JP2016502402A - サワードウ由来の微生物Lactobacillussanfranciscensis - Google Patents
サワードウ由来の微生物Lactobacillussanfranciscensis Download PDFInfo
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- JP2016502402A JP2016502402A JP2015540179A JP2015540179A JP2016502402A JP 2016502402 A JP2016502402 A JP 2016502402A JP 2015540179 A JP2015540179 A JP 2015540179A JP 2015540179 A JP2015540179 A JP 2015540179A JP 2016502402 A JP2016502402 A JP 2016502402A
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Images
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Abstract
Description
生地のpH値は、ガラス電極を用いて決定した。細胞数を決定するために、冷蔵および冷凍の生地を、緩衝ペプトン水中に段階希釈し、変性MRS寒天上に播いた。細菌数を決定するための変性MRS寒天は、酵母の生育を阻害するために、100mg/lのシクロヘキシミドを含有した。これらのプレートを、好気的に30℃で48時間インキュベートした。酵母数を決定するための変性MRS寒天は、100mg/lのクロラムフェニコールを含有した。これらのプレートを、好気的に30℃で48時間インキュベートした。
サンプル中に存在する細菌株を同定するために、形態学的に異なる分離株を、最も高い希釈段階のプレート(15〜150の細菌数)から拾って広げた。シングルコロニーを30℃で24時間、変性MRS緩衝液中でインキュベートした。冷蔵および冷凍の生地から、Dneasy Blood&Tissue Kit(Quiagen,Missiauga,Canada)を用いて、合計77コロニーからDNAを分離した。
マルトース、マンニット、ラクテート、グリセロール、アセテートおよびエタノールの濃度を、HPLCにより決定した。サンプル調製のために、生地を7%の過塩素酸で1:1希釈し、4℃で一晩インキュベートした。上清を直接分析した。Aminex HP87Xカラム(Biorad)上で分離を行ない、0.4mL分−1にて70℃で、5mMのH2SO4を用いて溶出を行なった。定量化は、屈折率(RI)検出および外部標準物質に基づいた。
細菌数およびpH値。
冷蔵生地中の細菌および酵母のpH値がより低くてより数が多いことは、デリバリー中の後発酵を示す。冷凍生地中の酵母数がより少ないことは、冷凍中に酵母が部分的に壊死することが原因であった。冷蔵生地中の細菌数は同様に、log8.3〜8.9の細菌形成単位(GFU)/gであった(表2)。冷凍全粒粉スターターはこの例外であり、冷蔵生地中よりも冷凍生地中の細菌数の方が多かった。
ランダム増幅多型DNA(RAPD)タイピングにより区別可能な10株の25分離株を、シーケンスのために選択した。20分離株がLactobacillus sanfranciscensisとして同定され、RAPDパターンd8に基づき異なる株に割り当てられた(表3)。いくつかの株は、同じ生地の冷蔵および冷凍サンプルにおいて分離された。いくつかの株は、全粒粉生地およびサワードウから分離することができた(株番号3、4、5、および6)。
サワードウ微生物叢の分類学的特徴付けは、代謝物の分析により裏付けられた。サワードウ中にマンニットおよびアセテートが存在することは、ヘテロ発酵性を導く細菌Lactobacillus sanfranciscensisが細菌叢で優勢であることを確証した(表4)。グリセロールおよびエタノールは、酵母の代謝に起因すると考えることができた。デリバリー中に起きた後発酵は、代謝物の分析においても確認することができた。ラクテート、グリセロールおよびエタノールは、冷蔵生地中に、さらにより高い濃度で存在した。したがって、マルトースおよび高いグルコース濃度は、冷凍生地においてのみ検出することができた。おそらく小麦粉と比較して全粒粉の緩衝能がより高いことにより、冷凍生地では、発酵性全粒粉スターターが最も多量のラクテートおよびアセテートを示した。
2つの試験された生地(全粒粉生地サンプル1と2、および生地サンプル3と4)は、細菌叢の組成物に関して有意差を示した。Lactobacillus sanfranciscensisのいくつかの異なる株が同定された。
Lactobacillus rossiae DSM26024株の分離
実施例1で記載したのと同様の条件下で,サワードウからLactobacillus rossiae DSM26024株をMRSプレート(1.5%寒天、0.15%L−システイン、pH5.4)上で分離し、コロニー形態および16S DNAシーケンシングによって同定した。
Lactobacillus株の培養
