JP5732387B2 - 口臭を予防および/または治療するための使用および方法 - Google Patents
口臭を予防および/または治療するための使用および方法 Download PDFInfo
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- JP5732387B2 JP5732387B2 JP2011512851A JP2011512851A JP5732387B2 JP 5732387 B2 JP5732387 B2 JP 5732387B2 JP 2011512851 A JP2011512851 A JP 2011512851A JP 2011512851 A JP2011512851 A JP 2011512851A JP 5732387 B2 JP5732387 B2 JP 5732387B2
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- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/225—Lactobacillus
- C12R2001/23—Lactobacillus acidophilus
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/225—Lactobacillus
- C12R2001/24—Lactobacillus brevis
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/225—Lactobacillus
- C12R2001/245—Lactobacillus casei
Description
(a)微生物を、1 x 107細胞/mlの出発細胞密度で15 g/lのペプチドを含有する合成培地中、嫌気的条件下、37℃で24時間培養すること;
(b)4000 x gで15分間の遠心分離により細胞を除去すること;および
(c)得られる上清中のペプチド濃度を決定すること;
に供する場合、以下の特性(b):
前記微生物が、24時間のインキュベーション後の上清中のペプチド濃度が、15 g/lの出発濃度と比較して少なくとも20%低下するように培養培地中のペプチド濃度の低下を誘導する特性、すなわち、該微生物が、アッセイ(a)における培地中のペプチド濃度を少なくとも20%低下させることができる特性、
を示すことを特徴とする、乳酸菌群に属する微生物に関する。
(a)微生物を、1 x 107細胞/mlの出発細胞密度で嫌気的条件下、37℃で24時間、100 mlの合成培地中で培養すること;
(b)続いて、細胞を4000 x gで15分間遠心分離し、20 mlのH2O中に再懸濁すること;
(c)続いて、細胞を-80℃に凍結し、減圧下で16時間凍結乾燥すること;
(d)10 mgの凍結乾燥された細菌をH2O中に再懸濁し、4000 x gで10分間遠心分離すること;
(e)7 g/lのペプチドを含有する1 mlの合成培地を細胞ペレットに添加し、細胞を培地中に再懸濁し、37℃で5分間好気的条件下でインキュベートした後、細胞を4000 x gで15分間の遠心分離により除去すること;ならびに
(f)得られる培地上清中のペプチド濃度を決定すること;
に供する場合、以下の特性(B):
凍結乾燥された細菌が、インキュベーション期間の開始時の培地の濃度(7 g/l)と比較して、得られる培地上清中のペプチド濃度の少なくとも20%の低下を誘導する特性、
をさらに示す。
グアニン: 0.1 g/l
シトシン: 0.1 g/l
チミジン: 0.1 g/l
2'-デオキシアデノシン: 0.1 g/l
2'-デオキシウリジン: 0.1 g/l
K2HPO4: 2 g/l
酢酸ナトリウム: 5 g/l
MgSO4・七水和物: 0.1 g/l
クエン酸水素二アンモニウム: 2 g/l
CaCl2・二水和物: 0.5 g/l
オレイン酸: 0.1%(w/v)
シアノコバラミン: 0.02 mg/l
リボフラビン: 10 mg/l
葉酸: 0.2 mg/l
ピリドキサル-5-リン酸一水和物: 10 mg/l
4-アミノ安息香酸: 0.2 mg/l
D(+)-ビオチン: 1 mg/l
アスコルビン酸: 500 mg/l
ニコチン酸: 10 mg/l
パントテン酸カルシウム: 10 mg/l
チアミン: 1 mg/l
コバルト(II)-硝酸六水和物: 500 mg/l
MnSO4・一水和物: 20 mg/l
MgSO4・七水和物: 500 mg/l
Na2MoO4: 0.04 mg/l
PTU-抽出物(Ohly, Deutsche Hefewerke Germany): 15 g/l(または他の場所に記載のように)
D-グルコース・一水和物: 10 g/l
を有する培地である。
平均分析:
・乾燥物質: 96%
・d.m.中のタンパク質(Nx6,25) 72.9%
・d.m.中の総窒素: 11.7%
・NaCl: 1.0%以下
・灰分: 10%
・pH(2%溶液中) 5.7
ビタミン(典型的):
・塩酸チアミンx HCl(B1): 1.2 mg/100 g
・リボフラビン(B2): 7.0 mg/100 g
・ピリドキシン x HCl(B6): 5.9 mg/100 g
・ニコチン酸: 47.8 mg/100 g
・ビオチン: 0.022 mg/100 g
・D-パントテン酸カルシウム: 17.9 mg/100 g
・葉酸: 3.7 mg/100 g
アミノ酸プロフィール(典型的):図7に示されるもの
を示すのが好ましい。
(a)微生物を、1 x 107細胞/mlの出発細胞密度で嫌気的条件下、37℃で24時間、100 mlの合成培地中で培養する;
(b)続いて、細胞を4000 x gで15分間遠心分離して、20 mlのH2O中に再懸濁する;
