JP6129294B2 - 新たに分離したバチルス・リケニフォルミス及びこれを利用したプロバイオティクス - Google Patents
新たに分離したバチルス・リケニフォルミス及びこれを利用したプロバイオティクス Download PDFInfo
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- JP6129294B2 JP6129294B2 JP2015504500A JP2015504500A JP6129294B2 JP 6129294 B2 JP6129294 B2 JP 6129294B2 JP 2015504500 A JP2015504500 A JP 2015504500A JP 2015504500 A JP2015504500 A JP 2015504500A JP 6129294 B2 JP6129294 B2 JP 6129294B2
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- bacillus licheniformis
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- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 238000009423 ventilation Methods 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
- 239000002351 wastewater Substances 0.000 description 1
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- 239000012138 yeast extract Substances 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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
- C12N1/20—Bacteria; Culture media therefor
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
- A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
- C02F3/342—Biological treatment of water, waste water, or sewage characterised by the microorganisms used characterised by the enzymes used
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2103/00—Nature of the water, waste water, sewage or sludge to be treated
- C02F2103/22—Nature of the water, waste water, sewage or sludge to be treated from the processing of animals, e.g. poultry, fish, or parts thereof
-
- 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
- C12R2001/07—Bacillus
- C12R2001/10—Bacillus licheniformis
Description
本発明の他の目的は、前記プロバイオティクス製剤を含む飼料添加剤を提供することである。
(1)試料の用意と菌株の分離
韓国の伝統食品であるテンジャン、メジュ、醤油などから名品醤類由来菌株を用意した。用意した試料を3%塩化ナトリウムが添加されたBHI個体培地(Difco、USA)に塗抹した後、37℃の条件で24時間培養した。各試料から分離された菌株は、コロニー観察によりグループ化して菌株を分離した。選別されたコロニーは3回にわたって新しい培地に移して培養する方法により再分離し、純粋培養した菌を20%グリセロールが添加された培地に入れ、−70℃以下で保存した。
水質汚染の主要原因であるアンモニアと亜硝酸を酸化させる微生物を1次選抜するため、アンモニアと亜硝酸に対する酸化評価を行った。
(1)1次選抜菌株のアンモニア定量評価
1次選抜菌株のアンモニア酸化による低減効果を評価するため、前記選抜菌株10種に対し、アンモニアの添加培地でのアンモニア消耗量を定量した。
1次選抜菌株の亜硝酸酸化による低減効果を評価するため、前記選抜菌株10種に対して、亜硝酸が添加された培地から定量して亜硝酸の消耗量を定量した。
水質環境は、好気、通性嫌気、嫌気的条件が共存し、特に嫌気的条件でアンモニアが硝酸に酸化するアナモックス(Anammox、嫌気的条件でアンモニアが硝酸に酸化する過程)が行われるため選抜菌株が多様な条件で生長することは重要なのである。そして、1次選抜菌株10種に対し、通性嫌気と嫌気条件においての生長性を評価した。
淡水条件と海水条件における菌株の活性を評価するため、塩化ナトリウムが添加された培地で2次選抜された菌株の生長性評価を行った。