本発明のLactobacillus株を、DSM培地225(サワードウ培地)またはmMRS5(Merothら、Appl.Microbiol.Environ.69:475)からなる培地中で培養した。組成物は、リットルあたり、10gのトリプトン、5gの肉エキス、5gの酵母、10gのマルトース、5gのフルクトース、5gのグルコース、5gのNa−アセテート×3H2O、3gのNH4Cl、2.6gのK2HPO4×3H2O、4gのKH2PO4、0.1gのMgSO4×7H2O、0.05gのMnSO4×4H2O、0.5gのシステイン−HCl、1mlのTween 80、1mlのビタミン混合物(コバラミン、葉酸、ニコチン酸アミド、ピリドキサールホスフェートおよびチアミンを、各0.2g/L)を含んだ。糖類は別々にオートクレーブした;ビタミン混合物を濾過滅菌し、オートクレーブ後に添加した。培地を120℃で20分間滅菌した。滅菌後のpHは好ましくは5.8であった。ガス雰囲気は、4%のO2、約5%のCO2、およびバランス窒素(balance nitrogen)を含んだ。ブレッディング温度は30℃であり、ブレッディング持続時間は24〜72時間であった。純粋培養を植菌に用いた。
1.細菌培養
本発明に係るLactobacillus sanfranciscensis(DSM 23090株、DSM 23091株、DSM 23092株、DSM 23093株、DSM 23174株、DSM 23200株、およびDSM 23201株)、本発明に係るLactobacillus rossiae DSM 26024株、および、Lactobacillus sanfranciscensis基準株DSM 20451(DSM GmbH,Braunschweig,Germany)を、0.25×107の細菌/mLで植菌後、Anaerogenパッケージ(Anaerogen,Basingstoke,Oxoid,UK)を用いて、嫌気的条件下で24時間、新しく添加された0.15%のL−システインを含むMRS培養液(pH5.4)中で、30℃で増殖させた。
代謝物の定量化のために、乳酸菌のMRS増殖培地をLC−MS/MS分析に供した。分析の前に、10 kDa Vivaspin 500フィルター(Sartorius Stedim biotech,Goettingen,Germany)を用いて、MRS培地を濾過した。
Dionex Ultra High Performance Liquid Chromatography UltiMate(登録商標)3000(Dionex,Idstein,Germany)
−ポンプ−HPG−3400SD
−デガッサー−SRD−3400
−オートサンプラー−WPS−3000TSL
−カラムオーブン−TCC−3000SD
API 4000 QTRAP,リニアイオントラップ四重極質量分析計(AB Sciex,Darmstadt,Germany):
−イオン化型−エレクトロスプレーイオン化(ESI)
−測定器制御−アナリストソフトウェア(AbSciex,Darmstadt,Germany)
−固定相:TSKgel Amide−80 3μm(150×2mm,Tosoh Bioscience,Stuttgart,Germany)
−固定相温度:40℃
−移動相:
溶離液A:アセトニトリル/5mM/Lのアンモニウムアセテート 水中(95+5)
溶離液B:5mM/Lのアンモニウムアセテート 水中(95+5)
データを平均値±標準偏差(SD)として表す。全ての統計的計算は、処置群と対応するコントロール群とを比較する統計プログラミングプラットフォームR(Statistical programming platform R)を用いて行ない、独立t検定を用いて分析した。様々な処置群と対応するコントロール群とを比較するデータは、1元配置分散分析(One−Way ANOVA)の後に適切な多重比較手段を用いて分析した。データが正規分布でないか、または、不連続なデータを含む場合は、ノンパラメトリック検定(マンフォイトニー(Mann−Whitney)/順位和検定、順位に関する分散分析(ANOVA on ranks))を用いた。p値が<0.05(*)または<0.01(**)である場合に有意差と見なした。主成分分析(PCA)は、Pearson,K.;On Lines and Planes of Closest Fit to Systems of Points in Space,Philosophical Magazine (1901),2 (11),559−572、および、Theodoridis,G.,Gika,H.G.,Wilson,I.D.;LC−MS−based methodology for global metabolite profiling in metabonomics/metabolomics,TrAC Trends in Analytical Chemistry (2008),27(3),251−260に記載されている。
4.1 サワードウ乳酸菌の増殖培地中の代謝物のLC−MS/MS分析
本発明に係るLactobacillus rossiae株(DSM 26024)、Lactobacillus sanfranfranciscensis株(DSM 23090、DSM 23091、DSM 23092、DSM 23093、DSM 23174、DSM 23200およびDSM 23210)、および、比較株、Lactobacillus sanfranciscensis基準株(DSM 20451)を、MRS培地で24時間培養した。増殖培地を回収して濾過し、LC−MS/MSを用いて分析した。
1.本発明の細菌株を含む、様々なパンのタイプおよびサワードウの、免疫調節特性の分析
サワードウブレッド(#1)、比較パンa(#2)と比較パンb(#3)、および、サワードウ(T)を分析した。サワードウブレッド(#1)は、本発明の乳酸菌を含むサワードウ(T)から作った。
サンプルを分配して急速冷凍し(−20℃)、それから凍結乾燥した。遊星ミルを用いて、凍結乾燥されたサンプルを細かく粉砕して合わせ、4つの混合サンプルを形成した(1、2、3およびT)。
