JP3900484B2 - Production method of lactic acid bacteria - Google Patents

Description

【００２７】
試験例１.「本発明の液体培養法の特徴、その１： Ｇｌｃ資化後も培地に分泌した乳酸を新たに資化することによって生育を続行することによって増大化状態に至る」
【００２８】
本発明の液体培地および公知の液体培地としてＢＬ液体培地を用いてＳｆを培養し、その経時的変化を解析した。表２に示す様に、本発明の液体培地における乳酸菌の増殖パターンをＢＬ液体培地の場合と比較すると両者の違いが顕著であり、本発明の特徴を次のように要約できる：
１）本発明の液体培地でＳｆを培養すると、菌体数は培地１ｍｌ当たり５００億 〜７００億に至り、ＢＬ液体培地に比べて約２０倍も増大化した。
２）本発明の培養条件で最大菌数に到達するまでの培養過程において、Ｓｆは培養１４時間目までは資化し易いＧｌｃを優先的に消費した。培地からＧｌｃを消費し尽くした１９時間目以降、培地に分泌され蓄積してきた乳酸濃度が減少に転じ、乳酸を資化するようになった。こうして、培地ｐHもアルカリ側へ移行し、やがて２７時間目で菌数が最大値に至った。
【００２９】
一方、ＢＬ液体培地では、培地のＧｌｃが消費された１９時間目で最大菌数３０億に到達した。それ以降の培養の継続でも菌数が増加しなかった他、培地に分泌された乳酸濃度の減少および培地ｐHのアルカリ化現象も見られなかった。
なお本発明の培養で見出された特徴と類似する現象は、培地のＧｌｃ濃度をあらかじめ低濃度に設定した場合、培地の炭素源として添加した乳酸が資化され、そして培地から乳酸消失に伴い培地ｐＨがアルカリ化したことがＳｔｒｅｐｔｏｃｏｃｃｕｓ ｍｕｔａｎｓで報告されている（Ｈｕ，Ｇ，および Ｓａｎｄｈａｍ，Ｈ.Ｊ.： Ａｒｃｈｉｖｅｓ ｏｆ Ｏｒａｌ Ｂｉｏｌｏｇｙ、１７巻、７２９−７４３頁、１９７２年）。このことから、本発明の培養において、最大菌数に至る過程で発生した乳酸量の減少は、菌体がＧｌｃに代わる新たな炭素源して乳酸を資化した現象と示唆される。
【００３０】
１）耐熱性は、本発明の条件では培養４〜８時間目の対数増殖期で低く（０.０４％以下）、培養２０〜２７時間目の定常期で高かった（４０％以上）。そして、最大菌数に至った２７時間目を超えて培養を継続した場合、３０時間目以降から急激に耐熱性が低くなった（２０％以下〜数％）。一方、ＢＬ液体培地では、同様に対数増殖期の培養４〜８時間目で低く（０.０４％以下）、最大菌数に到達した１９時間目は高い耐熱性であったが（３３％）、更に培養を継続しても１０〜１２％とほぼ一定の耐熱性が維持された。
本発明の培地で乳酸菌を培養すると、定常期の到達最大菌密度付近では高い耐熱性が得られる。その要因の一つは、ペプトンを高濃度添加することであり、Ｇｌｃを資化し終えた後も増殖するためにペプトンの栄養素を資化する。従って、最大菌数に到達した後、急激に耐熱性が低下する理由は、培地からペプトンの栄養素が減った為と推察される（表５参照）。
【００３１】
培養条件： ＢＬ液体培地１０ｍｌにＳｆを一白金耳量接種し、３０℃で一夜振盪培養した。この定常期の菌液１.０ｍｌを液体培地５０ｍｌ（綿栓付５００ｍｌ三角フラスコ使用）に接種し（接種菌数、０.１５ｘ１０^８個/ｍｌ）、３０℃で２００ｒｐｍの振盪条件で培養した。表２に示した各培養時間においてサンプリグし、その時点の培地ｐＨ、到達菌数、耐熱性および乳酸濃度などを分析した。なお、Ｇｌｃは他の培地成分と別に滅菌し、接種前に他の培地成分と混合した。
本発明の液体培地組成（重量％）：８.４３％炭酸マグネシウム、８.０％Ｇｌｃ、１２.０％ハイニュートＳＭＰおよび３.２％ＷＴ。
【００３２】
分析方法：
到達菌数の測定法は、培養液０.５ｍｌを下記の組成のリン酸バッファー４.５ｍｌで１０倍ずつ段階希釈し、その０.１ｍｌをＳＣＤ寒天培地「ダイゴ」（日本製薬（株）製）２〜３枚ずつ塗沫した。このプレートを３７℃で２４〜４０時間培養し、プレート上に生成した集落数を測定し、その平均値を液体培地１ｍｌ当たりに換算し、ｃｆｕと表現した（ｃｏｌｏｎｙ ｆｏｒｍｉｎｇ ｕｎｉｔ）。リン酸バッファーの組成：塩化ナトリウム７ｇ、リン酸二カリウム５.３１ｇ、リン酸一カリウム２.６５ｇ、ツイーン８０ ２.０ｇおよび脱イオン水で最終液量を１０００ｍｌ（ｐH６.８）に調整。
１） 培地に分泌された乳酸の濃度は、培養液に含まれるＬ−乳酸を特異的に定量分析する乳酸定量分析用試薬キット、Ｆ―キットＬ―乳酸（（株）Ｊ.Ｌ.インターナショルより購入）を用いて測定した。
２） D−グルコースの培地濃度の測定は、市販のグルコース定量分析用試薬キット、F−キットD−グルコース（（株）Ｊ.Ｌ.インターナショルより購入）を用いて行った。
３） 耐熱性は、到達菌数の測定時にリン酸バッファー（ｐH６.８）で段階希釈した液を６０℃、１５分間処理後、ＳＣＤ寒天培地に０.１ｍｌを塗沫し、熱処理後の生存菌数を測定した。耐熱性は熱未処理に対する菌数残存率％で表した。
【００３３】
【表２】

