JP2012161327A - う蝕を予防および/または治療するための手段ならびに方法 - Google Patents
う蝕を予防および/または治療するための手段ならびに方法 Download PDFInfo
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- JP2012161327A JP2012161327A JP2012089420A JP2012089420A JP2012161327A JP 2012161327 A JP2012161327 A JP 2012161327A JP 2012089420 A JP2012089420 A JP 2012089420A JP 2012089420 A JP2012089420 A JP 2012089420A JP 2012161327 A JP2012161327 A JP 2012161327A
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Abstract
【解決手段】ストレプトコッカス・ミュータンス(Streptococcusmutans)と特異的に結合できることを特徴とする乳酸菌のグループに属する微生物。または、前記能力を保持している、熱的に不活性化されているかまたは凍結乾燥されている前記微生物の類似体または断片。または、前記乳酸菌のグループに属する微生物またはその類似体もしくは断片を含んでなる、食品、飼料または飲料用の組成物および添加物。さらに、抗う蝕原性組成物もしくは医薬組成物、または抗う蝕原性食品もしくは飼料を製造するための前記微生物またはその類似体もしくは断片の使用、ならびに前記組成物または食品もしくは飼料を製造するための方法。
【選択図】なし
Description
(a) 前記微生物を静止期になるまで増殖させ、
(b) 前記微生物を、静止期へと増殖させたストレプトコッカス・ミュータンスと混合し、
(c) ステップ(b)で得られた混合物を、前記微生物とストレプトコッカス・ミュータンスとの凝集物の形成を可能にする条件下でインキュベートし、
(d) ペレットの出現により凝集物を検出する。
(i) 加熱処理に抵抗し、かつ/または
(ii) プロテアーゼ処理に抵抗し、かつ/または
(iii) カルシウム依存性であり、かつ/または
(iv) 4.5〜8.5のpH範囲で形成され、かつ/または
(v) 唾液の存在下で形成されるものである。
(a) 前記微生物を静止期になるまで増殖させ、
(b) 前記微生物を、静止期へと増殖させたストレプトコッカス・ミュータンスと混合し、
(c) ステップ(b)で得られた混合物を、前記微生物とストレプトコッカス・ミュータンスとの凝集物の形成を可能にする条件下でインキュベートし、
(d) ペレットの出現により凝集物を検出する。
(a) ホモ発酵乳酸菌
(i) グルコースからEmbden-Meyerhof経路を経て少なくとも85%の量で乳酸(好ましくは、乳酸のL-、D-またはDL-異性体)を産生する;
(ii) 45℃で生育するが、15℃では生育しない;
(iii) 長い桿状である;および
(iv) その細胞壁中にグリセロールテイコ酸を有する;
(b) ホモ発酵乳酸菌
(i) Embden-Meyerhof経路を経て乳酸(好ましくは、乳酸のL-またはDL-異性体)を産生する;
(ii) 15℃で生育し、45℃では変わり易い生育を示す;
(iii) 短い桿状またはコリネ型である;および
(iv) その細胞壁中にリビトールおよび/またはグリセロールテイコ酸を有する;
(c) ヘテロ発酵乳酸菌
(i) グルコースからペントース-リン酸経路を経て少なくとも50%の量で乳酸(好ましくは、乳酸のDL-異性体)を産生する;
(ii) 二酸化炭素とエタノールを産生する;
(iii) 15℃または45℃で変わり易い生育を示す;
(iv) 長いまたは短い桿状である;および
(v) その細胞壁中にグリセロールテイコ酸を有する。
(i) それはD-ラクトースを代謝するが、L-ソルボースおよび/またはD-サッカロースおよび/またはD-イヌリンを代謝しない;