ヒトの胃の中の状態をシミュレートするために、様々なサンプルの1g粉末を採取することにより(数回反復)、有効成分を10mlのHCl(150mM;pH2)中で抽出し、その後、インキュベーター内に置かれたオーバーヘッド回転子を用いて、37℃で2.5時間を超えてインキュベーションした。
懸濁液(HClと紛体)を生産するために、インキュベートしたサンプルを合わせて、8.25M NaOHを用いてpH値を7に調節した。サンプルを分配して−70℃で凍結乾燥した。
上清を生産するために、懸濁液を、4,000r/分の速度で10分を超えて遠心分離した。残渣(ペレット)上に生じた上清を取り出し、それからサンプルを合わせて、pH値を、a)に記載のとおりに7に調節した。その後、サンプルを分配して凍結乾燥した。
ペレットを生産するために、(b)からの残渣を、サンプルから抽出試薬を完全に除去するために10mlの0.9% NaCl溶液を用いて2回洗浄した。それから、ペレットを10mlの0.9% NaCl溶液で覆って、「濡れた(wet)」状態で冷凍した。
免疫調節またはHOCl解毒特性の分析を、全血テスト系を用いて行なった。3人の健康なヒトドナー由来の全血を用いた。活性化免疫細胞の反応中、活性酸素種(ROS)が放出され、それはとりわけ、HOCl=次亜塩素酸である。これは、病原体を死に導く。HOCl(次亜塩素酸)の産生の選択的指示薬(ACC;1−アミノシクロプロパン−1−カルボン酸)を用いて、この効果を、ガスクロマトグラフィーを用いて測定した。サンプル1、2、3およびTの、ペレット、懸濁液、および上清を、3人のドナー由来の血液を用いて全血系で分析した。
この方法は、用いられる被検物質が、HOClと反応することが可能かどうか把握することを可能にする。もしそうであれば、全血系で同定された抗酸化作用を、少なくとも部分的に、HOClとの反応に帰することができる。したがってHOClは、他の反応に関して無害にされた(「解毒された」)。
この場合では、様々な活性酸素種が産生されるように、全血中の免疫細胞を、ザイモサン(酵母の細胞壁成分)を用いて刺激した。これは、とりわけ、HOCl(ACCと反応してエチレンを産生する)を放出させた。産生されるエチレンの量は、免疫細胞の活性の基準となった。サンプル中に含まれるエチレンが少なければ少ないほど、免疫細胞の活性は低いことが分かった。
この場合では、反応サンプルにザイモサンを添加しなかった。用いられた被検物質がそれでもなお、その系において作用を示したら、すなわち、エチレンが産生されたら、免疫細胞を、その被検物質により刺激した。このようにして、免疫調節または免疫刺激作用を検出することができた。
Claims (11)
- DSM23090株、DSM23091株、DSM23200株、DSM23092株、DSM23093株、DSM23201株、DSM23174株、DSM23121株およびDSM26024株のいずれか1つから選択される、
Lactobacillus株。 - 請求項1に記載の少なくとも1つのLactobacillus株および担体を含む、
組成物。 - 請求項2に記載の組成物であって、
前記担体が、Lactobacillus株の培養に適切な培養培地である、
組成物。 - 請求項2または3に記載の組成物であって、
少なくとも1つのさらなる微生物を含む、
組成物。 - 請求項4に記載の組成物であって、
前記の少なくとも1つのさらなる微生物は、細菌および/または酵母であり、
具体的には、Lactobacillus sanfranciscensis株、Lactobacillus rossiae株、Lactobacillus plantarum株、Lactobacillus brevis株、Lactobacillus amyolyticus株、Lactobacillus amylovarus株、Lactobacillus delbruckii株、Lactobacillus pontis株、Lactobacillus acidophilus株、Lactobacillus lactis株 Gluconobacter oxydans株、Candida humilis株、Candida milleri株、Candida krusei株、Saccharomyces exiguus株、Saccharomyces barnetti株、Saccharomyces cerevisiae株および/またはSaccharomyces minor株から選択される、
組成物。 - 請求項1に記載の少なくとも1つのLactobacillus株または請求項2から5のいずれか一項に記載の組成物の使用であって、
ヒト用食品製品および補助食品製品または動物用飼料製品の製造のための、
使用。 - 請求項6に記載の使用であって、
前記の食品製品、補助食品製品または飼料製品が、発酵製品である、
使用。 - 請求項6または7に記載の使用であって、
前記食品製品が、サワードウまたは飲料、特にサワードウブレッドである、
使用。 - 請求項1に記載のLactobacillus株または請求項2から5のいずれか一項に記載の組成物であって、
ヒトまたは動物用医薬での、または化粧品での、使用のための、
組成物。 - 請求項9に記載の使用であって、
免疫応答の調節のための、またはスキンケアのための、
使用。 - 請求項1に記載のLactobacillus株の使用であって、
それからさらにLactobacillus株を培養するための、
使用。
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CN109321479A (zh) * | 2018-11-27 | 2019-02-12 | 宜宾五粮液股份有限公司 | 耐酸酿酒酵母及其用途 |
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