JP2020532293A - 新規バチルス・アミロリケファシエンス菌株、及びそれを用いた、大豆発酵物を製造する方法 - Google Patents
新規バチルス・アミロリケファシエンス菌株、及びそれを用いた、大豆発酵物を製造する方法 Download PDFInfo
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- fermented soybean
- bacillus amyloliquefaciens
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Abstract
Description
従来から大豆粕又は大豆蛋白濃縮物発酵に用いられることが知られているバチルス・アミロリケファシエンス(以下、「CJ823菌株」という,特許文献4参照)を準備し、次の方法で当該菌株の改良を行った。
実施例1で分離して得た各変異菌株のタンパク質分解能力及び抗菌力を次の方法で確認した。対照群としては、公知のバチルス・サブティリス(寄託番号KCCM11438P)及びバチルス・アミロリケファシエンス(CJ823菌株)を用いた(以下、それぞれ対照群1、対照群2という)。
実施例1で得た変異菌株のタンパク質分解能力を測定するために、2%(w/v)脱脂乳(Difco, USA)を含むYM寒天培地(yeast extract 3.0g,malt extract 3.0g,peptone 10.0g,agar 20.0g)に菌株を接種培養し、形成されるコロニーの直径(growth, G)を測定すると共に、基質が分解されて生成される透明環(clear zone, C)の大きさを測定した。
実施例1で得た変異菌株の抗菌力を測定するために、家畜に疾患を引き起こし得る代表的な病原菌であるサルモネラ・ティフィムリウム(Salmonella typhimurium, ATCC14028)を用いた。より具体的には、前記変異菌株をそれぞれGYP培地(培地の組成:glucose 10g,yeast extract 8g,polypeptone 2g,pH7.0)において、37℃の条件下、180rpmの攪拌速度で12時間培養した。各変異菌株の培養液をサルモネラ・ティフィムリウムが1×105CFU/mlで添加されたGYP寒天培地(培地の組成:glucose 10g,yeast extract 8g,polypeptone 2g,agar 15g,pH7.0)に1.5μLずつスポッティングし、37℃で15時間培養した。培養終了後、変異菌株のコロニーの周囲に形成される生育阻止円の大きさを測定し、抗菌力の活性力価を求めた。
実施例2で抗菌力に優れるものとして選択された菌株(CJ24−28、CJ24−29及びCJ24−34)のうち、透明環の大きさ(C)とコロニーの直径(G)が最も大きく、タンパク質分解能力に優れたCJ24−34菌株のサルモネラ以外の病原性微生物に対する抗菌力をさらに確認することにした。抗菌力の確認のために、ビブリオ・バルニフィカス(Vibrio vulnificus)、ビブリオ・パラヘモリティカス(Vibrio parahaemolyticus)、フォトバクテリウム・ダムセラエ(Photobacterium damsel)、リストネラ・アンギラルム(Listonella anguillarum)、ラクトコッカス・ガルビエ(Lactococcus garviaeae)及びエドワジエラ・タルダ(Edwardsiella tarda)の病原性微生物を用いた。これらは、敗血症、ビブリオコレラ、エビビブリオ症、ブリ類結節症、ヒラメ連鎖球菌症、ヒラメエドワジエラ症などを含む疾患を養魚に引き起こす恐れがあることが知られている。前記病原性微生物のうち、ビブリオ・バルニフィカス(Vibrio vulnificus)、ビブリオ・パラヘモリティカス(Vibrio parahaemolyticus)、フォトバクテリウム・ダムセラエ(Photobacterium damsel)及びラクトコッカス・ガルビエ(Lactococcus garviaeae)は慶尚大学校病原体資源銀行から分譲された菌株であり、リストネラ・アンギラルム(Listonella anguillarum)及びエドワジエラ・タルダ(Edwardsiella tarda)はKCTCから分譲された菌株である。各病原性微生物を表2に示す培地と温度で培養し、その後単一コロニーを選択してtest tubeに移し、当該液体培地でovernightして前培養した。前培養液を再び各病原菌の当該液体培地100mLに0.1%接種して約12時間本培養した。本培養液は、抗菌力テストのために各病原菌の寒天培地を準備する際に用いた。