【００３４】
試験例２ 「本発明の液体培養の特徴、その２： 増大化に及ぼす無機塩類の特異性」
本発明の液体培養条件において、高濃度の無機塩を添加した場合のＳｆ増大化に及ぼす特異性を検討し、得られた結果を表３に示した。
【００３５】
その結果、無機塩に関する特徴として以下の点が判明した：
１）ＷＴを３.２％、ＧｌｃおよびハイニュートRを各々１０％もの高濃度で培地に添加した場合、無機塩を添加しなくても１２０ｘ１０^８ｃｆｕ／ｍｌもの高い菌数が得られた。 従来の回分培養法ではこの程度の菌数が到達し得る上限であり、添加物を追加しても菌数を更に増加させ得なかった。 本発明者らは、この培地条件に対して、更に高濃度の無機塩添加による増大効果を検討した。 その結果、表３.に示した様に、炭酸カルシウムを６.００％あるいは炭酸マグネシウムを５.０６％もの高濃度添加した場合、更に増大することを見出した。 この他の無機塩としては、炭酸水素カリウムが無添加の条件に比べ菌数を若干高めたが、塩化カルシウムなどいずれも無添加の条件に比べて低い到達菌数となった。
【００３６】
２）特徴的なことは、難溶性の性質を有する硫酸カルシウム（石膏）、炭酸マグネシウム、あるいは炭酸カルシウムを添加した場合、いずれも耐熱性が無添加の場合とほぼ同様の５０％前後であった。一方、塩化ナトリウムなどの水溶性無機塩を添加した場合、いずれも耐熱性が低下した。 これら難溶性の無機塩を添加した場合、難溶性の性質のおかげで菌体に及ぼす作用が少ないおかげで耐熱性の低下を防止したと考えられるが、これら難溶性無機塩であっても添加濃度が高過ぎれば耐熱性を低下させる場合も見られた。
【００３７】
３）培地の乳酸濃度は、培養後も培地ｐHが高い状態を維持し、そして菌数が高いと云う両条件を満たした場合に高かった。 その中で、難溶性の炭酸マグネシウムを添加した場合が最も高い乳酸濃度となり、重質あるいは軽質いずれも同じ効果であった。一方、炭酸マグネシウムと同じマグネシウム塩であっても水溶性の塩化マグネシウムおよび硫酸マグネシウムは、無機塩無添加に比べて、到達菌数のみならず乳酸濃度も低かった。 ちなみに、菌数当たりの乳酸濃度（ｍｇ／億）は、塩化ナトリウム（０.７０）、塩化マグネシウム（０.３２）、炭酸マグネシウム（０.１４）、炭酸カルシウム（０.１１）、硫酸マグネシウム（０.０９３）の順に高く、本発明の液体培地の基本組成に塩化マグネシウムおよび硫酸マグネシウムを添加しても乳酸産生の促進効果があるとは認められなかった。
【００３８】
培養条件：ＢＬ液体培地１０ｍｌにＳｆを一白金耳量接種し、３０℃で一夜振盪培養した。 この定常期の菌液０.１ｍｌを液体培地１０ｍｌ（綿栓付５０ｍｌ試験管）に接種し（接種菌数０.２５ｘ１０^８個/ｍｌ）、３０℃で２１時間培養した。振盪速度は３００回／分と設定した。 グルコースは他の成分と別に滅菌し、接種前に残りの成分と混合した。
液体培地組成（重量％）：０.６M無機塩類、１０.０％Ｇｌｃ、１０.０ハイニュートRおよび３.２％ＷＴ。
【００３９】
【表３】

【００４０】
試験例３ 「本発明の液体培養の特徴、その３： Ｇｌｃおよびペプトンに対する補充培地栄養素の特異性は菌種により異なる」
本発明の液体培養条件における補充栄養素の特異性に関して、表４に示した結果より、
以下の特徴が見出された：
１）増大化に及ぼす補充栄養素の特異性は使用する菌種により異なることが判明した。 Ｓｆで効果が見られた成分はＷＴの他、ＡＫ、ＬＦＫ、胚芽培地、酵母末あるいは酵母エキスであった。 一方、Ｌ. ｃａｓｅｉ ＮＲＩＣ１９１７株に対して効果が見られた成分は、ＡＫおよび胚芽培地のみに限定された。 ＡＫを添加した場合、ＳｆとＬ. ｃａｓｅｉの両菌株ともほぼ同じ到達菌数であった。
【００４１】
２）酵母末の添加はＬ. ｃａｓｅｉ ＮＲＩＣ１９１７株の増大化に対し抑制的に作用し、更に酵母エキスもＳｆの場合に比べ高い効果が見られなかった。 酵母末の添加による抑制作用は、至適成分のＡＫが共存しても起こると予想された。 このことは、ＷＴを添加した場合、ＡＫほどの効果が見られなかった。 ＷＴは、ＡＫ５０％の他に酵母末３７％およびＬＦＫ１０％で構成されており、含有する酵母末によってＡＫの増大化効果が抑制されたと推察される。
【００４２】
３）Ｓｆの増殖は胚芽培地に比べてＬＦＫを添加した方が促進的に作用したが、Ｌ.ｃａｓｅｉ ＮＲＩＣ１９１７株の増殖はＬＦＫも胚芽培地と全く同じ到達菌数であった。ＬＦＫは胚芽培地においてＳｆを固体培養した麹であり、固体培養中に同菌株に対する増殖促進物質を分泌しており、そしてこの物質は異種のＬ. ｃａｓｅｉ菌株に対して増殖促進作用を及ぼさない性質と推察された。
【００４３】
培養条件：ＢＬ液体培地１０ｍｌにＳｆおよびＬａｃｔｏｂａｃｉｌｌｕｓ ｃａｓｅｉ ＮＲＩＣ１９１７株を各々一白金耳量接種し、３０℃で一夜振盪培養した。 これら定常期の菌液０.１ｍｌを液体培地１０ｍｌ（綿栓付５０ｍｌ試験管）に接種し、３０℃でＳｆを２０時間、Ｌ. ｃａｓｅｉ ＮＲＩＣ１９１７株を２７時間振盪培養した（振盪速度３００回／分）。なお、グルコースは他の培地成分とは別に滅菌し、接種する直前に他の成分と混合した。補充栄養素として使用した胚芽培地は、アスペルギルス・オリゼーおよび乳酸菌を固体培養して得たＡＫあるいはＬＦＫの培地であり、脱脂米胚芽と脱脂小麦胚芽の混合物である。
液体培地（重量％）：共通成分として５％炭酸カルシウム、１０％Ｇｌｃおよび５％ハイニュートＳを添加する他、補充栄養素を３.２％添加した。なお、補充栄養素も添加し、オートクレーブ滅菌後の培地ｐＨは次の通りである：無添加、７.５３；ＷＴ、７.０９；ＡＫ、７.０６；ＬＦＫ、７.２９；胚芽培地、７.４７；酵母末、７.１２；酵母エキス、６.６４。
【００４４】
【表４】

【００４５】
試験例４ 「本発明の液体培養の特徴、その４： 増大化は嫌気的培養条件よりも好気的な方が適」
増大化に及ぼす好気的および嫌気的振盪培養による影響に関し、表５に示した結果から、以下の特徴が判明した：
１）好気的培養条件の方が嫌気的条件に比べて高菌数化した。ただし、嫌気的条件でも２７０ｘ１０^８ｃｆｕ／ｍｌもの高い到達菌数になったことから、酸素があると生育し難い偏性嫌気性乳酸菌でもその属や種を問わず本発明の培養条件によって増大化し得ることを示唆する。
【００４６】
２）Ｓｆを液体培養によって増大化させるには、好気的および嫌気的いずれもの培養条件であっても、(1)炭酸マグネシウムおよび炭酸カルシウム混合物、(2)Ｇｌｃ、(3)ハイニュートＳ、および(4)ＷＴより構成された４要素が液体培地組成の必須要件であることが分かった。ちなみに、到達菌数が高いことおよび培地ｐＨがアルカリ性に近いことの両条件を満たす程、培地乳酸濃度も高くなった。
【００４７】
３）高い耐熱性を確保するためには、培地成分としてＧｌｃおよびハイニュートの両者が高濃度添加されていることが必要であり、特にペプトンの添加が重要である事が分った。
【００４８】
培養条件：ＢＬ液体培地１０ｍｌにＳｆを一白金耳量接種し、３０℃で一夜振盪培養した。 この定常期の菌液０.１ｍｌを液体培地１０ｍｌ（綿栓付５０ｍｌ試験管）に接種し（接種菌数、０.３５ｘ１０^８ｃｆｕ／ｍｌ）、綿栓して好気的培養条件とする一方、炭酸ガスを封入しゴム栓して嫌気的培養条件とした。 両者を３０℃で２０時間振盪培養した（振盪速度３００回／分）。 なお、グルコースは他の培地成分と一緒に滅菌した。
【００４９】
【表５】