(ii) それはイヌリンを代謝する;
(iii) それはL-ソルボースを代謝するが、D-ラクトースおよび/またはD-サッカロースおよび/またはイヌリンを代謝しない;および
(iv) それはL-ソルボース、D-ラクトースおよびイヌリンを代謝する。
(i) それは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) それは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) それは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) それはL-ソルボース、D-ラクトースおよびD-サッカロースを代謝するが、イヌリンを代謝しない。
occus epidermidis (DSMZ 1798) および/または Staphylococcus epidermidis (DSMZ 20044)と結合しない。
A ハッカ油 1.2部
Tinctura Arnicae 3.0部
Tinctura Myrrhae 3.0部
Tween 5.0部
B Spiritus 90% 50.0部
C 安息香酸ナトリウム 0.2部
甘味料(例: アスパルタン) 0.02部
蒸留水を十分量加えて100とする。
甘味料には、アスパルテーム、カンゾウ、ステビア、キシロース、ラカンカ(Momordica grosvenoriの果実)が含まれる。着色料には、カロテノイドおよびウコン色素、フラボノイド、カラメル色素、スピルリナ色素、クロロフィル、ムラサキサツマイモ色素、ムラサキイモ(purple yam)色素、シソ色素、およびブルーベリー色素が含まれる。
菌株の保存および増殖は通常の手順に従って行うことができる。例えば、菌株を-80℃で凍結ストックとして保存する。1 mlの培養物をMRS培地中で静止期(OD600/mL 4〜8)になるまで増殖させ、500μlの無菌50%グリセリン溶液と混合して凍結させる。S.ミュータンスの培養物はTSY培地中で静止期(OD600/mL 1〜2)になるまで増殖させ、上記のように処理する。S.ミュータンス(DSMZ 20523、血清型c; NCTC 10923、血清型e; NCTC 11060、血清型f;ならびに非血清型別分離株)および乳酸桿菌の培養は、密閉したファルコンチューブ内5 mlの容量で、振とうすることなく37℃で一晩行うことができる。
菌株の分類はそれらの炭水化物発酵パターンに基づいて行った。API 50 CH (bioMerieux, フランス) システムを用いてそれを測定し、APILAB PLUSソフトウェア・バージョン3.3.3 (bioMerieux, フランス)を用いて解析した。
乳酸桿菌とS.ミュータンスの混合は容量比3:1から60:1(S.ミュータンス:乳酸桿菌)で行ったが、これはコロニー形成単位の比1:50から1:2.5に相当する。1 ml中の波長600 nmで測定される光学密度は、好ましくはS.ミュータンスについて3×108コロニー形成単位、乳酸桿菌については好ましくは7×109コロニー形成単位を意味する。混合は15 mLファルコンチューブ内で2 mLの容量にて行った。培養懸濁物をPBSバッファーで希釈して上記の容量比としたが、最終容量は2 mLに保持した。この混合物を15秒間ボルテックス混合した。凝集は懸濁液の急速な濁りとして肉眼で見ることができる。チューブを静かに20分間放置すると、その後凝集物は目に見えるペレットとして沈降するのに対して、非凝集性の混合物は懸濁状態のままである。
MRS培地:
MRS混合物 (Difco, USA) 55 g/L
pH: 6.5
TSY培地:
TSY混合物 (Difco, USA) 30 g/L
酵母エキス (Deutsche Hefewerke, ドイツ) 3 g/L
PBSバッファー:
Na2HPO4*2H20 1.5 g/L
KH2PO4 0.2 g/L
NaCI 8.8 g/L
pHをHClで調整。
ラクトバチルス培養物を実施例1に記載のように増殖させた。
