JP7472038B2 - マンガン枯渇による真菌類増殖の抑制 - Google Patents
マンガン枯渇による真菌類増殖の抑制 Download PDFInfo
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- JP7472038B2 JP7472038B2 JP2020556878A JP2020556878A JP7472038B2 JP 7472038 B2 JP7472038 B2 JP 7472038B2 JP 2020556878 A JP2020556878 A JP 2020556878A JP 2020556878 A JP2020556878 A JP 2020556878A JP 7472038 B2 JP7472038 B2 JP 7472038B2
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
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Description
増殖を遅らせる」という用語は、真菌細胞の繁殖の減速を指す。これは、例えば、真菌の増殖を測定し、それを対照と比較することにより観察することができる。そのような対照は、例えば、マンガン捕捉剤(スカベンジャー)を適用しない製品であることができる。
- 製品中の遊離マンガンを低減する工程、および
- 遊離マンガン濃度が製品中約0.01 ppmより低い製品を得る工程
を含む方法を提供する。
- 製品中の遊離マンガンを製品中約0.01 ppm未満の濃度に減らす工程、および
- 製品中の遊離マンガンの濃度を測定し、そして0.01 ppm未満の値を得る工程
を含む方法を提供する。
本発明者らは、驚くべきことに、酵母やカビがマンガン枯渇により抑制されうることを発見した。1つの好ましい実施形態では、本発明は、製品、好ましくは食品中の酵母の増殖を抑制するまたは遅らせる方法であって、製品中の遊離マンガンを減らす工程を含む方法を提供する。別の好ましい実施形態では、本発明は、製品、好ましくは食品中のカビの増殖を抑制するまたは遅らせる方法であって、製品中の遊離マンガンを減らす工程を含む方法を提供する。
マンガンを除去する方法は当業界で既知である。マンガンは多数の坑内水、地下水および淡水中に良く見られる汚染物質である。廃水処理では、マンガンイオンはMn02への酸化、吸着、または炭酸塩としての沈殿により、廃水から化学的に除去することができる。
- 1以上の菌株をマンガン捕捉剤として選択する工程と、
- そのマンガン捕捉剤を添加することによって製品中の遊離マンガンを約0.03 ppmより低い濃度に減らす工程
とを含む方法を提供する。
〔(同一残基数)×100〕/〔(アラインメントの長さ)-(アラインメント中のギャップの総数)〕。
〔(同一残基数)×100〕/〔(アラインメントの長さ)-(アラインメント中のギャップの総数)〕
- マンガン捕捉剤として1以上の細菌株を選択する工程、および
- マンガン捕捉剤を添加することにより、製品中の遊離マンガンを製品中約0.01 ppm未満の濃度に低減する工程
を含み、ここで前記選択工程が1以上の細菌株のマンガン取込み活性を測定することを含む方法を提供する。
製品
本明細書に開示される方法は、高温および中温発酵乳製品、例えばヨーグルト製品などの発酵乳製品における酵母および/またはカビの繁殖を抑制または遅らせるのに特に有用である。「発酵乳製品」という用語は、関連する公的規制に従って一般的に定義されている用語であり、その基準は当該技術分野で周知である。例えば、ストレプトコッカス・サーモフィルス(Streptococcus thermophilus;サーモフィルス菌)とラクトバチルス・デルブリューキィ亜属ブルガリクス(Lactobacillus delbrueckii subsp. bulgaricus;ブリガリア菌)との共生培養がヨーグルト用のスターターカルチャーとして使用され、一方でラクトバチルス・アシドフィルス(Lactobacillus acidophillus;アシドフィルス菌)はアシドフィラスミルク(酸乳)の製造に使用される。他の中温乳酸菌は、クワルクまたはフロマージュ・フレ(フランスのフレッシュチーズ)を製造するために使用される。
本明細書に記載され特許請求される発明は、本発明の幾つかの態様の例示として意図されるため、開示される特定の態様により範囲が限定されることはない。任意の同等の態様も本発明の範囲内に入るものである。実際、本明細書中に示され記載されるものに加えて本発明の様々な変形は、下記の記載と次の実施例から当業者に明白になるであろう。そのような変形もまた、添付の特許請求の範囲内に入ると意図される。矛盾する場合、定義を含む本開示が優先する。
