JP6430931B2 - ラクトバチルス・ラムノサス株を使用した生物防除 - Google Patents
ラクトバチルス・ラムノサス株を使用した生物防除 Download PDFInfo
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- JP6430931B2 JP6430931B2 JP2015504932A JP2015504932A JP6430931B2 JP 6430931 B2 JP6430931 B2 JP 6430931B2 JP 2015504932 A JP2015504932 A JP 2015504932A JP 2015504932 A JP2015504932 A JP 2015504932A JP 6430931 B2 JP6430931 B2 JP 6430931B2
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- strain
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- lactobacillus rhamnosus
- lactobacillus
- cfu
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Description
更に、一部の乳酸菌はまた、抗微生物活性を有する代謝産物も産生する。
しかしながら、単一株として又は他の生物防除株と組み合わせて改善された抗真菌効果を有する生物防除作用物質がそれでも必要である。
本発明者らは、大規模なスクリーニングと研究によって、市販の工業用生物防除培養物及び一般的な化学保存料であるソルビン酸カリウムと比較して、特定のラクトバチルス・ラムノサス株が酵母及びカビに対して有意に高い効果を有することを見出した。
更に、本発明者らは、特定の群の乳酸菌が、他の群の乳酸菌と組み合わせたとき、有意な相乗的抗微生物効果を発揮することを見出した。組み合わせた二群の細菌の抗微生物効果は、驚くべきことに、前記二群の細菌の個別の効果の合計を上回る。
定義
本明細書中で使用されたとき、「乳酸菌(lactic acid bacterium)」という用語は、糖の発酵において主に生産される酸として乳酸を含む酸を生産する、グラム陽性微小好気性又は嫌気性細菌を指す。産業上最も有用な乳酸菌は、ラクトコッカス亜種(Lactococcus spp.)、ストレプトコッカス亜種(Streptococcus spp.)、ラクトバチルス亜種(Lactobacillus spp.)、ロイコノストック亜種(Leuconostoc spp.)、シュードロイコノストック亜種(Pseudoleuconostoc spp.)、ペディオコッカス亜種(Pediococcus spp.)、ブレビバクテリウム亜種(Brevibacterium spp.)、エンテロコッカス亜種(Enterococcus spp.)を含む目である「ラクトバチルス目(Lactobacillales)」に属する。これらは、単独で、又は他の乳酸菌と組み合わせて、食品用培養物としてしばしば使用される。
本明細書中、「均質化(homogenizing)」という用語は、可溶性懸濁物又は乳液を取得するための激しい混合を意味する。均質化が発酵の前に実施される場合、それは、乳脂肪が乳から分離しない程度に小さなサイズに分散するように実施されるべきである。これは、乳を高圧で小さなオリフィスを通して押し出すことにより達成され得る。
発明者らは、酵母及びカビなどの広範囲の微生物に対して最も効果的な株を見つけるために、200株のラクトバチルス・プランタルム(Lactobacillus plantarum)、ラクトバチルス・パラカゼイ及びラクトバチルス・ラムノサスの候補をスクリーニングした。
ラクトバチルス・パラカゼイ及びラクトバチルス・ラムノサスの中から17個の候補が、25℃で試験したとき、ベンチマークの乳酸菌と同等に、又はより顕著に、12個の指標のカビを阻害した。
酵母やカビに対する菌株の阻害効果は、以下の実施例に記載のように、適切な保存期間中、適切な温度で発酵乳製品を保存することによって判定され得る。
第三の態様において、本発明は、本発明の第一の態様によるラクトバチルス・ラムノサス株を含有する食料、飼料又は医薬製品、あるいは、本発明の第二の態様による抗真菌組成物に向けられる。
好ましい実施形態において、前記の食料、飼料又は医薬製品は、食料製品である。
