JP6800950B2 - 血栓溶解酵素の高生産のバチルス・サブチリス菌株 - Google Patents
血栓溶解酵素の高生産のバチルス・サブチリス菌株 Download PDFInfo
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- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 1
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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
- 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
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- 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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- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
- C12N9/54—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea bacteria being Bacillus
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/21—Serine endopeptidases (3.4.21)
- C12Y304/21062—Subtilisin (3.4.21.62)
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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
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- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Beans For Foods Or Fodder (AREA)
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Description
GTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCAGCGAAACCGCGAGGTTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTTAGGAGCCAGCCGCCGAAGGTGGGACAGATGATTGGGGTGAAGT
GTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCAGCGAAACCGCGAGGTTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTTAGGAGCCAGCCGCCGAAGGTGGGACAGATGATTGGGGTGAAGT
表1に示すように、分離した菌株は、グラム陽性であり、細胞のサイズは、ほぼ0.6〜0.7×1.4〜1.6μmであることを確認することができ、前記結果を総合して、バチルス・サブチリスGDN菌株の分類学上の位置を図2に示した。
希釈した試料100μlと0.1M Tris−HCl緩衝液200μl(10mM CaC12含む、pH7.8)、0.4%フィブリン溶液(pH7.0)200μlとを混合した後、37℃で30分間反応させた。反応終了後、反応停止液(0.22M酢酸ナトリウム、0.33M酢酸含有0.11Mトリクロロ酢酸)500μlを添加し、遠心分離して上澄み液を収得した。該収得した上澄み液に275nm波長の光を照射して吸光度を測定した(但し、血栓溶解酵素の活性1unitは、37℃で反応して1分間作り出す1μgのチロシン(tyrosine)の量と同じである)。
洗浄した大豆を室温で一晩浸漬した後、121℃で40分間高圧蒸気滅菌(autoclave)して、煮た大豆を作った。煮た大豆50g当たりバチルス・サブチリスGDN菌株胞子液(105胞子/ml)1mlの比率で煮た大豆が冷める前に接種して、発泡スチレン容器に入れ、被膜を行って、37℃で18時間培養した。その後、4℃で24時間熟成して、大豆発酵物を製造した。対照群としては、市販の宮城野菌を使用した。
表3に示すように、大豆発酵物で血栓溶解酵素の活性の測定結果、実施例のGDN菌で製造した大豆発酵物の血栓溶解酵素の活性は、対照群である宮城野菌に比べて、約5倍の酵素活性が増加したことを確認することができた。
前記の方法で得られた大豆発酵物について官能評価を行った。官能評価は、5段階評価法で行い、5点は非常に良好、4点は良好、3点は普通、2点は不良、1点は非常に不良として、下記の5項目について評価し、その結果を下記の表4に示した。
表4に示すように、官能評価を行った結果、GDN菌株で製造した大豆発酵物の官能評価は、3.6〜4.8点の高い点数が出て、納豆のような大豆発酵物で開発しても、立派な製品になりうるということを確認することができた。
当日製造された生おから(3種)をそのまま121℃で40分間高圧蒸気滅菌し、該滅菌したおから50g当たり105胞子/mlの胞子液を1mlの比率で冷める前に接種して、37℃で24時間培養した。
表3に示すように、互いに異なる3つの工場で製造したおからを発酵して生成されたおから発酵物の血栓溶解酵素の活性を測定した結果、おから発酵物が大豆発酵物よりも酵素生産量がほぼ1.5倍高いと確認された。これを通じて、産業廃棄物であるおからを用いて追加的な処理なしに固体培養を通じて効率的に血栓溶解酵素の生産に用いられうるということを確認することができた。
おからを30cm×24cm×6cmサイズのステンレストレー(stainless tray)に500gずつ入れ、121℃で40分間高圧蒸気滅菌した。該滅菌したおから1kg当たり105胞子/mlの胞子液20mlを冷める前に加えて接種し、ビニール被膜を行って、37℃で24時間培養して、おから発酵物を製造した。該製造したおから発酵物に3倍量の水を加え、よく撹拌して血栓溶解酵素を抽出した。これにCaCl2の最終濃度が0.1MになるようにCaCl2粉末を加え、30分間静置した後、遠心分離して沈殿を除去し、上澄み液であるおから抽出物を製造した。
表6に示すように、カルシウム(CaCl2)処理したおから抽出物の血清溶解酵素の活性が非常に高いと確認された。これを通じて、GDN菌株のおから発酵物に重量に比べて、3倍量の水を加えて均質化し、そこに最終濃度が0.1MになるようにCaCl2粉末を加え、30分間静置すれば、おからから由来した固形分が凝集して容易に沈殿で除去され、血栓溶解酵素は、清い上澄み液に残るので、簡単な操作によっても、血栓溶解酵素の精製が高収率でなされるという事実を確認することができた。
受託番号:KCTC13020BP
受託日:20160503
Claims (4)
- 配列番号1の16sRNAを含み、血栓溶解酵素を高生産するバチルス・サブチリスGDN菌株(Bacillus subtilis GDN、受託番号KCTC13020BP)を用いて発酵させたおから発酵物であって、3400〜4300fibrinolytic unit/gの血栓溶解酵素の活性を示すおから発酵物。
- 前記おから発酵物に含まれる前記血栓溶解酵素が納豆キナーゼである、請求項1に記載のおから発酵物。
- 請求項1に記載のおから発酵物を含む食品組成物。
- 請求項1に記載のおから発酵物を用いる段階を含む、血栓溶解酵素の製造方法。
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
KR10-2016-0079632 | 2016-06-24 | ||
KR1020160079632A KR101809509B1 (ko) | 2016-06-24 | 2016-06-24 | 혈전용해효소 고생산 바실러스 서브틸리스 균주 |
PCT/KR2017/001753 WO2017222141A1 (ko) | 2016-06-24 | 2017-02-17 | 혈전용해효소 고생산 바실러스 서브틸리스 균주 |
Publications (2)
Publication Number | Publication Date |
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JP2018525988A JP2018525988A (ja) | 2018-09-13 |
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