【００５０】
試験例５ 「本発明の液体培養の特徴、その５： 増大化にとって培養時の振盪が必須要件。炭酸カルシウム単独添加時の各種培地成分の濃度範囲。」
本発明の培養条件において高菌数化に及ぼす必須因子の解析を行ない、表６−２.に示した試験例の結果から、以下のことが判明した：
【００５１】
１）本発明の培地組成を用いて静置培養した場合、１００ｘ１０^８ｃｆｕ／ｍｌに届かない程の低い菌数であった。ちなみに、本発明の培地組成から逸脱した条件では、培地Ｎｏ.２２や２３の様に、振盪培養よりも静置培養の方が高い到達菌数になった例も見られた。
【００５２】
乳酸菌（Ｌｅｕｃｏｎｏｓｔｏｃ ｍｅｓｅｎｔｅｒｏｉｄｅｓ）を振盪することにより好気的に培養した場合、静置培養した場合に比べて、ＮＡＤＨオキシダーゼ活性が顕著に高いことが報告され、この酵素が酸素に対する終末電子受容体として働く為、振盪によって液体培地の酸素濃度を培地へ補給することによって酸素を消費しＡＴＰを生産し易くしてやり、その結果菌体濃度が増加することが報告されている（Ｃｏｎ，Ａ.ら：Ｊｏｕｒｎａｌ ｏｆ Ｇｅｎｅｒａｌ Ｍｉｃｒｏｂｉｏｌｏｇｙ，１３２巻、１７８９−１７９６頁、１９８６年）。 従って、本発明における振盪必須性の要因は、(1)水不溶性の炭酸カルシウムにとって培地を振盪することで培地における均一性を確保し易くなること、そして(2)ＮＡＤＨオキシダーゼ活性の増大化が考えられる。
【００５３】
２）本発明の培地組成では、振盪培養によって高い菌密度に到達した。静置培養によって低い菌密度であったにもかかわらず、振盪培養よりも培地乳酸濃度が高かった場合がＮｏ.１５、１７および２０で見られた。 培地ｐＨは静置培養の方が振盪培養よりも酸性側にシフトしており、菌体当たりの乳酸などの有機酸産生量は静置培養の方が高く、静置培養の方が嫌気的雰囲気の高いおかげて乳酸産生が活発化したと推察された。
【００５４】
３）ＷＴの添加量は３.２％が至適であり、それより高い濃度を添加すると、到達菌数が低下した。
４）炭酸カルシウムの添加濃度を増やすほど、Ｓｆの到達菌数が高くなった。
５）炭酸カルシウムおよびＧｌｃと共に、ペプトンとしてポリペプトンおよび補充栄養素として酵母エキスを用いても増大化した。
６）ガラクトースを炭酸カルシウム添加系においてＧｌｃの代わりに添加した場合、到達菌数は２００ｘ１０^８ｃｆｕ／ｍｌとＧｌｃと同様の増大状態に至ったものの、振盪培養後の培地ｐＨが培養前の培地ｐＨよりもアルカリ側に若干ではあるがシフトしていた。培地乳酸濃度から逆算すると、約０.２程度ｐＨが酸性側にシフトすべきであるので、培養中にアルカリ物質が分泌されたと思われた。
【００５５】
培養条件： ＢＬ液体培地１０ｍｌにＳｆを一白金耳量接種し、３０℃で一夜振盪培養した。この定常期の菌液０.１ｍｌを表６−１.に示した組成の液体培地１０ｍｌ（５０ｍｌ試験管）に接種した（接種菌数０.３５ｘ１０^８ｃｆｕ／ｍｌ）。 試験管に綿栓をして好気的に３０℃・２０時間培養したが、その際、振盪（振盪速度３００回／分）と静置の両条件で行った。 両培養条件とも培養を終了した後、直ちに到達菌数を測定した。 Ｄ−グルコースおよびガラクトース（Ｇａｌ）は他の培地成分と別に滅菌し、接種する前に混合した。更に、公知の液体培地としてブリッグス変法培地を用いた[培地組成：２.０％Ｇｌｃ,１.５％ポリペプトン、０.６％酵母エキス、０.５％塩化ナトリウム、０.１％レオドールＴＷ１２０、０.０５％可溶性澱粉、０.０５％L―システイン塩酸塩、０.２％軽質炭酸カルシウム、２０％トマトジュース、７.５％肝臓エキス]
【００５６】
【表６−１】

【００５７】
【表６−２】

【００５８】
試験例６ 「本発明の液体培養の特徴、その５： 炭酸マグネシウム単独添加系における各種培地成分の有効濃度範囲」
本発明の培養条件において高菌数化に及ぼす必須因子の解析を更に行ない、表６および７に示した試験結果から、増大化に必要な４つの培地成分の濃度範囲を以下の様に設定した：
(1) 炭酸マグネシウムや炭酸カルシウムは、各々２％以上の添加で増大化の効果が見られた。なお、これらの塩類を１３％より高濃度添加すると、培養タンクの底部への沈殿、培地滅菌中あるいは培養中の発泡現象などによって取り扱いが難しかった。
(2) 単糖類はいずれでも使用する菌株が資化し得るものであればいずれでも良く、Ｇｌｃの場合、４％以上の添加で増大化の効果が見られた。なお、Ｇｌｃを１５％より高濃度添加すると、培地の褐色化が大きくなり、凍結乾燥菌末への処理が難しくなった。
(3) ハイニュートは、３％以上の添加で増大化の効果が見られた。なお、ハイニュートを１３％以上もの高濃度添加すると、培地コストが大きくなり望ましくなかった。
(4) ＷＴは、０.５％以上の添加で増大化の効果が見られ、その至適濃度は３.２％であった。 なお、５％より添加量を増やすと不溶性残渣が多くなり取り扱いが難しくなり、その場合には麹エキスを使用すれば問題なかったものの、麹エキス製造費が増える問題があった。
【００５９】
培養条件： ＢＬ液体培地１０ｍｌにＳｆを一白金耳量接種し、３０℃で一夜振盪培養した。 この定常期の菌液０.５ｍｌをを表７.に示した組成の培地５０ｍｌ（綿栓付５００ｍｌ三角フラスコ）に接種し（接種菌数０.３８ｘ１０^８ｃｆｕ／ｍｌ）、３０℃・１９時間・２００ｒｐｍで振盪培養した。 Ｇｌｃは他の成分と別滅菌した。
【００６０】
【表７】

【００６１】
試験例７ 「本発明の液体培養の特徴、その５： 増大化に及ぼす糖質の特異性」
Ｓｆが資化して乳酸を産生できる糖質を選択し（ＢＥＲＧＥＹ'ｓ Ｍａｎｕａｌ ｏｆ Ｓｙｓｔｅｍａｔｉｃ Ｂａｃｔｅｒｉｏｌｏｇｙ、 Ｖｏｌｕｍｅ ２， Ｗｉｌｌｉａｍｓ ＆ Ｗｉｌｋｉｎｓ，１０６４頁、１９８６年）、増大化に及ぼす糖質の範囲を解析した。その結果、炭酸カルシウム添加条件では増大化に有効であったガラクトースが無効となった他、グリセリンおよびマンニトールが無効であったことを除けば、いずれの糖も増大化した。中でも、Ｇｌｃなどの六炭糖が最も高い効果を示し、二糖類ではマルトースが高い効果を示した。
【００６２】
試験方法
ＳｆをＢＬ液体培地５０ｍｌに一白金耳量を接種し、好気的に３０℃・１２０ｒｐｍの振盪条件で８時間培養した。到達菌数は３２ｘ１０^８ｃｆｕ／ｍｌであり、その時の培地ｐＨは４.６９であった。この定常期初期の菌液０.１ｍｌを１０ｍｌの本培養用培地に接種し、３０℃で１８時間振盪（３００回／分）しながら培養を行なった。本培養用の培地は、下記の糖類を８.０％添加したが、いずれも別滅菌し接種直前に他の成分と混合し、その共通成分（重量％）は次の通り：４.２２％炭酸マグネシウム、６.０％ハイニュートＳＭＰ、および３.２％ＷＴ（綿栓付き５０ｍｌ試験管）
【００６３】
【表８】

