JP3957132B2 - Separation medium for low turbidity soy sauce lactic acid bacteria, separation method for low turbidity soy sauce lactic acid bacteria using the same medium, and method for producing highly clear soy sauce using the same lactic acid bacteria - Google Patents

Description

【０００８】
【表２】

【０００９】
また本発明者は、醤油諸味から分離したさまざまな醤油乳酸菌株を用いて醤油醸造試験をおこなってみたところ、同じ麹を用いたにも関わらず、それぞれの諸味液汁の濁度はさまざまで、しかも醤油の濁度がその仕込み工程における乳酸醗酵の程度と密接に関係にあること(図２参照)を知った。
さらにまた、平成８年度及び９年度の全国各地で市販されていた濃口醤油９７品の乳酸濃度と濁度の関係を調べたところ、図３に示すように、醤油の濁度は０ｐｐｍから８０ｐｐｍくらいまでと、製品によってさまざまであったが、濁度の高い市販醤油にはその濁度の程度に相対して乳酸濃度が高いことを知った。
なお、図３の結果において、乳酸濃度が高い市販醤油が必ずしも濁度も高いとは言えないのは、仕込み工程以降の製成工程における各種清澄化剤の施用や濾過処理の適用によるものと思われる。
これらのことから、本発明者は、醤油乳酸菌株は低ｐＨ環境下における生存能が、その菌株ごとに大きく異なっており、この性質は特に主要な醗酵過程を終了してｐＨが低下した後熟過程（熟成過程）の諸味中において異なり、この菌株の性質が、結果的に醤油の濁度に影響を及ぼすことを知った。
【００１０】
仕込み工程においては、麹菌の生産したプロテアーゼなどの分解酵素群が諸味中の種々の成分の分解を進め、醤油の重要な呈味成分の生成に関与するが、このような乳酸醗酵の役割を終えて不要となった醤油乳酸菌の細胞の分解にも関与する。
しかしながら、この酵素による分解反応に際しては、醤油乳酸菌の細胞の生死が重要であり、生細胞は分解に対する耐性を示すのに対して、死細胞はすみやかに分解されるという、分解効率の差を示すことを、今回の研究を通じて知った。
【００１１】
また、本発明者は細胞表面の親水性度（または疎水性度）も菌株ごとで大きく異なっており、親水性度の低い（または疎水性度の高い）菌株の細胞は、恐らくは凝集しやすく、これらの分解酵素群の作用を受けにくいという性質を示すことを知った。
【００１２】
これらの知見より、低ｐＨ環境下での生存能が低く、かつ細胞表面の親水性度が高く（または疎水性度が低く）、かつ麹菌由来の分解酵素群により分解されやすい醤油乳酸菌株は、主たる醗酵を終えて後熟過程にはいった諸味中ではすみやかに死滅して、死細胞は酵素によってすみやかに分解されるため、その溶液画分である醤油に細胞の未分解かすが移行する割合が低く、その結果として醤油の濁度は低くなるはずであり、逆に低ｐＨ環境下での生存能が高く、細胞表面の親水性度が低く（または疎水性度が高く）、かつ麹菌由来の分解酵素群による分解を受けにくい醤油乳酸菌株の細胞は、主たる醗酵を終えて後熟過程にはいった諸味中でも生存し続け、酵素による分解を受けないために、未分解かすとして醤油中へ移行し、醤油の清澄度を低下させることが予想される。
さらに、この醤油乳酸菌株の細胞の分解を司る分解酵素群は、製麹工程において麹菌が生産するものであるが、麹菌によるこれらの分解酵素群の生産性が低下するような事態が発生すると、後熟工程における醤油乳酸菌株の細胞の分解性はより低下し、その結果として醤油の清澄度を著しく低下させ、場合によっては製品としての価値を著しく損なわせることもあり得る。
【００１３】
そこで、本発明では、低ｐＨ環境下での生存性の低さを確認するための簡便な「分離識別試験１」、麹菌由来の分解酵素群による分解の受けやすさを評価するための簡便な「分離識別試験２」、乳酸醗酵力の強さなどを間接的に評価するための簡便な「分離識別試験３」を考案し、これらを組み合わせることにより、乳酸醗酵能力は旺盛だが、低ｐＨ環境下での生存性が低く、細胞表面の親水性度が高く、麹菌由来の分解酵素による分解作用を受けやすいという性質を持つために、その菌株を用いて醤油を醸造すると、熟成過程において醤油乳酸菌株が早期に死滅し、麹菌由来の分解酵素の作用を受けて速やかに分解されるために、圧搾後の液汁画分への醤油乳酸菌の細胞の未分解かすの移行が少なく、結果的に清澄度の高い醤油をつくることができる、そのような醤油乳酸菌株（これを以下「低濁性醤油乳酸菌株」と呼ぶ）の分離識別法（図４）と、このような性質を持つ低濁性醤油乳酸菌株を用いた、清澄度の高い醤油の醸造法を提案する。
【００１４】
低ｐＨ環境下における細菌の生存性は、その細菌の細胞膜に存在し、細胞内外のプロトン（Ｈ+）濃度調節に関わる膜結合型ＡＴＰアーゼ（ｍｅｍｂｒａｎｅ−ｂｏｕｎｄ ＡＴＰ ａｓｅ）の活性と密接な関係にあり、しかも本酵素活性の強さは菌株によって大きく異なり、活性の弱い細菌株は低ｐＨ環境下における生存性が低く、逆に活性の強い細菌株は低ｐＨ環境下における生存性も高いことが既に知られており（Ｇ．Ｒ．Ｂｅｎｄｅｒ ｅｔ ａｌ．，Ｉｎｆｅｃｔ．Ｉｍｍｕｎ．，５３（２），３３１ （１９８６））、ネオマイシン（ｎｅｏｍｙｃｉｎ）などのアミノグリコシド系抗生物質はこの細胞内外のプロトン濃度の勾配（プロトン駆動力）に伴って細胞内に取り込まれた後に生育阻害作用を示すことから、種々の細菌株のこのような抗生物質の耐性能の強弱を調べることにより、その細菌が持つ膜結合型ＡＴＰアーゼの活性の強弱を評価することができる、すなわち高い耐性能を示す細菌株は膜結合型ＡＴＰアーゼ活性が弱く、低ｐＨ環境下における生存能が低く、逆に耐性能が弱い細菌株は膜結合型ＡＴＰアーゼ活性が強く、ゆえに低ｐＨ環境下における生存能は低いことが判っている（Ａ．Ｙｏｋｏｔａ ｅｔ ａｌ．，Ｂｉｏｓｃｉ．Ｂｉｏｔｅｃｈ．Ｂｉｏｃｈｅｍ．，５９（１０），２００４ （１９９５））。
【００１５】
本発明の「分離識別試験１」は、この知見を応用した、醤油乳酸菌株のネオマイシン耐性能を調べることを介して、その低ｐＨ環境下における生存性を簡便に評価する方法であり、上記の醤油乳酸菌株のネオマイシン耐性能を調べてみたところ、耐性能は菌株ごとにさまざまであり、しか小規模の醤油醸造試験において醤油の濁度が高い傾向を示した醤油乳酸菌株は概してネオマイシン耐性能が低く、逆に醤油の濁度が低い傾向を示した醤油乳酸菌株は概してネオマイシン耐性能が高いという傾向を示し、本法の有効性が確認された（表２）。
【００１６】
本法を用いて分離した、０．１２％（Ｗ／Ｖ）ネオマイシン耐性を示す乳酸菌株Ｓ−１６株と０．４５％（Ｗ／Ｖ）ネオマイシン感受性を示す乳酸菌株Ｓ−２株との、１％（Ｗ／Ｖ）グルコース、１％（Ｗ／Ｖ）ポリペプトン［日本製薬製］、０．４％（Ｗ／Ｖ）酵母エキス［ＤＩＦＣＯ製］、０．３％（Ｗ／Ｖ）燐酸二水素カリウム、０．２％（Ｗ／Ｖ）燐酸一水素二カリウム、０．１％（Ｗ／Ｖ）チオグリコール酸ナトリウム、３．３％（Ｗ／Ｖ）酢酸ナトリウム、１０％（Ｗ／Ｖ）塩化ナトリウムから構成される培地を用いたさまざまなｐＨ環境下における生存性を比較してみたところ、ｐＨ＝７．０条件下での増殖能には顕著な差異は認められず、またｐＨ＝４．０条件下においては共に速やかに死滅するという現象が観察されたが、ｐＨ＝５．０条件下においては、Ｓ−１６株が速やかに死滅してゆくのに対して、Ｓ−２株の死滅速度はＳ−１６株に較べて明らかに緩やかで、低ｐＨ環境下における生存能の高さを示した（表３）。
【００１７】
【表３】