(c)続いて、細胞を-80℃で凍結し、減圧下で16時間凍結乾燥する;
(d)10 mgの凍結乾燥された細菌を、ディープウェルプレート中のH2Oに再懸濁し、4000 x gで10分間遠心分離する;
(e)3 g/lのペプチドを含有する1 mlの合成培地をペレットに添加し、37℃で5分間インキュベートした後、細胞を4000 x gで15分間の遠心分離により除去する;
(f)次いで、上清を新しいディープウェルプレートに移した後、10〜100μl、好ましくは50μlの非滅菌ヒト唾液を接種し、37℃で6時間嫌気的にインキュベートし、その間、ディープウェルプレートを、酢酸鉛を含浸させた滅菌濾紙で被覆する;
(g)反応物中の微生物による硫化水素の産生を、濾紙の黒変を決定することによりモニターする;
にかけた場合、以下の特性(d):
本発明に従う微生物の存在下で、濾紙に含浸された酢酸鉛の黒変が、培地を前記微生物と共に予備インキュベートしなかった対照と比較して減少する特性、
も示す。濾紙の黒変の減少は、培地に接種するのに用いた非滅菌ヒト唾液中に含まれる細菌によるH2Sの産生の低下を示唆する。用語「H2Sの産生の低下」とは、対照と比較して少なくとも10%、より好ましくは少なくとも20%、さらにより好ましくは少なくとも30%および特に好ましくは少なくとも40%またはさらに少なくとも50%のH2S産生の低下を意味する。この低下を、例えば、濾紙の黒変を密度測定分析することにより測定することができる。あるいは、工程(f)および(g)における硫化水素の産生を、濾紙の使用により測定しないが、ガスクロマトグラフィーを用いるヘッドスペース分析により測定する。
(a)試験しようとする乳酸菌群に属する微生物を、1/2 TSY培地中、1:100(乳酸菌:ストレプトコッカス・サリバリウス)の細胞計数比でストレプトコッカス・サリバリウスと混合する;
(b)細胞懸濁液を37℃で12時間好気的にインキュベートする;
(c)対照として、消費されていない1/2 TSY培地またはMRSライト培地を用いる;
(d)指数増殖中の最大光密度(OD600, max)を決定する、および/または最大増殖速度(Vmax)を決定する;ならびに
(e)最大光密度(OD600, max)および/または最大増殖速度(Vmax)が対照と比較して少なくとも10%増加した場合、前記微生物を、ストレプトコッカス・サリバリウスの増殖を刺激することができる微生物と分類する、
を含む。
(a)試験しようとする乳酸菌群に属する微生物を、150μlの合成培地を含む96穴プレート中、37℃で24時間、嫌気的条件下で培養し、細胞を4000 x gで15分間の遠心分離によりペレット化し、上清を回収する工程;
(b)ストレプトコッカス・ミュータンスを、37℃で一晩、密閉された15 mlのFalconチューブ中、5 mlのTSY培地中で嫌気的に培養する工程;
(c)ストレプトコッカス・ミュータンス細胞培養物を、工程(a)の上清と、2:1の体積比で混合する工程;
(d)培養上清を、37℃で12時間、好気的にインキュベートする工程;
(e)対照として、消費されていない1/2 TSY培地またはMRSライト培地を用いる工程;
(f)指数増殖の間に最大光密度(OD600, max)を決定し、および/または最大増殖速度(Vmax)を決定する工程;ならびに
(g)最大光密度(OD600, max)および/または最大増殖速度(Vmax)が対照と比較して増加していない場合、前記微生物を、ストレプトコッカス・ミュータンスの増殖を刺激することができない微生物と分類する工程、
を含む。
(A)試験しようとする乳酸菌群に属する微生物を、1/2 TSY培地中、1:100の細胞計数比(ラクトバチルス:ストレプトコッカス・ミュータンス)でストレプトコッカス・サリバリウスと混合する工程;
(B)培養懸濁液を、37℃で12時間好気的にインキュベートする工程;
(C)対照として、消費されていない1/2 TSY培地またはMRSライト培地を用いる工程;
(D)指数増殖の間に最大光密度(OD600, max)を決定し、および/または最大増殖速度(Vmax)を決定する工程;ならびに
(E)最大光密度(OD600, max)および/または最大増殖速度(Vmax)が対照と比較して増加していない場合、前記微生物を、ストレプトコッカス・ミュータンスの増殖を刺激することができない微生物と分類する工程、
を含む。
(h)試験しようとする乳酸菌群に属する微生物を、150μlの合成培地を含む96穴プレート中、37℃で24時間、嫌気的条件下で培養し、細胞を4000 x gで15分間の遠心分離によりペレット化し、上清を回収する工程;
(i)ポルフィロモナス・ギンギバリスを、37℃で一晩、密閉された15 mlのFalconチューブ中の5 mlのFAB培地中で嫌気的に培養する工程;
(j)ポルフィロモナス・ギンギバリス細胞培養物を、工程(h)の上清と、2:1の体積比で混合する工程;
(k)培養上清を、37℃で45時間、嫌気的にインキュベートする工程;
(l)対照として、消費されていないFAB培地を用いる工程;
(m)インキュベーション(OD600)の10、15、21、39および45時間後に、光密度(OD600)を決定する工程;ならびに
(n)測定の各時間での光密度(OD600)が対照と比較して増加していない場合、前記微生物を、ポルフィロモナス・ギンギバリスの増殖を刺激することができない微生物と分類する工程、
を含む。
(H)試験しようとする乳酸菌群に属する微生物を、FAB培地中、1:100の細胞計数比(ラクトバチルス:ポルフィロモナス・ギンギバリス)でポルフィロモナス・ギンギバリスと混合する工程;
(I)培養懸濁液を37℃で45時間、好気的にインキュベートする工程;
(J)対照として消費されていないFAB培地を用いる工程;