【００６４】
試験例８「本発明の液体培養の特徴、その６： Ｓｆの増大化に及ぼすマルトースの同時滅菌・別滅菌、発育相、および炭酸マグネシウムの添加・無添加の影響」
Ｓｆの増大化に及ぼすマルトースの同時滅菌・別滅菌、発育相、および炭酸マグネシウムの添加・無添加の影響を検討し、その結果を表９に示した。マルトースを別滅菌した培地の方が同時滅菌した培地に比べて滅菌後の培地ｐＨが高くなり、ｐＨがアルカリ変化した分、別滅菌の培地の方が増大化し易いことが分った。更に、褐変物質（メラノイジン）が滅菌中に生成すると、菌が資化し得る糖の絶対量が減少する分、乳酸生成量が減少すると見込まれる。
炭酸マグネシウムを添加した培養条件における到達菌数は、種菌の発育相に影響せず、マルトースを別滅菌した培地の方が同時滅菌培地に比べて高く、培地の乳酸濃度も別滅菌培地の方が同時滅菌培地よりも著しく高かった。一方、炭酸マグネシウムを添加しない培養条件では、到達菌数は別滅菌培地の方が同時滅菌した培地よりも若干ではあるが高くなったが、逆に培地乳酸濃度は別滅菌培地の方が同時滅菌培地より約５０％前後に減少した。
【００６５】
試験方法
ＳｆをＢＬ液体培地１０ｍｌに一白金耳量を接種し、好気的に３０℃・６.５時間振盪培養し、対数増殖期の菌体を調製した。その到達菌数は３.６ｘ１０^８ｃｆｕ／ｍｌであり、その時の培地ｐＨは６.２３であった。この対数増殖期の菌液０.１ｍｌを１０ｍｌの本培養用培地に接種し、３０℃で１８時間振盪培養を行なった。 同様にＳｆをＢＬ液体培地に接種し、好気的に３０℃で２１.５時間振盪培養し、定常期の菌体を調製した。その到達菌数は１４.５ｘ１０^８ｃｆｕ／ｍｌであり、その時の培地ｐＨは４.３２であった。この定常期の菌液０.１ｍｌを１０ｍｌの本培養用培地に接種し、３０℃で２０時間振盪培養を行なった。更に、上記の定常期の菌液をリン酸バッファー（ｐＨ６.８）で１０倍希釈し、室温で３時間放置した後、この菌液０.１ｍｌを１０ｍｌの本培養用培地に接種し、３０℃で１７時間振盪培養を行なった。本培養用の培地は、マルトース一水和物を８.０％（ｗ/ｗ）添加したが、別滅菌の場合には、別滅菌したものを接種直前に他の成分と混合し、同時滅菌の場合には他の成分と一緒に滅菌し、その他の共通成分（重量％）は次の通り：４.２２％炭酸マグネシウム（添加、無添加の別を表９に示した）、６.０％ハイニュートＳＭＰ、および３.２％ＷＴ（綿栓付き５０ｍｌ試験管）。振盪速度、３００回転／分。
【００６６】
【表９】

【００６７】
試験例９「本発明の液体培養の特徴、その７： Ｓｆの増大化に必要なヒドロタルサイト濃度」
本発明の液体培養条件において、合成ヒドロタルサイトの有効濃度下限を検討し、その結果を表１０に示した。合成ヒドロタルサイトは、添加濃度が１.０％以上において増大化に効果を示し、それに付随して培地乳酸濃度も高くなった。
【００６８】
試験方法
ＳｆをＢＬ液体培地１０ｍｌに一白金耳量を接種し、好気的に３０℃・３００回転の振盪条件で２１.５時間培養し、定常期の菌体を調製した。その到達菌数は１４.５ｘ１０^８ｃｆｕ／ｍｌであり、その時の培地ｐＨは４.３２であった。この定常期の菌液０.１ｍｌを１０ｍｌの本培養用液体培地に接種し、３０℃で２０時間振盪（速度、３００回転／分）しながら培養を行なった。本培養用の液体培地の共通成分（重量％）は次の通り：６.０％ハイニュートＳＭＰ、８.０％Ｇｌｃ、３.２％ＷＴおよび合成ヒドロタルサイト（綿栓付き５０ｍｌ試験管） Ｇｌｃは別滅菌したものを接種直前に他の成分と混合した。
【００６９】
【表１０】

【００７０】
試験例１０ 「麹エキスを用いたＳｆの増大液体培養および凍結乾燥菌末の製造」
１.液体培養
前培養は、１.０％Ｇｌｃ、１.０％ポリペプトンおよび１.０％酵母エキス１％から構成されるＧＰＹ液体培地１０ｍｌ（綿栓付き５０ｍｌ試験管）にＳｆを一白金耳量接種し、３７℃・２４時間静置培養した。ＷＴエキスは、ＷＴを脱イオン水で１０％懸濁し３０分間煮沸処理した後、９０００ｒｐｍ・１０分間遠心分離して上清画分を回収し、これをＷＴエキス（１０％）として用いた。 本発明の培地組成（重量％）は、４.２％軽質炭酸マグネシウム、８.０％Ｇｌｃ、６.０％ハイニュートＳＭＰおよび３.２％ＷＴエキスである。但し、Ｇｌｃは他の成分と別にオートクレーブで滅菌し、接種する前に他の成分と混合した（培地液量、２００ｍｌ及び１００ｍｌ）。 比較例としてＢＬ液体培地を用いた（培地液量、１８６ｍｌ及び９６ｍｌ）。
本培養は、本発明の培地および比較例の培地それぞれ２００ｍｌ（２Ｌ−綿栓付き坂口フラスコ）及び１００ｍｌ（１Ｌ−綿栓付き坂口フラスコ）に前培養したＳｆの菌液を０.３８ｘ１０^８ｃｆｕ／ｍｌの菌数になるように接種し、３０℃で２４時間振盪培養した。振盪速度は毎分１５０往復であった。
【００７１】
その結果、本発明培地の到達菌数は、培地液量２００ｍｌの場合の到達菌数が４０５ｘ１０^８ｃｆｕ／ｍｌ、そして培地液量１００ｍｌの場合の到達菌数が４６５ｘ１０^８ｃｆｕ／ｍｌであった。本発明の液体培養によって得られた総菌数は１２７，５００ｘ１０^８ｃｆｕであり、比較例として用いたＢＬ培地で培養した場合よりも２２.２倍高く得られた。ＢＬ液体培地における到達菌数は、培地液量１８６ｍｌの場合の到達菌数が１７ｘ１０^８ｃｆｕ／ｍｌ、そして培地液量９６ｍｌの場合の到達菌数が２７ｘ１０^８ｃｆｕ／ｍｌであり、培養によって得られた総菌数は５，７５４ｘ１０^８ｃｆｕであった。
【００７２】
２.凍結乾燥末の製造
凍結乾燥末の調製は以下の通りである： 各培養液のｐＨを１Ｎ塩酸でｐＨ５.０に調整して難溶性の炭酸マグネシウムを溶解した後、９０００ｒｐｍ・５分間遠心分離して菌体沈殿画分を回収した。菌体は、リン酸バッファー（ｐＨ６.８）で懸濁し、遠心分離して回収する操作を２度行い、洗浄した。その後、安定化剤（組成：１０％スクムミルクおよび１％グルタミン酸ソーダ）で菌体を懸濁した。本発明培地の培養物の場合、安定化剤の菌懸濁液５０ｍｌ中、その８ｍｌを安定化剤で更に２倍希釈して凍結乾燥した結果、白色の凍結乾燥粉末が１.９５ｇ得られ、１２，９００ｘ１０^８ｃｆｕ／ｇの菌数となり、その耐熱性が２６.０％であった。 更に、残った全量（４２ｍｌ）を凍結乾燥した結果、白色の凍結乾燥粉末が６.４９ｇ得られ、２３，４００ｘ１０^８ｃｆｕ／ｇの菌数となり、その耐熱性が１７.２％であった。従って、２００ｍｌと１００ｍｌの培地を用いて遠心洗浄〜凍結乾燥工程を経た菌数歩留は１３８％であった。本発明の液体培養物を凍結乾燥して得られた菌末の総菌数は１７７，０２１ｘ１０^８ｃｆｕであり、比較例よりも２６.４倍も高い菌数が得られた。ＢＬ培地の培養物の場合、安定化剤の菌懸濁液９.２ｍｌ中、６.２ｍｌを凍結乾燥した結果、白色の凍結乾燥粉末が０.９６ｇ得られ、５，１８０ｘ１０^８ｃｆｕ／ｇの菌数であり、その耐熱性は２３.３％であった。残った３ｍｌを更に安定化剤で約３.３倍希釈して凍結乾燥した結果、白色の凍結乾燥粉末が１.１０ｇ得られ、１，５７０ｘ１０^８ｃｆｕ／ｇの菌数となり、その耐熱性が３０.９％であった。従って、２００ｍｌ培地と１００ｍｌ培地をプールし、全工程を経た菌数歩留は１１６％であり、ＢＬ液体培養物を凍結乾燥得られた菌末の総菌数は６，７００ｘ１０^８ｃｆｕであった。
【００７３】
試験例１１. 「ジャーファーメンターを用いたＳｆの増大液体培養」
ＢＬ液体培地５０ｍｌにＳｆを一白金耳量接種し、１３０ｒｐｍで３０℃一夜振盪培養した。
この液種１０ｍｌを本発明の液体培地１０００ｍｌに接種し、所定の培養を行なった。得られた結果を表１１に示した。攪拌しない条件では到達菌数が半分程度となり、ジャーの底に沈殿物が存在する状態で培養する状態となった。到達菌数は、本発明の液体培地組成において攪拌を行なって培養した場合、大気あるいは炭酸ガスの通気いずれにおいても同じとなった。一方、培地の乳酸濃度は、炭酸ガスを通気した嫌気的攪拌培養条件の方が大気を通気した好気的攪拌培養に比べて顕著に高かくなった。
【００７４】
本発明の液体培地組成（重量％）：４.２２％炭酸マグネシウム、８.０％Ｇｌｃ、６.０％ハイニュートＳＭＰ、３.２％ＷＴ（Ｇｌｃは他の培地成分と一緒に滅菌した。滅菌後の培地ｐＨは８.０７であった。）
ジャーファーメンター：東京理科器械（株）製 ジャー ファーメンターＭＢＦ、内容量２.５Ｌ培養条件：３０℃・２４時間培養。攪拌速度、５００ｒｐｍ。通気量、１ｖｖｍ
【００７５】
【表１１】