他の好ましい態様において、前記食料製品は、獣肉又は鶏肉である。
好ましくは、前記医薬製品は、本発明の第二の態様の抗真菌組成物を、病原性微生物を阻害し、当該病原性微生物に関連する症状を緩和するために、ヒト又は動物に投与するのに有用な製品である。そのような症状の例として、酵母感染に関連する症状が挙げられる。そのような態様において、前記医薬製品は、抗真菌組成物を含有する単位投与剤形であってもよい。好ましくは、当該単位投与剤形は、カプセル又は錠剤であってもよい。しかしながら、当該単位投与剤形は、粘膜又は皮膚への適用に適するものでもよく、その場合、ペースト、クリーム、軟膏などの形態である。
本発明の第六の態様は、食料、飼料又は医薬製品を調製するための、第一の態様によるラクトバチルス・ラムノサス株、あるいは、第二の態様による抗真菌組成物の使用に向けられる。好ましくは、第一の態様によるラクトバチルス・ラムノサス株又は第二の態様による抗真菌組成物の使用によって生産される食料製品は、カッテージ、フェタ、チェダーチーズ、パルメザン、モッツァレラ、エメンタール、ダンボ、ゴーダ、エダム、フェタタイプ、ブルーチーズ、塩漬けチーズ、カマンベールチーズ又はブリーチーズなどのチーズである。
本発明の実施形態は、制限されることのない例示を用いて、以下に説明される。
ラクトバチルス・ラムノサスCHCC5366を用いた場合の、P.ブレビコンパクツム、P.コミューン、A.ベルシコロル及びP.クラストスムに対する阻害作用を視覚的に試験するために、脂肪分1.5%のヨーグルトを調製した。
ラクトバチルス・ラムノサスCHCC5366を用いた場合の、デバリオマイセス・ハンセニイに対する阻害効果を定量的に試験するため、ヨーグルトを調製した。
Lb.パラカゼイCHCC14676及びLb.ラムノサスCHCC5366の単独及び組み合わせの阻害効果の半定量的試験のために、寒天アッセイを使用し、これはヨーグルトの製造プロセス及び製品を模倣するものである。
0.17%の脂肪を含む殺菌した乳を使用して、実施例1に記載の市販のスターター培養物を使用するヨーグルトの異なる3つのバッチを調製した。スターター培養物を、0013%で播種した。発酵前に、ソルビン酸カリウム(0.023%)を第一バッチに添加し、ラクトバチルス・ラムノサスCHCC5366(1x107CFU/g)を第二バッチに添加した。一方、第三バッチを、スターター培養物のみを含む基準として使用した。発酵を、pH4.55に達するまで、43℃で行った。pH4.55に達した後、ヨーグルトを7℃で保存した。
ペニシリウム・ソリタム
ペニシリウム・パリタンス
ペニシリウム・ディスカラー
ペニシリウム・スパスラタム
ペニシリウム・コミューン
ペニシリウム・クルストスム
ペニシリウム・パネウム
ペニシリウム・ロケフォルティ
の播種を達成した。
図4に示した結果は、ラクトバチルス・ラムノサスCHCC5366の存在が、7℃で60日間の保存の間、ヨーグルトにおいて、様々なカビの増殖及び典型的な緑/青色の形成を劇的に低減したことを明らかにした。カビの増殖及び色形成の範囲に対するラクトバチルス・ラムノサスCHCC5366の阻害効果は、0.023%のソルビン酸カリウムの添加によってもたらされる効果よりも、かなり強力であることを見出した。
組み合わせた状態のラクトバチルス・ラムノサスCHCC5366及びラクトバチルス・パラカゼイCHCC14676の、ムコール亜種に対する阻害効果を視覚的に試験するために、ギリシャヨーグルトを以下の通り実質的に調製した。
殺菌した脂肪分1.5%の乳に、市販のスターター培養物(Chr. Hansen A/S, Denmarkから入手可能なF-DVS YF-L901)を0.02%で播種した。1つのバッチには、基準としてスターター培養物のみ播種した。別のバッチには、更にラクトバチルス・ラムノサス株CHCC5366(5x106CFU/g)及びラクトバチルス・パラカゼイ株CHCC14676(5x106CFU/g)の組み合わせを播種した。
ヨーグルトを調製した翌日、二重反復試験のヨーグルトカップの表面混入株としてムコール亜種を、ヨーグルトの表面上のスポットあたり胞子100個/スポットの標的植菌濃度で塗布することによって播種した。カビの増殖を、視覚的に15日間22℃±1℃での保存後に評価した。
ラクトバチルス・ラムノサスCHCC5366とラクトバチルス・パラカゼイCHCC14676の組み合わせの、黒パンカビ、リゾプス・ストロニファーに対する阻害効果を視覚的に試験するために、ギリシャヨーグルトを以下の通り実質的に調製した。