(K)指数増殖の間に最大光密度(OD600, max)を決定し、および/または最大増殖速度(Vmax)を決定する工程;ならびに
(L)最大光密度(OD600, max)および/または最大増殖速度(Vmax)が対照と比較して増加していない場合、前記微生物を、ポルフィロモナス・ギンギバリスの増殖を刺激することができない微生物と分類する工程、
を含む。
(a)ストレプトコッカス・サリバリウスを、37℃で一晩、8 mlのTSY培地を含む6穴プレート中で嫌気的に培養する;
(b)試験しようとする乳酸菌群に属する微生物を、37℃で24時間、150μlの合成培地を含む96穴プレート中、嫌気的条件下で培養し、細胞を4000 x gで15分間の遠心分離によりペレット化し、上清を回収する;
(c)工程(a)のストレプトコッカス・サリバリウス細胞培養物を、2:1〜4:1の体積比で1/2 TSY培地中の工程(b)の上清と混合する;
(d)細胞懸濁液を37℃で12時間、好気的にインキュベートする;
(e)対照として、消費されていない1/2 TSYまたはMRSライト培地を用いる;
(f)指数増殖の間に最大光密度(OD600, max)を決定し、および/または最大増殖速度(Vmax)を決定する;ならびに
(g)前記微生物を、最大光密度(OD600, max)および/または最大増殖速度(Vmax)が対照と比較して少なくとも10%増加した場合、ストレプトコッカス・サリバリウスの増殖を刺激することができる微生物と分類する、
において起こる。
(h)ストレプトコッカス・サリバリウスを、37℃で一晩、8 mlのTSY培地を含む6穴プレート中で嫌気的に培養する工程;
(i)試験しようとする乳酸菌群に属する微生物を、37℃で24時間、150μlの合成培地を含む96穴プレート中、嫌気的条件下で培養し、細胞を4000 x gで15分間の遠心分離によりペレット化し、上清を回収する工程;
(j)上清を80℃で10分間、インキュベーター中でインキュベートした後、室温まで冷却する工程;
(k)工程(a)のストレプトコッカス・サリバリウス細胞培養物を、2:1の体積比で1/2 TSY培地中の工程(j)の上清と混合する工程;
(l)細胞懸濁液を37℃で12時間、好気的にインキュベートする工程;
(m)対照として、消費されていない1/2 TSYまたはMRSライト培地を用いる工程;
(n)指数増殖の間に最大光密度(OD600, max)を決定し、および/または最大増殖速度(Vmax)を決定する工程;ならびに
(o)前記微生物を、最大光密度(OD600, max)および/または最大増殖速度(Vmax)が対照と比較して少なくとも10%増加した場合、ストレプトコッカス・サリバリウスの増殖を刺激することができる微生物と分類する工程、
を含む。
(p)ストレプトコッカス・サリバリウスを、37℃で一晩、8 mlのTSY培地を含む6穴プレート中で嫌気的に培養する工程;
(q)試験しようとする乳酸菌群に属する微生物を、37℃で一晩、密閉した100 mlのボトル中、50 mlの合成培地中で嫌気的条件下で培養し、細胞を4000 x gで15分間の遠心分離によりペレット化し、上清を回収する工程;
(r)工程(q)の上清20 mlを-80℃で凍結し、減圧下で16時間凍結乾燥する工程;
(s)凍結乾燥された上清を20 mlのH2O中に再懸濁する工程;
(t)工程(a)のストレプトコッカス・サリバリウス細胞培養物を、2:1の体積比で1/2 TSY培地中の工程(s)の上清と混合する工程;
(u)細胞懸濁液を37℃で12時間、好気的にインキュベートする工程;
(v)対照として、消費されていない1/2 TSYまたはMRSライト培地を用いる工程;
(w)指数増殖の間に最大光密度(OD600, max)を決定し、および/または最大増殖速度(Vmax)を決定する工程;ならびに
(x)前記微生物を、最大光密度(OD600, max)および/または最大増殖速度(Vmax)が対照と比較して少なくとも10%増加した場合、ストレプトコッカス・サリバリウスの増殖を刺激することができる微生物と分類する工程、
を含む。
(a)同種発酵性ラクトバチルスであって、
(i)Embden-Meyerhof経路を介してグルコースから少なくとも85%の量の乳酸、好ましくは乳酸のL-、D-もしくはDL-異性体を産生する;
(ii)45℃の温度では増殖するが、15℃の温度では増殖しない;
(iii)長い棹状である;および
(iv)細胞壁中にグリセロールテイコ酸を有する、
前記ラクトバチルス;
(b)同種発酵性ラクトバチルスであって、
(i)Embden-Meyerhof経路を介して乳酸、好ましくは乳酸のL-もしくはDL-異性体を産生する;
(ii)15℃の温度で増殖し、45℃の温度でも可変的増殖を示す;
(iii)短い棹状またはコリネ型である;ならびに
(iv)その細胞壁中にリビトールおよび/もしくはグリセロールテイコ酸を有する、
前記ラクトバチルス;
(c)異種発酵性ラクトバチルスであって、
(i)ペントース-リン酸経路を介してグルコースから少なくとも50%の量の乳酸、好ましくは、乳酸のDL-異性体を産生する;
(ii)二酸化炭素およびエタノールを産生する;
(iii)15℃または45℃での可変的増殖を示す;
(iv)長いか、または短い棹状である;ならびに
(v)その細胞壁中にグリセロールテイコ酸を有する、
前記ラクトバチルス、
のそれぞれに属してもよいことが想定される。
(i)D-ラクトースを代謝するが、L-ソルボースおよび/もしくはD-サッカロースおよび/もしくはD-イヌリンを代謝しない、
(ii)イヌリンを代謝する、
(iii)L-ソルボースを代謝するが、D-ラクトースおよび/もしくはD-サッカロースおよび/もしくはイヌリンを代謝しない、ならびに
(iv)L-ソルボース、D-ラクトースおよびイヌリンを代謝する、
からなる群より選択される代謝フィンガープリントを有する。