【００７６】
試験例１２ 「Ｅ. ｆａｅｃａｌｉｓＪＣＭ５８０３株の増大液体培養 ＆ 凍結乾燥菌末の製造」
１. 液体培養
ＢＬ液体培地１０ｍｌ（シリコ栓付き試験管）にＥｎｔｅｒｏｃｏｃｃｕｓ ｆａｅｃａｌｉｓ ＪＣＭ５８０３株を一白金耳量接種し、３０℃一夜振盪培養した。 到達菌数は３９.５ｘ１０^８ｃｆｕ／ｍｌであった。その菌液０.５ｍｌを表９に示した２種類の組成の液体培地５０ｍｌ（綿栓付き５００ｍｌ三角フラスコ）へ接種し、３０℃・振盪速度１３０ｒｐｍで２４時間培養した。 なお、Ｇｌｃは他の成分と一緒にオートクレーブ滅菌した。培養後の分析結果を表１２に示した。本発明の培地２種類ではいずれも到達菌数が４００ｘ１０^８ｃｆｕ／ｍｌおよび３２５ｘ１０^８ｃｆｕ／ｍｌとなり高菌数化した。ちなみに、ＢＬ液体培地では液種の調製に用いた１０ｍｌ液体培地における到達菌数に比べて５０ｍｌの培地を用いた本培養では約３０％と減少した。本菌株は、ＢＬ液体培地で好気的振盪培養を行なうと、試験例で用いたＳｆに比べ酸素供給量が多いと増殖が阻害され易い傾向があると推察された。
【００７７】
【表１２】

【００７８】
２. 凍結乾燥
上記２種類の培養液４０.０ｍｌを１Ｎ塩酸で各々ｐＨ５.０に調整して不溶性炭酸塩を可溶化した後、９０００ｒｐｍ、５分間遠心分離して菌体を回収し、リン酸バッファー（ｐＨ６.８）３０ｍｌで懸濁した。 再び、９０００ｒｐｍ、５分間遠心分離して菌体を回収して洗浄菌体とした。安定化剤（組成：１０％スキムミルク ＆ １％グルタミン酸ソーダ；コッホ滅菌３０分処理済み）１５ｍｌで菌体を懸濁し、１００ｍｌ−ナスフラスコに全量を移し、凍結真空乾燥を一夜行なった。得られた凍結乾燥末を評価し、その結果を表１３に示した。 ２種類の本発明の培地はＢＬ液体培地に比べて、凍結乾燥して得られたＳｆの総菌数が炭酸マグネシウム含有培地で５.４８倍及び炭酸カルシウム含有培地で２.００倍も高かった。
【００７９】
【表１３】

【００８０】
試験例１３. 「Enterococcus faecium IFO3128の液体培養、及び凍結乾燥菌末の製造」
１.液体培養
種菌は、シリコ栓付き試験管にＢＬ液体培地１０ｍｌを入れ、２０分間オートクレーブ滅菌した後、表１４に記した菌株を一白金耳量接種し、３００回転／分の振盪条件で３０℃・２０時間培養して調製した。各培養液の菌数測定は、菌液をリン酸バッファー（ｐＨ６.８）で１０倍ずつ希釈した後、その０.１ｍｌをＢＬ寒天培地に２枚ずつ塗沫し、３７℃で好気培養してコロニーを形成させて行なった。得られた結果を表１４に示した。
【００８１】
【表１４】

【００８２】
上記の種菌０.５ｍｌをシリコ栓付き５００ｍｌ−三角フラスコに入れた液体培地５０ｍｌに接種し、３０℃で１２０ｒｐｍの振盪条件で２０時間培養した。得られた培養液は、種菌と同様の条件で菌数測定を行い、結果を表１５に示したが、比較例に比べて本発明の液体培地では菌数が増大となり、特に炭酸マグネシウムを添加した培地の方が炭酸カルシウムを添加培地よりも増大化した。
【００８３】
組成Ａ. ＢＬ液体培地（比較例）
組成Ｂ. ３.２％ＷＴエキス、６.０％ハイニュートＳＭＰ、８％Ｇｌｃ、４.２２％炭酸マグネシウム
組成Ｃ. ３.２％ＷＴエキス、５.０％ハイニュートＳＭＰ、１０％Ｇｌｃ、５.０％炭酸カルシウム
※ ＷＴエキスの調製：ＷＴを脱イオン水で１０重量％になるように懸濁し３０分間煮沸処理した後、９０００ｒｐｍ・１０分間遠心分離して上清画分を回収し、１０％ＷＴエキスとした。
【００８４】
【表１５】

