JP2005512591A - 新規のラクトバチルス属微生物及びその用途 - Google Patents
新規のラクトバチルス属微生物及びその用途 Download PDFInfo
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- JP2005512591A JP2005512591A JP2003556507A JP2003556507A JP2005512591A JP 2005512591 A JP2005512591 A JP 2005512591A JP 2003556507 A JP2003556507 A JP 2003556507A JP 2003556507 A JP2003556507 A JP 2003556507A JP 2005512591 A JP2005512591 A JP 2005512591A
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- lactobacillus paracasei
- lactobacillus
- kctc
- lactic acid
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- C12N1/20—Bacteria; Culture media therefor
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- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y10S435/822—Microorganisms using bacteria or actinomycetales
- Y10S435/853—Lactobacillus
Abstract
【解決手段】 具体的には、本発明は、キムチの発酵液から分離同定された新規のラクトバチルス属微生物であるラクトバチルスパラカゼイ(Lactobacillusparacaseiviro-01)及びこれを含む生菌剤、飼料添加剤、消臭剤及び食品添加物を提供する。本発明のラクトバチルスパラカゼイは、耐酸性、耐胆汁性及び人体安定性に優れ、病原性細菌抑制活性を持っており、腸内菌叢を変化させて有益な細菌を増加させ、下痢の発生度を減少させる効果があり、悪臭物質を分解する消臭効果及び肉類または魚調理時の軟肉効果と消臭効果がある。
Description
本発明者は、キムチの発酵液から耐酸性、耐胆汁性及び有害微生物抑制能力に優れた生理活性を有する新規のラクトバチルス属微生物を分離同定した。本発明の微生物は人に対して毒性がなくて非常に安全であり、前記微生物またはその発酵液は動物の胃腸管関連疾病の予防または治療のための生菌剤、飼料添加剤、消臭剤及び食品添加物として使用できる。
本発明は、前記ラクトバチルス属微生物またはその発酵液を含むことを特徴とする動物の腸疾患予防または治療のための生菌剤を提供する。
本発明は、前記ラクトバチルス属微生物またはその発酵液を含むことを特徴とする飼料添加剤を提供する。
本発明は、前記ラクトバチルス属微生物またはその発酵液を含むことを特徴とする消臭剤を提供する。
本発明は、前記ラクトバチルス属微生物またはその発酵液を含むことを特徴とする食品組成物を提供する。
本発明は、キムチ発酵液から分離した新規のラクトバチルス属(Lactobacillus sp.)微生物を提供する。本発明に係るラクトバチルス属微生物は、ラクトバチルスパラカゼイviro-01(Lactobacillus paracasei viro-01)であって、耐酸性、耐胆汁性、有害微生物抑制活性及び人体安定性に優れるという特性がある。
乳酸菌のみを分離し、これらの中からグラム陽性桿菌である菌株を選抜した後、ラクトバチルス選択培地で培養することによりラクトバチルス属微生物を分離し、その中でも酸生成能に優れた微生物を最終的に分離した。分離された微生物の同定は、形態学的、培養学的、生理学的特性に基づいてバージーマニュアル(Bergey's manual)の分類及び基準に応じて行うことができる。
1.低いpHのストレスで生き残り、胆汁酸に対しても生存力が優れる。具体的に、本発明のラクトバチルスパラカゼイviro-01はpH 3.0以下の環境と雄牛の胆汁(oxgall)濃度0.5%の環境でも生存可能な能力がある。
発酵が完了すると、発酵液を回収する。
発酵液の回収後、培養器内に残っている乳酸菌菌体及び沈殿物に精製水を加えて1倍〜10倍の割合、好ましくは1倍〜5倍の割合で希釈し、前記と同一の方法で再発酵を行う。再発酵が完了すると、発酵液に精製水を加えて1倍〜10倍、好ましくは1倍〜5倍の割合で希釈し熟成する。