ヨーグルトを調製した翌日、二重反復試験のヨーグルトのカップの表面混入株としてリゾプス・ストロニファーを、ヨーグルトの表面上のスポットあたり胞子100個/スポットの目標混入濃度で塗布することによって播種した。カビの増殖を、視覚的に42日間7℃±1℃で保存後に評価した。
組み合わせた状態でラクトバチルス・ラムノサスCHCC5366及びラクトバチルス・パラカゼイCHCC14676の、サッカロミセス・セレビシエに対する阻害効果を定量的に試験するために、白い塩漬けチーズを調製した。
組み合わせた状態でのラクトバチルス・ラムノサスCHCC5366とラクトバチルス・パラカゼイCHCC14676の、クリベロマイセス・マルキシアヌスに対する阻害効果の定量的に試験するために、缶に酵母混入株としてクリベロマイセス・マルキシアヌスを播種したことを除いて、白い塩漬けチーズを実施例1に記載の通り調製した。
組み合わせた状態でのラクトバチルス・ラムノサスCHCC5366とラクトバチルス・パラカゼイCHCC14676の、P.コミューンに対する阻害効果を視覚的に試験するために、白い塩漬けチーズを、実施例1に記載の通り調製した。
ラクトバチルス・ラムノサスCHCC5366の、P.クルストスムに対する阻害効果を視覚的に試験するために、白い塩漬けチーズを調製した。
ラクトバチルス・ラムノサス株CHCC5366のみ、又はラクトバチルス・パラカゼイCHCC14676と組み合わせて、カビP.パネウムに対する阻害効果を視覚的に試験するために、1.5%の脂肪分を含有するヨーグルトを調製した。
異なった保存温度において、ラクトバチルス・ラムノサス株CHCC5366のみ、又はラクトバチルス・パラカゼイCHCC14676と組み合わせて、カビP.クルストスムに対する阻害効果を視覚的に試験するために、1.5%の脂肪分を含有するヨーグルトを調製した。
出願人は、下記の寄託された微生物の試料が、当該特許が登録査定を受けるまでに、必ず当業者に利用可能となっているべきことを要求する。
以上の寄託は、特許手続を目的とする微生物の寄託の国際的承認についてのブダペスト条約に従い行われた。
EP0221499
EP0576780
EP1442113
US5,378,458
EP11161609.0
Kosikowski, F.V. and Mistry, V.V., "Cheese and Fermented Milk Foods", 1997, 3rd Ed. F.V. Kosikowski, L.L.C. Westport, CT
Claims (15)
- 受入番号DSM23035でドイツ微生物細胞培養コレクション(German Collection of Microorganisms and Cell Cultures)(DSMZ)に寄託されたラクトバチルス・ラムノサス株CHCC5366から成る群から選択されるラクトバチルス・ラムノサス(Lactobacillus rhamnosus)株。
- 請求項1に記載のラクトバチルス・ラムノサス株を含む抗真菌組成物。
- 請求項1に記載のラクトバチルス・ラムノサス株、及びラクトバチルス・パラカゼイ(Lactobacillus paracasei)株を少なくとも1種類含む抗真菌組成物。
- 前記少なくとも1種類のラクトバチルス・パラカゼイ株が、受入番号DSM24651のラクトバチルス・パラカゼイCHCC12777、及び受入番号DSM25612のラクトバチルス・パラカゼイCHCC14676から成る群から選択される、請求項3に記載の抗真菌組成物。
- 請求項1に記載のラクトバチルス・ラムノサス株、又は請求項2〜4のいずれか1項に記載の抗真菌組成物を含む食料、飼料又は医薬製品。
- 前記食料製品に抗真菌特性を付与するのに効果がある量の請求項1に記載のラクトバチルス・ラムノサス株を含む、請求項5に記載の食料製品。
- 前記食料製品が、果物及び果物由来の製品、野菜及び野菜由来の製品、穀物及び穀物由来の製品、乳製品、獣肉、鶏肉、及び水産物、並びにそれらの混合物から成る群から選択される、請求項5又は6に記載の食料製品。
- 前記食料製品が乳製品であり、さらに、前記乳製品が、ヨーグルトなどの中温性又は好熱性発酵乳製品である、請求項7に記載の食料製品。
- 食料、飼料又は医薬製品の製造方法であって、製造の過程で、請求項1に記載のラクトバチルス・ラムノサス株又は請求項2〜4のいずれか1項に記載の抗真菌組成物を、前記食料、飼料又は医薬製品に添加する工程、及びラクトバチルス・ラムノサス株の濃度を一定に保つか又は増加するように、前記製造の過程で製造パラメーターを制御する工程を含む、前記製造方法。