【００８５】
２.凍結乾燥
培地組成がＢとＣの培養液４０.０ｍｌを１Ｎ塩酸で各々ｐＨ５.０に調整して難溶性炭酸塩を可溶化した後、９０００ｒｐｍ、５分間遠心分離して菌体を回収し、リン酸バッファー（ｐＨ６.８）３０ｍｌで懸濁した。 再び、９０００ｒｐｍ、５分間遠心分離して菌体を回収して洗浄菌体とした。 安定化剤（組成：１０％スキムミルク ＆ １％グルタミン酸ソーダ；コッホ滅菌３０分処理済み）１０ｍｌで洗浄菌体を懸濁し、その全量を１００ｍｌ−ナスフラスコに入れ、凍結真空乾燥を一夜行なった。得られた凍結乾燥末の重量及び菌数を表１６に示した。いずれの菌株においても、本発明の液体培養条件で得られた菌液を凍結乾燥すると、顕著に高い菌数の凍結乾燥菌末が得られた。
【００８６】
【表１６】

【００８７】
試験例１４. 「乳酸桿菌の増大化液体培養および凍結乾燥菌末の製造」
１. 液体培養
ＢＬ液体培地１０ｍｌ（シリコ栓付き５０ｍｌ試験管）にＬａｃｔｏｂａｃｉｌｌｕｓｃａｓｅｉ ＮＲＩＣ１９１７株を一白金耳量接種し、３０℃で一夜振盪培養した（振盪速度、３００回転／分）。 到達菌数は３１ｘ１０^８ｃｆｕ／ｍｌであった。その定常期の菌液０.５ｍｌを本発明の液体培地５０ｍｌ（培地組成（重量％）：５.０％炭酸カルシウム、１０.０％Ｇｌｃ、５.０％ハイニュートＳＭＰ、および３.２％ＷＴ；綿栓付き５００ｍｌ三角フラスコ）へ接種し、３０℃・振盪速度１３０ｒｐｍで３０時間培養した。なお、Ｇｌｃは他の成分と一緒にオートクレーブ滅菌した。培養後の到達菌数は１５２ｘ１０^８ｃｆｕ／ｍｌであり、培地乳酸濃度は３８.９ｍｇ／ｍｌであった。
【００８８】
２. 凍結乾燥
上記の培養液４０.０ｍｌを１Ｎ塩酸でｐＨ５.０に調製して難溶性の炭酸カルシウムを溶解した後、９０００ｒｐｍ・５分間遠心分離して菌体を回収した。菌体は、リン酸バッファー（ｐＨ６.８）３０.０ｍｌで懸濁し、９０００ｒｐｍ・５分間遠心分離して菌体を回収して洗浄した。この洗浄菌体を安定化剤（組成；１０％スキムミルクおよび１％グルタミン酸ソーダ；コッホ滅菌３０分処理済み）１５.０ｍｌで懸濁し、全量を１００ｍｌ―ナスフラスコに移し、凍結真空乾燥を一夜行った。得られた凍結乾燥菌末は、重量が３.１５ｇ、菌数が６７５ｘ１０^８ｃｆｕ／ｇであった。
【００８９】
【発明の効果】
、(1)炭酸マグネシウム、炭酸カルシウムまたはヒドロタルサイトの単独またはこれらを含む無機塩混合物(2)単糖類または二糖類、(3)ペプトン、(4)麹、麹エキス、胚芽培地、酵母、または酵母エキスの単独またはこれらを含む混合物を液体培地に用い、乳酸菌を攪拌培養すると菌数の増大した乳酸菌培養物がえられることが判明した。[0001]
[Technical field to which the invention belongs]
  The present invention belongs to the field of lactic acid bacteria fermentation technology, and relates to a novel culture method in a liquid culture system of lactic acid bacteria and the production of the lactic acid bacteria powder.
[0002]
[Prior art]
  Inoculate a liquid medium with microorganisms such as lactic acid bacteria, culture while controlling the medium pH, etc., and after reaching a stationary phase where growth has stopped, a method of separating and recovering bacterial cells from the medium is a batch culture method (batch (Culture). This method is characterized in that the growth of lactic acid bacteria is easily suppressed during growth due to a change in the culture medium environment caused by a decrease in nutrients in the culture medium and an increase in secretions such as lactic acid. Kyota Hamada et al., “About Streptococcus faecalis, etc.¹⁰The number of bacteria is close to, and even if various growth-promoting substances known in the past are added, it is no longer possible to increase the yield and increase the number of bacteria. Kitahara, K., Research on lactic acid bacteria, The University of Tokyo Press, 1966, p. 494).
[0003]
  From the time to the present day, researches have been made on components that contribute to cell growth in the liquid culture method of lactic acid bacteria. For example, carrot extract (Japanese Patent Publication No. 37-3536), spinach extract (Japanese Patent Publication No. 37-3535). No.), black tea extract (Japanese Patent Publication No. 37-3534), Aspergillus culture filtrate (Japanese Patent Publication No. 36-14277), koji extract (Japanese Patent Publication No. 37-15292), calcium chloride (Japanese Patent Laid-Open No. 7-99968) Etc. are disclosed. In the batch culture method using such a growth promoting agent for lactic acid bacteria, there has been no report that the cells reached a state of 10 billion or more per 1 ml of the medium. However, increasing the number of lactic acid bacteria by batch culture increases the usefulness as a starter culture for producing dairy products, and research on the optimal culture conditions has been conducted earnestly.
[0004]
  The addition of about 0.5% of calcium carbonate to an agar medium for preservation of lactic acid bacteria has long been performed (edited by Yoshio Nei, Method for Preserving Microorganisms, University of Tokyo Press, 1977, p. 208). The significance of the addition of calcium carbonate has been analyzed as a 'neutralizing action' that prevents the pH of the medium from being lowered to the acidic side by lactic acid secreted by the lactic acid bacteria into the medium during the culture (Kitahara Tokuo, Research on Lactic Acid Bacteria, Tokyo University Press, 1966, page 371). There are many cases where calcium carbonate is added at a concentration of less than 1% in the liquid medium, and examples of addition at higher concentrations include 1 to 2% in the acetobacter agar medium (edited by Yoshio Nei, Ed. Law, The University of Tokyo Press, 1977, p. 173), Salmonella breeding composed of calcium carbonate 1.0%, proteose peptone 0.5%, bacto bile salt 0.1%, sodium thiosulfate 3.0% For example, Bacto Tetrationate Broth Base (Difco Manual, 10th edition, page 946), or JP-A-8-116872 in which 1.5% of calcium carbonate is added for the purpose of preventing a decrease in culture medium pH due to lactic acid as described above. It is limited to.
[0005]
  No example of using magnesium carbonate as a liquid medium component of lactic acid bacteria has been reported. The reason for this is thought to be that when magnesium carbonate is added to a liquid medium even in a small amount, the medium pH is more easily alkalized than the normal growth medium pH 7 to 6 of lactic acid bacteria, and promotion of medium browning and bacterial growth inhibition are expected. . In fact, as described in JP-A-8-116872, in order to improve the culture medium for lactic acid bacteria starter using milk as a nitrogen source and a carbon source, selection of a hardly soluble carbonate suitable for neutralization of lactic acid should be made. It is also clear from the fact that when magnesium carbonate is added, the culture medium pH is excluded from the optimum growth pH range of lactic acid bacteria.
[0006]
  On the other hand, inoculate microorganisms such as lactic acid bacteria in the liquid medium, monitor changes in the medium environment such as decrease in nutrients in the culture medium and increase in metabolites, and appropriate control such as supplementation of deficient nutrients and filtration removal of metabolites A culture method that maximizes the number of bacterial cells and the yield of the product by adding is called a fed-batch culture method (Taguchi, Nagai, microbial culture engineering, Kyoritsu Shuppan, 1985). Examples of fed-batch culture methods are as follows: Streptococcus faecalis AHU1256 strain is a 1% yeast extract, 1% polypeptone, 2% D-glucose, and 3.5% monophosphate. Example of efficient production of lactic acid in a liquid medium (pH 7.0) composed of dipotassium hydrogen using a device called a filter bed type reactor (Ohara, H. et al .: Journal of Fermentation and Bioengineering, Vol. 76, pages 73-75) 1993), as an example of increasing the number of lactic acid bacteria, using a culture apparatus equipped with a filter membrane made of alumina ceramic, as the main substrate of the initial medium, 0.6% glucose, 0.2% yeast extract and polypeptone 1 Lactococcus l with 0.0% Actis is cultured, and during the culture period, a fresh culture solution composed of 1.2% glucose and 0.4% yeast extract is intermittently supplied, and the culture solution is kept while the cells remain in the ceramic membrane tank. The bacterial cell concentration was increased by filtration separation (Suzuki, T: Journal of Fermentation and Bioengineering, Vol. 82, pages 264-271, 1996). In order to further increase the number of bacteria, the pH of the medium was lowered by lactic acid produced during the cultivation, whereas the acidified medium pH was adjusted to 7.0 by adding 5.0% of calcium carbonate. In addition, a method (Japanese Patent Laid-Open No. 8-56651) for increasing the number of bacteria by culturing and adjusting the pH of the medium with calcium carbonate several times has been reported. .
[0007]
[Problems to be solved by the invention]
  One of the objects of the present invention is to provide a technique capable of industrially producing lactic acid bacteria living in a liquid batch culture system at low cost and efficiently. Another object of the present invention is to obtain a high number of lactic acid bacteria powder by utilizing the production technique.
[0008]