(i)D-ラクトースを代謝するが、L-ソルボース、D-サッカロースおよびイヌリンを代謝しない、
(ii)L-ソルボース、D-ラクトースおよびイヌリンを代謝するが、D-サッカロースを代謝しない、
(iii)L-ソルボースを代謝するが、D-ラクトース、D-サッカロースおよびイヌリンを代謝しない、および
(iv)L-ソルボース、D-ラクトース、D-サッカロースを代謝するが、イヌリンを代謝しない、
からなる群より選択される代謝フィンガープリントを有する。
A ハッカ油 1.2重量部
アルニカチンキ 3.0重量部
ミルラチンキ 3.0重量部
Tween 5.0重量部
B 酒精90% 50.0重量部
C 安息香酸ナトリウム 0.2重量部
甘味料(例えば、アスパルテーム) 0.02重量部
蒸留水で100重量部に。
本発明およびその多くの利点のより良い理解を、例示目的のみで提供され、いかなる意味でも本発明の範囲を限定することを意図しない以下の実施例から得られるであろう。
TSY-培地:
TSY混合物(Difco, USA) 30 g/l
酵母抽出物(Deutsche Hefewerke, Germany) 3 g/l
MRSライト培地:
ペプトントリプチカーゼ: 1.0 g/l
酵母抽出物: 0.5 g/l
クエン酸水素ジアンモニウム: 0.2 g/l
酢酸ナトリウム: 0.5 g/l
MgSO4・七水和物: 0.050 g/l
MnSO4・一水和物: 0.025 g/l
D-グルコース一水和物: 1 g/l
K2HPO4: 0.2 g/l
オレイン酸: 0.1%(w/v)
合成培地:
グアニン: 0.1 g/l
シトシン: 0.1 g/l
チミジン: 0.1 g/l
2'-デオキシアデノシン: 0.1 g/l
2'-デオキシウリジン: 0.1 g/l
K2HPO4: 2 g/l
酢酸ナトリウム: 5 g/l
MgSO4・七水和物: 0.1 g/l
クエン酸水素ジアンモニウム: 2 g/l
CaCl2・二水和物: 0.5 g/l
オレイン酸: 0.1%(w/v)
シアノコバラミン: 0.02 mg/l
リボフラビン: 10 mg/l
葉酸: 0.2 mg/l
ピリドキサル-5-リン酸一水和物: 10 mg/l
4-アミノ安息香酸: 0.2 mg/l
D(+)-ビオチン: 1 mg/l
アスコルビン酸: 500 mg/l
ニコチン酸: 10 mg/l
パントテン酸カルシウム: 10 mg/l
チアミン: 1 mg/l
コバルト(II)-硝酸六水和物: 500 mg/l
MnSO4・一水和物: 20 g/l
MgSO4・七水和物: 500 mg/l
Na2MoO4: 0.04 mg/l
PTU抽出物(Ohly, Deutsche Hefewerke Germany): 15 g/l(または他の場所に記載)
D-グルコース一水和物: 10 g/l
FAB培地:
ペプトン混合物 15.0 g/l
酵母抽出物 10.0 g/l
チオグリコール酸ナトリウム 0.5 g/l
塩化ナトリウム 2.5 g/l
寒天No.1 0.75 g/l
L-システインHCl 0.5 g/l
レサズリン 0.001 g/l
重炭酸ナトリウム 0.4 g/l
ハエミン 0.005 g/l
ビタミンK 0.0005 g/l。
株の保存および増殖を、通常の手順に従って行うことができる。例えば、株を-80℃で凍結保存物として保存することができる。1 mlの培養液を、MRS培地中で定常期(OD600/mL 4-8)まで増殖させ、500μlの滅菌50%グリセリン溶液と混合し、凍結することができる。
株の分類学的分類を、その炭水化物発酵パターンに従って行った。これを、API 50 CH (bioMerieux, France)系を用いて決定し、APILAB PLUSソフトウェアバージョン3.3.3 (bioMerieux, France)を用いて分析した。
細菌を実施例1に記載のように培養した。ラクトバチルス(特に、DSM 19826)の上清を、4000 x gで15分間の遠心分離により取得した。ラクトバチルス上清とストレプトコッカス・サリバリウスの混合を、1/2 TSY培地中に2:1〜4:1(ストレプトコッカス・サリバリウス:ラクトバチルス上清)の体積比で行った。これを96穴プレート中で行った。培養上清を、BioTek PowerWaveマイクロプレート分光光度計中、37℃で12時間インキュベートした。対照として、消費されていない1/2 TSY培地またはMRSライト培地を、ラクトバチルス上清の代わりに用いた。
ストレプトコッカス・サリバリウス培養物を、実施例1に記載のように増殖させた。ストレプトコッカス・ミュータンス(DSM 20253)を、密閉された15 mlのFalconチューブ中で一晩、5 mlのTSY培地中で増殖させた。口内細菌を、2:1の体積比で、ラクトバチルス上清と混合し、増殖を実施例1に記載のようにアッセイした。対照として、口内細菌を、ラクトバチルス上清の代わりに、消費されていない1/2 TSY培地と共に培養した。
ストレプトコッカス・サリバリウス培養物を、実施例1に記載のように増殖させた。ポルフィロモナス・ギンギバリス(DSM 20709)を、密閉された15 mlのFalconチューブ中、37℃で一晩、5 mlのFAB培地中で嫌気的条件下で増殖させた。ポルフィロモナス・ギンギバリスを、ラクトバチルス上清(DSM 19826由来)と2:1の体積比で混合し、96穴プレート中で嫌気的に培養した。対照として、ポルフィロモナス・ギンギバリスを、ラクトバチルス上清の代わりに消費されていないFAB培地と共に培養した。