「動物」は、人を含む哺乳動物であり、好ましくは牛、馬、豚の家畜を含む。「腸疾患」は、これらに限定されるものではないが、例えば病原性微生物(大腸菌、サルモネラ、クロストリジウムなど)による感染性下痢、胃腸炎、炎症性腸疾患、神経性腸炎症候群、小腸微生物過成長症、腸給餌性下痢などを含む。
有効量のラクトバチルスパラカゼイviro-01は、好ましくは107cells/g以上を意味する。さらに好ましくは、107cells/g〜109cells/gを意味する。担体としては、例えば結合剤、賦形剤、分解剤、潤滑剤、コーティング剤、可溶化剤、分散剤、安定化剤、懸濁化剤、色素及び香料を使用することができる。
分解剤は、カルボキシメチルセルロースやカルボキシメチルセルロースカルシウム、低置換ヒドロキシプロピルセルロースなどのセルロース誘導体、及びカルボキシメチルスターチナトリウム、ヒドロキシプロピルスターチ、コーンスターチ、かたくり粉、米澱粉、部分的に前ゼラチン化された澱粉などの澱粉を含む。
コーティング剤は、ジメチルアミノエチルメタクリレート/メタクリル酸共重合体やポリビニールアセタールジエチルアミノアセテート、エチルアクリラート/メタクリル酸共重合体、エチルアクリラート/メチルメタクリレート/クロロトリメチルアンモニウムエチルメタクリレート共重合体、エチルセルロースなどの水不溶性重合体、メタクリル酸/エチルアクリラート共重合体、ヒドロキシプロピルメチルセルロースフタレート、ヒドロキシプロピルメチルセルロースアセテートサクシネートなどのトニック重合体、及びメチルセルロース、ヒドロキシプロピルメチルセルロース、ポリビニールピロリドン、ポリエチレングリコールなどの水溶性重合体を含む。
本発明は、ラクトバチルスパラカゼイviro-01またはその発酵液を含む飼料添加剤を提供する。本発明の飼料添加剤は、家畜の腸内有用な乳酸菌叢を増加させ、下痢の発生率を減少させ、増体率を増加させるという効果がある。
本発明に係る飼料添加剤は、本発明の乳酸菌またはその発酵液を含有したもので、好ましくは、果汁に乳酸菌を接種して製造した乳酸菌果汁発酵液または前記乳酸菌果汁発酵液を回収し、残った菌体及び沈殿物に水を加えて希釈した後、再発酵させた発酵液を含有する。さらに好ましくは、本質的に純粋な乳酸菌培養物を必要に応じて担体物質を添加した後、これを凍結乾燥、噴霧乾燥、カプセル化またはペレット化して使用することができる。本発明に係る飼料添加剤は、本発明に係る乳酸菌または本発明によって製造された乳酸菌培養液を1kg当り105〜1012cells、好ましくは107〜109cellsの量だけ含む。
(ラクトバチルス属微生物の分離及び同定)
1−1)キムチからのラクトバチルス属微生物の分離
伝統の方法通りに白菜キムチを製造して25℃で20日間発酵させた(pH 4.0±0.2)。前記発酵したキムチ液1mLを滅菌生理食塩水で101〜107まで段階的に希釈した後、各希釈液0.1mLをBHI(Brain Heart Infusion, Difco, USA)培地に接種して37℃で72時間培養した。培養された菌体を、乳酸生成によって培地の色が紫色から黄色に変わる乳酸菌測定用培地であるBCP(brom cresol purple)寒天培地で培養し、乳酸生成に優れた菌株を1次的に分離した。
1−2)分離した菌株の同定
前記実施例1−1で分離した菌株を滅菌果汁に接種した後発酵させ、自体的に発酵液のpHを3.0以下に低下させる菌株を選別した。選別された菌株に対し、API CHLキット(Bio Merioux社)を用いて供給会社の実験方法によって気質利用性の様相を分析した。APIキットによる分析結果は表1に示した通りである。
その結果、本発明に係る菌株は、16S rRNA塩基配列に基づいた分子系統分類学的分析においてラクトバチルス(Lactobacillus sp.)属に属する菌株であって、ラクトバチルスパラカゼイ(Lactobacillus paracasei)、ラクトバチルスカゼイ(Lactobacillus casei)、ラクトバチルスゼアエ(Lactobacillus zeae)、ラクトバチルスラムノーサス(Lactobacillus rhamnosus)を含む系統学的グループに最も近い有縁関係を示した(図1)。
(本発明のラクトバチルスパラカゼイviro-01の特性調査)
2−1)耐酸性及び耐胆汁性試験
pHを2.0、3.0とした0.85%の塩水に、前記実施例1で分離したラクトバチルスパラカゼイviro-01を懸濁した後、37℃で時間別に培養した。培養液を10進法で希釈してMRS培地に塗抹した後、現われたコロニーの数を計数した。その結果、試験菌株を全て生育し、これから本発明によるかラクトバチルスパラカゼイviro-01が耐酸性を有することを確認することができた。