実施例2で優れたタンパク質分解能力及び抗菌力が確認されて選択された変異菌株CJ24−28、CJ24−29及びCJ24−34の粘液質生成能を確認するために、前記菌株をGYP寒天培地で再び培養し、各コロニーの表現型を詳細に観察した。対照群として、実施例2の対照群2(バチルス・アミロリケファシエンス,CJ823菌株)を用いた。
実施例2及び実施例3で選択された変異菌株のうちタンパク質分解能力及び抗菌力に優れることが確認されたCJ24−34菌株を大豆蛋白濃縮物に接種して発酵させた場合の菌株の粘液質生成の有無と発酵能力を確認することにした。対照群として、実施例2の対照群1(バチルス・サブティリス:寄託番号KCCM11438P)及び対照群2(バチルス・アミロリケファシエンス:CJ823)を用いた。
本実施例においては、発酵物に含まれるペプチドサイズ及び分布を確認するために、実施例5の発酵大豆蛋白濃縮物のタンパク質分解性及び分子量分布度を分析した。これらは、それぞれSDS−PAGE及びGPC法で測定した。
原料(大豆蛋白濃縮物)及び実施例5で準備した発酵大豆蛋白濃縮物(CJ24−34の発酵大豆蛋白濃縮物及び対照群1の発酵大豆蛋白濃縮物)それぞれ100mgを8Mの尿素溶媒5mLで懸濁(suspension)し、超音波処理(sonication)し、その後遠心分離(8000rpm,10分)して上清を分離した。前記上清をbicinchoninic acidで定量し、SDS−PAGEのゲルに載せた。
GPCとは、分子量が異なる標準タンパク質を分析してそれぞれの保持時間(retention time)を確認し、次に分子量と保持時間の関係の標準曲線を算出することにより、測定しようとする試料中のタンパク質分子量分布度を求める方法である。より具体的には、特定分子量を有するタンパク質の保持時間を計算し、その時間に応じてクロマトグラムの部分を分け、全クロマトグラム面積中の各分子量範囲における部分面積比を計算するものである。
本出願によるバチルス・アミロリケファシエンスCJ24−34菌株が大豆粕に適用された場合も発酵能に優れるかを確認するために、発酵大豆粕の生産菌株として従来から用いられていた2種のバチルス・アミロリケファシエンス(KCCM11471P,KCCM11906P)を対照群として発酵能を確認した。前記KCCM11471Pを対照群3とし、前記KCCM11906Pを対照群4とする。
本出願によるCJ24−34菌株をGYP培地(培地の組成:glucose 10g/,yeast extract 8g/L,soy pepton 2g/L)で前培養し、前培養により得られた培養液を再びGYP培地に1%接種し、A660nm=6以上になるまで培養した。
本出願のCJ24−34変異菌株の同定のために、表10の塩基配列を用いて菌株の16S rRNA遺伝子の塩基配列を分析した。
受託番号:KCCM12038P
受託日:2017年6月15日
(配列表)
(構成1)
バチルス・アミロリケファシエンスCJ24−34(KCCM12038P)菌株を大豆粕又は大豆蛋白濃縮物に接種するステップと、
前記バチルス・アミロリケファシエンス菌株を培養して発酵させた発酵大豆粕又は発酵大豆蛋白濃縮物を得るステップとを含む、大豆発酵物を製造する方法。
(構成2)
前記大豆発酵物は、サルモネラ・ティフィムリウム(Salmonella typhimurium)、ビブリオ・バルニフィカス(Vibrio vulnificus)、ビブリオ・パラヘモリティカス(Vibrio parahaemolyticus)、フォトバクテリウム・ダムセラエ(Photobacterium damsel)、リストネラ・アンギラルム(Listonella anguillarum)及びエドワジエラ・タルダ(Edwardsiella tarda)からなる群から選択される少なくとも1つの病原菌に対して抗菌力を有する、構成1に記載の大豆発酵物を製造する方法。
(構成3)
前記大豆発酵物は、分子量30KDa以下の低分子ペプチドを40重量%以上含む、構成1又は2に記載の大豆発酵物を製造する方法。
(構成4)
菌株を大豆粕又は大豆蛋白濃縮物に接種するステップの前に、
大豆粕又は大豆蛋白濃縮物の水分含有量を調節し、熱処理するステップをさらに含む、構成1〜3のいずれか一項に記載の大豆発酵物を製造する方法。
(構成5)
前記大豆粕又は発酵大豆蛋白濃縮物の水分含有量を30〜80%(v/w)に調節し、前記熱処理を70〜130℃で10〜30分間行うものである、構成4に記載の大豆発酵物を製造する方法。
(構成6)
前記バチルス・アミロリケファシエンス菌株CJ24−34(KCCM12038P)は、10 5 〜10 9 CFU/gの菌数で接種するものである、構成1〜5のいずれか一項に記載の大豆発酵物を製造する方法。
(構成7)
前記培養は、20〜50℃で8〜72時間行うものである、構成1〜6のいずれか一項に記載の大豆発酵物を製造する方法。
(構成8)
前記得られた発酵大豆粕又は発酵大豆蛋白濃縮物を乾燥及び粉砕するステップをさらに含む、構成1〜7のいずれか一項に記載の大豆発酵物を製造する方法。