【００１８】
ちなみに、醤油乳酸菌株は、菌株ごとによってＬ−アルギニンをアルギニンデイミナーゼ経路（Ａｒｇｉｎｉｎｅ ｄｅｉｍｉｎａｓｅ（ＥＣ．３．５．３．６．） ｐａｔｈｗａｙ）を通して、シトルリン（Ｃｉｔｒｕｌｌｉｎｅ）を経て Ｌ−オルニチン（Ｌ−Ｏｒｎｉｔｈｉｎｅ） へと変換する機能の有無、すなわちアルギニン分解能の有無、アスパラギン酸デカルボキシラーゼ（Ａｓｐａｒｔａｔｅ ｄｅｃａｒｂｏｘｙｌａｓｅ（ＥＣ．４．１．１．１２））作用によってアスパラギン酸をアラニンへと変換する機能の有無、すなわちアスパラギン酸分解能の有無、チロシンデカルボキシラーゼ（Ｔｙｒｏｓｉｎｅ ｄｅｃａｒｂｏｘｙｌａｓｅ）（ＥＣ．４．１．１．２５）作用によってチロシンをチラミン（Ｔｙｒａｍｉｎｅ）へと変換する機能の有無、すなわちチロシン分解性の有無、ヒスチジンデカルボキシラーゼ（Ｈｉｓｔｉｄｉｎｅ ｄｅｃａｒｂｏｘｙｌａｓｅ）（ＥＣ．４．１．１．２２）作用によってヒスチジンをヒスタミン（Ｈｉｓｔａｍｉｎｅ）へと変換する機能の有無、すなわちヒスチジン分解性の有無などが異なり、結果的にアミノ酸分解能の多様性を有することが知られており（内田金治ら、醸協誌、７７、７４０（１９８２））、中でもチロシンやヒスチジンの分解性を有する菌株が醤油中で増殖すると、チラミンやヒスタミンなどの、いわゆる「生体アミン」を生成したり、またアルギニン分解能を有する菌株が醤油諸味中で増殖し、しかも醤油乳酸菌ファージに感染した場合には、醤油乳酸菌細胞の溶菌現象に伴ってその代謝中間体であるシトルリンが放出され、これが火入れ工程においてエタノールと化学反応を起こして、カルバミン酸エチルを生成させてしまう事が内田らによって報告されている（飯塚ら、調味科学、２０(５)、１７(１９７３)（内田金治ら、平成４年度日本農芸化学会大会要旨集、ｐ３３６ （１９９２））。
【００１９】
このような種々のアミノ酸の分解性の有無に従った醤油乳酸菌株の群分類と、これらの菌株を用いて醸造した生醤油の濁度との関係を調べたところ、アスパラギン酸分解性を有する菌株やアルギニン分解性を有する菌株に比べて、これらのアミノ酸分解性を持たない菌株を用いて醸造した生醤油の濁度は、低い傾向を示すことを見出した（図５参照）。
【００２０】
乳酸菌におけるアスパラギン酸の取込みとアスパラギン酸デカルボキシラーゼによるアラニンへの変換に関しては、Ｌａｃｔｏｂａｃｉｌｌｕｓ属細菌Ｍ３株を用いたＫ．Ａｂｅらの報告（Ｊ．Ｂｉｏｌ．Ｃｈｅｍ．，２７１（６），３０７９〜３０８４（１９９６））があるが、この報告によると、細胞外のアスパラギン酸は、まず最初は、膜貫通型蛋白質ＡｓｐＴを介して、細胞内ＯＨ^１−の排出を伴いながら細胞内へと取込まれ、アスパラギン酸デカルボキシラーゼ作用により、アスパラギン酸１分子に対して、細胞内のプロトン（Ｈ^＋）１分子を消費することを介してアラニンへと変換されるが、その後このアスパラギン酸の取込み様式は代謝産物であるアラニンの細胞外への排出を伴う「アスパラギン酸／アラニンアンチポーター」方式へと変化し、この取込みの際に生ずる電位差を利用してＡＤＰからＡＴＰを生産する。
すなわち、アスパラギン酸分解性菌株のアスパラギン酸デカルボシキラーゼ反応は、細胞内外に電位差を形成させる形で基質(アスパラギン酸)を取込み、しかも細胞内のプロトン（Ｈ^＋）を消費する形で働くため、プロトン濃度勾配の形成を助長するものと見られ、このことは低ｐＨ環境下での細胞の生存性を高めることを意味する。
さらには、本酵素が酸性アミノであるアスパラギン酸を中性アミノ酸であるアラニンに変換する作用を触媒することから、アミノ酸濃度の高い醤油諸味では相当のｐＨ上昇をももたらすことから、このようなアスパラギン酸分解性醤油乳酸菌株が醤油諸味中で乳酸発酵に寄与した場合、同時にアスパラギン酸の分解反応を引き起こす結果、乳酸発酵に伴う醤油諸味のｐＨ低下が起こりにくく、しかも後熟過程におけるｐＨが下がった醤油諸味中でもより長期に活動し、生存することができるようになり、このことが結果的に生醤油の濁度を高めさせることとなる。
このことは、チロシンやヒスチジンの分解性に関与するチロシンデカルボキシラーゼや、ヒスチジンデカルボキシラーゼを有する菌株についても同様である。また、アルギニンの分解性に関わるアルギニンデイミナーゼ経路に関しては、アンモニアを生成する反応であるために、醤油諸味中での乳酸発酵に伴う細胞外ｐＨの低下を抑制する結果、諸味中で他の醤油乳酸菌株に比べてより長く生存し続けることができることが予想される。
これらの事実から、プロトン濃度勾配の形成に関与するアスパラギン酸分解性やチロシン分解性、ヒスチジン分解性を有さず、かつアルギニン分解性も持たない、すなわちアミノ酸分解性を持たない醤油乳酸菌株は、後熟過程における醤油諸味のような低ｐＨ環境下での生存性が弱く、アミノ酸分解による乳酸発酵に伴うｐＨ低下の中和も起きないために、速やかに死滅する結果として、生醤油を濁らせにくいものと考えられる。
【００２１】
更に、このような醤油乳酸菌株のアスパラギン酸分解性を司るアスパラギン酸デカルボキシラーゼの構造遺伝子は、プラスミドとして核外に位置し、３μｇ／ｍｌ臭化エチジウム及び５％（Ｗ／Ｖ）塩化ナトリウムを加えたＭＲＳ培地［ＤＩＦＣＯ社製］に接種し、３０℃で４日間培養するか、あるいは臭化エチジウムを用いずとも、０〜５％（Ｗ／Ｖ）程度の、塩化ナトリウムしか含まない、いわゆる低塩培地中で同条件にて培養するだけで、このプラスミドを脱落したキュアリング株を容易に、かつ高頻度に取得することができることが既に報告されていること（Ｔ．Ｈｉｇｕｃｈｉ ｅｔ ａｌ．，Ｂｉｏｓｃｉ．Ｂｉｏｔｅｃｈｎｏｌ．Ｂｉｏｃｈｅｍ．，６２（８），１６０１〜１６０３（１９９８））から、醤油諸味から分離した、アスパラギン酸分解性のみを有し、チロシン及びヒスチジン、アルギニン分解性は持たない醤油乳酸菌株を一度低塩培地に接種するという処理を施してプラスミドを脱落させ、アスパラギン酸分解性を欠落させることにより、その醤油乳酸菌株の他の醸造特性には影響を与えることなく、その菌株を用いて醸造した際の醤油の濁度のみを低減化させることも可能である。
ちなみに、本発明において示した、低ｐＨ環境下における菌株の生存性を評価するための寒天培地はナトリウム濃度が低く、ここでいうところの低塩培地に相当するため、本寒天培地で１．２ｍｇ／ｍｌネオマイシン耐性を観察した後、培地上に形成された耐性菌株のコロニーを釣り上げれば、接種前にはアスパラギン酸分解性を有していた菌株であったとしても、アスパラギン酸分解性を欠落した菌株を高頻度に取得することが可能であるということも確認された。
【００２２】
さて醤油諸味から分離した、さまざまなアミノ酸分解性を有する醤油乳酸菌株７６株について、ネオマイシン耐性能を調べてみたところ、アルギニン分解能を有する菌株は分解能を持たない菌株に較べてネオマイシンにより感受的であり、アスパラギン酸分解能を有する菌株は分解能を持たない菌株に較べてネオマイシンにより耐性である事から、本法で規定した０．１２％（Ｗ／Ｖ）濃度のネオマイシンに耐性を示す菌株は、そのほとんどがアルギニン分解能を持たない菌株となる事も判明した（図５参照）。
すなわち、本法を用いて分離される醤油乳酸菌菌株には、アルギニン分解能を有さず、ゆえにシトルリンの生成が起こらない優良菌株が多い事も確認された。
【００２３】
醤油乳酸菌のアミノ酸分解性とそれらの菌株を用いて醸造した生醤油の濁度を測定した。結果を図６に示す。
【００２４】
また、乳酸醗酵の役割を果たし終えた後の醤油乳酸菌の細胞は、諸味中に含まれる、麹菌由来の各種分解酵素群の作用により分解されるものと思われるが、麹と０．５％（Ｗ／Ｖ）安息香酸ナトリウムを含む０．１Ｍ燐酸カリウム緩衝液を混合した後に濾紙濾過する事によって調製した、麹菌由来の各種分解酵素群を含む「麹粗抽出液」による各種醤油乳酸菌株の細胞の分解性を調べてみたところ、その分解性も菌株によってさまざまに異なり、すなわち分解しやすい醤油乳酸菌株と分解しにくい醤油乳酸菌株とが存在するが、分解しやすい醤油乳酸菌株を用いて醸造した醤油の濁度は低く、分解しにくい醤油乳酸菌株を用いて醸造した醤油の濁度は高くなる事が確認された。すなわち、すなわち、０．５％（Ｗ／Ｖ）安息香酸ナトリウムを含む０．１Ｍ燐酸カリウム緩衝液（ｐＨ７．０）と醤油麹とを均一に混合した後、濾過して得られた無菌の醤油麹粗抽出液に、被検菌を懸濁して、６００ｎｍにおける吸光度を０．５に調整した後、３０℃で７日間、１００ｒｐｍの条件で振盪しながら放置し、再び６００ｎｍにおける吸光度(ハ)を測定して、（０．５- ハ )×１００／ ０．５を算出し、その値が１５以上、特に２５以上の菌株が、清澄な生醤油を得るのに適していることが判る（表４参照）。
【００２５】
【表４】

【００２６】
更に、本試験に供する菌株を８〜１８％（Ｗ／Ｖ）塩化ナトリウムを含む液体培地にて前培養（静置培養）した際に、培地中での増殖に伴う細胞沈降性が高く、培養液の上部が澄むという性質を示す菌株（たとえばＳ−２株）と、これとは逆に沈降性が低いために、培養液の上部にまで細胞が懸濁した状態となる菌株（たとえばＳ−１６株、Ｎ−４株）とが存在し（図１０参照）、前者、すなわち細胞沈降性の高い菌株を用いて醸造した醤油の濁度は高く、これに対して後者、すなわち細胞沈降性の低い菌株を用いて醸造した醤油の濁度は概して低い事も確認された。
すなわち、長さ約１６０ｍｍ、内径約１５ｍｍの試験管に、１０％(Ｗ／Ｖ)塩化ナトリウムを含む液体培地（例えば、下記Ａの培地組成参照）１０ｍｌを入れ、これに被検菌を接種し、３０℃で４８時間静置培養し、上層部４〜６ｍｌを液面を揺らさぬようにして静かに採取し、均一に攪拌後６００ｎｍにおける吸光度(イ)を測定し、また、採取した上層部全量を元に戻して培養液全体を均一に攪拌後６００ｎｍにおける吸光度(ロ)を測定し、（イ×１００）／ロを算出し、その値が３０以上である菌株(例えばＳ−１６株は４３、Ｎ−４株は６５の値を示す)が、清澄な生醤油を得るのに適していることが判明した（表５参照）(図１０のＮ−４株が濁濁状態にある)。
Ａ．培地組成
１％(％は断りない限り、いずれもＷ／Ｖ)グルコース、０．４％酵母エキス、１％ポリペプトン、０．３％燐酸二水素カリウム、０．２％燐酸一水素カリウム、３．０％酢酸ナトリウム、０．１％チオグリコール酸ナトリウム、１０％塩化ナトリウム、ｐＨ７．２。
【００２７】
【表５】

