもの、多糖類の分解物とサポニン類を組み合わせたものが多糖類の分解物単独で用いるより優れた増殖抑制効果のあることを見出し本発明を完成させるに至った。
【０００７】
本発明における多糖類の分解物とは、グアーガム、ローカストビーンガム、タマリンドガム、ペクチン、キサンタンガム、プルランなどの多糖類を酵素または酸によって分解したものを指す。具体的には、グアーガム、ローカストビーンガムについてはアスペルギルス属菌やリゾープス属菌等に由来する酵素ガラクトマンナナーゼによる分解が好ましく、ペクチンについてはアスペルギルス属菌の生産する酵素ペクチナーゼによる分解が好ましい。タマリンドガム及びキサンタンガムについては微生物由来のセルラーゼやキシラナーゼによる分解が好ましく、プルランについてはプルラナーゼによる分解が好ましい。
また本発明の多糖類の分解物は加水分解することによって得られた低分子化したものであり、酵素の反応時間又は酸分解の反応時間を変えることにより分子量を変化させることができる。例えば、グアーガムの加水分解物ではマンノース直鎖の鎖長が３０〜２００単位、または５〜２９単位の範囲内に８０％以上分布していることがよい。
【０００８】
グアーガムの加水分解物の鎖長とは、本分解物の主鎖はマンノースであり、その結合している数を指す。また、その他の多糖類の加水分解も同様で鎖長が３０〜２００単位、好ましくは５〜２９単位の範囲内に８０％以上分布していることがよい。それらの測定法は特に限定するものではないが、例えば分解されたグアーガムを水に溶解し、８０３Ｄ型（東ソー（株）製）の高速液体クロマトグラフィーを用い、水を移動相にしてＧ３０００ＰＷ（東ソー（株）製）のカラムにてゲル濾過を行い、示差屈折計にて検出することにより測定できる。
【０００９】
本発明に用いられるタンニン類は、緑茶、ウーロン茶又は紅茶の水もしくはアルコール抽出物の限外濾過および逆浸透膜処理、又は酢酸可溶画分より得ることができるが、柿しぶやりんごなど他の原料起源のものまたは化学合成品でもよい。タンニン類としては、（＋）−カテキン，（＋）−ガロカテキン，（−）−ガロカテキンガレート，（−）−エピカテキン，（−）−エピカテキンガレート，（−）−エピガロカテキン，（−）−エピガロカテキンガレート，遊離型テアフラビン，テアフラビンモノガレートＡ，テアフラビンモノガレートＢおよびテアフラビンジガレートが挙げられる。得られたこれらのタンニン類を本発明に用いる場合は単独で、もしくは二種以上の混合物として、さらにはタンニン類を含む粗抽出物でも使用できる。
【００１０】
本発明で使用するグリセリン脂肪酸エステルはグリセリンと脂肪酸のエステルであり、脂肪酸としては、炭素数８〜１４の直鎖脂肪酸が好ましく、カプリル酸、カプリン酸、ラウリン酸が挙げられる。グリセリン脂肪酸エステルにはモノ、ジ、トリエステルがあり、モノエステルを使用するのが好ましく、一例を挙げるとグリセリンモノカプリル酸エステル、グリセリンモノカプリン酸エステル、グリセリンモノラウリン酸エステルである。
本発明で使用するグリセリン脂肪酸エステルは、単独もしくは二種以上の混合物でもかまわない。
本発明で使用するポリグリセリン脂肪酸エステルの脂肪酸は、炭素数８〜１４の直鎖脂肪酸が好ましく、カプリル酸、カプリン酸、ラウリン酸、ミリスチン酸が挙げられる。
本発明で使用するポリグリセリン脂肪酸エステルのポリグリセリンはグリセリンの重合したものであり、一般にジ、トリ、ペンタ、ヘキサ、オクタ、デカグリセリンがあげられる。本発明で使用するポリグリセリン脂肪酸エステルは、単独または二種以上の混合物でもかまわない。
【００１１】
本発明に使用するサポニン類は、キラヤサポニン、ユッカサポニン、ビートサポニン、大豆サポニン、茶サポニン、杜仲茶サポニンであり、単独または二種以上の混合物でもかまわない。
本発明の多糖類の分解物の有効量は、哺乳動物、鳥類に対して１日当たり０．０１〜１０ｇ／体重ｋｇ、好ましくは０．０５〜５ｇ／体重ｋｇ、また多糖類の分解物と合わせて用いることのできるタンニン類は１日当たり０．０００１ｇ〜１．０ｇ／体重ｋｇ、好ましくは０．００１〜０．５ｇ／体重ｋｇ、多糖類の分解物とタンニン類の混合比は、６：１〜１０００：１、好ましくは１０：１〜５０：１である。多糖類の分解物と合わせて用いることのできるグリセリン脂肪酸エステル及び／又はポリグリセリン脂肪酸エステルは０．００１〜５ｇ／体重ｋｇ、好ましくは０．００５〜０．５ｇ／体重ｋｇであり、多糖類の分解物とグリセリン脂肪酸エステルおよびポリグリセリン脂肪酸エステルの混合比は、８：１〜４００：１、好ましくは１５：１〜６０：１である。多糖類の分解物と合わせて用いることのできるサポニン類の有効量は０．０００１〜１．０ｇ／体重ｋｇ、好ましくは０．００１〜０．５ｇ／体重ｋｇであり、多糖類の分解物とサポニン類との混合比は６：１〜７００：１、好ましくは１５：１〜７０：１である。
【００１２】
本発明のグラム陰性菌増殖抑制剤は、通常の哺乳動物及び鳥類の飼料製造工程中のいずれかにおいて、飼料原料中に液状、粉末状、もしくは顆粒状の多糖類の分解物、タンニン類、グリセリン脂肪酸エステルまたはポリグリセリン脂肪酸エステル、サポニン類を添加、混合して、粉末状、スラリー状、もしくはペレット状等、適宜の形態の製品に加工するか、または飼料製品に直接添加、混合することによって製造できる。本発明品の有効成分である多糖類の分解物、タンニン類、グリセリン脂肪酸エステルまたはポリグリセリン脂肪酸エステル、サポニン類の飼料への添加量は、動物に投与する際、上述の有効量となるように適宜添加すればよい。
本発明の動物とは、牛、馬、羊、山羊、豚、犬、猫等の哺乳動物および鶏、あひる、うずら等の鳥類である。また本発明の畜産物とは、それら動物から得られる生産物であり、乳、畜肉、内臓や鶏等から得られる卵、肉、内臓等を指す。
以下、本発明を実施例により詳細に説明するが、これにより特に限定されるものではない。
【００１３】
【実施例】
実施例１
水９００部にクエン酸を加えてｐＨを３に調整した。これにアスペルギルス由来のガラクトマンナナーゼ０．２部とグアーガム粉末１００部を添加混合して４０〜４５℃で２４時間酵素反応を行った。反応後９０℃、１５分間加熱して酵素を失活させた。濾過により不純物を除去し得られた透明な溶液を減圧濃縮した後、噴霧乾燥し本発明品のグアーガム分解物６５部が得られた。高速液体クロマトグラフィーで測定した結果、該ガラクトマンナンの糖鎖の８０％以上はマンノースの鎖長が５０〜１５０単位の範囲内に包含されていた。
【００１４】
実施例２
同様の方法で、反応時間のみを４８時間と変えることにより、マンノース直鎖の短い本発明品のグアーガム分解物（マンノースの鎖長の８０％以上が５〜２５単位の範囲内に包含される）６８部が得られた。
実施例３
水９００部にクエン酸を加えてｐＨを３に調整した。これにアスペルギルス由来のガラクトマンナナーゼ０．２部とローカストビーンガム粉末１００部を添加混合して４０〜４５℃で６時間酵素を作用させた。反応後、９５℃、１５分間加熱して酵素を失活させた。そして濾過分離して不純物を除き、得られた溶液を凍結乾燥し、ローカストビーンガム分解物６４部を得た。
【００１５】
実施例４
水９００部にクエン酸を加えてｐＨを３に調整した。これにアスペルギルス由来のペクチナーゼ０．１部とペクチン粉末（エステル化度７０％）１００部を添加混合して３０〜３５℃で８時間酵素を作用させた。反応後、９５℃、１５分間加熱して酵素を失活させた。そして濾過分離して不純物を除き、得られた溶液を凍結乾燥し、ペクチン分解物６４部を得た。
実施例５
実施例１で得られたグアーガム分解物と、タンニン類として緑茶の熱水抽出物の限外濾過膜処理画分を凍結乾燥した粉末を用いて、重量比で１９：１となるように混合し本発明品を得た。
【００１６】
実施例６
実施例２で得られたグアーガム分解物と、タンニン類として緑茶の熱水抽出物の酢酸エチル可溶画分を凍結乾燥した粉末を用いて、重量比で１９：１となるように混合し本発明品を得た。
実施例７
実施例２で得られたグアーガム分解物と、タンニン類として緑茶のエタノール抽出物の酢酸エチル可溶画分からさらにカラムクロマトグラフィーを用いて精製した（−）−エピガロカテキンガレートを用いて、重量比で１９：１となるように混合し本発明品を得た。
【００１７】
実施例８
実施例２で得られたグアーガム分解物と、タンニン類として市販のインスタント紅茶の熱水抽出液のクロロホルム可溶画分より得た粗テアフラビンを用いて、重量比で１９：１となるように混合し本発明品を得た。