口内細菌、すなわちStreptococcus salivarius subsp. thermophilus (OrganoBalanceにより分離し、API 50 CH (Biomerieux, フランス)をメーカーの使用説明書に従って用いて同定した); Streptococcus oralis (DSMZ 20066); Streptococcus oralis (DSMZ 20395); Streptococcus oralis (DSMZ 20627); Staphylococcus epidermidis (DSMZ 1798); Staphylococcus epidermidis (DSMZ 20044);Streptococcus mitis (DSMZ 12643); Streptococcus sanguinis (DSMZ 20567)を、密閉した15 mLファルコンチューブ内の5 mLのBHI培地中で37℃にて一晩増殖させた。好ましくは、口内細菌のそれぞれをラクトバチルス培養物と容量比3:1で混合して、実施例3に記載のようにして凝集をアッセイした。凝集/非凝集の各試験につき、試験の結果をすぐに確認するために上記細菌の1種のみを使用することが好ましい。
BHI混合物 (Difco, USA) 37 g/L
pH: 7.2
細菌を実施例1のように増殖させた。
増殖させた乳酸桿菌培養物を飽和水蒸気中121℃、2バールで20分間インキュベートした(オートクレーブ処理した)。オートクレーブ処理済みの培養物を室温まで冷ましてから、乳酸桿菌を、増殖させたS.ミュータンス培養物と容量比1:3で混合し、対照実験も含めて実施例3のように凝集をアッセイした。
細菌を実施例1のように増殖させた。
0.5 mlの乳酸桿菌と1.5 mlのS.ミュータンスを3200 g、10分の遠心分離により回収し、上清を捨てた。細胞を、異なるpH値に調整した種々のPBSバッファー中にその最初の容量(それぞれ0.5 mlと1.5 ml)で再懸濁させた。バッファーのpH値は0.5 pH単位きざみで7.0から3.0までのpH値に調整した。凝集挙動アッセイに使用することになっている、それぞれのpH値のバッファーに培養物を再懸濁させた。
細菌を実施例1のように増殖させた。
乳酸桿菌培養物の1 mlのアリコートを3200 g、10分の遠心分離により回収した。上清を捨て、ペレットを室温で真空下に2時間凍結乾燥させた。得られた各試験ラクトバチルス株の乾燥ペレットを室温および4℃で、それぞれ1日、1週間、2週間、3週間および4週間保存した。保存期間後、凍結乾燥ペレットを1 mlのPBSバッファー、pH 7.0中に再懸濁させた。再懸濁した乳酸桿菌を新たに増殖させたS.ミュータンス培養物と容量比1:3で混合し、対照実験も含めて実施例3のように凝集をアッセイした。
細菌を実施例1のように増殖させた。
用いたプロテアーゼはプロナーゼE、プロテイナーゼK、トリプシン、キモトリプシンであった(すべてドイツのSigma社から入手した)。乳酸桿菌の1 mlのアリコートを3200 g、10分の遠心分離にかけて細胞を回収し、ペレットを1 mlのPBSバッファー(pH 7.0)中に再懸濁させることにより、細胞をPBSバッファーで洗浄した。その後、細胞を上記のように再度回収し、それぞれのプロテアーゼを2.5 mg/mLの最終濃度で含有するPBSバッファー(pH 7.0)中に再懸濁させた。この懸濁液を37℃で1時間インキュベートした。その後細胞を上記のように洗浄してPBSバッファー(pH 7.0)中に再懸濁させた。
細菌を実施例1のように増殖させた。
乳酸桿菌の1 mlのアリコートを上記のように1 mlの200 mM EDTA溶液で2回洗浄した。その後細胞を回収し、1 mlのPBSバッファー(pH 7.0)中に再懸濁させた。
細菌を実施例1のように増殖させた。
S.ミュータンス培養物の2 mlアリコートを上記のように回収し、2 mlの唾液中に再懸濁させた。唾液は2人のボランティアにより提供されたものであり、採取後すぐに使用した。
ロゼンジ組成物は好ましくはDE-C2 36 45 147、8頁の実施例4に記載されるとおりに製造するが、ここでは、実施例4に挙げられた成分に加えて、本発明の微生物をロゼンジ1 mgあたり102〜1012、好ましくは103〜108細胞の量で添加する。