【００２８】
上記値が３０未満である菌株(Ｓ−２株は３の値を示す)(図１０のＳ−２株が透明状態にある)（細胞沈降性の高い菌株）は細胞表面が疎水的で細胞どうしが凝集しやすいために、麹菌由来の各種分解酵素の作用を受けにくく、また凝集した場合には、酵素作用を受ける細胞当たりの表面積が小さく、清澄度の高い生醤油は得にくくなる。
【００２９】
１０％塩化ナトリウム添加液体培地におけるさまざまな醤油乳酸菌の増殖と細胞沈降性を測定した。結果を図７に示す。
【００３０】
１８％塩化ナトリウム添加液体培地におけるさまざまな醤油乳酸菌の増殖と細胞沈降性を測定した。結果を図８に示す。
【００３１】
図７及び図８の結果から、液体培地に含まれる塩化ナトリウムの濃度は、８〜１２％、特に約１０％が好ましく、１３％以上では、培地自体の比重が大きくなるために、同じ醤油乳酸菌の細胞であっても、細胞が沈降しにくくなる、すなわち菌株の差異に基づいた細胞沈降性の差異を目視にて見極めにくくなる傾向がある。
【００３２】
本発明では、このような知見を基に、低ｐＨ環境下における醤油乳酸菌株の生存性を評価し、分離取得するための方法として、下記（１）〜（５）の手段を単独又は組合わせることを特徴とする低濁性醤油乳酸菌の分離法を提供する。
（１）長さ１６０ｍｍ、内径約１５ｍｍの試験管に、８〜１２％（Ｗ／Ｖ）塩化ナトリウムを含む液体培地１０ｍｌを入れ、これに被検菌を接種し、３０℃で４８時間静置培養し、上層部４〜６ｍｌを液面を揺らさぬようにして静かに採取し、均一に攪拌後６００ｎｍおける吸光度（イ）を測定し、また、採取した上層部全量を元に戻して培養液全体を均一に攪拌後６００ｎｍにおける吸光度（ロ）を測定し、（イ×１００）／ロを算出し、その値が３０以上である菌株を分離する。
（２）０．５％（Ｗ／Ｖ）安息香酸ナトリウムを含む０．１Ｍ燐酸カリウム緩衝液（ｐＨ７．０）と醤油麹とを均一に混合した後、濾過して得られた無菌の醤油麹粗抽出液に、被検菌を懸濁して、６００ｎｍにおける吸光度を０．５に調整した後、３０℃で７日間、１００ｒｐｍの条件で振盪しながら放置し、再び６００ｎｍにおける吸光度（ハ）を測定して、（０．５−ハ）×１００／０．５を算出し、その値が１５以上である菌株を分離する。
（３）被検菌を、０．２〜０．２５％（Ｗ／Ｖ）の塩化コバルトを含み、食塩を含まない寒天培地に接種、培養し、生育する菌株を分離する。
（４）被検菌を、０．１２％（Ｗ／Ｖ）以上のネオマイシン（力価６５０Ｕ／ｍｇ）を含む寒天培地に接種、培養し、生育する菌株を分離する。
（５）基礎培養基に、アスパラギン酸、アルギニン、チロシン、ヒスチジン又はフェニルアラニンを０．２〜１．０％（Ｗ／Ｖ）加えて殺菌し、これに被検菌を接種培養し、分解生成物の有無を観察し、いずれのアミノ酸も分解性を有しない菌株を分離する。
【００３３】
具体的には、ネオマイシン耐性能の差異を利用する方法、すなわち「分離識別試験１」が挙げられる。また、アスパラギン酸、アルギニン、チロシン、ヒスチジン又はフェニルアラニンのアミノ酸の分解性の有無を評価するための方法として、「分離識別試験２」が挙げられる。また、細胞の分解性を評価するための方法として、８〜１２％（Ｗ／Ｖ）塩化ナトリウムを含む液体培地中で静置培養した際の、増殖に伴う細胞沈降性の差異を利用する方法と、醤油麹菌の生産する分解酵素群溶液（麹粗抽出液）を利用する方法、すなわち「分離識別試験３」および防腐性の高い分解酵素群溶液を調製する方法、さらには細胞の生育能や醗酵能の高さを間接的に評価する方法、すなわち「分離識別試験４」、あるいはこれらを組み合わせて使用することにより、目的とする低ｐＨ環境となる後熟過程でより早期に死滅して、さらに麹菌由来の分解酵素群の作用を受けて、すみやかに分解される醤油乳酸菌株を、簡便かつ迅速に分離識別する。
【００３４】
図４は、以下の４つの手順（分離識別試験１＋分離識別試験２＋分離識別試験３＋分離識別試験４）を組合わせ低濁性醤油乳酸菌株を分離識別法を示す。
【００３５】
分離識別試験１（０．１２％（Ｗ／Ｖ）ネオマイシンを含む寒天培地を用いたネオマイシン耐性能評価試験による低濁性醤油乳酸菌株の分離識別試験）
１．０ｇグルコース、１．０ｇポリペプトン［極東化学社製］、０．４ｇ酵母エキス［ＤＩＦＣＯ製］、０．３ｇ燐酸二水素カリウム、０．２ｇ燐酸一水素二カリウム、０．１ｇチオグリコール酸ナトリウム、３．３ｇ酢酸ナトリウム、１．２〜１．５ｇ寒天［和光純薬社製］を加え、蒸留水に溶かして、これに水酸化ナトリウム水溶液を加え、溶液のｐＨを７．０に調整した後に全量を９５ｍｌにし、加圧加熱殺菌器で１２１℃、１５分間加熱殺菌する。一方、２．４％（Ｗ／Ｖ）のネオマイシン硫酸塩［ナカライテスク社製試薬で、力価は６５０Ｕ／ｍｇ］水溶液５ｍｌを準備し、これをワットマン社製シリンジフィルター（２５ｍｍＧＤ／Ｘ）を用いて除菌濾過し、無菌化する。
加熱殺菌の済んだ寒天溶液９５ｍｌを４６±２℃にまで冷却した後、無菌条件下でネオマイシン硫酸塩水溶液５ｍｌを加え、よくかき混ぜてから、その１０ｍｌずつを無菌シャーレに分注して、寒天培地を作製する（０．１２％（Ｗ／Ｖ）ネオマイシン硫酸塩を含む寒天培地）。
１．０ｇグルコース、１．０ｇポリペプトン［極東化学社製］、０．４ｇ酵母エキス［ＤＩＦＣＯ製］、０．３ｇ燐酸二水素カリウム、０．２ｇ燐酸一水素二カリウム、０．１ｇチオグリコール酸ナトリウム、３．３ｇ酢酸ナトリウムを蒸留水に溶かし、水酸化ナトリウム水溶液を用いてｐＨ＝７．０に調整した後に全量を１００ｍｌとし、加圧加熱殺菌器で殺菌した液体培地（ネオマイシン硫酸塩を含まない）５〜１０ｍｌ中で培養して、増殖活性を安定化させた供試菌株を、この０．１２％（Ｗ／Ｖ）ネオマイシン硫酸塩を含む寒天培地の表面に塗抹して、３０℃条件下で７日間嫌気培養する。本培地上で生育して、菌集落を形成した菌株のみを低濁性醤油乳酸菌株と評価して、次の分離識別試験２に供する。
【００３６】
分離識別試験２（アスパラギン分解性、アルギニン分解性、チロシン分解性、ヒスチジン分解性の有無を指標とした低濁性醤油乳酸菌株の分離識別試験）
２．０ｇグルコース、１．０ｇポリペプトン［日本製薬社製］、０．３ｇ酵母エキス［ＤＩＦＣＯ社製］、０．０５〜０．２ｇ燐酸水素二カリウム、３．３ｇ酢酸ナトリウム、０．１ｇチオグリコール酸ナトリウム、５ｇ塩化ナトリウムを蒸留水に溶かして、水酸化ナトリウム水溶液を用いてｐＨ＝７．２に調整した後に全量を１００ｍｌにして、加圧加熱殺菌器で、１２１℃、１５分間殺菌した液体培地５〜１０ｍｌ中に、試験に供する醤油乳酸菌株を接種して、３０℃条件下で２日間培養する。
０．１〜０．２ｇグルコース、０．５ｇ肉エキス[ＤＩＦＣＯ製］、０．５ｇ酵母エキス[ＤＩＦＣＯ製］、０．５gポリペプトン[日本製薬社製]、０．１ｇチオグリコール酸ナトリウム、１５ｇ塩化ナトリウム、０．０００５gピリドキシン塩酸塩を蒸留水に溶かし、０．６％（Ｗ／Ｖ）ブロモクレゾールパープルを溶かしたエタノール溶液１mlを加え、更にこれに２．０gのＬ―アスパラギン酸ナトリウム一水和物を溶かし、水酸化ナトリウム水溶液でｐＨ＝７．２に調整した後に全量を１００ｍｌにして、加圧加熱殺菌器で１２１℃、１５分間殺菌した液体培地（以下、アスパラギン酸培地と称する）、１．０ｇのＬ―アルギニン塩酸塩を溶かし、水酸化ナトリウム水溶液でｐＨ＝７．２に調整した後に全量を１００ｍｌにし、加圧加熱殺菌器で１２１℃、１５分間殺菌した液体培地（以下、アルギニン培地と称する）、１．０ｇのＬ−ヒスチジン塩酸塩一水和物を溶かし、水酸化ナトリウム水溶液でｐＨ＝７．２に調整した後に全量を１００ｍｌにして、加圧加熱殺菌器で１２１℃、１５分間殺菌した液体培地（以下、ヒスチジン培地と称する）の、計３種類の培地を作製し、この３種類の培地の培地にて培養した菌株の培養液を接種し、３０℃条件下で３〜７日間培養しながら、菌株の増殖に伴う培地の濁濁の程度と培地の黄変（菌株未接種の培地の色調はいずれも紫色で、それぞれの培地中で増殖しながらそれぞれのアミノ酸を分解すると、培地が黄色に変色する）を観察する。アスパラギン酸培地、アルギニン培地、ヒスチジン培地の計３種類のいずれかの培地についても黄変が観察されない菌株を選択する。
更に、１．０ｇグルコース、０．３ｇ酵母エキス［ＤＩＦＣＯ社製］、１．０ｇポリペプトン［日本製薬社製］、１．０ｇ燐酸一水素二カリウム、０．１ｇチオグリコール酸ナトリウム、１５ｇ塩化ナトリウム、２．２ｇ寒天を蒸留水に溶かして、水酸化ナトリウム水溶液でｐＨ＝７．２に調整してから、全量を１００ｍｌにした後に加圧加熱殺菌器で１２１℃、１５分間殺菌してから、４５〜５０℃まで冷却する。
あらかじめ擂り鉢などを使って粒子を細かくすりつぶし、更に乾熱殺菌器で１３０℃、３時間殺菌しておいた０．２〜０．３ｇのＬ−チロシンを、無菌条件下でこの殺菌済みの培地に加え、よく混合しながら、１０ｍｌづつ殺菌済みのシャーレに分注して固める（以下、チロシン培地と称する）。
上記３種類の培地のいずれにおいても黄変が認められなかった菌株をこのチロシン培地に接種し、３０℃条件下で７〜１４日間兼気培養し、このチロシン培地上に形成されたコロニーのうち、コロニー周辺部に透明帯が形成されていないものを低濁性醤油乳酸菌として取得する。
【００３７】
分離識別試験３（醤油麹粗抽出液を用いた醤油乳酸菌細胞の消化試験による低濁性醤油乳酸菌株の分離識別試験）
蒸した大豆５００ｇおよび炒った小麦５００ｇを混ぜ合わせ、そこに醤油麹菌（アスペルギルス オリゼ（Ａｓｐｅｒｇｉｌｌｕｓ ｏｒｙｚａｅ））を接種して、２〜４日間加湿培養してつくった醤油麹１ｋｇに、０．５％（Ｗ／Ｖ）安息香酸ナトリウムを含む０．１Ｍの燐酸カリウム緩衝液（ｐＨ＝７．０）１Ｌを加えてよくかき混ぜ、３０℃に保温した状態で１〜２時間放置した後、東洋濾紙社製Ｋ濾紙（ＡＤＶＡＮＴＥＣ−ＴＯＹＯ、Ｎｏ．２）を用いて濾過をおこない、濾液をさらにローター回転数１５０００ｒｐｍ、４℃冷却条件下で約１０分間遠心分離して、その上澄液を回収する（これを以後「醤油麹粗抽出液」と呼ぶ）。醤油麹粗抽出液は、さらにワットマン社製シリンジフィルター（２５ｍｍＧＤ／Ｘ）を用いて、除菌濾過をおこない、無菌化する。
試験に供する醤油乳酸菌株は、まず１％（Ｗ／Ｖ）グルコース、１％ポリペプトン［日本製薬製］、０．４％酵母エキス［ＤＩＦＣＯ製］、０．３％（Ｗ／Ｖ）燐酸二水素カリウム、０．２％（Ｗ／Ｖ）燐酸一水素二カリウム、０．１％（Ｗ／Ｖ）チオグリコール酸ナトリウム、３．３％（Ｗ／Ｖ）酢酸ナトリウム、１０％（Ｗ／Ｖ）塩化ナトリウムから構成される液体培地（ｐＨ＝７．０）中で、３０℃、４〜７日間静置した後、培養液の上部が透明に澄んだ菌株、すなわち細胞沈降性の高い菌株は除き、培養液の上部までが細胞の懸濁によって濁った状態を示す菌株、すなわち細胞沈降性の低い菌株のみを、１．０％（Ｗ／Ｖ）グルコース、０．３％（Ｗ／Ｖ）酵母エキス［ＤＩＦＣＯ製]、１０％（Ｖ／Ｖ）濃口生醤油か ら構成され、最終濃度が１５％になるように塩化ナトリウムを加えた液体培地（殺菌後のｐＨ＝７．０）中で、３０℃、７日間静置培養した後、ローター回転数７０００〜１００００ｒｐｍ、４℃冷却条件下で１０分間遠心分離して菌体を回収、これを１０％塩化ナトリウム水溶液中に撹拌しては、同条件にて遠心分離することにより菌体をよく洗浄する。
このようにして準備した供試菌株の菌体を無菌条件下で、先の除菌処理した醤油麹粗抽出液に懸濁して、分光光度計での６００ｎｍの吸光度が０．５となるように、醤油麹粗抽出液に懸濁する菌体量を調節する。
このようにして調製した、６００ｎｍの吸光度が０．５を示すように醤油乳酸菌株の菌体を懸濁した醤油麹粗抽出液１０ｍｌを３７℃に保温した状態で無菌的に７日間振盪（１００〜２００ｒｐｍ．）し、その後再び分光光度計を用いて６００ｎｍの吸光度（Ａ）を測定し、これをもとに７日間の吸光度の減少率、すなわち（０．５−Ａ）／０．５を算出、この値が１５以上である菌株を次なる分離識別試験３の供試菌株とする。
複数回の評価試験をおこなう場合には、第二回目以降の供試菌株のなかに、既に初回の試験で評価してある菌株１〜２種類を加えて試験をおこなうことにより、試験に供する醤油麹やその粗抽出液の成分誤差に起因する試験結果の誤差を小さくすることができる。
【００３８】
分離識別試験４（０．２〜０．２５％（Ｗ／Ｖ）塩化コバルトを含む無塩寒天培地を用いた塩化コバルト耐性能評価試験による低濁性醤油乳酸菌株の分離識別試験）
１．０ｇグルコース、１．０ｇポリペプトン［極東化学社製］、０．４ｇ酵母エキス［ＤＩＦＣＯ製］、０．３ｇ燐酸二水素カリウム、０．２ｇ燐酸一水素二カリウム、０．１ｇチオグリコール酸ナトリウム、３．３ｇ酢酸ナトリウム、１．２〜１．５ｇ寒天［和光純薬社製］を蒸留水に溶かして、水酸化ナトリウム水溶液を加え、溶液のｐＨを７．０に調整した後に全量を９５ｍｌとし、加圧加熱殺菌器で、１２１℃、１５分間殺菌する。一方、４〜５％（Ｗ／Ｖ）塩化コバルト（ＣｏＣｌ2・６Ｈ2Ｏ）水溶液を準備し、これをワットマン社製シリンジフィルター（２５ｍｍＧＤ／Ｘ）を用いて除菌濾過し、無菌化する。
加熱殺菌の済んだ寒天溶液９５ｍｌを４６±２℃にまで冷却した後、これに無菌条件下で塩化コバルト水溶液５ｍｌを加え、よくかき混ぜてから、その１０ｍｌずつをシャーレに分注して、寒天培地を作製する（０．２〜０．２５％（Ｗ／Ｖ）塩化コバルトを含む寒天培地）。
１．０ｇグルコース、１．０ｇポリペプトン［極東化学社製］、０．４ｇ酵母エキス［ＤＩＦＣＯ製］、０．３ｇ燐酸二水素カリウム、０．２ｇ燐酸一水素二カリウム、０．１ｇチオグリコール酸ナトリウム、３．３ｇ酢酸ナトリウムを蒸留水に溶かして、水酸化ナトリウム水溶液を用いてｐＨ＝７．０に調整し、全量を１００ｍｌに調整、加圧加熱殺菌器を用いて殺菌した液体培地（塩化コバルトを含まない液体培地）５〜１０ｍｌ中で培養して、増殖活性を安定化させた供試菌株を、この０．２〜０．２５％（Ｗ／Ｖ）塩化コバルトを含む寒天培地の表面に塗抹して、３０℃条件下で、７日間嫌気培養する。本培地上で生育して、菌集落を形成した菌株のみを低濁性醤油乳酸菌株と評価する。
【００３９】
実施例１（本法により低濁性醤油乳酸菌株と評価された醤油乳酸菌株と、それ以外の醤油乳酸菌株を用いた醤油醸造試験）
天然仕込みの醤油諸味から分離した醤油乳酸菌群の中から、上記の分離識別試験１、２、３を経て選択された低濁性醤油乳酸菌株２株（Ｓ−１６株及びＮ−４株）と、同試験２及び同試験３の寒天培地上で生育せず、菌集落の形成が観察されなかった醤油乳酸菌株１株（Ｓ−２株）を用いた小規模の醤油仕込み試験をおこなった。
諸味の調製は、関根らの方法（醤研、１３、１４９ （１９８７））に従った。それぞれの醤油乳酸菌株は、１．０％（Ｗ／Ｖ）グルコース、０．３％（Ｗ／Ｖ）酵母エキス［ＤＩＦＣＯ製］、１０％濃口生醤油から構成され、調製後の最終濃度が１５％になるように塩化ナトリウムを加えた液体培地（殺菌後のｐＨ＝７．０）中で、３０℃、７日間静置培養した培養液を用い、諸味への接種量は接種後の諸味中の菌数が１０5ｃｆｕ／ｇになるように調 節した。
醤油乳酸菌株による醤油の濁りを観察しやすいように、仕込み期間は８０日間とし、諸味の品温管理も仕込み直後より７日目までの間を１５℃、８日目より１４日目までの間を２０℃、１５日目より２０日目までの間を２５℃とし、２１日目に諸味のｐＨが５．０になったのを確認したうえで醤油主醗酵酵母（チゴサッカロマイセス ルーキシー（Ｚｙｇｏｓａｃｃｈａｒｏｍｙｃｅｓ ｒｏｕｘｉｉ））の培養液を、接種後の諸味中の菌数が１０5ｃｆ ｕ／ｇになるように接種するか（表５の中の「処理１」）、あるいは市販の９９％以上未変性エタノールを、添加後の諸味中の濃度が３％になるように添加して（表５の中の「処理２」）、その後は７０日目まで３０℃で、さらに７１日目より８０日目まで２５℃とした。
このようにして８０日間仕込んだ諸味を東洋濾紙社製の濾紙（ＡＤＶＡＮＴＥＣ−ＴＯＹＯ、Ｎｏ．２）で濾過して、濾液を回収し、それぞれの濁度を濁度計で測定した。結果を表６に示す。
【００４０】
【表６】