- 前記ラクトバチルス・ラムノサス株の濃度が、前記食料、飼料又は医薬製品に対して、少なくとも1x106cfu/g又はそれぞれ少なくとも1x106cfu/ml、あるいは、前記食料、飼料又は医薬製品の表面に対して、少なくとも1x105cfu/cm2である、請求項9に記載の方法。
- 請求項9に記載の製造方法であって、
(a)前記食料、飼料又は医薬製品の製造の過程で、請求項2〜4のいずれか1項に記載の抗真菌組成物を、ラクトバチルス・ラムノサス株及び/又は少なくとも1種類のラクトバチルス・パラカゼイ株が、前記食料、飼料又は医薬製品に対して、それぞれ少なくとも1x106cfu/g又は少なくとも1x106cfu/ml、又は前記食料、飼料又は医薬製品の表面に対して、少なくとも1x105cfu/cm2の濃度となるように添加する工程、並びに
(b)ラクトバチルス・ラムノサス株及び/又は少なくとも1種類のラクトバチルス・パラカゼイ株の濃度が、増加するか又は一定に保たれるように、前記製造の過程で製造パラメーターを制御する工程、
を含む、前記製造方法。 - 前記方法が、1つ以上の発酵工程を含む、請求項9〜11のいずれか1項に記載の方法。
- 前記方法が、ラクトバチルス(Lactobacillus)、ストレプトコッカス(streptococcus)、ラクトコッカス(Lactococcus)及びロイコノストック(Leuconostoc)から選択される属の少なくとも1種類の株を含むスターター培養物を用いて乳基質を発酵させる工程を含む、請求項12に記載の方法。
- 食料、飼料又は医薬製品を製造するための、請求項1に記載のラクトバチルス・ラムノサス株又は請求項2〜4のいずれか1項に記載の抗真菌組成物の使用。
- 食料製品及び飼料製品中の酵母やカビの増殖を阻害するための、請求項1に記載のラクトバチルス・ラムノサス株又は請求項2〜4のいずれか1項に記載の抗真菌組成物の使用。
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- 2013-04-09 JP JP2015504932A patent/JP6430931B2/ja active Active
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AR090631A1 (es) | 2014-11-26 |
KR20140148479A (ko) | 2014-12-31 |
US20150079057A1 (en) | 2015-03-19 |
EP2836587B1 (en) | 2017-03-01 |
PL2836587T3 (pl) | 2017-08-31 |
CN104284975B (zh) | 2016-08-24 |
WO2013153074A1 (en) | 2013-10-17 |
MX2014012050A (es) | 2015-01-16 |
BR112014024918B1 (pt) | 2022-04-19 |
MX351628B (es) | 2017-10-23 |
ES2630711T3 (es) | 2017-08-23 |
EA033656B1 (ru) | 2019-11-13 |
PT2836587T (pt) | 2017-05-26 |
UA115330C2 (uk) | 2017-10-25 |
EA201491847A1 (ru) | 2015-05-29 |
EP2836587A1 (en) | 2015-02-18 |
BR112014024918A2 (ja) | 2017-06-20 |
HUE034259T2 (en) | 2018-02-28 |
US9485992B2 (en) | 2016-11-08 |
DK2836587T3 (en) | 2017-05-08 |
KR101947110B1 (ko) | 2019-02-13 |
JP2015515273A (ja) | 2015-05-28 |
EA201891030A3 (ru) | 2019-03-29 |
CN104284975A (zh) | 2015-01-14 |
EA036321B1 (ru) | 2020-10-26 |
EA201891030A2 (ru) | 2018-09-28 |
HK1206391A1 (en) | 2016-01-08 |
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