実施例９
実施例１で得られたグアーガム分解物と、グリセリンモノカプリル酸エステルを用いて、重量比で１９：１となるように混合し本発明品を得た。
【００１８】
実施例１０
実施例２で得られたグアーガム分解物と、グリセリンモノラウリン酸エステルを用いて、重量比で１９：１となるように混合し本発明品を得た。
実施例１１
実施例２で得られたグアーガム分解物とキラヤサポニン（商品名：キラヤニンＣ−１００、部分加水分解サポニンとして約１０％、丸善製薬（株）製）を用いて、重量比で１９：１となるように混合し本発明品を得た。
【００１９】
実施例１２
実施例２で得られたグアーガム分解物とユッカサポニン（部分加水分解サポニンとして約２０％）を用いて、重量比で１９：１となるように混合し本発明品を得た。
実施例１３
実施例２で得られたグアーガム分解物とビートサポニン（部分加水分解サポニンとして約６０％）を用いて、重量比で１９：１となるように混合し本発明品を得た。
【００２０】
実施例１４
実施例２で得られたグアーガム分解物と大豆サポニン（部分加水分解サポニンとして約６０％）を用いて、重量比で１９：１となるように混合し本発明品を得た。
実施例１５
実施例２で得られたグアーガム分解物と茶の実から得た茶サポニン（部分加水分解サポニンとして約５０％）を用いて、重量比で１９：１となるように混合し本発明品を得た。
実施例１６
実施例２で得られたグアーガム分解物と杜仲茶サポニン（部分加水分解サポニンとして約５０％）を用いて、重量比で１９：１となるように混合し本発明品を得た。
実施例１７
実施例３で得られたローカストビーンガム分解物と、タンニン類として緑茶の熱水抽出物の酢酸エチル可溶画分を凍結乾燥した粉末を用いて、重量比で１９：１となるように混合し本発明品を得た。
【００２１】
実施例１８
実施例４で得られたペクチン分解物とキラヤサポニン（商品名：キラヤニンＣ−１００、部分加水分解サポニンとして約１０％、丸善製薬（株）製）を用いて、重量比で１９：１となるように混合し本発明品を得た。
実施例１９
水溶性食物繊維のプルランをプルラナーゼで処理したプルラン分解物とキラヤサポニン（商品名：キラヤニンＣ−１００、部分加水分解サポニンとして約１０％、丸善製薬（株）製）を用いて、重量比で１９：１となるように混合し本発明品を得た。
【００２２】
実施例２０
実施例２で得られたグアーガム分解物と、緑茶の熱水抽出物の酢酸エチル可溶画分を凍結乾燥した粉末およびグリセリンモノカプリル酸エステルを用いて、重量比で１８：１：１となるように混合し本発明品を得た。
実施例２１
実施例２で得られたグアーガム分解物と、緑茶の熱水抽出物の酢酸エチル可溶画分を凍結乾燥した粉末およびキラヤサポニン（商品名：キラヤニンＣ−１００、部分加水分解サポニンとして約１０％、丸善製薬（株）製）を用いて、重量比で１８：１：１となるように混合し本発明品を得た。
【００２３】
試験例１
サルモネラ・エンテリティディス ＩＦＯ−３３１３とサルモネラ・チフィムリウム ＩＦＯ−１２５２９、サルモネラ・ダブリン ＮＩＡＨ−１２０１、ビブリオ・パラファエモリティカス、シュードモナス・マレイ、ブルセラ・スイスを本発明品含有培地にて次のように培養した。肉汁培地に、炭素源として実施例１〜４で得られた本発明品を０．５％となるように加え、対照として、肉汁培地にグルコースを０．５％加えたものを用いた。３７℃で４８時間培養を行い、培養液のｐＨ低下により菌の生育を確認した。結果を表１に示す。表中の菌の増殖の程度は、次のように表示した。−（増殖なし：ｐＨ６．０以上）、＋−（弱い増殖：ｐＨ５．５１−６．００）、＋（増殖：ｐＨ５．０１−５．５０）、＋＋（よく増殖：ｐＨ５．００以下）
【００２４】
【表１】
表１の結果より、本発明品を添加すると試験菌の増殖はほとんどないことがわかる。
試験例２
サルモネラ・エンテリティディス ＩＦＯ−３３１３とサルモネラ・チフィムリウム ＩＦＯ−１２５２９およびサルモネラ・ダブリン ＮＩＡＨ−１２０１、ビブリオ・パラファエモリティカス、シュードモナス・マレイ、ブルセラ・スイスを本発明品含有培地にて次のように培養した。肉汁培地に、炭素源としてグルコース０．５％と実施例５〜２１で得られた本発明品を０．５％となるように加えて、３７℃で４８時間培養を行い、培養液のｐＨ低下により菌の生育を確認した。対照として、グルコース０．５％を含む肉汁培地を用い、比較として、緑茶の熱水抽出物の限外濾過膜処理画分を凍結乾燥した粉末（Ａ）、緑茶の熱水抽出物の酢酸エチル可溶画分を凍結乾燥した粉末（Ｂ）、（−）−エピガロカテキンガレート（Ｃ）、粗テアフラビン（Ｄ）、グリセリンモノカプリル酸エステル（Ｅ）、グリセリンモノラウリン酸エステル（Ｆ）、キラヤサポニン（Ｇ）、ユッカサポニン（Ｈ）、ビートサポニン（Ｉ）、大豆サポニン（Ｊ）、茶サポニン（Ｋ）、杜仲茶サポニン（Ｌ）、ＢとＥを同重量比で混合したもの（Ｍ）、ＢとＧを同重量比で混合したもの（Ｎ）をそれぞれ０．０２５％となるように加えて同様に培養した。結果を表２および表３に示す。表中の菌の増殖の程度は、次のように表示した。−（増殖なし：ｐＨ６．０１以上）、＋−（弱い増殖：ｐＨ５．５１−６．００）、＋（増殖：ｐＨ５．０１−５．５０）、＋＋（よく増殖：ｐＨ５．００以下）
【００２６】
【表２】
【表３】
表２および表３の結果より、多糖類の分解物にタンニン類、グリセリン脂肪酸エステルまたはポリグリセリン脂肪酸エステル、サポニン類を併用すると、試験菌の増殖はないのはもちろんのこと、増殖阻害効果のあることがわかる。
試験例３
１５頭の分娩直後の成牛を３頭ずつ５群に分け、基本飼料のみを与えた群をＡ群、基本飼料１ｋｇに実施例１、７、１０、１２で調製した本発明品１０ｇを添加した群をそれぞれＢ、Ｃ、Ｄ、Ｅ群とし５週間飼育した。牛由来のサルモネラ・タブリンをＢＨＩ培地で培養し集菌後、生理食塩水で１×１０5 個／ｍｌとなるように調製した菌液を１頭当たり１００ｍｌ哺乳壜にて、各飼料で飼育後１週間目に経口感染させた。感染後２および４週間目の糞便を採取し、サルモネラ選択培地（栄研化学（株）製）にてサルモネラ菌の菌数を測定した。結果を表４に示す。
【００２９】
【表４】
表４より、本発明品を添加した群でサルモネラ菌は検出されなかった。また、グアーガム分解物とタンニン類、グリセリン脂肪酸エステルまたはポリグリセリン脂肪酸エステル、サポニン類を併用すると、増殖抑制効果は顕著であった。
試験例４
１５頭の搾乳牛を３頭ずつ５群に分け、基本飼料のみを与えた群をＡ群、基本飼料１ｋｇに実施例１、７、１０、１２で調製した本発明品１０ｇを添加した群をそれぞれＢ、Ｃ、Ｄ、Ｅ群とし５週間飼育した。牛由来のサルモネラ・タブリンをＢＨＩ培地で培養し集菌後、生理食塩水で１×１０5 個／ｍｌとなるように調製した菌液を１頭当たり１００ｍｌ哺乳壜にて、各飼料で飼育後１週間目に経口感染させた。感染後２および４週間目の牛乳を採取し、牛乳１ｍｌをサルモネラ増菌培地で増菌を行った後、サルモネラ選択培地（栄研化学（株）製）に塗抹してサルモネラ菌の有無を判定した。サルモネラ菌が多数検出された場合＋＋、わずかの場合＋、全く検出されなかった場合−と表示した。結果を表５に示す。
【００３１】
【表５】
表５より、本発明品を添加した群の牛乳中にはサルモネラ菌は検出されなかった。また、グアーガム分解物とタンニン類、グリセリン脂肪酸エステルまたはポリグリセリン脂肪酸エステル、サポニン類を併用すると、増殖抑制効果は顕著であった。
試験例５
２０日令の子豚１５頭を３頭ずつ５群に分け、基本飼料として哺乳期子豚育成用飼料（昭和産業（株）製）のみを与えた群をＡ群、基本飼料１ｋｇ実施例２、６、１１、１３で調製した本発明品１０ｇを添加した飼料を与えた群をそれぞれＢ、Ｃ、Ｄ、Ｅ群とし、５週間飼育した。豚由来のサルモネラ・チフィムリウムをＢＨＩ培地で培養し集菌後、生理食塩水で１×１０5 個／ｍｌとなるように菌液を調製した。１頭当たり１００ｍｌ哺乳壜にて、各飼料で飼育後１週間目に経口感染させた。感染後２および４週間目の糞便を採取し、サルモネラ選択培地（栄研化学（株）製）にてサルモネラ菌の菌数を測定した。また同時にデスオキシコレート培地（栄研化学（株）製）にて糞便中の大腸菌の菌数も測定した。結果を表６に示す。
【００３３】
【表６】
表６より、本発明品を添加した群でサルモネラ菌は検出されず、大腸菌は減少した。また、グアーガム分解物とタンニン類、グリセリン脂肪酸エステルまたはポリグリセリン脂肪酸エステル、サポニン類を併用することにより増殖抑制効果は顕著であった。
試験例６