【００４１】
表６に示すとおり、Ｓ−２株、Ｓ−１６株およびＮ−４株はいずれも、諸味中でほぼ同程度の生育を示したが、Ｓ−１６株およびＮ−４株で醸造した諸味の濾液の濁度は、醤油主醗酵酵母液を加えた「処理１」では、Ｓ−２株で醸造した諸味の濾液に較べて低い値を示した。また、２１日目に酵母のかわりにエタノールを添加した諸味（表６の中の「処理２」）では後熟過程（本試験における４０日目以降）の諸味中でのＳ−２株、Ｓ−１６株、Ｎ−４株の生存性に差異は認められず、Ｓ−２株、Ｓ−１６株、Ｎ−４株それぞれの諸味の濾液の濁度にも差異は認められず、このことからも、先に記したとおり、後熟過程の諸味中での醤油乳酸菌株の生存性が醤油の濁度と密接に関係していることがわかる。ゆえに、本発明を用いて、使用する醤油乳酸菌株を選択することはきわめて有意義である。
【００４２】
【発明の効果】
本発明によれば野生の乳酸菌の中から、乳酸発酵力は旺盛で、低pH環境下での生存能が低く、かつ細胞表面の親水性度が高く（疎水性度が低く）、しかも麹菌由来の酵素群により分解され易い性質を有する低濁性醤油乳酸菌を非常に簡単に、しかも確実に得ることができる。
また、上記低濁性醤油乳酸菌を野生の乳酸菌の中から非常に簡単にしかも確実に分離できる培地を提供することができる。さらにまた本発明は、予め選択された、または特に育種した、性質の優秀な醤油乳酸菌を人為的に、醤油麹および／または諸味に添加し、仕込工程における乳酸発酵を安定して行わせる醤油醸造法において、該醤油乳酸菌として上記「低濁性醤油乳酸菌」を用い、仕込み工程以降の製成工程における各種清澄化剤の施用や濾過処理をすることなく、清澄度の高い醤油を容易に得ることができる。
【図面の簡単な説明】
【図１】生醤油中の混濁物質を遠心分離して得られたものの顕微鏡による画像を示す図。
【図２】醤油諸味から単離したさまざまな醤油乳酸菌株を用いて仕込んだ醤油諸味の仕込後３カ月目における諸味液汁の濁度と乳酸濃度との関係を示す図。
【図３】全国各地の市販濃口醤油製品の乳酸濃度と濁度の関係を示す図。
【図４】低濁性醤油乳酸菌株の分離識別法を示す図。
【図５】さまざまなアミノ酸分解性を有する醤油乳酸菌株のネオマイシン耐性を示す図。
【図６】醤油乳酸菌株のアミノ酸分解性とそれらの菌株を用いて醸造した生醤油の濁度の関係を示す図。
【図７】１０％塩化ナトリウム添加液体培地におけるさまざまな醤油乳酸菌株の増殖と細胞沈降性の関係を示す図。
【図８】１８％塩化ナトリウム添加液体培地におけるさまざまな醤油乳酸菌株の増殖と細胞沈降性の関係を示す図。
【図９】醤油の濁度が醤油の色調に及ぼす影響を示す図。
【図１０】従来の醤油乳酸菌株と本発明により分離された醤油乳酸菌の液体培養液の透明度を示す図。[0001]
[Technical field to which the invention belongs]
The present invention has a strong lactic acid fermenting ability among wild lactic acid bacteria, low viability in a low pH environment, high cell surface hydrophilicity (low hydrophobicity), and The present invention relates to a medium for obtaining a low-turbidity soy lactic acid bacterium having properties that are easily degraded by an enzyme group in a very simple and reliable manner, and a method for separating a low-turbidity soy lactic acid bacterium using the same medium.
Further, the present invention provides a soy sauce brewing method in which a soy lactic acid bacterium having excellent properties selected in advance or particularly bred is artificially added to soy sauce koji and / or moromi to stably carry out lactic acid fermentation in the preparation process. The above-mentioned “low-turbidity soy sauce lactic acid bacteria” as the soy sauce lactic acid bacteria, and a method for easily obtaining soy sauce with high clarity.
[0002]
[Prior art]
The clarity of soy sauce is one of the important elements of soy sauce that influences the taste through the characteristic of “watching” when used for food.
FIG. 9 shows the effect of the turbidity of soy sauce on the color tone of the soy sauce. It can be seen that the soy sauce having a lower turbidity, that is, a higher clarity, has a higher chromaticity and a brighter color tone.
The cause of clarification of soy sauce by turbidity is, for example, Bacillus genus bacteria, Staphylococcus genus bacteria and Micrococcus genus bacteria, which are mixed in the soy sauce making process. , There is a report on so-called miscellaneous bacterial cell residue (Naruyuki Kitahara et al., J. Ferment. Technol., 47 (1), 1-7 (1969)). The number of cases of soy sauce clarification reduction due to the contamination with such miscellaneous bacteria has been significantly reduced.
In recent years, various clarifiers for food additives and filtration processing equipment have been developed and marketed, so the clarification of soy sauce is relatively easy by using these methods and equipment in the production process. It became possible to improve.
However, it is also true that soy sauce clarification is limited by such methods and equipment, and maintaining the clarification of raw soy sauce after the pressing process does not use these methods or equipment. From this point of view, it can be said that it is important for producing cheaper soy sauce with high clarity. In addition, consumer demand for soy sauce clarification has recently become higher and more rigorous, and the establishment of a high-clarity raw soy sauce brewing method backed by scientific grounds. Highly sought after.
[0003]
Moreover, recently, a soy sauce brewing method that has been pre-selected or especially bred, artificially added to soy sauce lactic acid bacteria and / or moromi, and stably lactic acid fermentation in the preparation process, As the soy sauce lactic acid bacteria, it is known to use soy sauce lactic acid bacteria having high cohesiveness to obtain soy sauce with a high degree of clarity (Tatsuro Ueki, Kazunori Oba, Yoshiharu Noda, 1998 Japan Society for Biotechnology, 1998) Abstract of the lecture: 1028 “Aggregation of salt-tolerant lactic acid bacteria isolated from soy sauce moromi” and JP 2000-245443).
However, a low-turbidity soy lactic acid bacterium having a low viability in a low pH environment, a high degree of hydrophilicity on the cell surface (low hydrophobicity), and a property that is easily degraded by enzymes derived from Aspergillus oryzae, It is not known at all what kind of soy sauce is obtained when it is added and used in the soy sauce preparation process, especially about obtaining soy sauce with high clarity. Moreover, it is not known that a medium for obtaining such a lactic acid bacterium and a low-turbidity soy sauce lactic acid strain using the medium are obtained.
[0004]
[Problems to be solved by the invention]
The present invention improves the clarification of soy sauce by devising the brewing process without using various clarifiers for food additives or the use of high-performance filtration equipment, that is, increasing the turbidity in soy sauce. The purpose is to supply soy sauce that is low in appearance and beautiful for appetite and is appetizing stably and inexpensively.
[0005]
[Means for Solving the Problems]
As a result of intensive studies to solve the above problems, the present inventors have finally completed the present invention. That is, the present invention provides a crude koji obtained by uniformly mixing a 0.1 M potassium phosphate buffer solution (pH 7.0) containing 0.5% (W / V) sodium benzoate and soy sauce cake and then filtering the mixture. It is the culture medium for isolation | separation of the low turbidity soy sauce lactic acid bacteria which consists of an extract. Moreover, this invention is a culture medium for isolation | separation of the low turbidity soy sauce lactic acid bacteria which consists of an agar medium which contains 0.2-0.25% (W / V) cobalt chloride and does not contain salt. Moreover, this invention is a culture medium for isolation | separation of the low turbidity soy sauce lactic acid bacteria which consists of an agar medium containing neomycin (650 U / mg) 0.12% (W / V) or more. Moreover, this invention is the following (1)- (5) The method of separating low turbidity soy sauce lactic acid bacteria characterized by combining these means alone or in combination.
(1) Into a test tube having a length of 160 mm and an inner diameter of about 15 mm, 10 ml of a liquid medium containing 8 to 12% (W / V) sodium chloride is inoculated, inoculated with the test bacteria, and left at 30 ° C. for 48 hours. Cultivate and gently collect 4-6 ml of upper layer without shaking the liquid level, measure the absorbance (b) at 600 nm after stirring uniformly, and restore the total amount of the collected upper layer to the original culture solution. After the whole is uniformly stirred, the absorbance (b) at 600 nm is measured, (b x 100) / b is calculated, and a strain having a value of 30 or more is isolated.
(2) Aseptic soy sauce cake obtained by uniformly mixing a 0.1M potassium phosphate buffer (pH 7.0) containing 0.5% (W / V) sodium benzoate and soy sauce cake and then filtering the mixture. Suspend the test bacteria in the crude extract and adjust the absorbance at 600 nm to 0.5, then leave it at 30 ° C. for 7 days with shaking at 100 rpm, and measure the absorbance (c) at 600 nm again. Then, (0.5−c) × 100 / 0.5 is calculated, and a strain whose value is 15 or more is isolated.
(3) The test strain is inoculated and cultured in an agar medium containing 0.2 to 0.25% (W / V) cobalt chloride and no salt, and the growing strain is isolated.
(4) The test strain is inoculated and cultured in an agar medium containing 0.12% (W / V) or more of neomycin (titer 650 U / mg), and the growing strain is isolated.
(5) 0.2-1.0% (W / V) of aspartic acid, arginine, tyrosine, histidine or phenylalanine is added to the basic culture medium to sterilize, inoculate and inoculate the test bacteria, The presence or absence is observed, and a strain in which any amino acid is not degradable is isolated.
The present invention also provides a soy sauce brewer that artificially adds a preselected or particularly bred soy lactic acid bacterium having excellent properties to soy sauce koji and / or moromi to stably perform lactic acid fermentation in the preparation process. In the method, the soy sauce lactic acid bacterium is a turbid soy sauce lactic acid bacterium isolated by the above-described method.
[0006]
DETAILED DESCRIPTION OF THE INVENTION
The present inventor obtained a total of 97 kinds of commercially available dark mouth soy sauce (dark mouth round soybean soy sauce), dark mouth raw soy sauce brewed by the present applicant (including dark mouth round soybean soy sauce), and turbidity components contained in fresh mouth raw soy sauce by centrifugation. It was recovered, and through morphological observation and component analysis using a microscope, it was found that one of the main components was cell residue of soy sauce lactic acid bacteria (cocci) (see FIG. 1).
That is, the present inventor found that the turbid substance of raw soy sauce was precipitated and separated by a centrifugal separation method (15000 rpm. × 10 min.) And observed with a microscope. Knew.
Soy sauce lactic acid bacteria are important microorganisms that cause lactic acid bacteria fermentation in the soy sauce moromi preparation process, and are taxonomically included in one genus and one species of Tetragenococcus halophilus, but Uchida reports According to the strain, assimilation of various saccharides (K. Uchida, J. Gen. Appl. Microbiol., 28, 215 (1982)) and amino acid degradability (for example, arginine degradability, Uchida Kin J. et al., 1992 Annual Meeting of the Agricultural Chemical Society of Japan, p336 (1992)), it has been found that the resistance to various chemical substances and physical environmental conditions is quite different. Among the properties of such strains, for example, when soy sauce is brewed using such strains, such as sugar assimilation and amino acid degradability, there are many that affect the properties of the soy sauce. .
The present inventor conducted a small-scale soy sauce brewing test (preparation period of 6 months) using various soy sauce lactic acid strains isolated from soy sauce moromi as a seed lactic acid strain (Table 1). Despite using the same product temperature control, the turbidity of fresh soy sauce after pressing showed various values (ppm) (Table 2).
[0007]
[Table 1]