本例は、最少培地中でのデバリオミセス・ハンゼニィ(Debaryomyces hansenii)のマンガン需要量を実証する。腐敗したヨーグルト(株1)およびクワルク(株2)からそれぞれ単離された2種の酵母D.ハンゼニィ(D. hansenii)を使用した。それらの株を異なるマンガン濃度を有する既知組成培地中で増殖させた。
本実施例は、発酵乳中のデバリオミセス・ハンゼニィ(Debaryomyces hansenii)のマンガン需要量を証明する。
本実施例は、様々な細菌のマンガン除去活性と、デバリオミケス属とロドツロラ属に対するそれらの抑制効果を示す。低マンガン濃度と高マンガン濃度を有する発酵乳製品での抑制効果を評価した。
本実施例は、スターターカルチャーを用いて調製した発酵乳における異なるマンガン濃度の影響を評価する。
様々なカビに対する抑制効果に対するマンガンの影響を評価した。発酵乳製品の製造プロセスと製法を模倣した寒天アッセイを使用した。L.ラムノサス(L. rhamnosus)とL.パラカゼイ(L. paracasei)をマンガン捕捉剤として併用した。
発酵乳サンプルの調製:
マンガン捕捉剤を含む発酵乳製品と含まない発酵乳製品に、異なるマンガン濃度を添加し、マンガンレベルに加算を行った(参照製品には0、0.006および6 ppmのマンガン、そしてサンプルには0、0.000006、0.00006、0.0006、0.006、0.06、0.6および6 ppmのマンガン)。
2種のデバリオミセス・ハンゼニィ(株1と株2)と1つのクリプトコッカス・フラギコラ(Cryptococcus fragicola)を含む3つの標的汚染物質を、1×103、102および101 CFU/スポットの濃度でスポットした。プレートを7±1℃でインキュベートし、酵母の増殖について定期的に検査した。
酵母寒天アッセイの結果が図6に与えられ、それはスターターカルチャーのみ(参照、最上行)またはマンガン捕捉剤(最下行)と共に発酵させた乳から調製したプレート上の3種の異なる酵母の増殖を示す。写真の上部に示される通り、異なるマンガン濃度を添加した。3つの標的汚染物〔A欄:デバリオミケス・ハンゼニイ(Debaryomyces hansenii (株2)、B欄:クリプトコッカス・フラギコラ(Cryptococcus fragicola)、C欄:デバリオミケス・ハンゼニイ(株1)〕を、3種類の濃度: 1×103 cfu/スポット (プレート上の最上行)、1×102 cfu/スポット (プレート上の中央行)および1×101 cfu/スポット(プレート上の最下行)で添加した。
図7は、スターターカルチャーのみで(参照、最上行)または付加的にマンガン捕捉剤と共に(最下行)発酵させた乳より調製したプレート上での3種のカビの繁殖を示す。写真の上部に示す通り、異なるマンガン濃度を更に添加した。3種の標的汚染物質(A:ペニシリウム・ブレビコンパクタム(Penicillium brevicompactum)、B:ペニシリウム・クラストサム(Penicillium crustosum)およびC:ペニシリウム・ソリタム(Penicillium solitum))を500胞子/スポットの濃度で添加した。プレートを7±1℃で25日間インキュベートした。
実施例8では、2種類の濃度、6 ppmと0.6 ppmのマンガンを使用した。6 ppmの濃度は酵母の増殖に対して抑制/毒性効果を有することが示され、一方で0.6 ppmは標準濃度として用いられ、これはマンガン捕捉剤として作用する1以上の細菌株により引き起こされるマンガン欠損(枯渇)状態を無効にするのに十分な濃度である。
ヨーグルトでは、ヨーグルトにマンガン捕捉剤である最大0.89 mg/mLのEDTAを添加することにより、1以上の細菌株(捕捉剤1)から選択されたマンガン捕捉剤の抑制を再生させることが可能であった。前記濃度では、参照ヨーグルトは、マンガン捕捉剤1を添加した場合に観察されるものと同様な抑制を示した。図14の最下行のネガティブコントロール(EDTAを添加しない)は、マンガン捕捉剤1の存在下で正常な抑制を示し、そしてマンガンを添加すると酵母の増殖が回復した。
Claims (16)
- 発酵乳製品中の真菌類の増殖を抑制または遅らせる方法であって、前記方法が、
マンガン捕捉剤としてマンガントランスポーターを含む1つ以上の細菌株を選択する工程、及び