豚１５頭を３頭ずつ５群に分け、基本飼料として豚育成用飼料（昭和産業（株）製）のみを与えた群をＡ群、基本飼料１ｋｇ実施例２、６、１１、１３で調製した本発明品１０ｇを添加した飼料を与えた群をそれぞれＢ、Ｃ、Ｄ、Ｅ群とし、５週間飼育した。豚由来のサルモネラ・チフィムリウムをＢＨＩ培地で培養し集菌後、生理食塩水で１×１０5 個／ｍｌとなるように菌液を調製した。１頭当たり１００ｍｌ哺乳壜にて、各飼料で飼育後１週間目に経口感染させた。感染後５日目に屠殺し、大腿筋、胃、十二指腸、盲腸、直腸、肝臓、脾臓を採取し、各検体１ｇをサルモネラ増菌培地で増菌を行った後、サルモネラ選択培地（栄研化学（株）製）に塗抹してサルモネラ菌の有無を判定した。サルモネラ菌が多数検出された場合＋＋、わずかの場合＋、全く検出されなかった場合−と表示した。結果を表７に示す。
【００３５】
【表７】
表７より、本発明品を添加した群から得られた大腿筋、肝臓、脾臓にサルモネラ菌は検出されず、また、胃、十二指腸、盲腸、直腸の消化管ではサルモネラ菌の増殖抑制効果が見られた。さらに、グアーガム分解物とタンニン類、グリセリン脂肪酸エステルまたはポリグリセリン脂肪酸エステル、サポニン類を併用することにより増殖抑制効果は顕著であった。
試験例７
孵化直後の幼雛６０羽を５羽ずつ１２群に分け、基本飼料にて１週間飼育した。その後、２週間基本飼料のみを与えた群をＡ群、基本飼料１ｋｇ実施例１〜２１で調製した本発明の組成物５ｇをそれぞれ添加した飼料を与えた群をそれぞれＢ〜ｖ群とした。各飼料で１週間飼育後、サルモネラ菌の感染を行った。鶏由来のサルモネラ・エンテリティディスをＢＨＩ培地で培養し集菌後、生理食塩水で１×１０5 個／ｍｌとなるように調製した菌液を１羽当たり１ｍｌ注射器にて経口感染させた。感染前日、感染後１、２、４、６および８日目の糞便を採取し、サルモネラ選択培地（栄研化学（株）製）にてサルモネラ菌の菌数を測定した。結果を表８〜表１０に示す。
【００３７】
【表８】
【表９】
【表１０】
表８〜表１０より、本発明品を添加した群でサルモネラ菌は検出されなかった。また、多糖類の分解物とタンニン類、グリセリン脂肪酸エステルまたはポリグリセリン脂肪酸エステル、サポニン類を併用することによりサルモネラ菌の増殖抑制効果は顕著であった。
【００４１】
試験例８
ブロイラー６０羽を５羽ずつ１２群に分け、基本飼料にて１週間飼育した。その後、２週間基本飼料のみを与えた群をＡ群、基本飼料１ｋｇと実施例１〜２０で調製した本発明の組成物５ｇをそれぞれ添加した飼料を与えた群をそれぞれＢ〜Ｕ群とした。各飼料で１週間飼育後、サルモネラ菌の感染を行った。鶏由来のサルモネラ・エンテリティディスをＢＨＩ培地で培養し集菌後、生理食塩水で１×１０5 個／ｍｌとなるように調製した菌液を１羽当たり１ｍｌ注射器にて経口感染させた。感染後５日目に屠殺し、大腿筋、十二指腸、盲腸、直腸、肝臓、脾臓を採取し、各検体１ｇをサルモネラ増菌培地で増菌を行った後、サルモネラ選択培地（栄研化学（株）製）に塗抹してサルモネラ菌の有無を判定した。サルモネラ菌が多数検出された場合＋＋、わずかの場合＋、全く検出されなかった場合−と表示した。結果を表１１〜表１３に示す。
【００４２】
【表１１】
【表１２】
【表１３】
表１１〜表１３より、本発明品を添加した群から得られた大腿筋、肝臓、脾臓にサルモネラ菌は検出されず、十二指腸、盲腸、直腸の消化管ではサルモネラ菌の増殖抑制効果が見られた。また、多糖類の分解物とタンニン類、グリセリン脂肪酸エステルまたはポリグリセリン脂肪酸エステル、サポニン類を併用することによりサルモネラ菌の増殖抑制効果は顕著であった。
試験例９
産卵鶏６０羽を５羽ずつ１２群に分け、基本飼料にて１週間飼育した。その後、２週間基本飼料のみを与えた群をＡ群、基本飼料１ｋｇと実施例１〜２１で調製した本発明の組成物５ｇをそれぞれ添加した飼料を与えた群をそれぞれＢ〜ｖ群とした。各飼料で１週間飼育後、サルモネラ菌の感染を行った。鶏由来のサルモネラ・エンテリティディスをＢＨＩ培地で培養し集菌後、生理食塩水で１×１０4 個／ｍｌとなるように調製した菌液を１羽当たり１ｍｌ注射器にて経口感染させた。感染後１０日目の卵を採取し、卵殻表面と卵黄中のサルモネラ菌を測定した。卵殻表面の場合、卵１個につき１０ｍｌの生理食塩水で卵殻表面を洗い出しその液について、また卵黄の場合１０倍希釈液について、それぞれサルモネラ増菌培地で増菌を行った後、サルモネラ選択培地（栄研化学（株）製）に塗抹してサルモネラ菌の有無を判定した。サルモネラ菌が多数検出された場合＋＋、わずかの場合＋、全く検出されなかった場合−と表示した。結果を表１４〜表１６に示す。
【００４６】
【表１４】
【表１５】
【表１６】
表１４〜表１６より、本発明品を添加したすべての群から得られた鶏卵の卵殻表面、卵黄中にサルモネラ菌は検出されなかった。従って、本発明品によるサルモネラ菌の鶏卵内部および外部の汚染は予防可能となった。本発明の実施態様ならびに目的生成物を挙げれば以下の通りである。
（１）多糖類の分解物を含有するグラム陰性菌増殖抑制剤。
（２）多糖類がグアーガムである前記（１）記載のグラム陰性菌増殖抑制剤。
（３）多糖類がローカストビーンガムである前記（１）記載のグラム陰性菌増殖抑制剤。
（４）多糖類がキサンタンガムである前記（１）記載のグラム陰性菌増殖抑制剤。
（５）多糖類がペクチンである前記（１）記載のグラム陰性菌増殖抑制剤。
（６）多糖類がプルランである前記（１）記載のグラム陰性菌増殖抑制剤。
（７）分解物が酵素による分解物である前記（１）〜（６）記載のグラム陰性菌増殖抑制剤。
（８）分解物が酸による分解物である前記（１）〜（６）記載のグラム陰性菌増殖抑制剤。
（９）多糖類の分解物とタンニン類と併用することを特徴とするグラム陰性菌増殖抑制剤。
（１０）タンニン類が緑茶の熱水抽出物である前記（９）記載のグラム陰性菌増殖抑制剤。
（１１）タンニン類が緑茶の酢酸エチル可溶画分である前記（９）記載のグラム陰性菌増殖抑制剤。
（１２）タンニン類が（＋）−カテキン，（＋）−ガロカテキン，（−）−ガロカテキンガレート，（−）−エピカテキン，（−）−エピカテキンガレート，（−）−エピガロカテキン，（−）−エピガロカテキンガレート，遊離型テアフラビン，テアフラビンモノガレートＡ，テアフラビンモノガレートＢおよびテアフラビンジガレートの化合物群より選ばれる一種または二種以上の化合物である前記（９）記載のグラム陰性菌増殖抑制剤。
（１３）タンニン類が（−）−エピガロカテキンガレードである前記（９）記載のグラム陰性菌増殖抑制剤。
（１４）多糖類の分解物とグリセリン脂肪酸エステルまたはポリグリセリン脂肪酸エステルとを併用することを特徴とするグラム陰性菌増殖抑制剤。
（１５）グリセリン脂肪酸エステルがモノ、ジ、トリエステルである前記（１４）記載のグラム陰性菌増殖抑制剤。
（１６）ポリグリセリン脂肪酸エステルの脂肪酸が、炭素数８〜１４のカプリル酸、カプリン酸、ラウリン酸、ミリスチン酸の直鎖脂肪酸である前記（１４）記載のグラム陰性菌増殖抑制剤。
（１７）多糖類の分解物とサポニン類とを併用することを特徴とするグラム陰性菌増殖抑制剤。
（１８）サポニン類がキラヤサポニン、ユッカサポニン、ビートサポニン、大豆サポニン、茶サポニン、杜仲茶サポニンより選ばれる一種または二種以上のサポニンである前記（１７）記載のグラム陰性菌増殖抑制剤。
（１９）多糖類の分解物とタンニン類とグリセリン脂肪酸エステルまたはポリグリセリン脂肪酸エステルとを併用することを特徴とするグラム陰性菌増殖抑制剤。
（２０）多糖類の分解物とタンニン類とサポニン類とを併用することを特徴とするグラム陰性菌増殖抑制剤。
（２１）畜産物からグラム陰性菌の増殖を抑制することを特徴とする前記（１）〜（２０）記載のグラム陰性菌増殖抑制剤。
（２２）畜産物が哺乳動物及び鳥類から得られる生産物である前記（２１）記載のグラム陰性菌抑制剤。
（２３）畜産物が牛乳である前記（２１）記載のグラム陰性菌抑制剤。
（２４）畜産物が鶏卵である前記（２１）記載のグラム陰性菌抑制剤。
（２５）畜産物が動物の内臓である前記（２１）記載のグラム陰性菌抑制剤。
（２６）畜産物が動物の消化管である前記（２１）記載のグラム陰性菌抑制剤。
（２７）畜産物が動物の筋肉である前記（２１）記載のグラム陰性菌抑制剤。
（２８）グラム陰性菌がサルモネラ菌である前期（１）〜（２７）記載のグラム陰性菌抑制剤。
【００５０】
【発明の効果】