[0008]
[Table 2]

[0009]
In addition, the present inventor conducted a soy sauce brewing test using various soy sauce lactic acid bacterial strains isolated from soy sauce moromi, and the turbidity of each moromi soup was various, even though the same koji was used. I learned that the turbidity of soy sauce is closely related to the degree of lactic acid fermentation in the charging process (see Fig. 2).
Furthermore, when the relationship between lactic acid concentration and turbidity of 97 soy sauce soy sauces sold nationwide in 1996 and 1997 was investigated, as shown in FIG. 3, the turbidity of soy sauce was about 0 to 80 ppm. Until now, although it was different depending on the product, we learned that commercial soy sauce with high turbidity has a high lactic acid concentration relative to the degree of turbidity.
In addition, in the result of FIG. 3, it can be said that commercial soy sauce with high lactic acid concentration does not necessarily have high turbidity due to application of various clarifiers and application of filtration in the production process after the preparation process. It is.
From these facts, the present inventors have found that soy sauce lactic acid strains differ greatly in viability in a low pH environment from one strain to another, and this property is particularly matured after the main fermentation process is finished and the pH is lowered. It was found that the properties of this strain differed in the moromi of the process (ripening process), and consequently the turbidity of soy sauce was affected.
[0010]
In the preparation process, a group of degrading enzymes such as protease produced by koji molds promotes the decomposition of various components in moromi and contributes to the generation of important taste components in soy sauce. It is also involved in the degradation of soy sauce lactic acid bacteria cells that are no longer needed.
However, in the degradation reaction by this enzyme, life and death of soy sauce lactic acid bacteria cells are important, and live cells exhibit resistance to degradation, whereas dead cells are rapidly degraded, showing a difference in degradation efficiency. I learned that through this research.
[0011]
In addition, the inventor of the present invention has greatly different cell surface hydrophilicity (or hydrophobicity) from strain to strain, and cells of strains with low hydrophilicity (or high hydrophobicity) are likely to aggregate, I knew that it was difficult to be affected by these degradation enzymes.
[0012]
From these findings, soy sauce lactic acid strains that have low viability in a low pH environment, have high cell surface hydrophilicity (or low hydrophobicity), and are easily decomposed by a group of koji mold-degrading enzymes, After the main fermentation, it is quickly killed in the ripening process after the main fermentation, and the dead cells are quickly degraded by the enzyme, so the rate of undegraded cell migration to soy sauce, which is the solution fraction, is low. As a result, the turbidity of soy sauce should be low, and conversely, it has high viability in low pH environment, low hydrophilicity of cell surface (or high hydrophobicity), and degradation derived from Aspergillus oryzae Cells of soy sauce lactic acid bacterial strains that are less susceptible to degradation by the enzyme group continue to survive in the various flavors that have entered the post-ripening process after the main fermentation, and in order not to be degraded by the enzyme, move into soy sauce as undegraded debris, Soy sauce Kiyosumi It is expected to reduce the.
Furthermore, the group of degrading enzymes responsible for cell degradation of this soy sauce lactic acid strain is produced by koji molds in the koji making process, but when a situation occurs in which the productivity of these group of degrading enzymes by koji molds decreases, The degradability of the cells of the soy sauce lactic acid strain in the post-ripening process is further reduced, and as a result, the clarification of the soy sauce is remarkably lowered, and in some cases, the value as a product can be significantly impaired.
[0013]
Therefore, in the present invention, a simple “separation discrimination test 1” for confirming low viability under a low pH environment, and a simple method for evaluating the susceptibility to degradation by a group of gonococcal degrading enzymes. Invented “Separation Identification Test 2” and simple “Separation Identification Test 3” for indirectly evaluating the strength of lactic acid fermentation, etc. Soy sauce lactic acid bacteria when brewing soy sauce using this strain because of its low viability under the surface, high hydrophilicity on the cell surface, and the ability to be easily degraded by the degrading enzyme derived from Aspergillus oryzae. Since the strain died early and is rapidly degraded by the action of a degrading enzyme derived from Aspergillus, the transfer of undegraded debris of soy lactic acid bacteria to the juice fraction after squeezing is small, resulting in clarification Making high-quality soy sauce The soy sauce lactic acid strain (hereinafter referred to as “low turbidity soy lactic acid strain”) separation and identification method (FIG. 4), and a low turbidity soy lactic acid strain having such properties, Propose a method of brewing soy sauce with high clarity.
[0014]
The viability of a bacterium under a low pH environment is closely related to the activity of a membrane-bound ATPase that exists in the cell membrane of the bacterium and is involved in the regulation of proton (H +) concentration inside and outside the cell. In addition, the strength of this enzyme activity varies greatly depending on the strain, and bacterial strains with weak activity have low viability in low pH environments, and conversely strong bacterial strains have high viability in low pH environments. Already known (GR Bender et al., Infect. Immun., 53 (2), 331 (1986)), aminoglycoside antibiotics such as neomycin have a gradient in the proton concentration inside and outside the cell. Since it has a growth inhibitory action after being taken into cells with (proton driving force), It is possible to evaluate the strength of the activity of the membrane-bound ATPase possessed by the bacteria by examining the strength of the resistance of a particular antibiotic, that is, a bacterial strain exhibiting high performance has a weak membrane-bound ATPase activity. It has been found that bacterial strains with low viability in low pH environments and weakly resistant performance have strong membrane-bound ATPase activity, and thus have low viability in low pH environments (A. Yokota et al. Biosci. Biotech. Biochem., 59 (10), 2004 (1995)).
[0015]
The “separation discrimination test 1” of the present invention is a method for simply evaluating the viability in a low pH environment through examining the neomycin resistance performance of a soy sauce lactic acid strain applying this knowledge. When we examined the neomycin resistance of soy lactic acid strains, the resistance varies depending on the strain. However, the soy lactic acid strains that showed high turbidity of soy sauce in small-scale soy sauce brewing tests generally have neomycin resistance. Soy sauce lactic acid bacterial strains that were low and, conversely, showed a tendency of low turbidity of soy sauce generally showed a tendency of high resistance to neomycin, confirming the effectiveness of this method (Table 2).
[0016]
Lactobacillus strain S-16 showing 0.12% (W / V) neomycin resistance and 0.45% (W / V) lactic acid strain S-2 showing neomycin sensitivity, isolated using this method, 1% (W / V) glucose, 1% (W / V) polypeptone [Nippon Pharmaceutical Co., Ltd.], 0.4% (W / V) yeast extract [manufactured by DIFCO], 0.3% (W / V) diphosphate Potassium hydrogen, 0.2% (W / V) dipotassium monohydrogen phosphate, 0.1% (W / V) sodium thioglycolate, 3.3% (W / V) sodium acetate, 10% (W / V) ) When comparing the viability under various pH environments using a medium composed of sodium chloride, no significant difference was observed in the growth ability under the condition of pH = 7.0. Under 4.0 conditions, both were observed to die quickly. Under the condition of pH = 5.0, the S-16 strain died rapidly, whereas the death rate of the S-2 strain was clearly slower than that of the S-16 strain, The high viability was shown (Table 3).
[0017]
[Table 3]