細菌を実施例1に記載のように増殖させた。(DSM 19827の)ラクトバチルス上清を、インキュベーター中、80℃で10分間インキュベートした。上清を室温まで冷却した後、ラクトバチルス上清を、増殖させたストレプトコッカス・サリバリウス培養物と1:2の体積比で混合し、対照実験を含む実施例1に記載のように刺激をアッセイした(刺激を、実施例4および5に概略されたように口腔病原菌を用いて同様にアッセイした。口腔病原菌に対するラクトバチルスの非刺激行動は熱処理により影響されないことが証明された)。ストレプトコッカス・サリバリウスに対する刺激活性に対する熱処理による影響を観察することはできなかった。その結果を図4に示す。
ストレプトコッカス・サリバリウスを実施例1に記載のように増殖させた。ラクトバチルスを、密閉された100 mlのボトル(Schott, Germany)中、37℃で一晩、50 mlの合成培地中で嫌気的に培養した。ラクトバチルス上清を、4000 x gで15分間の遠心分離により取得した。20 mlの上清を-80℃に凍結し、減圧下で16時間凍結乾燥した。凍結乾燥された上清を20 mlのH2O中に再懸濁した。再懸濁された上清を、96穴プレート中の1/2 TSY培地中、2:1(ストレプトコッカス・サリバリウス:再懸濁された上清)の比でストレプトコッカス・サリバリウス培養物と混合した。増殖刺激を、対照実験を含む実施例1に記載のようにアッセイした。刺激活性は、凍結乾燥により低下しなかった。
ラクトバチルス(DSM 19827)を実施例1に記載のように培養した。主培養物を、15 g/lのペプチド(PTU抽出物)を含有する合成培地中で培養した。培地に10μlの培養懸濁液を接種し、37℃で24時間、嫌気的に培養した。次いで、ペプチド濃度を決定したところ、それが24時間後に少なくとも20%低下したことがわかった。
ラクトバチルス(DSM 19827)を、37℃で24時間、100 mlの合成培地中で培養した。全培養物を、4000 x gで15分間遠心分離し、20 mlのH2O中に再懸濁した。20 mlの再懸濁されたラクトバチルスを-80℃に凍結し、減圧下で16時間凍結乾燥した。
ラクトバチルスを、実施例8に記載のように培養し、凍結乾燥した。実験のために、10 mgの凍結乾燥されたラクトバチルスを、ディープウェルプレート中のH2O中に再懸濁し、4000 x gで10分間遠心分離した。3 g/lのペプチドを含有する1 mlの合成培地をペレットに添加し、37℃で5分間インキュベートした後、細胞を遠心分離により除去した。培地のpHはインキュベーションにより変化しなかった。次いで、培地に50μlの非滅菌ヒト唾液を接種し、37℃で6時間嫌気的にインキュベートした。ディープウェルプレートを、酢酸鉛を含浸させた滅菌濾紙で被覆した。唾液由来の微生物による硫化水素の産生を、濾紙の黒変によりモニターした。
好ましくは、ロゼンジ組成物を、DE C2 36 45 147の8頁の実施例4に記載のように調製するが、前記実施例4に記載の成分に加えて、乳酸菌群に属する上記微生物を、ロゼンジ1 mgあたり102〜1012、好ましくは103〜108個の細胞の量で添加する。
好ましくは、ロゼンジ組成物を、DE C2 36 45 147の8頁の実施例5に記載のように調製するが、前記実施例5に記載の成分に加えて、乳酸菌群に属する上記微生物を、ロゼンジ1 mgあたり102〜1012、好ましくは103〜108個の細胞の量で添加する。
好ましくは、歯磨き剤組成物を、DE C2 36 45 147の8頁の実施例3に記載のように調製するが、前記実施例3に記載の成分に加えて、乳酸菌群に属する上記微生物を、歯磨き剤1 mgあたり102〜1012、好ましくは103〜108個の細胞の量で添加する。
好ましくは、チョークに基づく歯磨き剤組成物を、教科書「Kosmetik」、W. Umbach(編)、第2版、Thieme Verlag, 1995の205頁の7.1.4.4章「Rezepturbeispiel」に記載のように調製するが、205頁の前記章に記載の成分に加えて、乳酸菌群に属する上記微生物を、チョークに基づく歯磨き剤1 mgあたり102〜1012、好ましくは103〜108個の細胞の量で添加する。
好ましくは、ケイ酸/フッ化ナトリウム歯磨き剤組成物に基づくゲル歯磨き剤を、教科書「Kosmetik」、W. Umbach(編)、第2版、Thieme Verlag, 1995の205頁の7.1.4.4章「Rezepturbeispiel」に記載のように調製するが、205頁の前記章に記載の成分に加えて、乳酸菌群に属する上記微生物を、ケイ酸/フッ化ナトリウムに基づくゲル歯磨き剤1 mgあたり102〜1012、好ましくは103〜108個の細胞の量で添加する。
好ましくは、歯石に対する歯磨き剤組成物を、教科書「Kosmetik」、W. Umbach(編)、第2版、Thieme Verlag, 1995の206頁の7.1.4.4章「Rezepturbeispiel」に記載のように調製するが、206頁の前記章に記載の成分に加えて、乳酸菌群に属する上記微生物を、歯石に対する歯磨き剤1 mgあたり102〜1012、好ましくは103〜108個の細胞の量で添加する。
好ましくは、チューイングガム組成物を、DE C2 36 45 147の9頁の実施例6に記載のように調製するが、前記実施例6に記載の成分に加えて、乳酸菌群に属する上記微生物を、チューイングガム1 mgあたり102〜1012、好ましくは103〜108個の細胞の量で添加する。
好ましくは、濃縮口内洗浄液組成物を、教科書「Kosmetik」、W. Umbach(編)、第2版、Thieme Verlag, 1995の206頁の7.1.4.4章「Rezepturbeispiel」に記載のように調製するが、206頁の前記章に記載の成分に加えて、乳酸菌群に属する上記微生物を、歯石に対する歯磨き剤1 mgあたり102〜1013個の細胞の量で添加する。
フィルムの調製:
1. 水相
・水を60℃に加熱する
・アスパルテーム(甘味料)を攪拌下で添加する
・アスパルテームを完全に溶解する