本発明のグラム陰性菌増殖抑制剤は哺乳動物および鳥類のグラム陰性菌の増殖を抑制する。また、それから得られる畜産物についても増殖を抑制する。しかも本発明品の有効成分が食品工業で多用されている多糖類の分解物であることからその安全性は極めて高い。また、タンニン類、グリセリン脂肪酸エステルまたはポリグリセリン脂肪酸エステル、サポニン類を多糖類の分解物と併用することによりその増殖抑制効果は極めて高くなり産業上有用である。[0001]
[Industrial application fields]
The present invention relates to a gram-negative bacterial growth inhibitor for mammals and birds and livestock products obtained therefrom.
[0002]
[Prior art]
Bacteria are classified into Gram-positive bacteria and Gram-negative bacteria based on Gram staining due to differences in the surface structure of the bacteria. Gram-negative bacteria include Shigella, Salmonella typhi, Salmonella, Salmonella, Escherichia coli, Klebsiella, Serratia, Proteus, Plague, Yersinia, Vibrio parahaemolyticus, Chlamydia, Haemophilus influenzae, Brucella, Legionella, Campylobacter, etc. There are many pathogenic bacteria. Among them, Escherichia coli and Salmonella are typical food poisoning bacteria and are also common infectious diseases of humans and animals. In the case of Salmonella infection, in animals, it is found in mammals such as cattle, horses, sheep, goats, pigs, dogs and cats, and in birds such as chickens, ducklings, and quails. In cattle, diarrhea and arthritis. In the case of chickens, broiler chick dysentery and diarrhea / sepsis in young chicks within 10 days of age occur, and in severe cases, death occurs. Salmonella infection is an important public health problem because it has a high incidence and damage, and beef cattle and chickens can also be a source of human infection. The occurrence of Salmonella food poisoning due to Salmonella enteritidis has increased worldwide in the past, and it has been revealed that the cause is contamination of eggs, which has become a major social problem. In addition, Escherichia coli propagates in the body and foods, producing toxins and causing diarrhea.
[0003]
It is unfavorable for hygiene and health that such gram-negative bacteria invade and proliferate into the body of animals and humans. Disinfectants, antibacterial agents, cleaning agents, vaccines and the like are known as countermeasures against these gram-negative bacteria. For example, methods for preventing and treating livestock and chicken salmonella infections include not only cleaning and disinfection of barns and poultry farms, but also administration of antibiotics and other synthetic antibacterial agents together with feed. (Japanese Patent Laid-Open No. 62-294623). However, such a method using a drug has problems such as the appearance of drug-resistant bacteria and the residue of the drug on livestock products, and is not a preferable method for human health. Japanese Examined Patent Publication No. 64-7049 discloses an attempt to eliminate Salmonella in the intestinal tract by orally administering an anaerobic culture of cecal contents, but the effect is not clear and anaerobic culture, etc. Requires special equipment and is not practical. Furthermore, in Europe and the United States, infection control methods using vaccines have been implemented, but it takes a lot of time to vaccinate, and in the case of chickens, there are problems such as effects on subsequent growth and egg production due to stress such as fasting. It ’s not the right way.
[0004]
Recently, in the case of chicken Salmonella infection, methods for inhibiting the growth of Salmonella using fructooligosaccharides or galactooligosaccharides have been disclosed (Japanese Patent Laid-Open Nos. 3-501971 and 5-208912). However, these oligosaccharides have drawbacks such that they are unstable to heating and acid when they are made into feed, and the effect of inhibiting growth against bacteria is limited to the results at the test tube level and the effect is weak.