(構成9)
バチルス・アミロリケファシエンスCJ24−34菌株(KCCM12038P)。
(構成10)
バチルス・アミロリケファシエンスCJ24−34菌株(KCCM12038P)、前記菌株の培養物、前記培養物の濃縮物、又は前記培養物の乾燥物を含み、
サルモネラ・ティフィムリウム(Salmonella typhimurium)、ビブリオ・バルニフィカス(Vibrio vulnificus)、ビブリオ・パラヘモリティカス(Vibrio parahaemolyticus)、フォトバクテリウム・ダムセラエ(Photobacterium damsel)、リストネラ・アンギラルム(Listonella anguillarum)及びエドワジエラ・タルダ(Edwardsiella tarda)からなる群から選択される少なくとも1つの病原菌に対して抗菌力を有する、抗菌性組成物。
(構成11)
構成1〜8のいずれか一項に記載の方法により製造された大豆発酵物。
(構成12)
構成11に記載の大豆発酵物を含む動物飼料組成物。
Claims (12)
- バチルス・アミロリケファシエンスCJ24−34(KCCM12038P)菌株を大豆粕又は大豆蛋白濃縮物に接種するステップと、
前記バチルス・アミロリケファシエンス菌株を培養して発酵させた発酵大豆粕又は発酵大豆蛋白濃縮物を得るステップとを含む、大豆発酵物を製造する方法。 - 前記大豆発酵物は、サルモネラ・ティフィムリウム(Salmonella typhimurium)、ビブリオ・バルニフィカス(Vibrio vulnificus)、ビブリオ・パラヘモリティカス(Vibrio parahaemolyticus)、フォトバクテリウム・ダムセラエ(Photobacterium damsel)、リストネラ・アンギラルム(Listonella anguillarum)及びエドワジエラ・タルダ(Edwardsiella tarda)からなる群から選択される少なくとも1つの病原菌に対して抗菌力を有する、請求項1に記載の大豆発酵物を製造する方法。
- 前記大豆発酵物は、分子量30KDa以下の低分子ペプチドを40重量%以上含む、請求項1又は2に記載の大豆発酵物を製造する方法。
- 菌株を大豆粕又は大豆蛋白濃縮物に接種するステップの前に、
大豆粕又は大豆蛋白濃縮物の水分含有量を調節し、熱処理するステップをさらに含む、請求項1〜3のいずれか一項に記載の大豆発酵物を製造する方法。 - 前記大豆粕又は発酵大豆蛋白濃縮物の水分含有量を30〜80%(v/w)に調節し、前記熱処理を70〜130℃で10〜30分間行うものである、請求項4に記載の大豆発酵物を製造する方法。
- 前記バチルス・アミロリケファシエンス菌株CJ24−34(KCCM12038P)は、105〜109CFU/gの菌数で接種するものである、請求項1〜5のいずれか一項に記載の大豆発酵物を製造する方法。
- 前記培養は、20〜50℃で8〜72時間行うものである、請求項1〜6のいずれか一項に記載の大豆発酵物を製造する方法。
- 前記得られた発酵大豆粕又は発酵大豆蛋白濃縮物を乾燥及び粉砕するステップをさらに含む、請求項1〜7のいずれか一項に記載の大豆発酵物を製造する方法。
- バチルス・アミロリケファシエンスCJ24−34菌株(KCCM12038P)。
- バチルス・アミロリケファシエンスCJ24−34菌株(KCCM12038P)、前記菌株の培養物、前記培養物の濃縮物、又は前記培養物の乾燥物を含み、
サルモネラ・ティフィムリウム(Salmonella typhimurium)、ビブリオ・バルニフィカス(Vibrio vulnificus)、ビブリオ・パラヘモリティカス(Vibrio parahaemolyticus)、フォトバクテリウム・ダムセラエ(Photobacterium damsel)、リストネラ・アンギラルム(Listonella anguillarum)及びエドワジエラ・タルダ(Edwardsiella tarda)からなる群から選択される少なくとも1つの病原菌に対して抗菌力を有する、抗菌性組成物。 - 請求項1〜8のいずれか一項に記載の方法により製造された大豆発酵物。
- 請求項11に記載の大豆発酵物を含む動物飼料組成物。
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PCT/KR2018/010074 WO2019045493A1 (en) | 2017-08-31 | 2018-08-30 | NOVEL BACILLUS AMYLOLIC FACTOR STRAIN AND PROCESS FOR PREPARING A FERMENTED SOY PRODUCT USING THE SAME |
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