・例えば、Kollicoat IR(ポリビニルアルコール上のポリエチレングリコール)もしくはPVP(ポリビニルピロリドン)もしくはアルギン酸塩などの天然ポリマーなどのポリマー性水溶性フィルム形成剤を、それらが溶解するまで攪拌しながら添加する
・10分後、気泡の残りを除去する
・最終芳香フィルムあたり102〜1012、好ましくは103〜108個の細胞の量の乳酸菌群に属する上記微生物を、混合物の冷却後に添加する;あるいは、乳酸菌群に属する上記微生物の突然変異体もしくは誘導体または乳酸菌群に属する上記微生物の類似体もしくは断片を添加してもよい。
・メントールをペパーミント油中に溶解する
・ポリソルベート80をペパーミント油-メントール混合物に攪拌下で添加する
・次いで、この混合物を攪拌下でプロピレングリコールに添加する
・任意の着色料(色素、レーキなど)を添加してもよい。
・攪拌下で、油相を水相とゆっくりと混合する。
・切断装置を用いて薄いフィルムを機械的に作製する。
本発明の他の実施形態および使用は、本明細書の考慮および本明細書に開示された本発明の実施から、当業者には明らかであろう。いかなる理由でも、全ての刊行物、全ての米国および外国の特許ならびに全ての米国および外国の特許出願を含む本明細書に引用される全ての参考文献は、あらゆる目的で具体的かつ完全に参照により本明細書に組み入れられるものとする。本明細書および実施例は以下の特許請求の範囲により示される本発明の真の範囲および精神を用いてのみ典型的に考慮されることが意図される。
[1] 下記アッセイ(a):
(a)微生物を、1 x 10 7 細胞/mlの出発細胞密度で15 g/lのペプチドを含有する合成培地中、嫌気的条件下、37℃で24時間培養すること;
(b)4000 x gで15分間の遠心分離により細胞を除去すること;および
(c)得られる上清中のペプチド濃度を決定すること;
に供する場合、
以下の特性(b):
前記微生物が、24時間のインキュベーション後の上清中のペプチド濃度が、15 g/lの出発濃度と比較して少なくとも20%低下するように培養培地中のペプチド濃度の低下を誘導する特性であって、該微生物が、アッセイ(a)における培地中のペプチド濃度を少なくとも20%低下させることができる特性、
を示すことを特徴とする、乳酸菌群に属する微生物。
[2] 下記アッセイ(A):
(a)微生物を、1 x 10 7 細胞/mlの出発細胞密度で嫌気的条件下、37℃で24時間、100 mlの合成培地中で培養すること;
(b)続いて、細胞を4000 x gで15分間遠心分離し、20 mlのH 2 O中に再懸濁すること;
(c)続いて、細胞を-80℃に凍結し、減圧下で16時間凍結乾燥すること;
(d)10 mgの凍結乾燥された細菌をH 2 O中に再懸濁し、4000 x gで10分間遠心分離すること;
(e)7 g/lのペプチドを含有する1 mlの合成培地を細胞ペレットに添加し、細胞を培地中に再懸濁し、37℃で5分間好気的条件下でインキュベートした後、細胞を4000 x gで15分間の遠心分離により除去すること;ならびに
(f)得られる培地上清中のペプチド濃度を決定すること;
に供する場合、
以下の特性(B):
凍結乾燥された細菌が、インキュベーション期間の開始時の培地の濃度(7 g/l)と比較して、得られる培地上清中のペプチド濃度の少なくとも20%の低下を誘導する特性、
をさらに示す、1に記載の微生物。
[3] 前記微生物が、ストレプトコッカス・サリバリウスの増殖を刺激することができるが、ストレプトコッカス・ミュータンスおよび/もしくはポルフィロモナス・ギンギバリスの増殖は刺激しないことをさらに特徴とする、1または2に記載の微生物。
[4] 前記微生物がラクトバチルス属に属する微生物である、1〜3のいずれか1項に記載の微生物。
[5] 前記ラクトバチルスが、ラクトバチルス・アシドフィルスである、4に記載の微生物。
[6] 7 g/lのペプチドを含有する合成培地に添加し、好気的条件下、37℃で5分間インキュベートされた場合、前記培地のペプチド濃度を少なくとも20%低下させる特性を示す、1〜5のいずれか1項に記載の微生物の不活化形態。
[7] 1〜5のいずれか1項に記載の微生物または6に記載の不活化形態を含む組成物。
[8] 前記組成物が歯磨き剤、チューイングガム、ロゼンジ剤、口内洗浄液、マウスリンス、デンタルフロスまたはデンタルテープである、7に記載の組成物。
[9] 口臭および/または臭い息を低下させるための、1〜5のいずれか1項に記載の微生物、6に記載の不活化形態または7もしくは8に記載の組成物の使用。
[10] ストレプトコッカス・サリバリウスの増殖を刺激することができるが、ストレプトコッカス・ミュータンスおよび/もしくはポルフィロモナス・ギンギバリスの増殖を刺激しないことを特徴とする、乳酸菌群に属する微生物。
[11] 前記微生物が、ラクトバチルス属に属する微生物である、10に記載の微生物。
[12] 前記ラクトバチルスが、ラクトバチルス・アシドフィルスである、11に記載の微生物。
[13] 10〜12のいずれか1項に記載の微生物の不活化形態。
[14] 10〜12のいずれか1項に記載の微生物の培養上清。
[15] 10〜12のいずれか1項に記載の微生物、13に記載の不活化形態または14に記載の培養上清を含む組成物。
[16] 前記組成物が、歯磨き剤、チューイングガム、ロゼンジ剤、口内洗浄液、マウスリンス、デンタルフロスまたはデンタルテープである、15に記載の組成物。
[17] 口臭および/または臭い息を低下させるための、10〜12のいずれか1項に記載の微生物、13に記載の不活化形態、14に記載の培養上清または15もしくは16に記載の組成物の使用。
Claims (13)
- 下記アッセイ(a):
(a)ラクトバチルス・アシドフィルスを、1 x 107細胞/mlの出発細胞密度で15 g/lの抽出物ペプチドを含有する合成培地中、嫌気的条件下、37℃で24時間培養すること;