[0005]
[Problems to be solved by the invention]
Therefore, under such circumstances, there has been a strong demand for a safe substance that eliminates Gram-negative bacteria or suppresses growth from mammals, birds, and livestock products obtained therefrom.
[0006]
[Means for Solving the Problems]
As a result of intensive studies to solve the above-mentioned problems, the present inventors have found an effect of suppressing the growth of Gram-negative bacteria in the body by the oral intake of a polysaccharide degradation product by mammals or birds. Moreover, it discovered that the livestock product obtained from these mammals, birds, etc. also has an inhibitory effect. Furthermore, a combination of these polysaccharide degradation products and tannins, a combination of polysaccharide degradation products and glycerin fatty acid esters or polyglycerin fatty acid esters, and a combination of polysaccharide degradation products and saponins As a result, the present invention was completed.
[0007]
The polysaccharide degradation product in the present invention refers to a product obtained by degrading a polysaccharide such as guar gum, locust bean gum, tamarind gum, pectin, xanthan gum, or pullulan with an enzyme or an acid. Specifically, for guar gum and locust bean gum, degradation by the enzyme galactomannanase derived from Aspergillus or Rhizopus is preferred, and for pectin, degradation by the enzyme pectinase produced by Aspergillus is preferred. Decomposition with a cellulase or xylanase derived from microorganisms is preferable for tamarind gum and xanthan gum, and decomposition with pullulanase is preferable for pullulan.
The degradation product of the polysaccharide of the present invention is a low molecular weight product obtained by hydrolysis, and the molecular weight can be changed by changing the reaction time of the enzyme or the reaction time of acid decomposition. For example, in the hydrolyzate of guar gum, it is preferable that the chain length of the mannose linear chain is distributed 80% or more within the range of 30 to 200 units, or 5 to 29 units.
[0008]
The chain length of the hydrolyzate of guar gum refers to the number of bonded main chains of mannose, which are bonded. In addition, the hydrolysis of other polysaccharides is the same, and the chain length is preferably 30 to 200 units, and preferably 80% or more in the range of 5 to 29 units. The measurement method is not particularly limited. For example, dissolved guar gum is dissolved in water, and high-performance liquid chromatography of 803D type (manufactured by Tosoh Corp.) is used to make G3000PW (Tosoh Corp.) using water as the mobile phase. It is possible to measure by performing gel filtration on a column manufactured by Co., Ltd. and detecting with a differential refractometer.
[0009]
Tannins used in the present invention can be obtained from ultrafiltration and reverse osmosis membrane treatment of water or alcoholic extract of green tea, oolong tea or black tea, or acetic acid soluble fraction, but other raw materials such as amber and apples. It may be of origin or chemically synthesized. Tannins include (+)-catechin, (+)-gallocatechin, (−)-gallocatechin gallate, (−)-epicatechin, (−)-epicatechin gallate, (−)-epigallocatechin, (− ) -Epigallocatechin gallate, free theaflavin, theaflavin monogallate A, theaflavin monogallate B and theaflavin digallate. When these obtained tannins are used in the present invention, they can be used singly or as a mixture of two or more, and even a crude extract containing tannins.
[0010]
The glycerin fatty acid ester used in the present invention is an ester of glycerin and a fatty acid, and the fatty acid is preferably a linear fatty acid having 8 to 14 carbon atoms, and examples thereof include caprylic acid, capric acid, and lauric acid. The glycerin fatty acid ester includes mono-, di- and triesters, and it is preferable to use monoesters. For example, glycerin monocaprylate, glycerin monocaprate, and glycerin monolaurate.
The glycerin fatty acid ester used in the present invention may be a single type or a mixture of two or more types.
The fatty acid of the polyglycerol fatty acid ester used in the present invention is preferably a linear fatty acid having 8 to 14 carbon atoms, and examples thereof include caprylic acid, capric acid, lauric acid, and myristic acid.
The polyglycerol of the polyglycerol fatty acid ester used in the present invention is a polymer of glycerol, and generally includes di, tri, penta, hexa, octa, and decaglycerol. The polyglycerin fatty acid ester used in the present invention may be a single type or a mixture of two or more types.
[0011]
The saponins used in the present invention are Quillaja saponin, Yucca saponin, beet saponin, soybean saponin, tea saponin, and Tochu tea saponin, and may be used alone or as a mixture of two or more.
The effective amount of the polysaccharide degradation product of the present invention is 0.01 to 10 g / kg body weight, preferably 0.05 to 5 g / kg body weight per day for mammals and birds, and is combined with the polysaccharide degradation product. The tannins that can be used are 0.0001 g to 1.0 g / kg body weight per day, preferably 0.001 to 0.5 g / kg body weight, and the mixing ratio of polysaccharide degradation products and tannins is 6: 1. ~ 1000: 1, preferably 10: 1 to 50: 1. The glycerin fatty acid ester and / or polyglycerin fatty acid ester that can be used in combination with the degradation product of the polysaccharide is 0.001 to 5 g / kg body weight, preferably 0.005 to 0.5 g / kg body weight. The mixing ratio of the degradation product, glycerin fatty acid ester and polyglycerin fatty acid ester is 8: 1 to 400: 1, preferably 15: 1 to 60: 1. The effective amount of saponins that can be used in combination with the polysaccharide degradation product is 0.0001 to 1.0 g / kg body weight, preferably 0.001 to 0.5 g / kg body weight. The mixing ratio with saponins is 6: 1 to 700: 1, preferably 15: 1 to 70: 1.
[0012]
The gram-negative bacterial growth inhibitor of the present invention is a liquid, powdery or granular polysaccharide degradation product, tannins, glycerin in feed raw materials in any of the normal mammal and bird feed production processes. Manufactured by adding and mixing fatty acid esters, polyglycerin fatty acid esters, and saponins, and processing them into products in an appropriate form such as powder, slurry, or pellets, or adding and mixing directly to feed products it can. The amount of polysaccharide degradation products, tannins, glycerin fatty acid esters or polyglycerin fatty acid esters, and saponins added to the feed, which are the active ingredients of the product of the present invention, is the above-mentioned effective amount when administered to animals. What is necessary is just to add suitably.
The animals of the present invention include mammals such as cows, horses, sheep, goats, pigs, dogs and cats, and birds such as chickens, ducks and quails. The livestock product of the present invention is a product obtained from these animals, and refers to eggs, meat, internal organs and the like obtained from milk, livestock meat, internal organs, chickens and the like.
EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, it is not specifically limited by this.
[0013]
【Example】
Example 1
Citric acid was added to 900 parts of water to adjust the pH to 3. To this, 0.2 part of galactomannanase derived from Aspergillus and 100 parts of guar gum powder were added and mixed, and an enzyme reaction was carried out at 40 to 45 ° C. for 24 hours. After the reaction, the enzyme was inactivated by heating at 90 ° C. for 15 minutes. The transparent solution obtained by removing impurities by filtration was concentrated under reduced pressure, and then spray-dried to obtain 65 parts of guar gum decomposition product of the present invention. As a result of measurement by high performance liquid chromatography, 80% or more of the sugar chains of the galactomannan were included in the range of 50 to 150 units of mannose chain length.
[0014]
Example 2
In the same manner, by changing only the reaction time to 48 hours, the guar gum degradation product of a short mannose linear product of the present invention (80% or more of the chain length of mannose is included in the range of 5 to 25 units). 68 parts were obtained.
Example 3
Citric acid was added to 900 parts of water to adjust the pH to 3. To this, 0.2 part of galactomannanase derived from Aspergillus and 100 parts of locust bean gum powder were added and mixed, and the enzyme was allowed to act at 40 to 45 ° C. for 6 hours. After the reaction, the enzyme was inactivated by heating at 95 ° C. for 15 minutes. And it filtered and isolate | separated, the impurity was removed, the obtained solution was freeze-dried, and 64 parts of locust bean gum decomposition products were obtained.