2−2)有害微生物抑制活性の測定
本発明に係るラクトバチルスパラカゼイviro-01の有害微生物抑制活性を測定した。有害微生物としては、大腸菌(Escherichia coli、ATCC-25922)、大腸菌O157(Escherichia coli O157、ATCC-43895)、ブドウ球菌(Staphylococcus aureus、ATCC-25923)、ビブリオ菌(Vibrio parahaemolyticus、KCTC-2471)、サルモネラ菌(Salmonella typhy、KCTC-2424)、緑膿菌(Pseudomonas aeruginosa、KCTC-1636)をBHI液体培地で増菌させて試験に使用し、カンジダ菌(Candida albicans、KCTC-7965)はSDB(Sabouraud Dextrose broth)を使用した。
30秒後、2分後、5分後、24時間後に菌数を測定して初期菌数に対する減少率を調査した。
実験結果、表2のように、カンジダ菌以外に他の6種の菌に対する相当の殺菌効果を示し、特にビブリオ菌に対する効果は非常に卓越であって30秒後から速い菌減少効果を示し、5分後には95.6%が減少し、24時間後には完全死滅した。
また、サルモネラ菌に対しても大きい殺菌効果を示し、他の大腸菌、ブドウ球菌、緑膿菌に対しても高い殺菌力を示した。
本発明に係るラクトバチルスパラカゼイviro-01のバチルス菌に対する殺菌効果を測定した。試験菌株は、食中毒菌として知られているバチルスセレウス(Bacillus cereus)及びバチルスサブチリス(Bacillus subtilis)を使用した。前記試験菌株の種培養液を栄養寒天(nutrient agar)培地に塗抹して36℃で24時間培養した後、滅菌生理食塩水で懸濁して試験菌液として使用した。この際、試験菌液は1.5×108cfu/mL程度に希釈した。前記試験菌液を段階別に(102、103、104)希釈し、ここに本発明に係るラクトバチルスパラカゼイviro-01培養液を同量添加した後、30分、1時間、6時間及び24時間処理し、ここに栄養寒天(nutrient agar)培地を分注した後、37℃で48時間培養して試験菌の生育状態を観察した。前記試験菌液に本発明のラクトバチルスパラカゼイviro-01を添加していない場合を対照群とした。
本発明に係るラクトバチルスパラカゼイviro-01の抗生剤に対する耐性及び感受性を試験した。抗生剤耐性試験は、韓国食品医薬品安全庁から公認を受けた(株)科学技術分析センタ(大田広域式大徳区)に依頼して行った。
実験結果、本発明に係るラクトバチルスパラカゼイviro-01は、セファレキシ、フルメキン、フラゾリドン、スペクチノマイシン及びカナマイシンに対して耐性を示した。また、ゲンタマイシン、ネオマイシンに対しては中程度の耐性を示した。一方、テトラサイクリン、チアムリン、クロラムフェニコールに対しては耐性を示していない(表4)。
(本発明のラクトバチルスパラカゼイviro-01を用いた乳酸菌果汁発酵液の製造及び安全性の検査)
本発明に係るラクトバチルスパラカゼイviro-01を用いて発酵製品を製造し、その安全性を検査した。前記発酵製品は、成熟した果物の汁に本発明に係るラクトバチルスパラカゼイを接種させて発酵させ、乳酸菌果汁発酵液を製造した。本発明に係るラクトバチルスパラカゼイviro-01をMRSブロスに接種して37℃で48時間培養し、滅菌リンゴ果汁50%、オレンジ汁20%、ブドウ汁20%及び棗汁10%からなる混合果汁に2%を接種して30℃で嫌気的に発酵させた。前記混合果汁は精製水で5ブリックスとなるように希釈して糖度を5%に調節した。72時間経過後、pHを測定してpHが3.0に到達すると、酸度が4.0となるまで24時間毎に1時間ずつ曝気しながら30℃で約7日間発酵させた。
(本発明のラクトバチルスパラカゼイviro-01を用いた乳酸菌果汁再発酵液の製造)
前記実施例3の乳酸菌果汁発酵液を回収した後、残っている菌体及び沈殿物を用いて再発酵を行った。残っている菌体及び沈殿物に精製水を加えて5倍に希釈し、30℃で嫌気的に発酵させた。72時間経過後、pHを測定してpHが3.0に到達すると、酸度が4.0となるまで24時間毎に1時間ずつ曝気しながら30℃で約7日間発酵させた。発酵が完了すると、発酵液に精製水を加えて5倍に希釈し熟成させた。
(本発明のラクトバチルスパラカゼイviro-01の母豚及び子豚に対する下痢予防及び増体効果)
本発明に係るラクトバチルスパラカゼイviro-01の母豚及び子豚に対する下痢予防及び増体効果を調査した。韓国「安城」所在の母豚250匹規模の一括飼育農場で各群当り母豚3匹、子豚30匹ずつ3群に分け、2日〜10日以上本発酵液を経口投与した。この際、1群は本発明の発酵液を10日間投与した後、10日以後からは飲水に0.2%を添加して投与した。