ここで該抽出物ペプチドは、
乾燥物質: 96%
乾燥物質中のタンパク質(Nx6.25) 72.9%
乾燥物質中の総窒素: 11.7%
NaCl: 1.0%以下
灰分: 10%
pH(2%溶液中) 5.7
塩酸チアミンx HCl(B1): 1.2 mg/100 g
リボフラビン(B2): 7.0 mg/100 g
ピリドキシン x HCl(B6): 5.9 mg/100 g
ニコチン酸: 47.8 mg/100 g
ビオチン: 0.022 mg/100 g
D-パントテン酸カルシウム: 17.9 mg/100 g
葉酸: 3.7 mg/100 g
を含み、
アミノ酸プロフィールが以下に示すもの
である、
(b)4000 x gで15分間の遠心分離により細胞を除去すること;および
(c)得られる上清中の前記抽出物ペプチド濃度を決定すること;
に供する場合、
以下の特性(b):
前記ラクトバチルス・アシドフィルス微生物が、24時間のインキュベーション後の上清中の前記抽出物ペプチド濃度が、15 g/lの出発濃度と比較して少なくとも20%低下するように培養培地中の前記抽出物ペプチド濃度の低下を誘導する特性であって、該ラクトバチルス・アシドフィルス微生物が、アッセイ(a)における培地中の前記抽出物ペプチド濃度を少なくとも20%低下させることができる特性、
を示すことを特徴とする、ラクトバチルス・アシドフィルス。 - 下記アッセイ(A):
(a) ラクトバチルス・アシドフィルスを、1 x 107細胞/mlの出発細胞密度で嫌気的条件下、37℃で24時間、100 mlの合成培地中で培養すること;
(b)続いて、細胞を4000 x gで15分間遠心分離し、20 mlのH2O中に再懸濁すること;
(c)続いて、細胞を-80℃に凍結し、減圧下で16時間凍結乾燥すること;
(d)10 mgの凍結乾燥された細菌をH2O中に再懸濁し、4000 x gで10分間遠心分離すること;
(e)7 g/lの請求項1に特定した抽出物ペプチドを含有する1 mlの合成培地を細胞ペレットに添加し、細胞を培地中に再懸濁し、37℃で5分間好気的条件下でインキュベートした後、細胞を4000 x gで15分間の遠心分離により除去すること;ならびに
(f)得られる培地上清中の前記抽出物ペプチド濃度を決定すること;
に供する場合、
以下の特性(B):
凍結乾燥された細菌が、インキュベーション期間の開始時の培地の濃度(7 g/l)と比較して、得られる培地上清中の前記抽出物ペプチド濃度の少なくとも20%の低下を誘導する特性、
をさらに示す、請求項1に記載のラクトバチルス・アシドフィルス微生物。 - 前記ラクトバチルス・アシドフィルス微生物が、ストレプトコッカス・サリバリウスの増殖を刺激することができるが、ストレプトコッカス・ミュータンスおよび/もしくはポルフィロモナス・ギンギバリスの増殖は刺激しないことをさらに特徴とする、請求項1または2に記載のラクトバチルス・アシドフィルス微生物。
- 7 g/lの請求項1に特定した抽出物ペプチドを含有する合成培地に添加し、好気的条件下、37℃で5分間インキュベートされた場合、前記培地の前記抽出物ペプチド濃度を少なくとも20%低下させる特性を示す、請求項1〜3のいずれか1項に記載のラクトバチルス・アシドフィルス微生物の不活化形態。
- 請求項1〜3のいずれか1項に記載のラクトバチルス・アシドフィルス微生物または請求項4に記載の不活化形態を含む組成物。
- 前記組成物が歯磨き剤、チューイングガム、ロゼンジ剤、口内洗浄液、マウスリンス、デンタルフロスまたはデンタルテープである、請求項5に記載の組成物。
- 口臭および/または臭い息を低下させるための薬剤の製造における、請求項1〜3のいずれか1項に記載のラクトバチルス・アシドフィルス微生物、請求項4に記載の不活化形態または請求項5もしくは6に記載の組成物の使用。
- ストレプトコッカス・サリバリウスの増殖を刺激することができるが、ストレプトコッカス・ミュータンスおよび/もしくはポルフィロモナス・ギンギバリスの増殖を刺激しないことを特徴とする、ラクトバチルス・アシドフィルス微生物。
- 請求項8に記載のラクトバチルス・アシドフィルス微生物の不活化形態。
- 請求項8に記載のラクトバチルス・アシドフィルス微生物の培養上清。
- 請求項8に記載のラクトバチルス・アシドフィルス微生物、請求項9に記載の不活化形態または請求項10に記載の培養上清を含む組成物。
- 前記組成物が、歯磨き剤、チューイングガム、ロゼンジ剤、口内洗浄液、マウスリンス、デンタルフロスまたはデンタルテープである、請求項11に記載の組成物。
- 口臭および/または臭い息を低下させるための薬剤の製造における、請求項8に記載のラクトバチルス・アシドフィルス微生物、請求項9に記載の不活化形態、請求項10に記載の培養上清または請求項11もしくは12に記載の組成物の使用。
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Application Number | Priority Date | Filing Date | Title |
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EP08010641A EP2133414A1 (en) | 2008-06-11 | 2008-06-11 | Uses and methods for preventing and /or treating oral malodour |
EP08010641.2 | 2008-06-11 | ||
PCT/EP2009/003586 WO2009149816A1 (en) | 2008-06-11 | 2009-05-19 | Use and methods for preventing and/or treating oral malodour |