[0018]
By the way, the soy sauce lactic acid strain is obtained by passing L-arginine through the arginine deiminase pathway (Arginine deiminase (EC.3.5.3.6.6) pathway) and citrulline (L-Ornithine). ) Presence / absence of a function for converting to arginine, ie, presence / absence of arginine resolution, presence / absence of a function for converting aspartic acid to alanine by the action of aspartate decarboxylase (EC 4.1.1.12), ie, asparagine Tyrosine is converted to Tyramine by the action of tyrosine decarboxylase (EC 4.1.1.25) with or without acid resolving power. Presence / absence of a function to convert, ie, presence / absence of tyrosine degradation, presence / absence of a function to convert histidine to histamine by the action of histidine decarboxylase (EC 4.1.1.22), ie, histidine degradation It is known that the presence or absence of sex is different, and as a result, it has a variety of amino acid resolution (Kanaji Uchida et al., Shukyo Journal, 77, 740 (1982)). Among them, there are strains having tyrosine and histidine degradability. When grown in soy sauce, so-called “biological amines” such as tyramine and histamine are produced, or when a strain with arginine degradability grows in soy sauce moromi and is infected with soy sauce lactic acid bacteria phage, soy sauce lactic acid bacteria cells As a metabolic intermediate, It has been reported by Uchida et al. (Iizuka et al., Seasoning Science, 20 (5), 17 (1973) that rurin is released and this undergoes a chemical reaction with ethanol in the firing process to produce ethyl carbamate. (Kinji Uchida et al., Abstracts of Annual Meeting of Japanese Society for Agricultural Chemistry, p. 336 (1992)).
[0019]
As a result of examining the relationship between the group classification of soy sauce lactic acid strains according to the presence or absence of degradability of various amino acids and the turbidity of raw soy sauce brewed using these strains, the strain having aspartic acid degradability It was found that the turbidity of raw soy sauce brewed using these non-amino acid-degradable strains was lower than that of arginine-degradable strains (see FIG. 5).
[0020]
Regarding the uptake of aspartate in lactic acid bacteria and the conversion to alanine by aspartate decarboxylase, K. using Lactobacillus genus bacteria strain M3. According to Abe et al. (J. Biol. Chem., 271 (6), 3079-3084 (1996)), according to this report, extracellular aspartic acid firstly transmembrane protein AspT. Via intracellular OH ^1- Is taken into the cell accompanied by the excretion of the intracellular proton, and as a result of the action of aspartate decarboxylase, an intracellular proton (H ⁺ ) It is converted to alanine through the consumption of one molecule, but this aspartic acid uptake mode is then changed to the “aspartic acid / alanine antiporter” system, which involves excretion of the metabolite alanine out of the cell. ATP is produced from ADP by utilizing the potential difference generated during the change.
That is, the aspartic acid decarboxylase reaction of an aspartic acid-degrading strain takes in a substrate (aspartic acid) in such a way that a potential difference is formed inside and outside the cell, and also the intracellular proton (H ⁺ ) In a manner that consumes a), it is likely to promote the formation of a proton concentration gradient, which means increased cell viability in a low pH environment.
Furthermore, since this enzyme catalyzes the action of converting aspartic acid, which is an acidic amino acid, to alanine, which is a neutral amino acid, soy sauce with a high amino acid concentration also causes a considerable pH increase. When acid-decomposable soy sauce lactic acid bacteria contributed to lactic acid fermentation in soy sauce moromi, it simultaneously caused a decomposition reaction of aspartic acid, so that the pH of soy sauce moromi caused by lactic acid fermentation was less likely to decrease, and the pH in the post-ripening process was lowered It will be able to survive and survive for a longer period even in soy sauce moromi, which will increase the turbidity of raw soy sauce.
The same applies to tyrosine decarboxylase involved in tyrosine and histidine degradability and strains having histidine decarboxylase. In addition, the arginine deiminase pathway related to the degradation of arginine is a reaction that produces ammonia, and as a result, it suppresses the decrease in extracellular pH associated with lactic acid fermentation in soy sauce moromi. It is expected that they can continue to live longer than lactic acid strains.
From these facts, soy sauce lactic acid strains that do not have aspartic acid degradability, tyrosine degradability, histidine degradability, and arginine degradability, which are involved in the formation of proton concentration gradient, Viability is low in low pH environment such as soy sauce moromi in the post-ripening process, and neutralization of pH reduction due to lactic acid fermentation due to amino acid degradation does not occur, resulting in quick dying, so that raw soy sauce is hard to become turbid It is considered a thing.
[0021]
Furthermore, the structural gene of aspartate decarboxylase responsible for the aspartate degradability of such a soy sauce lactic acid strain is located outside the nucleus as a plasmid, and 3 μg / ml ethidium bromide and 5% (W / V) sodium chloride are added. Inoculated into MRS medium [manufactured by DIFCO] and cultured at 30 ° C. for 4 days, or without ethidium bromide, containing only sodium chloride at about 0 to 5% (W / V), so-called low It has already been reported that a curing strain from which this plasmid has been eliminated can be easily and frequently obtained by simply culturing in a salt medium under the same conditions (T. Higuchi et al., Biosci). Biotechnol.Biochem., 62 (8), 1601-1603 (1998)), separated from soy sauce moromi, By only inoculating soy sauce lactic acid strains that have only aspartic acid degradability, tyrosine and histidine, and no arginine degradability, once the low salt medium has been treated, the plasmid is dropped, and aspartic acid degradability is lost. It is also possible to reduce only the turbidity of soy sauce when brewing using the strain without affecting the other brewing characteristics of the soy sauce lactic acid strain.
Incidentally, since the agar medium for evaluating the viability of the strain under the low pH environment shown in the present invention has a low sodium concentration and corresponds to the low salt medium here, 1.2 mg in this agar medium. / Ml After observing neomycin resistance, if a colony of resistant strains formed on the medium is picked up, even if the strain had aspartic acid degradability before inoculation, it lacks aspartic acid degradability It was also confirmed that the obtained strain can be obtained with high frequency.
[0022]
As a result of examining the resistance of neomycin to 76 soy lactic acid bacterial strains with various amino acid-degrading properties isolated from soy sauce moromi, the strain with arginine resolution is more sensitive to neomycin than the strain without resolution. Since strains with aspartic acid degradability are more resistant to neomycin than non-degradable strains, most of the strains resistant to neomycin at the 0.12% (W / V) concentration defined in this method are Was found to be a strain having no arginine resolution (see FIG. 5).
That is, it was confirmed that the soy sauce lactic acid bacteria strain isolated using this method has many excellent strains that do not have arginine degradability and therefore do not produce citrulline.
[0023]
The amino acid degradability of soy sauce lactic acid bacteria and the turbidity of raw soy sauce brewed using these strains were measured. The results are shown in FIG.
[0024]
In addition, the cells of soy sauce lactic acid bacteria after completing the role of lactic acid fermentation are considered to be decomposed by the action of various degrading enzyme groups derived from koji mold contained in moromi, but 0.5% ( W / V) Cells of various soy sauce lactic acid bacterial strains prepared by mixing 0.1M potassium phosphate buffer containing sodium benzoate and then filtering through filter paper, and containing various crude enzymes derived from Aspergillus oryzae. The degradability of the soy sauce lactic acid strains that are easy to degrade and the soy sauce lactic acid strains that are difficult to degrade exist, but they were brewed using a soy sauce lactic acid strain that is easy to degrade. It was confirmed that the turbidity of soy sauce brewed using a soy sauce lactic acid strain that is difficult to decompose is high. That is, aseptic soy sauce obtained by uniformly mixing a 0.1M potassium phosphate buffer solution (pH 7.0) containing 0.5% (W / V) sodium benzoate and soy sauce cake and then filtering. After suspending the test bacteria in the crude extract and adjusting the absorbance at 600 nm to 0.5, the mixture was allowed to stand at 30 ° C. for 7 days with shaking at 100 rpm, and the absorbance at 600 nm (c) was again measured. Measured and calculated (0.5-ha) × 100 / 0.5, and it is found that a strain having a value of 15 or more, particularly 25 or more is suitable for obtaining a clear raw soy sauce (Table 4).
[0025]
[Table 4]

[0026]
Furthermore, when the strain to be used in this test is pre-cultured (stationary culture) in a liquid medium containing 8 to 18% (W / V) sodium chloride, the cell sedimentation accompanying growth in the medium is high, and the culture A strain (for example, S-2 strain) showing the property that the upper part of the liquid is clear, and a strain (for example, S-) in which the cells are suspended in the upper part of the culture solution due to low sedimentation. 16 strains and N-4 strains) (see FIG. 10), and the former, that is, soy sauce brewed using a strain having high cell sedimentation, has high turbidity, whereas the latter, that is, cell sedimentation It was confirmed that the turbidity of soy sauce brewed using low strains was generally low.
That is, in a test tube having a length of about 160 mm and an inner diameter of about 15 mm, 10 ml of a liquid medium containing 10% (W / V) sodium chloride (for example, refer to the medium composition of A below) is inoculated with a test bacteria. Incubate statically at 30 ° C. for 48 hours, gently collect 4-6 ml of the upper layer without shaking the liquid surface, measure the absorbance (ii) at 600 nm after stirring uniformly, and collect the upper layer After returning the total amount to the original culture and stirring the whole culture solution uniformly, the absorbance (b) at 600 nm is measured, and (ii x 100) / b is calculated. A strain having a value of 30 or more (for example, S-16 strain is 43, N-4 strain shows a value of 65), but was found to be suitable for obtaining clear raw soy sauce (see Table 5) (N-4 strain in FIG. 10 is in a turbid state) .
A. Medium composition
1% (W / V unless otherwise noted) glucose, 0.4% yeast extract, 1% polypeptone, 0.3% potassium dihydrogen phosphate, 0.2% potassium monohydrogen phosphate, 3.0% Sodium acetate, 0.1% sodium thioglycolate, 10% sodium chloride, pH 7.2.
[0027]
[Table 5]