(本発明のラクトバチルスパラカゼイviro-01発酵液を給与した離乳子豚の腸内微生物変化の測定)
本発明の乳酸菌発酵液の給与による離乳子豚の腸内微生物変化を調査するために韓国全北大学校畜産学科で産学協同によって現場飼育実験を行った。実験は、本発明の乳酸菌発酵液を給与していない対照区、市販抗生剤0.1%を給与した処理区、及び飲水に0.1%の本発明の乳酸菌発酵液を給与した処理区をそれぞれ設定し、各処理区当り雄雌それぞれ6匹ずつ12匹を配置した。実験期間を8週間として腸内微生物の数を比較した。
(本発明の消臭剤組成物の脱臭能力試験)
本発明の乳酸菌発酵液を精製水で150倍希釈した後、脱臭試験用装置内の悪臭発生源の分解程度を調査した。悪臭発生源としては、メチルメルカプタンを50ppm、アンモニア、トリメチルアミン、硫化水素をそれぞれ60ppmずつ使用し、これを、脱臭試験用装置に悪臭ガス濃度をガステックで測定して調節しながら注入した。
実験結果、塩基性臭気物であるアンモニアとトリメチルアミンに対する脱臭力は6時間後に初期濃度に比べてそれぞれ85%、80%の除去率を示し、酸性臭気物であるメチルメルカプタンと硫化水素に対する脱臭力は6時間後に初期濃度に比べてそれぞれ24%、47%の除去率を示した(表7)。
(本発明の乳酸菌発酵液の肉類及び魚類における軟肉効果及び消臭効果)
本発明の乳酸菌発酵液で製造した天然乳酸菌食品添加物を肉類及び魚類の調理に添加したときの不快臭低減効果と軟肉作用効果を試験した。
Claims (10)
- ラクトバチルスパラカゼイviro-01(KCTC 10132BP)。
- ラクトバチルスパラカゼイviro-01(KCTC 10132BP)の発酵液。
- ラクトバチルスパラカゼイviro-01(KCTC 10132BP)またはその発酵液を含む動物の腸疾患の予防または治療のための生菌剤。
- 前記腸疾患は病原性微生物による感染性下痢、胃腸炎、炎症性腸疾患、神経性腸炎症候群、小腸微生物過成長症及び腸給餌性下痢であることを特徴とする請求項3記載の生菌剤。
- ラクトバチルスパラカゼイviro-01(KCTC 10132BP)またはその発酵液を含む飼料添加剤。
- ラクトバチルスパラカゼイviro-01(KCTC 10132BP)またはその発酵液を含む消臭剤。
- ラクトバチルスパラカゼイviro-01(KCTC 10132BP)またはその発酵液を含む食品組成物。
- ラクトバチルスパラカゼイviro-01(KCTC 10132BP)の発酵液の製造方法。
- 果汁にラクトバチルスパラカゼイviro-01(KCTC 10132BP)を接種し、これを温度範囲15℃〜45℃で嫌気的に発酵させた後、酸度が2.0〜5.0となるまで曝気しながら20℃〜40℃で発酵させることを特徴とする請求項8記載の製造方法。
- 1次乳酸菌発酵液を回収した後、残っている菌体及び沈殿物に精製水を加えて1倍〜10倍の割合で希釈し、これを再発酵させた後、ここに精製水を加えて1倍〜10倍の割合で希釈し熟成させることを特徴とする請求項8記載の製造方法。
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
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KR2001/0085666 | 2001-12-27 | ||
KR1020010085666A KR100356672B1 (ko) | 2001-12-27 | 2001-12-27 | 신규한 락토바실러스 속 미생물 및 그 용도 |
PCT/KR2002/000914 WO2003055987A1 (en) | 2001-12-27 | 2002-05-15 | Novel lactobacillus sp. strain and use thereof |
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US (1) | US7026161B2 (ja) |
EP (1) | EP1468075B1 (ja) |
JP (1) | JP2005512591A (ja) |
KR (1) | KR100356672B1 (ja) |
AT (1) | ATE313621T1 (ja) |
AU (1) | AU2002258275A1 (ja) |
DE (1) | DE60208238D1 (ja) |
WO (1) | WO2003055987A1 (ja) |
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-
2001