ロゼンジ組成物は好ましくはDE-C2 36 45 147、8頁の実施例5に記載されるとおりに製造するが、ここでは、実施例4に挙げられた成分に加えて、本発明の微生物をロゼンジ1 mgあたり102〜1012、好ましくは103〜108細胞の量で添加する。
歯磨剤組成物は好ましくはDE-C2 36 45 147、8頁の実施例3に記載されるとおりに製造するが、ここでは、実施例4に挙げられた成分に加えて、本発明の微生物を歯磨剤1 mgあたり102〜1012、好ましくは103〜108細胞の量で添加する。
チョークを基剤とする歯磨剤組成物は好ましくは参考書"Kosmetik", W. Umbach (編集), 第2版, Thieme Verlag, 1995の205頁、7.1.4.4章 "Rezepturbeispiel" に記載されるとおりに製造するが、ここでは、205頁の7.1.4.4章に挙げられた成分に加えて、本発明の微生物をチョークを基剤とする歯磨剤1 mgあたり102〜1012、好ましくは103〜108細胞の量で添加する。
ケイ酸/フッ化ナトリウムに基づくゲル歯磨剤は好ましくは参考書"Kosmetik", W. Umbach (編集), 第2版, Thieme Verlag, 1995の205頁、7.1.4.4章 "Rezepturbeispiel" に記載されるとおりに製造するが、ここでは、205頁の7.1.4.4章に挙げられた成分に加えて、本発明の微生物をケイ酸/フッ化ナトリウムに基づくゲル歯磨剤1 mgあたり102〜1012、好ましくは103〜108細胞の量で添加する。
歯石予防の歯磨剤組成物は好ましくは参考書"Kosmetik", W. Umbach (編集), 第2版, Thieme Verlag, 1995の206頁、7.1.4.4章 "Rezepturbeispiel" に記載されるとおりに製造するが、ここでは、206頁の7.1.4.4章に挙げられた成分に加えて、本発明の微生物を歯石予防の歯磨剤1 mgあたり102〜1012、好ましくは103〜108細胞の量で添加する。
チューインガム組成物は好ましくはDE-C2 36 45 147、9頁の実施例6に記載されるとおりに製造するが、ここでは、実施例4に挙げられた成分に加えて、本発明の微生物をチューインガム1 mgあたり102〜1012、好ましくは103〜108細胞の量で添加する。
濃縮マウスウォッシュ組成物は好ましくは参考書"Kosmetik", W. Umbach (編集), 第2版, Thieme Verlag, 1995の206頁、7.1.4.4章 "Rezepturbeispiel" に記載されるとおりに製造するが、ここでは、206頁の7.1.4.4章に挙げられた成分に加えて、本発明の微生物を濃縮マウスウォッシュ組成物1 mlあたり102〜1013細胞の量で添加する。
フィルムの製造
1. 水相
- 水を60℃に温める;
- アスパルテーム(甘味料)を撹拌しながら添加する;
- アスパルテームを完全に溶解させる;
- 水溶性の高分子フィルム形成剤、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)またはPVP(ポリビニルピロリドン)または天然ポリマー(例:アルギネート)を、それが溶解するまで撹拌しながら添加する;
- 10分後、残っている泡を取り除く;
- この混合物が冷めてから、本発明の微生物を最終アロマフィルムあたり102〜1012、好ましくは103〜108細胞の量で添加する;あるいはまた、本発明の微生物の変異体もしくは誘導体、または本発明の微生物の類似体もしくは断片を添加することもできる;
2. 油相
- メントールをペパーミント油中に溶解させる;
- 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撹拌しながら添加する;
- この混合物をその後プロピレングリコールに撹拌しながら添加する;
- 任意成分として着色剤(顔料、レーキなど)を添加してもよい;
3.
- 撹拌しながら油相を水相に徐々に添加する;
4.