[0015]
Example 4
Citric acid was added to 900 parts of water to adjust the pH to 3. To this, 0.1 part of pectinase derived from Aspergillus and 100 parts of pectin powder (esterification degree 70%) were added and mixed, and the enzyme was allowed to act at 30 to 35 ° C. for 8 hours. After the reaction, the enzyme was inactivated by heating at 95 ° C. for 15 minutes. Then, it was separated by filtration to remove impurities, and the resulting solution was freeze-dried to obtain 64 parts of a pectin degradation product.
Example 5
Using the lyophilized powder of the guar gum degradation product obtained in Example 1 and the ultrafiltration membrane treated fraction of green tea hot water extract as tannins, the mixture was mixed to a weight ratio of 19: 1. The product of the present invention was obtained.
[0016]
Example 6
Using the guar gum decomposition product obtained in Example 2 and a powder obtained by freeze-drying an ethyl acetate soluble fraction of hot water extract of green tea as tannins, the mixture was mixed at a weight ratio of 19: 1. An invention was obtained.
Example 7
Using the (-)-epigallocatechin gallate further purified by column chromatography from the guar gum degradation product obtained in Example 2 and the ethyl acetate soluble fraction of the ethanol extract of green tea as tannins, the weight ratio To 19: 1 to obtain a product of the present invention.
[0017]
Example 8
Using the guar gum degradation product obtained in Example 2 and the crude theaflavin obtained from the chloroform soluble fraction of a commercial instant black tea hot water extract as tannins, the mixture was mixed at a weight ratio of 19: 1. The present invention product was obtained.
Example 9
Using the guar gum degradation product obtained in Example 1 and glycerin monocaprylate, the mixture was mixed at a weight ratio of 19: 1 to obtain the product of the present invention.
[0018]
Example 10
Using the guar gum decomposition product obtained in Example 2 and glycerin monolaurate, the mixture was mixed at a weight ratio of 19: 1 to obtain a product of the present invention.
Example 11
Using the guar gum degradation product obtained in Example 2 and Kirayasaponin (trade name: Kirayanin C-100, approximately 10% as partially hydrolyzed saponin, manufactured by Maruzen Pharmaceutical Co., Ltd.), the weight ratio is 19: 1. The product of the present invention was obtained by mixing as described above.
[0019]
Example 12
Using the guar gum degradation product obtained in Example 2 and Yucca saponin (about 20% as partially hydrolyzed saponin), the mixture was mixed at a weight ratio of 19: 1 to obtain the product of the present invention.
Example 13
Using the guar gum degradation product obtained in Example 2 and beet saponin (about 60% as partially hydrolyzed saponin), the mixture was mixed at a weight ratio of 19: 1 to obtain the product of the present invention.
[0020]
Example 14
Using the guar gum degradation product obtained in Example 2 and soybean saponin (about 60% as partially hydrolyzed saponin), the mixture was mixed at a weight ratio of 19: 1 to obtain the product of the present invention.
Example 15
Using the guar gum degradation product obtained in Example 2 and tea saponin obtained from tea seeds (about 50% as partially hydrolyzed saponin), the mixture was mixed at a weight ratio of 19: 1 to obtain the product of the present invention. It was.
Example 16
Using the guar gum degradation product obtained in Example 2 and Tochu tea saponin (about 50% as partially hydrolyzed saponin), the mixture was mixed at a weight ratio of 19: 1 to obtain the product of the present invention.
Example 17
Using a powder obtained by freeze-drying the ethyl acetate soluble fraction of the hot water extract of green tea as tannins and mixing the locust bean gum decomposition product obtained in Example 3 to a weight ratio of 19: 1. The present invention product was obtained.
[0021]
Example 18
Using the pectin degradation product obtained in Example 4 and Kirayasaponin (trade name: Kirayanin C-100, approximately 10% as partially hydrolyzed saponin, manufactured by Maruzen Pharmaceutical Co., Ltd.), the weight ratio is 19: 1. The product of the present invention was obtained by mixing as described above.
Example 19
Using a pullulan degradation product obtained by treating pullulan of water-soluble dietary fiber with pullulanase and Kirayasaponin (trade name: Kirayanin C-100, approximately 10% as a partially hydrolyzed saponin, manufactured by Maruzen Pharmaceutical Co., Ltd.), a weight ratio of 19 Was mixed to obtain a product of the present invention.
[0022]
Example 20
Using the guar gum degradation product obtained in Example 2 and the powder obtained by freeze-drying the ethyl acetate soluble fraction of the hot water extract of green tea and glycerin monocaprylate, the weight ratio is 18: 1: 1. The product of the present invention was obtained by mixing as described above.
Example 21
The guar gum degradation product obtained in Example 2, the ethyl acetate-soluble fraction of green tea hot water extract and freeze-dried powder and Kirayasaponin (trade name: Kirayanin C-100, approximately 10% as partially hydrolyzed saponin) , Manufactured by Maruzen Pharmaceutical Co., Ltd.) to obtain a product of the present invention by mixing at a weight ratio of 18: 1: 1.
[0023]
Test example 1
Salmonella Enteritidis IFO-3313 and Salmonella typhimurium IFO-12529, Salmonella Dublin NIAH-1201, Vibrio parafaemoliticus, Pseudomonas malea, Brucella Swiss in the medium containing the present invention as follows Cultured. The product of the present invention obtained in Examples 1 to 4 as a carbon source was added to the broth medium so as to be 0.5%, and as a control, 0.5% glucose was added to the broth medium. Culturing was carried out at 37 ° C. for 48 hours, and the growth of the bacteria was confirmed by lowering the pH of the culture solution. The results are shown in Table 1. The degree of bacterial growth in the table was indicated as follows. -(No growth: pH 6.0 or higher), +-(Weak growth: pH 5.51-6.00), + (Growth: pH 5.01-5.50), ++ (Well grown: pH 5.00 or lower)
[0024]
[Table 1]
From the results in Table 1, it can be seen that there is almost no growth of the test bacteria when the product of the present invention is added.
Test example 2
Salmonella Enteritidis IFO-3313, Salmonella typhimurium IFO-12529, Salmonella Dublin NIAH-1201, Vibrio parafaemoliticus, Pseudomonas malea, Brucella Swiss in the medium containing the present invention as follows Cultured. To the broth medium, 0.5% glucose as a carbon source and the product of the present invention obtained in Examples 5 to 21 were added to 0.5%, and cultured at 37 ° C. for 48 hours. The growth of the bacteria was confirmed by the decrease. As a control, a broth culture medium containing 0.5% glucose was used, and as a comparison, a powder obtained by freeze-drying the ultrafiltration membrane treated fraction of green tea hot water extract (A), ethyl acetate of green tea hot water extract Freeze-dried powder (B), (-)-epigallocatechin gallate (C), crude theaflavin (D), glycerin monocaprylate (E), glycerin monolaurate (F), kirayasaponin (G), Yucca saponin (H), beet saponin (I), soybean saponin (J), tea saponin (K), Tochu tea saponin (L), a mixture of B and E in the same weight ratio (M), A mixture of B and G in the same weight ratio (N) was added to a concentration of 0.025%, followed by culturing in the same manner. The results are shown in Table 2 and Table 3. The degree of bacterial growth in the table was indicated as follows. -(No growth: pH 6.01 or higher), +-(weak growth: pH 5.51-6.00), + (growth: pH 5.01-5.50), ++ (well growth: pH 5.00 or lower)
[0026]
[Table 2]
[Table 3]
From the results in Tables 2 and 3, when tannins, glycerin fatty acid esters or polyglycerin fatty acid esters, and saponins are used in combination with polysaccharide degradation products, there is no growth of the test bacteria, and there is a growth inhibitory effect. I understand that.
Test example 3
15 adult cattle immediately after parturition are divided into 5 groups of 3 each, and the group fed with only basic feed is group A, and 10 g of the present invention product prepared in Examples 1, 7, 10 and 12 is added to 1 kg of basic feed These groups were designated as groups B, C, D, and E, and were bred for 5 weeks. Salmonella tabulin derived from cattle is cultured in BHI medium and collected, then 1 × 10 5 with physiological saline. Five The bacterial solution prepared so that the number of cells was 1 ml / ml was orally infected in a 100 ml suckling bottle per head one week after feeding with each feed. Feces were collected at 2 and 4 weeks after infection, and the number of Salmonella was measured using Salmonella selective medium (Eiken Chemical Co., Ltd.). The results are shown in Table 4.