前記製品中の遊離マンガンを製品中0.01 ppmより低い濃度に低減する工程を含み、前記製品中の遊離マンガンを低減する工程が、1つ以上の前記マンガン捕捉剤を添加することを含む、方法。 - 前記製品中の遊離マンガンの濃度を測定しそして0.01 ppm未満の値を得る工程を更に含む、請求項1に記載の方法。
- 前記真菌類が酵母またはカビである、請求項1又は2に記載の方法。
- 前記真菌類が、トルラスポラ属(Torulaspora spp.)、クリプトコッカス属(Cryptococcus spp.)、サッカロミセス属(Sacharomyces spp.)、ヤロウィア属(Yarrowia)、デバリオミセス属(Debaryomyces spp.)、カンジダ属(Candida spp.)およびロドツロラ属(Rhodoturola spp.)から成る群より選択される酵母であり、または前記真菌類がアスペルギルス属(Aspergillus spp.)、クラドスポリウム属(Cladosporium spp.)、ジダイメラ属(Didymella spp)またはペニシリウム属(Penicillium spp.)から成る群より選択されるカビである、請求項1~3のいずれか一項に記載の方法。
- 前記製品が好熱性または中温性発酵食品である、請求項1~4のいずれか一項に記載の方法。
- 前記製品中の遊離マンガンが0.005 ppm未満の濃度に低減される、請求項1~5のいずれか一項に記載の方法。
- 前記選択工程が、前記1つ以上の細菌株が配列番号1~3のいずれか1つの配列と少なくとも90%の配列同一性を有するマンガントランスポーターを含むかどうかを決定することを含む、請求項1~6のいずれか一項に記載の方法。
- 前記選択工程が、前記1つ以上の細菌株が配列番号1~3のいずれか1つの配列と少なくとも95%の配列同一性を有するマンガントランスポーターを含むかどうかを決定することを含む、請求項7に記載の方法。
- 前記選択工程が、前記1つ以上の細菌株が配列番号1~3のいずれか1つの配列と少なくとも96%の配列同一性を有するマンガントランスポーターを含むかどうかを決定することを含む、請求項7に記載の方法。
- 前記選択工程が、前記1つ以上の細菌株が配列番号1~3のいずれか1つの配列と少なくとも97%の配列同一性を有するマンガントランスポーターを含むかどうかを決定することを含む、請求項7に記載の方法。
- 前記選択工程が、前記1つ以上の細菌株が配列番号1~3のいずれか1つの配列と少なくとも98%の配列同一性を有するマンガントランスポーターを含むかどうかを決定することを含む、請求項7に記載の方法。
- 前記選択工程が、前記1つ以上の細菌株が配列番号1~3のいずれか1つの配列と少なくとも99%の配列同一性を有するマンガントランスポーターを含むかどうかを決定することを含む、請求項7に記載の方法。
- 前記選択工程が、前記1つ以上の細菌株が配列番号1~3のいずれか1つの配列と100%の配列同一性を有するマンガントランスポーターを含むかどうかを決定することを含む、請求項7に記載の方法。
- 前記選択工程が、前記1つ以上の細菌株がスーパーオキシドジスムターゼを含まないこと、又は、マンガンスーパーオキシドジスムターゼを含まないことを決定することを含む、請求項1~13のいずれか一項に記載の方法。
- 前記選択工程が、前記1つ以上の細菌株のマンガン取込み活性を測定することを含む、請求項1~14のいずれか一項に記載の方法。
- 前記遊離マンガンを低減する工程が、ラクトバチルス・プランタラム(Lactobacillus plantarum)、ラクトバチルス・ファーメンタム(Lactobacillus fermentum)、ラクトバチルス・ロイテリ(Lactobacillus reuteri)、ラクトバチルス・サケイ(Lactobacillus sakei)、ラクトバチルス・ブレビス(Lactobacillus brevis)、ラクトバチルス・カゼイ(Lactobacillus casei)、ラクトバチルス・パラカゼイ(Lactobacillus paracasei)、ラクトバチルス・サリバリウス(Lactobacillus salivarius)、ラクトバチルス・アリメンタリウス(Lactobacillus alimentarius)、ペディオコッカス・アシディラクティチ(Pediococcus acidilactici)、ラクトバチルス・ラムノサス(Lactobacillus rhamnosus)およびラクトバシラス・ケフィリ(Lactobacillus kefiri)から成る群より任意に選択される、1つ以上の乳酸菌をマンガン捕捉剤として添加することを含む、請求項1~15のいずれか一項に記載の方法。
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