- カッター装置を使って薄いフィルムを機械的に形成させる。
本発明の他の実施形態および用途は、ここに開示した本発明の仕様および実施を考慮することにより、当業者には明らかであろう。全ての刊行物、全ての米国および外国特許、全ての米国および外国特許出願を含めて、何らかの理由のためにここに引用した文献はどれも、あらゆる目的のために参考として具体的かつ全体的に援用されるものである。本発明の仕様および実施例は単なる例示とみなされ、本発明の真の範囲および精神は特許請求の範囲によって示されるものとする。
DSM 16668
DSM 16669
DSM 16670
DSM 16671
DSM 16672
DSM 16673
Claims (33)
- ストレプトコッカス・ミュータンス(Streptococcus mutans)と特異的に結合できることを特徴とする乳酸菌のグループに属する微生物であって、前記特異的結合が、
(i) 加熱処理に抵抗する、および/または
(ii) プロテアーゼ処理に抵抗する、および/または
(iii) カルシウム依存性である、および/または
(iv) 4.5〜8.5のpH範囲で形成される、および/または
(v) 唾液の存在下で形成される、
ことを特徴とする、上記微生物。 - 前記特異的結合が、
(a) 前記微生物を静止期になるまで増殖させ、
(b) 前記微生物を、静止期へと増殖させたストレプトコッカス・ミュータンスと混合し、
(c) ステップ(b)で得られた混合物を、前記微生物とストレプトコッカス・ミュータンスとの凝集物の形成を可能にする条件下でインキュベートし、
(d) ペレットの出現により凝集物を検出する、
ことによりアッセイされる、請求項1に記載の微生物。 - ラクトバチルス属(Lactobacillus)に属する微生物である、請求項1または2に記載の微生物。
- 前記ラクトバチルスがラクトバチルス・パラカゼイ(Lactobacillus paracasei)またはラクトバチルス・ラムノサス(Lactobacillus rhamnosus)である、請求項3に記載の微生物。
- DSMZ受託番号DSM 16667、DSMZ受託番号DSM 16668、DSMZ受託番号DSM 16669、DSMZ受託番号DSM 16670、DSMZ受託番号DSM 16671、DSMZ受託番号DSM 16672、およびDSMZ受託番号DSM 16673を有するラクトバチルス・パラカゼイもしくはラクトバチルス・ラムノサス、またはストレプトコッカス・ミュータンスと特異的に結合する能力を保持するその変異体もしくは誘導体からなる群より選択される、請求項4に記載の微生物。
- 前記微生物がストレプトコッカス・ミュータンス血清型c(DSMZ 20523)および/または血清型e(NCTC 10923)および/または血清型f(NCTC 11060)と結合することができる、請求項1〜5のいずれか1項に記載の微生物。
- 前記加熱処理が4〜121℃の温度で少なくとも20分間実施される、請求項1〜6のいずれか1項に記載の微生物。
- 前記プロテアーゼ処理がプロナーゼE、プロテイナーゼK、トリプシンおよびキモトリプシンからなる群より選択されるプロテアーゼによる処理である、請求項1〜6のいずれか1項に記載の微生物。
- 熱的に不活性化されているかまたは凍結乾燥されている、請求項1〜8のいずれか1項に記載の微生物の類似体または断片であって、ストレプトコッカス・ミュータンスと特異的に結合する能力を保持している、上記類似体または断片。
- ストレプトコッカス・ミュータンスと特異的に結合できることを特徴とする乳酸菌のグループに属する微生物、または該微生物の変異体、誘導体、類似体もしくは断片を含んでなる組成物。
- 前記微生物が請求項1〜8のいずれか1項に記載の微生物であり、前記類似体または断片が請求項9に記載の類似体または断片である、請求項10に記載の組成物。
- 化粧上許容される担体または賦形剤をさらに含む化粧用組成物である、請求項10または11に記載の組成物。
- 製薬上許容される担体または賦形剤をさらに含む医薬組成物である、請求項10または11に記載の組成物。
- 経口的に許容される担体または賦形剤をさらに含む食品または飼料組成物である、請求項10または11に記載の組成物。
- 歯磨剤、チューインガム、ロゼンジ、マウスウォッシュ、マウスリンス、またはデンタルフロスである、請求項10〜14のいずれか1項に記載の組成物。
- 抗う蝕活性を有する、請求項10〜15のいずれか1項に記載の組成物。
- 抗う蝕原性組成物を製造するための、ストレプトコッカス・ミュータンスと特異的に結合できることを特徴とする乳酸菌のグループに属する微生物または該微生物の変異体、誘導体、類似体もしくは断片の使用。
- 前記微生物が請求項1〜8のいずれか1項に記載の微生物であり、前記類似体または断片が請求項9に記載の類似体または断片である、請求項17に記載の使用。
- 前記抗う蝕原性組成物が歯磨剤、チューインガム、ロゼンジ、マウスウォッシュ、マウスリンス、またはデンタルフロスである、請求項17または18に記載の使用。
