JP6663977B2 - バチルスサブチリスとバチルスリケニフォルミスを含む飼料添加剤、前記添加剤を含む飼料組成物及び前記飼料添加剤の製造方法 - Google Patents
バチルスサブチリスとバチルスリケニフォルミスを含む飼料添加剤、前記添加剤を含む飼料組成物及び前記飼料添加剤の製造方法 Download PDFInfo
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- JP6663977B2 JP6663977B2 JP2018501973A JP2018501973A JP6663977B2 JP 6663977 B2 JP6663977 B2 JP 6663977B2 JP 2018501973 A JP2018501973 A JP 2018501973A JP 2018501973 A JP2018501973 A JP 2018501973A JP 6663977 B2 JP6663977 B2 JP 6663977B2
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- feed additive
- bacillus licheniformis
- lactic acid
- bacillus
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Classifications
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- 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
- 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
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/10—Feeding-stuffs specially adapted for particular animals for ruminants
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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
- C12N1/205—Bacterial isolates
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- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
- C12R2001/10—Bacillus licheniformis
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- 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
- C12R2001/125—Bacillus subtilis ; Hay bacillus; Grass bacillus
-
- 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
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S426/00—Food or edible material: processes, compositions, and products
- Y10S426/807—Poultry or ruminant feed
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- General Health & Medical Sciences (AREA)
- Virology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Medicinal Chemistry (AREA)
- General Engineering & Computer Science (AREA)
- Fodder In General (AREA)
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Description
本出願の他の目的は、本明細書に記載のバチルスサブチリス及びバチルスリケニフォルミスを含む飼料添加剤が含まれた飼料組成物を提供することである。
本発明の一様態は、バチルスサブチリス及びバチルスリケニフォルミスを含む、乳脂肪及び乳量増加用の飼料添加剤に関する。
初期乳酸対比酢酸転換率=(酢酸生成量/初期乳酸量)×100
乳酸消費量対比転換率(%)=(酢酸生成量/乳酸消費量)×100
伝統醤類である味噌、コチュジャンなどの試料を確保し、確保された試料を段階的に希釈してBHI(Brain Heart Infusion、Difco)固体培地に塗抹した後、37℃で24時間培養した。各試料から分離された菌株を新しい培地に移して培養する方法で純粋分離し、純粋分離して培養された菌を総重量対比20重量%グリセロールが添加された培地に入れて−70℃以下で保存した。このうち、下記方法でセルラーゼの活性が優秀な菌株と、乳酸を分解して酢酸を生産する能力が優秀である菌株を分離した。
前記分離された菌株を同定するために、一次的に形態学的、生化学的調査を行った。形態的な特徴はグラム染色の結果、分離された菌株はすべてグラム陽性であり、電子顕微鏡の撮影結果で桿菌であることを確認した(図1)。
菌株をより正確に同定するため、DNA塩基配列による分子系統分類学的方法を実施した。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′AGAGTTTGATCMTGGCTCAG 3′)と1492R(5′GGTTACCTTGTTACGACTT 3′)を用いて16s rDNAの遺伝子増幅を行った。遺伝子増幅する際の全反応液は20μlに合わせて、94℃で1分、56℃で1分、72℃で1分を合計30回繰り返して、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1〜3に示す。前記分析の結果、CJBL215とCJBL219はバチルスリケニフォルミス、CJBS62はバチルスサブチリスと99%の相同性を示す微生物として同定された。前記分離された菌株を各々「バチルスリケニフォルミスCJBL215」、「バチルスリケニフォルミスCJBL219」、「バチルスサブチリスCJBS62」で命名した。前記方法で同定された本発明の新たな微生物は、韓国微生物保存センター(KCCM)に2017年6月22日付でバチルスリケニフォルミスCJBL215、バチルスリケニフォルミスCJBL219、バチルスサブチリスCJBS62を、各々KCCM12040P、KCCM12041P、KCCM12039Pの寄託名で寄託した。