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JP2011525351A JP2011525351A (ja) | 2011-09-22 |
JP2011525351A5 JP2011525351A5 (ja) | 2013-11-21 |
JP5732387B2 true JP5732387B2 (ja) | 2015-06-10 |
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US (1) | US8506953B2 (ja) |
EP (2) | EP2133414A1 (ja) |
JP (1) | JP5732387B2 (ja) |
KR (1) | KR101648992B1 (ja) |
CN (2) | CN104277998B (ja) |
AU (1) | AU2009256978B2 (ja) |
CA (1) | CA2724769A1 (ja) |
ES (1) | ES2574135T3 (ja) |
PL (1) | PL2300598T3 (ja) |
RU (2) | RU2515113C2 (ja) |
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-
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-
2009
- 2009-05-19 EP EP09761379.8A patent/EP2300598B1/en active Active
- 2009-05-19 CN CN201410437155.1A patent/CN104277998B/zh active Active
- 2009-05-19 US US12/997,035 patent/US8506953B2/en active Active
- 2009-05-19 JP JP2011512851A patent/JP5732387B2/ja active Active
- 2009-05-19 CN CN200980121838.3A patent/CN102057036B/zh active Active
- 2009-05-19 WO PCT/EP2009/003586 patent/WO2009149816A1/en active Application Filing
- 2009-05-19 KR KR1020117000589A patent/KR101648992B1/ko active IP Right Grant
- 2009-05-19 AU AU2009256978A patent/AU2009256978B2/en not_active Ceased
- 2009-05-19 PL PL09761379.8T patent/PL2300598T3/pl unknown
- 2009-05-19 CA CA2724769A patent/CA2724769A1/en not_active Abandoned
- 2009-05-19 ES ES09761379.8T patent/ES2574135T3/es active Active
- 2009-05-19 RU RU2010154397/10A patent/RU2515113C2/ru active
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2011
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Also Published As
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KR20110019420A (ko) | 2011-02-25 |
WO2009149816A1 (en) | 2009-12-17 |
AU2009256978B2 (en) | 2015-07-30 |
CN102057036A (zh) | 2011-05-11 |
RU2515113C2 (ru) | 2014-05-10 |
CN104277998A (zh) | 2015-01-14 |
CA2724769A1 (en) | 2009-12-17 |
US20110097284A1 (en) | 2011-04-28 |
RU2014106603A (ru) | 2015-08-27 |
PL2300598T3 (pl) | 2016-10-31 |
RU2010154397A (ru) | 2012-07-20 |
RU2743057C2 (ru) | 2021-02-15 |
KR101648992B1 (ko) | 2016-08-17 |
ZA201100199B (en) | 2012-03-28 |
EP2133414A1 (en) | 2009-12-16 |
US8506953B2 (en) | 2013-08-13 |
JP2011525351A (ja) | 2011-09-22 |
EP2300598A1 (en) | 2011-03-30 |
CN102057036B (zh) | 2017-11-17 |
AU2009256978A1 (en) | 2009-12-17 |
EP2300598B1 (en) | 2016-04-13 |
ES2574135T3 (es) | 2016-06-15 |
CN104277998B (zh) | 2021-02-02 |
BRPI0914726A2 (pt) | 2015-10-20 |
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