- 2001-12-27 KR KR1020010085666A patent/KR100356672B1/ko not_active IP Right Cessation
-
2002
- 2002-05-15 AU AU2002258275A patent/AU2002258275A1/en not_active Abandoned
- 2002-05-15 US US10/500,348 patent/US7026161B2/en not_active Expired - Fee Related
- 2002-05-15 WO PCT/KR2002/000914 patent/WO2003055987A1/en active IP Right Grant
- 2002-05-15 AT AT02728241T patent/ATE313621T1/de not_active IP Right Cessation
- 2002-05-15 EP EP02728241A patent/EP1468075B1/en not_active Expired - Lifetime
- 2002-05-15 DE DE60208238T patent/DE60208238D1/de not_active Expired - Lifetime
- 2002-05-15 JP JP2003556507A patent/JP2005512591A/ja active Pending
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JP2007097455A (ja) * | 2005-10-03 | 2007-04-19 | Katsuya Fukami | イカの塩辛および塩辛の製法法 |
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JP2010521136A (ja) * | 2007-03-16 | 2010-06-24 | キリンホールディングス株式会社 | 腸内細菌叢改善用組成物 |
JP2011505162A (ja) * | 2007-12-04 | 2011-02-24 | コンパニ・ジェルベ・ダノン | 抗真菌剤としてのエル・カゼイ亜種パラカゼイの使用 |
JP4736102B2 (ja) * | 2008-05-16 | 2011-07-27 | 株式会社千葉喜商店 | シュウマイ |
JP2009273452A (ja) * | 2008-05-16 | 2009-11-26 | Chibaki Shoten:Kk | シュウマイ |
JP2011030737A (ja) * | 2009-07-31 | 2011-02-17 | Hodogaya Chem Co Ltd | 消臭剤 |
JP2015502148A (ja) * | 2011-11-17 | 2015-01-22 | アワーホーム カンパニー リミテッドOurhome Co.,Ltd. | キムチの熟成遅延のための混合乳酸菌培養液組成物及びこれを用いたキムチ製造方法 |
US9615594B2 (en) | 2011-11-17 | 2017-04-11 | Ourhome Co., Ltd. | Mixed lactic acid bacterial culture fluid composition for delayed ripening of kimchi and method for making kimchi using same |
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Also Published As
Publication number | Publication date |
---|---|
EP1468075A1 (en) | 2004-10-20 |
KR20020023886A (ko) | 2002-03-29 |
EP1468075B1 (en) | 2005-12-21 |
WO2003055987A1 (en) | 2003-07-10 |
EP1468075A4 (en) | 2005-01-26 |
US20050019894A1 (en) | 2005-01-27 |
DE60208238D1 (de) | 2006-01-26 |
AU2002258275A1 (en) | 2003-07-15 |
US7026161B2 (en) | 2006-04-11 |
ATE313621T1 (de) | 2006-01-15 |
KR100356672B1 (ko) | 2002-10-19 |
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