複合消化酵素活性を有する菌株を選別するために、分離された醤類由来菌に対してマンナーゼ(mannase)、セルラーゼの二つの酵素の活性を評価した。酵素活性の評価は、各酵素の基質が含まれた培地を使用して、透明環(clear zone)の形成程度で酵素活性能力を測定した。
1%マンナン(logust bean gum、sigma、USA)が添加された基質培地(Yeast extract 3g/L、Peptone 5g/L、KH2PO4 1g/L、Agar 20g/L、Ph5)を製造した。補酵素液を1.5μlずつ基質培地に点滴した後、37℃で15〜18時間反応した後、透明環を形成程度に応じて酵素活性能力を測定し、その結果を表2に示した。
1%CMC(carboxymethylcellulose)基質が添加されたYM培地を製造した。補酵素液を1.5μlずつ基質培地に点滴して、37℃で15〜18時間反応させた。透明環を測定するために0.2%コンゴレッド(Congo red)溶液で30分間染色し、1M NaCl水溶液で脱色した。菌株周囲の基質が分解されて生じる透明環の形成程度に応じて酵素の活性能力を測定し、その結果を表2に示した。
前記評価に基づいて、選抜菌株の中でマンナーゼ活性が優秀であり、かつ、セルラーゼ活性が最も優秀であったバチルスサブチリスCJBS16、CJBS62菌株を選抜した。
選抜された菌株の培養上澄み液におけるセルロース分解能を確認するために、菌株を24時間培養し、10,000rpmで5分間遠心分離した上澄み液を、0.2μmシリンジフィルターを用いてフィルタリングした。この上澄み液をセルロース(Cellulose)が含有された培地(Carboxymethylcellulose sodium salt10g/L、Bacto agar15g/L)に20μl投入して、37℃で1日間反応した後、0.4%コンゴレッド溶液を入れて20分間染色した。染色後、1M NaCl溶液で脱色して透明環の大きさを測定し、培養上澄み液内のセルロース分解能を確認し、その結果を表3に示した。選抜されたバチルスサブチリスCJBS62菌株が培養上澄み液の評価においても最も優秀であったことを確認した。
前記選抜されたCJBS16、CJBL215、CJBL219及びCJBS62をBHI(Brain Heart Infusion、Difco)培地に接種し、37℃と200rpmで16時間培養して菌を活性化させた。15mMの乳酸を含有した培地(15mM乳酸、Yeast extract 10g/L、Peptone 20g/L、NaCl 10g/L)に前記菌を5%接種し、37℃と200rpmで48時間培養した。
初期乳酸対比酢酸転換率=(酢酸生成量/初期乳酸量)×100
選抜菌株の培地内乳酸消費量と酢酸生成量を定量した。
前記培養液を遠心分離して得られた上澄み液を0.2μmィルターでフィルタリングして準備した。フィルタリングされた培養上澄み液をマイクロセントリフージチューブ(microcentrifuge tube)に入れて、Cedex Bio(ROCHE)の乳酸bio(ROCHE)装置を用いて乳酸の含量を測定し、その結果を表5に示した。
前記培養液を遠心分離して得られた上澄み液1mlに25%メタリン酸(metaphosphoric acid)0.2mlを添加した。10,000rpmで5分間遠心分離して得られた上澄み液を0.2μmフィルターでフィルタリングした後、GC(Agilent Technologies 7890A)を用いて酢酸の含量を分析し、その結果を表5、図4及び図5に示した。
消費された乳酸対比酢酸転換率=(酢酸生成量/乳酸消費量)×100
β−溶血は、有害菌のうちリン脂質酵素を生産し、赤血球によって供給されるリン脂質を加水分解して赤血球を溶血させる作用である。分離菌株の溶血性を調べるために、血液寒天平板培地(sheep blood 5%、(株)ハニルコメッド、韓国)を用いた。用意した血液寒天平板培地に画線(streaking)した後、37℃で24時間培養して溶血有無を確認した結果、図5に示すように溶血性を示さなかったことを確認した。
選抜菌株における抗生剤の感受性を確認するための実験を行った。選抜菌株をBHI(Brain Heart Infusion、Difco)培地に接種して、37℃と200rpmで16時間培養した。培養された菌を滅菌した綿棒でつけて、Mueller HintonIIAgar plate(Difco)に塗抹する。菌が塗抹された平板培地に抗生剤ディスクを載せて、37℃で15〜18時間培養する。抗生剤検査のために、Ampicillin、Clindamycin、Gentamicin、Kanamycin、Tetracyclin、Vancomycin、Erythromycin、Ampcillin/Sulbactam、Chloramphenicol、Streptomycinのディスク(OXOID)を準備して実験を行った。培養後、抗生剤ディスク周囲の生長阻害領域(clear zone)の形成程度によって、抗生剤の感受性を確認した。選抜菌株における抗生剤の感受性を検査した結果、選抜菌株は前記抗生剤に耐性を有しないことを確認した(表6)。
乾物消化率(%)=(培養前の基質量−培養後の基質量)/培養前の基質量×100
通常的に搾乳牛が高温ストレス環境にさらされた場合には、一般的に搾乳牛から生産される乳量と乳脂肪が減少する。しかし、本発明の飼料添加剤を提供した搾乳牛では、高温ストレス環境にさらされた時にも乳脂肪及び乳量が減少されず、むしろ乳脂肪及び乳量が改善され得る。
Claims (6)
- バチルスサブチリス及びバチルスリケニフォルミスを含む、乳脂肪及び乳量増加用の飼料添加剤であって、前記バチルスサブチリスは、バチルスサブチリスCJBS62(Bacillus subtilis CJBS62(KCCM12039P))を含むことを特徴とする、乳脂肪及び乳量増加用の飼料添加剤。