[0028]
A strain having the above value of less than 30 (S-2 strain shows a value of 3) (S-2 strain in FIG. 10 is in a transparent state) (a strain with high cell sedimentation) has a hydrophobic cell surface and a cell Since they are easy to aggregate, they are not easily affected by various degrading enzymes derived from Aspergillus oryzae, and when they are aggregated, the surface area per cell subjected to the enzymatic action is small, and it becomes difficult to obtain raw soy sauce with high clarity.
[0029]
The growth and cell sedimentation of various soy lactic acid bacteria in a liquid medium supplemented with 10% sodium chloride were measured. The results are shown in FIG.
[0030]
The growth and cell sedimentation of various soy lactic acid bacteria in a liquid medium supplemented with 18% sodium chloride were measured. The results are shown in FIG.
[0031]
From the results of FIG. 7 and FIG. 8, the concentration of sodium chloride contained in the liquid medium is preferably 8 to 12%, particularly about 10%, and if it is 13% or more, the specific gravity of the medium itself becomes large. Even in this case, the cells tend not to settle, that is, the difference in cell sedimentation based on the difference in the strain tends to be difficult to visually determine.
[0032]
In the present invention, based on such knowledge, as a method for evaluating the viability of the soy sauce lactic acid strain under a low pH environment and obtaining it, the following (1) to (1) to (5) A method for separating lactic acid bacteria having a low turbidity soy sauce characterized by combining the above means alone or in combination.
(1) Into a test tube having a length of 160 mm and an inner diameter of about 15 mm, 10 ml of a liquid medium containing 8 to 12% (W / V) sodium chloride is inoculated, inoculated with the test bacteria, and left at 30 ° C. for 48 hours. Cultivate and gently collect 4-6 ml of upper layer without shaking the liquid level, measure the absorbance (b) at 600 nm after stirring uniformly, and restore the total amount of the collected upper layer to the original culture solution. After the whole is uniformly stirred, the absorbance (b) at 600 nm is measured, (b x 100) / b is calculated, and a strain having a value of 30 or more is isolated.
(2) Aseptic soy sauce cake obtained by uniformly mixing a 0.1M potassium phosphate buffer (pH 7.0) containing 0.5% (W / V) sodium benzoate and soy sauce cake and then filtering the mixture. Suspend the test bacteria in the crude extract and adjust the absorbance at 600 nm to 0.5, then leave it at 30 ° C. for 7 days with shaking at 100 rpm, and measure the absorbance (c) at 600 nm again. Then, (0.5−c) × 100 / 0.5 is calculated, and a strain whose value is 15 or more is isolated.
(3) The test strain is inoculated and cultured in an agar medium containing 0.2 to 0.25% (W / V) cobalt chloride and no salt, and the growing strain is isolated.
(4) The test strain is inoculated and cultured in an agar medium containing 0.12% (W / V) or more of neomycin (titer 650 U / mg), and the growing strain is isolated.
(5) 0.2-1.0% (W / V) of aspartic acid, arginine, tyrosine, histidine or phenylalanine is added to the basic culture medium to sterilize, inoculate and inoculate the test bacteria, The presence or absence is observed, and a strain in which any amino acid is not degradable is isolated.
[0033]
Specifically, a method using a difference in neomycin resistance, that is, “separation discrimination test 1” can be mentioned. In addition, as a method for evaluating the presence or absence of amino acid decomposability of aspartic acid, arginine, tyrosine, histidine or phenylalanine, “Separation Identification Test 2” can be mentioned. In addition, as a method for evaluating the degradability of cells, a method utilizing the difference in cell sedimentation associated with proliferation in stationary culture in a liquid medium containing 8 to 12% (W / V) sodium chloride. And a method using a degradation enzyme group solution (raw crude extract) produced by soy sauce koji mold, that is, “Separation Identification Test 3” and a method for preparing a highly preservative degradation enzyme group solution, By using the method of indirectly evaluating the high fermentative ability, that is, "separation identification test 4", or a combination of these, it is killed earlier in the post-ripening process that becomes the target low pH environment, Furthermore, soy sauce lactic acid strains that are rapidly decomposed by the action of a group of degrading enzymes derived from Aspergillus are easily and quickly separated and identified.
[0034]
FIG. 4 shows a method for separating and identifying low-turbidity soy lactic acid bacterial strains by combining the following four procedures (separation identification test 1 + separation identification test 2 + separation identification test 3 + separation identification test 4).
[0035]
Isolation discrimination test 1 (Separation discrimination test of low turbidity soy sauce lactic acid strain by neomycin resistance evaluation test using agar medium containing 0.12% (W / V) neomycin)
1.0 g glucose, 1.0 g polypeptone [manufactured by Kyokuto Chemical Co., Ltd.], 0.4 g yeast extract [manufactured by DIFCO], 0.3 g potassium dihydrogen phosphate, 0.2 g dipotassium monohydrogen phosphate, 0.1 g sodium thioglycolate 3.3 g sodium acetate, 1.2 to 1.5 g agar [manufactured by Wako Pure Chemical Industries, Ltd.] was added and dissolved in distilled water, and an aqueous sodium hydroxide solution was added thereto to adjust the pH of the solution to 7.0. Later, the total volume is 95 ml, and heat sterilization is performed at 121 ° C. for 15 minutes in a pressure heat sterilizer. On the other hand, 5 ml of 2.4% (W / V) neomycin sulfate [Nacalai Tesque reagent, titer 650 U / mg] aqueous solution was prepared, and this was used with Whatman syringe filter (25 mmGD / X). Filter the bacteria and sterilize.
After cooling the heat-sterilized agar solution (95 ml) to 46 ± 2 ° C, add 5 ml of neomycin sulfate aqueous solution under aseptic conditions, stir well, and then dispense 10 ml each into a sterile petri dish. (Agar medium containing 0.12% (W / V) neomycin sulfate).
1.0 g glucose, 1.0 g polypeptone [manufactured by Kyokuto Chemical Co., Ltd.], 0.4 g yeast extract [manufactured by DIFCO], 0.3 g potassium dihydrogen phosphate, 0.2 g dipotassium monohydrogen phosphate, 0.1 g sodium thioglycolate 3.3g Sodium acetate dissolved in distilled water, adjusted to pH = 7.0 using aqueous sodium hydroxide solution, adjusted to pH = 100ml, sterilized with pressure heat sterilizer (not containing neomycin sulfate) ) The test strain that has been cultured in 5 to 10 ml to stabilize the growth activity is smeared on the surface of this agar medium containing 0.12% (W / V) neomycin sulfate, Incubate anaerobically for 7 days. Only the strain that has grown on this medium and formed a bacterial colony is evaluated as a low-turbidity soy sauce lactic acid strain, and subjected to the following separation and identification test 2.
[0036]
Separation discrimination test 2 (separation discrimination test of low-turbidity soy sauce lactic acid bacteria using asparagine degradability, arginine degradability, tyrosine degradability, histidine degradability as an index)
2.0 g glucose, 1.0 g polypeptone [manufactured by Nippon Pharmaceutical Co., Ltd.], 0.3 g yeast extract [manufactured by DIFCO], 0.05 to 0.2 g dipotassium hydrogen phosphate, 3.3 g sodium acetate, 0.1 g thioglycol Dissolved sodium acid, 5g sodium chloride in distilled water, adjusted to pH = 7.2 using sodium hydroxide aqueous solution, made up to 100ml, and sterilized with pressure heat sterilizer at 121 ° C for 15 minutes A 5-10 ml medium is inoculated with a soy sauce lactic acid strain to be used for the test and cultured at 30 ° C. for 2 days.
0.1-0.2 g glucose, 0.5 g meat extract [manufactured by DIFCO], 0.5 g yeast extract [manufactured by DIFCO], 0.5 g polypeptone [manufactured by Nippon Pharmaceutical Co., Ltd.], 0.1 g sodium thioglycolate, 15 g chloride Dissolve sodium, 0.0005 g pyridoxine hydrochloride in distilled water, add 1 ml of ethanol solution with 0.6% (W / V) bromocresol purple, and add 2.0 g of sodium L-aspartate monohydrate to this. A liquid medium (hereinafter referred to as an aspartic acid medium) that was dissolved in an aqueous solution of sodium hydroxide and adjusted to pH = 7.2 with an aqueous sodium hydroxide solution and sterilized at 121 ° C. for 15 minutes with a pressure heat sterilizer, 1 0.0 g of L-arginine hydrochloride is dissolved and adjusted to pH = 7.2 with an aqueous sodium hydroxide solution to make a total volume of 100 ml. Dissolve a liquid medium (hereinafter referred to as arginine medium) sterilized at 121 ° C. for 15 minutes, 1.0 g of L-histidine hydrochloride monohydrate, and adjust the pH to 7.2 with an aqueous sodium hydroxide solution. A total of three types of liquid culture medium (hereinafter referred to as histidine medium) sterilized at 121 ° C. for 15 minutes in a pressurized heat sterilizer with 100 ml, and the strains cultured in the medium of these three types of medium Inoculate the culture solution and culture for 3 to 7 days at 30 ° C., while the degree of turbidity of the medium accompanying the growth of the strain and yellowing of the medium (the color of the medium not inoculated with the strain is purple, When the respective amino acids are decomposed while growing in each medium, the medium turns yellow. A strain in which no yellowing is observed in any of the three types of media, aspartic acid medium, arginine medium, and histidine medium, is selected.
Furthermore, 1.0 g glucose, 0.3 g yeast extract [manufactured by DIFCO], 1.0 g polypeptone [manufactured by Nippon Pharmaceutical Co., Ltd.], 1.0 g dipotassium monohydrogen phosphate, 0.1 g sodium thioglycolate, 15 g sodium chloride, After dissolving 2.2 g agar in distilled water and adjusting to pH = 7.2 with an aqueous sodium hydroxide solution, the total volume was adjusted to 100 ml, and then sterilized with a pressure heating sterilizer at 121 ° C. for 15 minutes, then 45 Cool to ~ 50 ° C.
Finely pulverize the particles using a mortar or the like, and further sterilize in a dry heat sterilizer at 130 ° C. for 3 hours. 0.2 to 0.3 g of L-tyrosine is sterilized under aseptic conditions. In addition, with good mixing, dispense 10 ml each into a sterilized petri dish and harden (hereinafter referred to as tyrosine medium).
Among the colonies formed on the tyrosine medium, the tyrosine medium was inoculated with a strain in which no yellowing was observed in any of the above three types of medium, and was cultivated at 30 ° C. for 7 to 14 days. In addition, a turbidity-free soy sauce lactic acid bacterium is obtained in a colony periphery.
[0037]
Separation discrimination test 3 (separation discrimination test of low turbidity soy sauce lactic acid bacteria by digestion test of soy sauce lactic acid bacteria cells using soy sauce koji crude extract)
Mix 500g of steamed soybeans and 500g of roasted wheat, inoculate them with soy sauce koji (Aspergillus oryzae), and 0.5% W / V) Add 1 L of 0.1 M potassium phosphate buffer solution (pH = 7.0) containing sodium benzoate, stir well, leave it at 30 ° C. for 1-2 hours, and then manufactured by Toyo Roshi Kaisha, Ltd. Filtration is performed using K filter paper (ADVANTEC-TOYO, No. 2), and the filtrate is further centrifuged for about 10 minutes under a rotor rotation speed of 15000 rpm and 4 ° C., and the supernatant is recovered (this is Hereinafter referred to as “soy sauce crude extract”. The crude soy sauce extract is further sterilized by performing sterilization filtration using a Whatman syringe filter (25 mm GD / X).
The soy sauce lactic acid strains to be used for the test were 1% (W / V) glucose, 1% polypeptone (manufactured by Nippon Pharmaceutical), 0.4% yeast extract (manufactured by DIFCO), 0.3% (W / V) dihydrogen phosphate. Potassium, 0.2% (W / V) dipotassium monohydrogen phosphate, 0.1% (W / V) sodium thioglycolate, 3.3% (W / V) sodium acetate, 10% (W / V) Except for strains that have been left to stand at 30 ° C for 4-7 days in a liquid medium (pH = 7.0) composed of sodium chloride, and the upper part of the culture solution is clear and clear, that is, strains with high cell sedimentation properties. Only strains showing a state in which the upper part of the culture solution becomes cloudy due to the suspension of cells, that is, strains with low cell sedimentation properties, are 1.0% (W / V) glucose and 0.3% (W / V) yeast. Extract [made by DIFCO], composed of 10% (V / V) concentrated raw soy sauce, After stationary culture at 30 ° C. for 7 days in a liquid medium (pH after sterilization = 7.0) with sodium chloride added so that the final concentration becomes 15%, the rotor rotation speed is 7000 to 10000 rpm, and 4 ° C. cooling. Centrifugation is carried out for 10 minutes under the conditions, and the bacterial cells are collected, stirred in a 10% aqueous sodium chloride solution, and then thoroughly washed by centrifuging under the same conditions.
The cells of the test strain prepared in this way are suspended under sterile conditions in the previous sterilized soy sauce cake crude extract so that the absorbance at 600 nm in the spectrophotometer is 0.5. Adjust the amount of cells suspended in the crude soy sauce extract.
10 ml of the soy sauce crude extract in which the cells of the soy sauce lactic acid strain were suspended so that the absorbance at 600 nm shows 0.5 was prepared and shaken aseptically for 7 days in a state kept at 37 ° C. (100 ˜200 rpm.), And then the absorbance (A) at 600 nm is measured again using a spectrophotometer. Based on this, the decrease rate of absorbance for 7 days, that is, (0.5−A) /0.5 is calculated. The strain having this value of 15 or more is used as a test strain for the following separation and identification test 3.
In the case of performing multiple evaluation tests, soy sauce to be used for the test by adding one or two types of strains already evaluated in the first test to the second and subsequent test strains. It is possible to reduce the error of the test result caused by the component error of the cocoon or its crude extract.
[0038]
Separation discrimination test 4 (separation discrimination test of low turbidity soy sauce lactic acid strain by cobalt chloride tolerance evaluation test using a salt-free agar medium containing 0.2 to 0.25% (W / V) cobalt chloride)
1.0 g glucose, 1.0 g polypeptone [manufactured by Kyokuto Chemical Co., Ltd.], 0.4 g yeast extract [manufactured by DIFCO], 0.3 g potassium dihydrogen phosphate, 0.2 g dipotassium monohydrogen phosphate, 0.1 g sodium thioglycolate 3.3 g sodium acetate, 1.2-1.5 g agar [manufactured by Wako Pure Chemical Industries, Ltd.] was dissolved in distilled water, an aqueous sodium hydroxide solution was added to adjust the pH of the solution to 7.0, and the total volume was 95 ml. And sterilize at 121 ° C. for 15 minutes in a pressure heating sterilizer. On the other hand, a 4 to 5% (W / V) aqueous solution of cobalt chloride (CoCl2 · 6H2O) is prepared, which is sterilized by sterilization using a Whatman syringe filter (25 mmGD / X).
After cooling 95 ml of the heat-sterilized agar solution to 46 ± 2 ° C., add 5 ml of cobalt chloride aqueous solution under aseptic conditions, stir well, and then dispense 10 ml each into a petri dish. (0.2 to 0.25% (W / V) agar medium containing cobalt chloride).
1.0 g glucose, 1.0 g polypeptone [manufactured by Kyokuto Chemical Co., Ltd.], 0.4 g yeast extract [manufactured by DIFCO], 0.3 g potassium dihydrogen phosphate, 0.2 g dipotassium monohydrogen phosphate, 0.1 g sodium thioglycolate Dissolve 3.3 g sodium acetate in distilled water, adjust to pH = 7.0 using aqueous sodium hydroxide solution, adjust the total volume to 100 ml, and sterilize using pressurized heat sterilizer (cobalt chloride) Liquid culture medium not containing the test strains cultured in 5 to 10 ml to stabilize the growth activity on the surface of the agar medium containing 0.2 to 0.25% (W / V) cobalt chloride. Smear and incubate under anaerobic conditions at 30 ° C for 7 days. Only strains that have grown on this medium and formed bacterial colonies are evaluated as low turbidity soy sauce lactic acid strains.
[0039]
Example 1 (soy sauce brewing test using soy sauce lactic acid strains evaluated as low turbidity soy sauce lactic acid strains by this method and other soy sauce lactic acid strains)
Two low-turbidity soy lactic acid bacterial strains (S-16 and N-4) selected from the soy sauce lactic acid bacteria group isolated from natural soy sauce moromi, through the above-mentioned separation identification tests 1, 2, and 3; A small-scale soy sauce preparation test was conducted using one soy sauce lactic acid bacterial strain (S-2 strain) that did not grow on the agar medium of the same test 2 and the same test 3, and no formation of fungal colonies was observed.
Moromi was prepared according to the method of Sekine et al. (Soken, 13, 149 (1987)). Each soy sauce lactic acid strain is composed of 1.0% (W / V) glucose, 0.3% (W / V) yeast extract [manufactured by DIFCO], 10% concentrated soy sauce, and the final concentration after preparation is 15 % In a liquid medium (pH after sterilization = 7.0) in which sodium chloride was added so that the amount of the moromi was inoculated at 30 ° C. for 7 days. The number of bacteria was adjusted to 10 5 cfu / g.
In order to make it easy to observe the turbidity of soy sauce due to the soy sauce lactic acid strain, the preparation period is 80 days, and the temperature control of the moromi taste is also from 15 days until the 7th day immediately after the preparation, and from the 8th day to the 14th day. 20 ° C., 25 ° C. from the 15th day to the 20th day, and after confirming that the pH of the moromi became 5.0 on the 21st day, the soy sauce main fermentation yeast (Zygosaccharomyces rouxii (Zygosaccharomyces rouxii) )) Is inoculated so that the number of bacteria in the moromi after inoculation is 10 5 cfu / g ("Treatment 1" in Table 5), or commercially available 99% or more unmodified ethanol, It was added so that the concentration in the moromi after the addition would be 3% ("Treatment 2" in Table 5), then 30 ° C until the 70th day, and 25 ° C from the 71st to the 80th day It was.
The moromi prepared for 80 days in this way was filtered through a filter paper (ADVANTEC-TOYO, No. 2) manufactured by Toyo Roshi Kaisha, and the filtrate was collected. The turbidity of each was measured with a turbidimeter. The results are shown in Table 6.
[0040]
[Table 6]