[Means for Solving the Invention]
  The liquid culture equipment for microorganisms introduced by companies for a long time is usually a production equipment for batch culture, and requires new equipment for fed-batch culture. As a result of intensive studies on a method for efficiently growing lactic acid bacteria in a liquid medium, the present inventors have found a method capable of realizing an increase even in a batch culture method. That is, the present invention increases the number of bacteria by liquid culture of lactic acid bacteria by stirring and culturing in a liquid medium to which the following four nutrients are added.
[0009]
  The four nutrients used in the liquid medium of the present invention and the amount of addition thereof are as follows: (1) One type of magnesium carbonate, calcium carbonate or hydrotalcite, or a mixture of two or more types, and magnesium carbonate or hydrotalcite alone. 1% (w / w) or more in the case, and 2% (w / w) or more when calcium carbonate is used alone, (2) 4% (w / w) or more monosaccharide or disaccharide, ( 3) 3% (w / w) or more of peptone, and 4) 0.5% (w / w) or more of koji or koji extract or one of yeast or yeast extract, or a mixture of two or more.
[0010]
  The method for growing lactic acid bacteria of the present invention can be applied to various techniques in terms of increasing the number of lactic acid bacteria. A suitable example is the production of lactic acid bacteria powder. Thus, in the present invention, after bringing lactic acid bacteria into an increased state under the liquid culture conditions described above, acid is added to the medium and the liquid medium pH is adjusted to 4-6 to dissolve the hardly soluble carbonate, Lactic acid bacteria can be separated from the medium and recovered. In that case, microbial cells and stabilizers may be mixed, or lactic acid bacteria powder can be obtained by freeze-drying and further pulverizing.
[0011]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, the features of the method of the present invention will be described in more detail.
  The present invention aims to provide a liquid culture method for increasing the number of lactic acid bacteria, and in addition to studying medium components such as peptone, glucose, and yeast extract and their concentration ranges that have been used for a long time, optimization of inorganic salts For the purpose of aiming, we focused on calcium carbonate and have been intensively studied. And magnesium carbonate was discovered as a substance which exhibits the effect over calcium carbonate, and it came to complete this invention. Although the following are illustrated as a culture-medium raw material of lactic acid bacteria in this invention, it is not limited to these.
[0012]
1) As a hardly soluble carbonate, magnesium carbonate, calcium carbonate, or hydrotalcite [molecular formula, Mg₆Al₂(OH)₁₆CO₃・ 4H₂O, commercially available under the name of hydrotalcite or synthetic hydrotalcite]. When magnesium carbonate or hydrotalcite is added alone, the effect is obtained by addition of 1% by weight or more, and when calcium carbonate is added alone, the effect is obtained by addition of 2% by weight or more, preferably in the range of 2 to 13% by weight. It is. It may be used in a mixed state with both or other inorganic salts. The essence of magnesium carbonate is hydrous basic magnesium carbonate or hydrous normal magnesium carbonate, containing 40.0 to 44.0% magnesium oxide (MgO), and its molecular formula is (MgCO₃)₄・ Mg (OH)₂・ 5H₂O.
[0013]
2) Examples of monosaccharides include hexoses such as glucose (hereinafter referred to as Glc), mannose, fructose, galactose, and pentoses such as ribose. These monosaccharides are effective when added in an amount of 4% by weight or more, but are preferably in the range of 5 to 15% by weight. These saccharides are preferably sterilized separately from other medium components for the purpose of preventing medium browning and mixed at the time of inoculation. Galactose shows an effect of increasing the number of bacteria in a calcium carbonate-added medium, but does not show an effect in a magnesium carbonate-added medium.
[0014]
3) As peptone, soybean peptide (High New SMP, High New R, High New S, High New SMS, High New PM, etc., manufactured by Fuji Oil Co., Ltd.), or polypeptone (milk casein enzymatic degradation product, Nippon Pharmaceutical ( Co., Ltd.), Trypticase Peptone (BBL, USA), Polypeptone Peptone (Mixed milk casein peptone and Meat peptone, USA BBL), Polypeptone S (Delipidated soybean enzyme degradation product, Nippon Pharmaceutical Co., Ltd.) Manufactured), phyton (manufactured by BBL, USA), soyton (manufactured by Difco), polypeptone P1 (degraded animal meat enzyme, manufactured by Nippon Pharmaceutical Co., Ltd.), thiotone (manufactured by BBL, USA), proteose peptone (manufactured by Difco) ), Polypeptone Y (egg yolk protein enzyme degradation product, manufactured by Nippon Pharmaceutical Co., Ltd.) and the like. The effect is obtained at an addition amount of 3% by weight or more, but it is preferably in the range of 5 to 13% by weight. Many examples of using hynewt as an alternative to peptone are disclosed in Fuji Oil Co., Ltd.'s "Soy Peptide Microbial Growth Promotion Material". Among them, YPD medium is used as an example of a concentration of 1% or more. (Glc 1%, high newt 2%, and yeast extract 2%) and Sabouraud medium (Glc 4%, high newt 1%), which are used at a concentration of 2% or less.
[0015]
4) As a mixture of koji and yeast, powerful wakamoto (hereinafter referred to as WT. Okamoto Pharmaceutical Co., Ltd. OTC drug) is exemplified. As an agate, a culture obtained by solid culture of the filamentous fungus Aspergillus oryzae in the embryo medium (hereinafter referred to as AK), or a culture obtained by solid culture of the lactic acid bacteria in the embryo medium (hereinafter referred to as LFK). The effect is obtained by adding 0.5% by weight or more of an extract collected from these straws, or an embryo medium, yeast powder or yeast extract used for AK or LFK production alone or in a mixture, preferably 2 It is in the range of ˜5% by weight. Among these, the optimum component is AK, and the highest effect is obtained when used alone. In addition, when using a cocoon or germ medium, it is possible to use even in a high concentration state by preparing and using an extract excluding insoluble residues in water, and further, since insoluble residues are not brought into the lyophilized powder. desirable.
[0016]
Needless to say, the additives in 1) to 4) can be added in a larger amount than the above concentration range. However, it should be noted that [1] foaming occurs during autoclaving of the medium. (To avoid this, it is desirable to foam by preheating the medium before sterilization), [2] When magnesium carbonate is used alone, the medium pH becomes too alkaline, or the medium permeates. Care must be taken because the pressure becomes too high, [3] Browning of the medium tends to proceed, [4] Raw material costs are too high, [5] There is no problem if koji extract and yeast extract are used When WT, AK, LFK, germ medium or yeast powder is used as a medium component, the ratio of insoluble solids to the amount of bacterial cells increases, so these insoluble residues are triturating with respect to lyophilized powder. Although with the significance of a certain, and the like that result in low cell concentration of lactic acid bacteria during lyophilization.
[0017]
5) In the present invention, as a culture condition for lactic acid bacteria, it is essential to stir and shake the medium. As long as this point is ensured, the effects of the present invention can be sufficiently confirmed by setting the most favorable culture conditions for the strain to be used regardless of the culture temperature, culture time, anaerobic or aerobic.