- バチルスサブチリス及びバチルスリケニフォルミスを含む、乳脂肪及び乳量増加用の飼料添加剤であって、前記バチルスリケニフォルミスは、バチルスリケニフォルミスCJBL215(Bacillus licheniformis CJBL215(KCCM12040P))及びバチルスリケニフォルミスCJBL219(Bacillus licheniformis CJBL219(KCCM12041P))からなる群より選択された一つ以上を含むことを特徴とする、乳脂肪及び乳量増加用の飼料添加剤。
- 前記飼料添加剤1g当たり、前記バチルスサブチリス及びバチルスリケニフォルミスを各々1×107cfu以上含むことを特徴とする、請求項1又は2に記載の乳脂肪及び乳量増加用の飼料添加剤。
- 前記バチルスリケニフォルミスは、乳酸含有培地で9時間〜48時間培養され酢酸を生成し、前記バチルスリケニフォルミスの初期乳酸量対比酢酸転換率は30%〜70%であることを特徴とする、請求項1又は2に記載の乳脂肪及び乳量増加用の飼料添加剤。
- 前記バチルスサブチリス及び前記バチルスリケニフォルミスの配合重量比が1:9〜9:1であることを特徴とする、請求項1又は2に記載の乳脂肪及び乳量増加用の飼料添加剤。
- 請求項1ないし5のいずれか一つに記載の飼料添加剤を含む、飼料組成物。
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Application Number | Priority Date | Filing Date | Title |
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KR1020170088646A KR101934400B1 (ko) | 2017-07-12 | 2017-07-12 | 바실러스 서브틸리스 및 바실러스 리체니포미스를 포함하는 사료 첨가제, 상기 첨가제를 포함하는 사료 조성물 및 상기 사료첨가제의 제조 방법 |
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PCT/KR2017/008446 WO2019013382A1 (ko) | 2017-07-12 | 2017-08-04 | 바실러스 서브틸리스 및 바실러스 리체니포미스를 포함하는 사료 첨가제, 상기 첨가제를 포함하는 사료 조성물 및 상기 사료첨가제의 제조 방법 |
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RU2018128578A (ru) | 2016-01-07 | 2020-02-10 | Аскус Байосайенсиз, Инк. | Способы улучшения продукции молока путем введения микробных консорциумов |
CN110913702A (zh) | 2017-04-28 | 2020-03-24 | 埃斯库斯生物科技股份公司 | 利用微生物的人造生物集合在反刍动物中用于支持谷物加强剂和/或能量加强剂饮食的方法 |
KR102299266B1 (ko) * | 2019-03-07 | 2021-09-08 | 씨제이제일제당 주식회사 | 바실러스 서브틸리스 cjbs303 및 이를 포함하는 조성물 |
EP4326087A1 (en) * | 2021-04-22 | 2024-02-28 | Native Microbials, Inc. | Compositions and methods for improving milk yield and milk compositional characteristics in ruminants |
CN115251246B (zh) * | 2021-04-30 | 2024-06-11 | 中国农业大学 | 一种降低猪肉滴水损失的饲料添加剂 |
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MXPA06001722A (es) | 2003-08-14 | 2006-05-19 | Bio Balance Corp | Cepas de bacterias, composiciones que las incluyen y el uso de probioticos de las mismas. |
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JP5459980B2 (ja) * | 2008-05-21 | 2014-04-02 | 土田 誠 | 健康食品並びに飼料並びに肥料の製造方法及びその製造用の複合発酵菌体物 |
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RU2437563C1 (ru) | 2010-06-09 | 2011-12-27 | Ольга Николаевна Дегтярёва | Пробиотическая кормовая добавка |
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CN102640855A (zh) | 2012-04-27 | 2012-08-22 | 杨洪星 | 一种微生态类饲料添加剂 |
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US20190014795A1 (en) | 2019-01-17 |
JP2019524049A (ja) | 2019-09-05 |
BR112020000540A2 (pt) | 2020-07-21 |
US20200275680A1 (en) | 2020-09-03 |
BR112020000540B1 (pt) | 2023-04-11 |
CA3069404A1 (en) | 2019-01-17 |
KR101934400B1 (ko) | 2019-03-25 |
WO2019013382A1 (ko) | 2019-01-17 |
CA3069404C (en) | 2022-03-15 |
US11638432B2 (en) | 2023-05-02 |
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