[0029]
[Table 4]
From Table 4, Salmonella was not detected in the group to which the product of the present invention was added. Moreover, when a guar gum decomposition product, tannins, glycerin fatty acid ester or polyglycerin fatty acid ester, and saponins were used in combination, the growth inhibitory effect was remarkable.
Test example 4
15 milking cows are divided into 5 groups each of 3 cows, a group to which only the basic feed is given, a group A, and a group in which 10 g of the present invention product prepared in Examples 1, 7, 10 and 12 is added to 1 kg of the basic feed. Each group was bred for 5 weeks in groups B, C, D and E. Salmonella tabulin derived from cattle is cultured in BHI medium and collected, then 1 × 10 5 with physiological saline. Five The bacterial solution prepared so that the number of cells was 1 ml / ml was orally infected in a 100 ml suckling bottle per head one week after feeding with each feed. Milk was collected at 2 and 4 weeks after infection, 1 ml of milk was enriched with Salmonella enrichment medium, and then smeared on Salmonella selective medium (Eiken Chemical Co., Ltd.) to determine the presence of Salmonella. . When a large number of Salmonella was detected, ++, a slight amount +, and a case where no Salmonella was detected were displayed. The results are shown in Table 5.
[0031]
[Table 5]
From Table 5, Salmonella was not detected in the milk of the group to which the product of the present invention was added. Moreover, when a guar gum decomposition product, tannins, glycerin fatty acid ester or polyglycerin fatty acid ester, and saponins were used in combination, the growth inhibitory effect was remarkable.
Test Example 5
15 pigs of 20-day age were divided into 5 groups of 3 each, and the group fed only feed for growing piglets (manufactured by Showa Sangyo Co., Ltd.) as basic feed was group A, 1 kg of basic feed Example 2 The groups fed with 10 g of the product of the present invention prepared in 6, 11 and 13 were fed with groups B, C, D and E, respectively, and were bred for 5 weeks. Salmonella typhimurium derived from swine was cultured in BHI medium, collected, and then 1 × 10 with physiological saline Five A bacterial solution was prepared so that the number of cells / ml was reached. Oral infection was performed 1 week after breeding with each feed at 100 ml suckling per head. Feces were collected at 2 and 4 weeks after infection, and the number of Salmonella was measured using Salmonella selective medium (Eiken Chemical Co., Ltd.). At the same time, the number of Escherichia coli in feces was also measured using a desoxycholate medium (Eiken Chemical Co., Ltd.). The results are shown in Table 6.
[0033]
[Table 6]
From Table 6, Salmonella was not detected in the group to which the product of the present invention was added, and E. coli decreased. Moreover, the growth inhibitory effect was remarkable by using together a guar gum decomposition product, tannins, glycerol fatty acid ester or polyglycerol fatty acid ester, and saponins.
Test Example 6
15 pigs were divided into 5 groups of 3 each, and a group fed with only feed for raising pigs (manufactured by Showa Sangyo Co., Ltd.) as a basic feed was prepared in Group A, 1 kg of basic feed in Examples 2, 6, 11, and 13. The groups to which 10 g of the present invention product was added were fed as groups B, C, D and E, respectively, and were bred for 5 weeks. Salmonella typhimurium derived from swine was cultured in BHI medium, collected, and then 1 × 10 with physiological saline Five A bacterial solution was prepared so that the number of cells / ml was reached. Oral infection was performed 1 week after breeding with each feed at 100 ml suckling per head. Slaughtered 5 days after infection, collected thigh muscle, stomach, duodenum, cecum, rectum, liver, spleen, and 1 g of each sample was enriched with Salmonella enrichment medium, followed by Salmonella selective medium (Eiken Chemical) And the presence or absence of Salmonella was determined. When a large number of Salmonella was detected, ++, a slight amount +, and a case where no Salmonella was detected were displayed. The results are shown in Table 7.
[0035]
[Table 7]
From Table 7, Salmonella was not detected in the thigh muscle, liver and spleen obtained from the group to which the product of the present invention was added, and the growth inhibitory effect of Salmonella was observed in the gastrointestinal tract of the stomach, duodenum, cecum and rectum. . Further, the combined use of guar gum degradation products with tannins, glycerin fatty acid esters or polyglycerin fatty acid esters, and saponins resulted in a remarkable growth inhibitory effect.
Test Example 7
Sixty young chicks immediately after hatching were divided into 12 groups of 5 chicks and bred for 1 week on basic feed. Then, the group which gave only the basic feed for 2 weeks was made into the A group, and the group which gave the feed which respectively added 5 g of the composition of this invention prepared in 1 kg of basic feed 1kg Examples 1-21 was set as Bv group. After feeding on each feed for 1 week, infection with Salmonella was performed. Chicken-derived Salmonella enteritidis is cultured in BHI medium, collected, and then 1 × 10 with physiological saline Five The bacterial solution prepared so as to be the number of cells / ml was orally infected with a 1 ml syringe per bird. Feces on the day before infection, 1, 2, 4, 6 and 8 days after infection were collected, and the number of Salmonella was measured using Salmonella selective medium (Eiken Chemical Co., Ltd.). The results are shown in Tables 8-10.
[0037]
[Table 8]
[Table 9]
[Table 10]
From Table 8 to Table 10, Salmonella was not detected in the group to which the product of the present invention was added. Further, the combined use of polysaccharide degradation products with tannins, glycerin fatty acid esters or polyglycerin fatty acid esters, and saponins resulted in a remarkable effect of inhibiting the growth of Salmonella.
[0041]
Test Example 8
60 broiler chickens were divided into 12 groups of 5 each and were bred for 1 week on basic feed. Thereafter, the group fed only with the basic feed for 2 weeks was group A, and the groups fed with 1 kg of the basic feed and 5 g of the composition of the present invention prepared in Examples 1 to 20, respectively, were designated as groups B to U. . After feeding on each feed for 1 week, infection with Salmonella was performed. Chicken-derived Salmonella enteritidis is cultured in BHI medium, collected, and then 1 × 10 with physiological saline Five The bacterial solution prepared so as to be the number of cells / ml was orally infected with a 1 ml syringe per bird. On day 5 after the infection, the thigh muscle, duodenum, cecum, rectum, liver, and spleen were collected, and 1 g of each specimen was enriched with Salmonella enrichment medium, and then Salmonella selective medium (Eiken Chemical Co., Ltd.). ), And the presence or absence of Salmonella was determined. When a large number of Salmonella was detected, ++, a slight amount +, and a case where no Salmonella was detected were displayed. The results are shown in Tables 11 to 13.
[0042]
[Table 11]
[Table 12]
[Table 13]
From Tables 11 to 13, Salmonella was not detected in the thigh muscle, liver and spleen obtained from the group to which the product of the present invention was added, and the growth inhibitory effect of Salmonella was observed in the duodenum, cecum and rectal digestive tract. Further, the combined use of polysaccharide degradation products with tannins, glycerin fatty acid esters or polyglycerin fatty acid esters, and saponins resulted in a remarkable effect of inhibiting the growth of Salmonella.
Test Example 9
Sixty laying hens were divided into 12 groups of 5 hens and bred for 1 week on basic feed. Thereafter, the group fed only with the basic feed for 2 weeks was group A, and the groups fed with 1 kg of the basic feed and 5 g of the composition of the present invention prepared in Examples 1 to 21 were fed with groups B to v, respectively. . After feeding on each feed for 1 week, infection with Salmonella was performed. Chicken-derived Salmonella enteritidis is cultured in BHI medium, collected, and then 1 × 10 with physiological saline Four The bacterial solution prepared so as to be the number of cells / ml was orally infected with a 1 ml syringe per bird. Eggs on the 10th day after infection were collected, and the surface of the eggshell and Salmonella in the yolk were measured. In the case of the eggshell surface, the eggshell surface is washed with 10 ml of physiological saline for each egg, and in the case of egg yolk, the 10-fold diluted solution is enriched with the Salmonella enrichment medium, and then the Salmonella selection medium ( Eiken Chemical Co., Ltd.) was smeared and the presence or absence of Salmonella was determined. When a large number of Salmonella was detected, ++, a slight amount +, and a case where no Salmonella was detected were displayed. The results are shown in Tables 14-16.