2次選抜された菌株3種に対する水質浄化能力をヒラメ稚魚で評価した。ヒラメ稚魚に市販飼料を満腹で供給した後、実験水槽(64L)でランダムに配置した。実験魚を鉢した後、全ての実験飼育水槽の飼育水を綺麗に換水し、0時間、24時間、48時間、72時間、92時間、120時間目に各々50mlずつ飼育水をサンプリングした。2次選抜菌株の投与の24時間の間隔で約3mlに1x105CFU菌数になるよう希釈した後、水槽に5日間投与した。対照区の場合は同量の海水を投与した。サンプルを採取してアンモニアの濃度を分析し、分析して残った海水サンプルは−20℃で保管した後、亜硝酸塩と硝酸塩の分析に用いた。水質分析は、アンモニア、亜硝酸塩、硝酸塩の3つの項目を分析した。これらの分析は水質分析器(RQflex 10、Merck、ドイツ)を利用し、キットの試薬と各々反応した後分析した。
(1)形態学的、生化学的特性の調査
最終選抜された菌株の種定のため、1次的に形態学的、生化学的調査を行った。形態的特徴において、グラム染色結果グラム陽性であり、電子顕微鏡撮影の結果桿菌であることを確認した(図6)。生化学的特性を分析するためAPI50 CHB system(Biomerieux、フランス)で菌株の糖発酵パターンを分析した(表2)。
正確な菌株同定をするため、DNA塩基配列による分子系統分類学的方法を行った。塩基配列分析はPCR premix(バイオニア、韓国)とユニバーサルプライマー27F(5’AGAGTTTGATCMTGGCTCAG3’:配列番号2)、及び1492R(5’GGYTACCTTGTTACGACTT 3’:配列番号3)を用いて、16s rDNAの遺伝子を増幅した。遺伝子増幅の際、総反応液は20μlに合わせて、94℃で1分、56℃で1分、72℃で1分を30回繰り返し、増幅されたDNA塩基配列を分析した。分離菌株の分析された16s rDNAの塩基配列は配列番号1に示す。前記分析結果、バチルス・リケニフォルミスと96%の相同性を有する微生物として同定された。よって、選抜された前記菌株をバチルス・リケニフォルミスCJMPB283に命名し、前述の方法により同定された本発明の新たな微生物は、2012年3月22日、ブタペスト条約上の国際寄託機関である韓国微生物保存センターに、"Bacillus licheniformis CJMPB283"として寄託された(寄託番号KCCM11270P)。
(1)分離菌株の消化酵素活性
1)助酵素液の採取
前記分離した菌株をBHI液体培地で、24時間培養した後、培養液を酵素活性の分析のための助酵素液にして、下記の通り各酵素別に各々の基質が入っている培地を用いて基質分解の程度を判定した。
脱脂粉乳(skim milk、Sigma、USA)を2%添加したYM(Difco、USA)培地を製造した。上記で採取した助酵素液2μlを基質培地に分株し、37℃で15時間反応した後、透明環(clear zone)形成の程度により酵素活性能力を測定した。
1%CMC(carboxyl methyl cellulose)基質が添加されたYM培地を製造した。上記で採取した助酵素液を2μlずつ基質培地に分株し、37℃で15時間反応した後、0.2%のコンゴーレッド(Congo red)水溶液で30分間染色し、1M NaCl水溶液で脱色した。助酵素液周囲の基質が分解されて生じる透明環(clear zone)の形成程度により酵素の活性能力を測定した。
1%の可溶性デンプン(soluble starch)基質が添加されたYM培地を製造した。上記で採取した助酵素液を3μlずつ基質培地に分株し、37℃で15時間反応した。I2とKIが各々0.1%、2%添加された水溶液で染色した後、助酵素液周囲の基質が分解されて生じる透明環(clear zone)の形成程度により酵素の活性能力を測定した。
バチルスは、必須栄養源の中で一つ以上の栄養源が枯渇するなどのストレスを受けると生存のため内生胞子を形成する。内生胞子は、紫外線、高温、低温、乾燥、及び高圧などの極限環境に抵抗性を有するため、内生胞子の形成はバチルスの生存率を維持することにおいて非常に重要である。よって、バチルス・リケニフォルミスCJMPB283を長時間培養し、内生胞子の形成能を確認した。
Claims (6)
- 消化酵素としてプロテアーゼ、セルラーゼ、及びアミラーゼ全てを生産し、アンモニアと亜硝酸の酸化能力を有する、バチルス・リケニフォルミスKCCM11270P(Bacillus licheniformis KCCM11270P)。
- 請求項1に記載のバチルス・リケニフォルミスKCCM11270Pの培養液、その濃縮液、及びその乾燥物からなる群より選択された少なくとも一つの、バチルス・リケニフォルミスKCCM11270Pの培養物。
- 請求項1に記載のバチルス・リケニフォルミスKCCM11270P、又は請求項2に記載の培養物を含む、プロバイオティクス製剤。
- 請求項3に記載のプロバイオティクス製剤を含む、水産養殖用の飼料添加剤。
- 請求項1に記載のバチルス・リケニフォルミスKCCM11270P(bacillus licheniformis KCCM11270P)、その培養液、その濃縮液、又はその乾燥物を含む、水産養殖用の水質改善剤。
- 養殖場において養殖前、又は養殖段階中に、請求項5に記載の水質改善剤を撒布する、前記養殖場の水質改善方法。
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PCT/KR2013/002829 WO2013151364A1 (ko) | 2012-04-05 | 2013-04-04 | 신규 분리한 바실러스 리체니포미스 및 이를 이용한 프로바이오틱스 |
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