- う蝕を予防するための、ストレプトコッカス・ミュータンスと特異的に結合できることを特徴とする乳酸菌のグループに属する微生物または該微生物の変異体、誘導体、類似体もしくは断片の使用。
- 前記微生物が請求項1〜8のいずれか1項に記載の微生物であり、前記類似体または断片が請求項9に記載の類似体または断片である、請求項20に記載の使用。
- ストレプトコッカス・ミュータンスと特異的に結合できることを特徴とする乳酸菌のグループに属する微生物または該微生物の変異体、誘導体、類似体もしくは断片を、化粧上、製薬上または経口的に許容される担体または賦形剤と共に製剤化することを含んでなる、抗う蝕原性組成物の製造方法。
- ストレプトコッカス・ミュータンスと特異的に結合できることを特徴とする乳酸菌のグループに属する微生物または該微生物の変異体、誘導体、類似体もしくは断片を、食品または飼料に添加することを含んでなる、抗う蝕原性食品または飼料の製造方法。
- 前記微生物が請求項1〜8のいずれか1項に記載の微生物であり、前記類似体または断片が請求項9に記載の類似体または断片である、請求項22または23に記載の方法。
- ストレプトコッカス・ミュータンスと特異的に結合できることを特徴とする乳酸菌のグループに属する微生物または該微生物の変異体、誘導体、類似体もしくは断片を含んでなる、食品、飼料または飲料用の添加物。
- 前記微生物が請求項1〜8のいずれか1項に記載の微生物であり、前記類似体または断片が請求項9に記載の類似体または断片である、請求項25に記載の食品、飼料または飲料用の添加物。
- ストレプトコッカス・ミュータンスと特異的に結合できることを特徴とする乳酸菌のグループに属する微生物または該微生物の変異体、誘導体、類似体もしくは断片を提供することを含んでなる、抗う蝕原性組成物の製造方法。
- 前記微生物が請求項1〜8のいずれか1項に記載の微生物である、請求項27に記載の方法。
- 前記類似体または断片が請求項9に記載の類似体または断片である、請求項27に記載の方法。
- 前記抗う蝕原性組成物が歯磨剤、チューインガム、ロゼンジ、マウスウォッシュ、マウスリンス、またはデンタルフロスである、請求項27〜29のいずれか1項に記載の方法。
- ストレプトコッカス・ミュータンスと特異的に結合できることを特徴とする乳酸菌のグループに属する微生物または該微生物の変異体、誘導体、類似体もしくは断片を提供することを含んでなる、う蝕の予防方法。
- 前記微生物が請求項1〜8のいずれか1項に記載の微生物である、請求項31に記載の方法。
- 前記類似体または断片が請求項9に記載の類似体または断片である、請求項31に記載の方法。
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PL2267112T3 (pl) | 2016-11-30 |
ATE465239T1 (de) | 2010-05-15 |
EP2267112A1 (en) | 2010-12-29 |
CN101056972B (zh) | 2012-07-04 |
US20080193427A1 (en) | 2008-08-14 |
AU2005281796A1 (en) | 2006-03-16 |
CN101056972A (zh) | 2007-10-17 |
EP1634948A1 (en) | 2006-03-15 |
ES2344654T3 (es) | 2010-09-02 |
US8192978B2 (en) | 2012-06-05 |
EP1797170A1 (en) | 2007-06-20 |
DE602005020820D1 (de) | 2010-06-02 |
US20200085727A1 (en) | 2020-03-19 |
ES2579879T3 (es) | 2016-08-17 |
JP2008512104A (ja) | 2008-04-24 |
JP5143559B2 (ja) | 2013-02-13 |
PL1797170T3 (pl) | 2010-09-30 |
JP5632874B2 (ja) | 2014-11-26 |
CN101948773A (zh) | 2011-01-19 |
WO2006027265A1 (en) | 2006-03-16 |
US20170258708A1 (en) | 2017-09-14 |
US11419811B2 (en) | 2022-08-23 |
EP2267112B1 (en) | 2016-05-11 |
JP2015006185A (ja) | 2015-01-15 |
EP1797170B1 (en) | 2010-04-21 |
DK1797170T3 (da) | 2010-07-19 |
EP2218774A2 (en) | 2010-08-18 |
AU2005281796C1 (en) | 2014-01-23 |
AU2005281796B2 (en) | 2011-06-16 |
US20120237454A1 (en) | 2012-09-20 |
EP2218774A3 (en) | 2010-09-22 |
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