[0046]
[Table 14]
[Table 15]
[Table 16]
From Table 14 to Table 16, Salmonella was not detected in the eggshell surface and egg yolk of chicken eggs obtained from all groups to which the product of the present invention was added. Therefore, the contamination inside and outside the eggs of Salmonella by the product of the present invention can be prevented. The embodiment of the present invention and the target product are as follows.
(1) Gram-negative bacterial growth inhibitor containing a polysaccharide degradation product.
(2) The gram-negative bacterial growth inhibitor according to the above (1), wherein the polysaccharide is guar gum.
(3) The gram-negative bacterial growth inhibitor according to (1), wherein the polysaccharide is locust bean gum.
(4) The gram-negative bacterial growth inhibitor according to (1) above, wherein the polysaccharide is xanthan gum.
(5) The gram-negative bacterial growth inhibitor according to the above (1), wherein the polysaccharide is pectin.
(6) The gram-negative bacterial growth inhibitor according to (1) above, wherein the polysaccharide is pullulan.
(7) The gram-negative bacterial growth inhibitor according to (1) to (6), wherein the degradation product is a degradation product by an enzyme.
(8) The gram-negative bacterial growth inhibitor according to the above (1) to (6), wherein the degradation product is a degradation product due to an acid.
(9) A gram-negative bacterial growth inhibitor, which is used in combination with a polysaccharide degradation product and tannins.
(10) The gram-negative bacterial growth inhibitor according to (9) above, wherein the tannin is a hot water extract of green tea.
(11) The gram-negative bacterial growth inhibitor according to the above (9), wherein the tannin is an ethyl acetate soluble fraction of green tea.
(12) Tannins are (+)-catechin, (+)-gallocatechin, (−)-gallocatechin gallate, (−)-epicatechin, (−)-epicatechin gallate, (−)-epigallocatechin, ( -)-Gram-negative bacterial growth according to (9) above, which is one or more compounds selected from the group consisting of compounds of epigallocatechin gallate, free theaflavin, theaflavin monogallate A, theaflavin monogallate B and theaflavin digallate Inhibitor.
(13) The gram-negative bacterial growth inhibitor according to the above (9), wherein the tannin is (−)-epigallocatechin garade.
(14) A Gram-negative bacterial growth inhibitor characterized by using a degradation product of polysaccharide and glycerin fatty acid ester or polyglycerin fatty acid ester in combination.
(15) The gram-negative bacterial growth inhibitor according to the above (14), wherein the glycerin fatty acid ester is a mono, di, or triester.
(16) The gram-negative bacterial growth inhibitor according to the above (14), wherein the fatty acid of the polyglycerol fatty acid ester is a linear fatty acid of caprylic acid, capric acid, lauric acid, and myristic acid having 8 to 14 carbon atoms.
(17) A Gram-negative bacterial growth inhibitor characterized by using a polysaccharide degradation product and saponins in combination.
(18) The gram-negative bacterial growth inhibitor according to (17), wherein the saponins are one or more saponins selected from Kiraya saponin, Yucca saponin, beet saponin, soybean saponin, tea saponin, and Tochu tea saponin.
(19) A gram-negative bacterial growth inhibitor characterized by using a degradation product of a polysaccharide, tannins, and a glycerin fatty acid ester or a polyglycerin fatty acid ester.
(20) A gram-negative bacterial growth inhibitor characterized by using a polysaccharide degradation product, tannins, and saponins in combination.
(21) The gram-negative bacterial growth inhibitor according to (1) to (20) above, wherein the growth of gram-negative bacteria from livestock products is suppressed.
(22) The Gram-negative bacterium inhibitor as described in (21) above, wherein the livestock product is a product obtained from mammals and birds.
(23) The Gram-negative bacterium inhibitor as described in (21) above, wherein the livestock product is milk.
(24) The Gram-negative bacterium inhibitor as described in (21) above, wherein the livestock product is a chicken egg.
(25) The Gram-negative bacterial inhibitor according to (21), wherein the livestock product is an internal organ of an animal.
(26) The Gram-negative bacterium inhibitor as described in (21) above, wherein the livestock product is an animal digestive tract.
(27) The Gram-negative bacterium inhibitor as described in (21) above, wherein the livestock product is animal muscle.
(28) The gram-negative bacterium inhibitor according to the previous period (1) to (27), wherein the gram-negative bacterium is Salmonella.
[0050]
【The invention's effect】
The gram-negative bacterial growth inhibitor of the present invention suppresses the growth of gram-negative bacteria in mammals and birds. Moreover, the growth of livestock products obtained therefrom is also suppressed. Moreover, since the active ingredient of the product of the present invention is a degradation product of polysaccharides frequently used in the food industry, its safety is extremely high. In addition, when tannins, glycerin fatty acid esters or polyglycerin fatty acid esters, and saponins are used in combination with the degradation products of polysaccharides, the growth inhibitory effect becomes extremely high, which is industrially useful.
Claims (2)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP26141294A JP4127864B2 (en) | 1994-09-29 | 1994-09-29 | Gram-negative bacterial growth inhibitor |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP26141294A JP4127864B2 (en) | 1994-09-29 | 1994-09-29 | Gram-negative bacterial growth inhibitor |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH0899884A JPH0899884A (en) | 1996-04-16 |
| JP4127864B2 true JP4127864B2 (en) | 2008-07-30 |
Family
ID=17361515
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP26141294A Expired - Fee Related JP4127864B2 (en) | 1994-09-29 | 1994-09-29 | Gram-negative bacterial growth inhibitor |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP4127864B2 (en) |
Families Citing this family (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1165628A (en) * | 1996-02-14 | 1997-11-26 | 浙江农业大学 | Triterpenoid saponin extracted from tea oil cake and application as additive |
| EP1129628B1 (en) * | 1998-11-09 | 2008-06-25 | Taiyo Kagaku Co., Ltd. | Poultry producibility improver and poultry producibility improvement method |
| DE19961182B4 (en) * | 1999-12-18 | 2006-01-12 | Südzucker AG Mannheim/Ochsenfurt | Galactomannan oligosaccharides and process for their preparation and their use |
| JP2002114690A (en) * | 2000-10-12 | 2002-04-16 | Taiyo Kagaku Co Ltd | Deodorant for excrement |
| DE10104055A1 (en) * | 2001-01-31 | 2002-08-14 | Suedzucker Ag | Use of carbohydrates to eliminate intestinal infections in animals |
| ATE388723T1 (en) * | 2001-07-30 | 2008-03-15 | Dsm Ip Assets Bv | COMPOSITION CONTAINING EPIGALLOCATECHIN GALLATE |
| FR2834619B1 (en) * | 2002-01-14 | 2005-07-22 | Innovation Dev En Nutrition An | FOOD SUPPLEMENT FOR USE IN THE FORMULATION OF FEEDS FOR RUMINANT ANIMALS |
| ES2195796B1 (en) * | 2002-05-30 | 2005-02-01 | Institut De Recerca I Tecnologia Agroalimentaries | NATURAL RUBBER BASED PREMIX FOR ANIMAL FEEDING. |
| EP2110024A1 (en) * | 2008-04-14 | 2009-10-21 | Institut de Recerca i Tecnologia Agroalimentaires | An enzymatic pre-mixture against Gram negative bacteria colonization in the animal intestinal tract |
| JP6130620B2 (en) * | 2011-02-07 | 2017-05-17 | 太陽化学株式会社 | Gene expression promoter |
| WO2015061755A1 (en) | 2013-10-25 | 2015-04-30 | Phibro Animal Health Corporation | Combination and/or composition comprising bacillus, and yucca, quillaja or both and a method for making and using |
| EP3145521A1 (en) * | 2014-04-09 | 2017-03-29 | BioMar Group A/S | Compound or composition for use in the prevention and/or treatment of an ectoparasitic copepod infestation or infection in fish |
| MA41080B1 (en) * | 2017-09-25 | 2019-08-30 | Univ Sidi Mohamed Ben Abdellah | New manufacturing processes for locust bean gum |
-
1994
- 1994-09-29 JP JP26141294A patent/JP4127864B2/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| JPH0899884A (en) | 1996-04-16 |
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