[0041]
As shown in Table 6, the S-2 strain, the S-16 strain and the N-4 strain all showed almost the same growth in moromi, but the moromi brewed with the S-16 and N-4 strains. As for the turbidity of the filtrate, the “treatment 1” to which the soy sauce main fermentation yeast solution was added showed a lower value than the moromi filtrate brewed with the S-2 strain. In addition, in the moromi ("treatment 2" in Table 6) in which ethanol was added instead of yeast on the 21st day, the S-2 strain and S in the moromi in the post-ripening process (from the 40th day in this test) No difference was observed in the viability of the -16 strain and the N-4 strain, and no difference was observed in the turbidity of the moromi filtrates of the S-2 strain, the S-16 strain, and the N-4 strain. Therefore, as described above, it can be seen that the viability of the soy sauce lactic acid strain in the moromi in the post-ripening process is closely related to the turbidity of the soy sauce. Therefore, it is very meaningful to select the soy sauce lactic acid strain to be used using the present invention.
[0042]
【The invention's effect】
According to the present invention, among wild lactic acid bacteria, lactic acid fermenting ability is strong, viability under low pH environment is low, cell surface has a high degree of hydrophilicity (low hydrophobicity), and is derived from Aspergillus. A low-turbidity soy sauce lactic acid bacterium having the property of being easily decomposed by the enzyme group can be obtained very easily and reliably.
Moreover, the culture medium which can isolate | separate the said low-turbidity soy sauce lactic acid bacteria from wild lactic acid bacteria very easily and reliably can be provided. Furthermore, the present invention is a soy sauce brewing in which soy lactic acid bacteria having excellent properties selected in advance or specifically bred are artificially added to soy sauce koji and / or moromi to stably perform lactic acid fermentation in the preparation process. In the method, the above-mentioned “low turbidity soy lactic acid bacterium” is used as the soy sauce lactic acid bacterium, and soy sauce with a high degree of clarification can be easily obtained without applying various clarifiers and filtration treatment in the production process after the preparation process. Can do.
[Brief description of the drawings]
FIG. 1 is a view showing an image obtained by centrifuging a turbid substance in raw soy sauce using a microscope.
FIG. 2 is a graph showing the relationship between the turbidity of moromi soup and the lactic acid concentration in the third month after the soy sauce moromi prepared using various soy lactic acid strains isolated from soy sauce moromi.
FIG. 3 is a diagram showing the relationship between lactic acid concentration and turbidity of commercially available concentrated soy sauce products in various parts of the country.
FIG. 4 is a view showing a method for separating and identifying a low-turbidity soy lactic acid bacterium strain.
FIG. 5 is a graph showing neomycin resistance of soy sauce lactic acid strains having various amino acid degradability.
FIG. 6 is a graph showing the relationship between the amino acid degradability of soy sauce lactic acid strains and the turbidity of raw soy sauce brewed using those strains.
FIG. 7 is a graph showing the relationship between the growth of various soy sauce lactic acid strains and cell sedimentation in a liquid medium supplemented with 10% sodium chloride.
FIG. 8 is a graph showing the relationship between the growth of various soy sauce lactic acid bacterial strains and cell sedimentation in a liquid medium supplemented with 18% sodium chloride.
FIG. 9 is a graph showing the influence of turbidity of soy sauce on the color tone of soy sauce.
FIG. 10 is a view showing the transparency of a liquid culture solution of a conventional soy sauce lactic acid strain and soy sauce lactic acid bacteria isolated according to the present invention.

Claims (5)

  A low-grade crude extract obtained by filtering a 0.1M potassium phosphate buffer solution (pH 7.0) containing 0.5% (W / V) sodium benzoate and soy sauce cake and then filtering the mixture. Medium for separation of turbid soy lactic acid bacteria.   A medium for separation of low-turbidity soy lactic acid bacteria comprising an agar medium containing 0.2 to 0.25% (W / V) cobalt chloride and not containing sodium chloride.   A medium for separation of low-turbidity soy lactic acid bacteria comprising an agar medium containing neomycin (650 U / mg) of 0.12% (W / V) or more. A method for separating a low-turbidity soy lactic acid bacterium characterized by combining the following means (1) to (5) alone or in combination.
(1) Into a test tube having a length of 160 mm and an inner diameter of about 15 mm, 10 ml of a liquid medium containing 8 to 12% (W / V) sodium chloride is inoculated. Cultivate and gently collect 4-6 ml of upper layer without shaking the liquid surface, measure the absorbance (b) at 600 nm after stirring uniformly, and restore the total amount of the collected upper layer to the original culture solution. After the whole is uniformly stirred, the absorbance (b) at 600 nm is measured, (b x 100) / b is calculated, and a strain having a value of 30 or more is isolated.
(2) Aseptic soy sauce cake obtained by uniformly mixing a 0.1M potassium phosphate buffer solution (pH 7.0) containing 0.5% (W / V) sodium benzoate and soy sauce cake, followed by filtration. Suspend the test bacteria in the crude extract and adjust the absorbance at 600 nm to 0.5, then leave it at 30 ° C. for 7 days with shaking at 100 rpm, and measure the absorbance at 600 nm again. Then, (0.5−c) × 100 / 0.5 is calculated, and a strain whose value is 15 or more is isolated.
(3) The test bacteria are inoculated and cultured in an agar medium containing 0.2 to 0.25% (W / V) cobalt chloride and no salt, and the growing strain is isolated.
(4) The test strain is inoculated and cultured in an agar medium containing 0.12% (W / V) or more of neomycin (titer 650 U / mg), and the growing strain is isolated.
(5) 0.2-1.0% (W / V) of aspartic acid, arginine, tyrosine, histidine or phenylalanine is added to the basic culture medium to sterilize, inoculate and inoculate the test bacteria, The presence or absence is observed, and a strain in which any amino acid is not degradable is isolated.   In a soy sauce brewing method in which a soy lactic acid bacterium having excellent properties selected in advance or specifically bred is artificially added to soy sauce koji and / or moromi so as to stably carry out lactic acid fermentation in the preparation process. A method for producing soy sauce with high clarity, characterized in that the low-turbidity soy lactic acid bacterium obtained in claim 4 is used.

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