For example, the effect of the present invention can be sufficiently confirmed by setting the amount of carbonate added in the vicinity of the lower limit of the present invention and bubbling the medium using carbon dioxide gas instead of the air during tank culture.
[0018]
6) The lactic acid bacteria that can be used in the present invention and whose effects can be confirmed may be any bacterial species belonging to Enterococcus faecium or E. faecalis, and these are currently taxonomically previously referred to as Streptococcus faecalis and are still present. The name may be used, and any name of the strain may be used. Therefore, in this example, in addition to S. faecalis (hereinafter referred to as Sf) used in the commercially available live cell preparation WT, E. faecalis JCM5803 strain and E. faecium IFO3128 strain were also used. Furthermore, Lactobacillus casei (NRIC1917 strain is used in the examples) is exemplified as other bacterial species applicable to the present invention. It goes without saying that the effects of the present invention can be sufficiently confirmed even if a breeding strain having the above-mentioned strain as a parent strain is used, or a symbiotic liquid culture in which these strains are mixed and cultured.
[0019]
  The theoretical background that can increase lactic acid bacteria by liquid culture of the present invention is described: In the liquid culture system of lactic acid bacteria, focusing on the fact that the growth of lactic acid bacteria is likely to be suppressed due to the fact that the medium pH is lowered by lactic acid secreted into the medium. In addition to promoting the growth of growth by adding abundant sources of C and N, it is possible to increase by culturing with the addition of factors that inhibit lactic acid production. It was an idea.
[0020]
  The mechanism by which lactic acid bacteria increase when lactic acid production is suppressed is as follows. (1) When liquid culture using Glc as a carbon source is performed using homolactic lactic acid fermentation bacteria such as Sf, between shaking culture and stationary culture It is inferred from the remarkable difference found (edited by Tomoo Kitahara, Research on Lactic Acid Bacteria, The University of Tokyo Press, pages 403-407, 1966). That is, none of the strains produce any product other than lactic acid in static culture, but the production of lactic acid decreases by increasing the oxygen concentration of the medium by shaking culture, and instead produces a large amount of acetic acid and acetoin. Changed. ATP was newly generated in the biosynthesis process of acetic acid and used as an energy source for growth, and growth was promoted under this aerobic condition. Therefore, it was assumed that increasing the amount of oxygen supplied to the cells by aerobically shaking the liquid medium was an essential factor for increase (= increase factor-A).
[0021]
  In addition, (2) homolactic fermentation bacteria such as Sf produce 2 moles of lactic acid and 2 moles of ATP per mole of Glc under normal culture conditions. However, even in the case of homolactic fermentation, in the alkaline pH range near the upper limit of growth, lactic acid production decreases, so that formic acid, acetic acid, ethanol, etc. increase, so-called 'heterolactic fermentation' is performed. In this process, ATP is generated. It has been reported that the excessively generated energy enables vigorous growth in an alkaline pH range (Rhee, SK and Pack, MY: Journal of Bacteriology, 144, 217-221). 1980). Therefore, it was assumed that setting the liquid medium pH in the vicinity of the upper limit growth range of the strain was an essential factor for increase (= increase factor-B).
[0022]
  In addition, as a source of nutrients necessary for growth, (1) monosaccharide, (2) peptone, and (3) straw, straw extract, germ medium, yeast, or a mixture containing these alone or in combination Add as high a concentration as possible to the liquid medium (= increase factor-C).
[0023]
  In the liquid culture conditions of the present invention, the increasing factor-C is a factor that stimulates growth, and promotes lactic acid production accompanying the effect. Therefore, the culture system is loaded with both factors -A and -B that suppress lactic acid production, and the culture system is cultured while suppressing lactic acid production. By inventing this method, it was possible to actually increase the number of bacteria, and the present hypothesis is that the above is the mechanism for increasing the number of bacteria under the liquid culture conditions of the present invention.
[0024]
【Example】
Comparative Example 1 “It does not increase even if Sf is batch-cultured by adding poorly soluble carbonate to a known liquid medium.”
Even if 5% by weight of calcium carbonate is added to the BL liquid medium and Sf is cultured, the effect is such that the number of bacteria increases slightly, and this increase in the number of bacteria is achieved by adding the same concentration of magnesium sulfate. However, it was difficult to say that the number of bacteria increased due to the specific action of calcium carbonate. Further, even when 5% of calcium carbonate was added to the BL medium, the high heat resistance of Sf and the high medium lactic acid concentration, which are the characteristics of the present invention, were not observed at all.
  On the other hand, Sf was cultured by adding 2 to 5% by weight of calcium carbonate or magnesium carbonate to a rice bran or soy flour medium containing a high concentration of carbon source and nitrogen source, which is comparable to the present invention. The increase effect depending on the addition concentration was not observed at all, and the high lactic acid production effect which is one of the characteristics of the present invention was not observed.
[0025]
Experimental method: One platinum loop of Sf was inoculated into 10 ml of BL liquid medium, and cultured overnight at 30 ° C. with shaking. 0.1 ml of this stationary-phase bacterial solution was inoculated into 10 ml of liquid medium (50 ml test tube with cotton plug) having the composition shown in Table 1 (the number of bacteria, 0.21 × 10 6⁸cfu / ml), and cultured with shaking at 30 ° C. for 24 hours (shaking speed: 300 times / min). The number of bacteria reached, the heat resistance, and the lactic acid concentration of the medium of the obtained bacterial solution were analyzed by the method described in Test Example 1.
Preparation method of rice bran medium and soybean powder medium: Prepared according to the method disclosed in JP-A-8-56651. That is, rice bran powder and defatted soybean powdered with a coffee mill were suspended in deionized water at a rate of 30% (w / w) and heated at 50 ° C. for 1 hour. Thereafter, the mixture was centrifuged at 9000 rpm for 5 minutes to collect the supernatant fraction, which was overlaid with 4 sheets of gauze and filtered. Carbonate was added to the obtained filtrate, and then autoclaved to obtain a medium.
BL liquid medium: BL agar medium, “Nissui” manufactured by Nissui Pharmaceutical Co., Ltd., 5.8 g suspended in 100 ml of deionized water, filtered through industrial tissue, Kimwipe S-200, manufactured by Crecia Co., Ltd. Was prepared by autoclaving for 20 minutes.
[0026]
[Table 1]

[0027]
Test Example 1. “Characteristics of the liquid culture method of the present invention, Part 1: After assimilating Glc, the lactic acid secreted into the medium is newly assimilated to continue to grow and reach an increased state”
[0028]
  Sf was cultured using the liquid medium of the present invention and a BL liquid medium as a known liquid medium, and the change with time was analyzed. As shown in Table 2, when the growth pattern of lactic acid bacteria in the liquid medium of the present invention is compared with that of the BL liquid medium, the difference between the two is remarkable, and the characteristics of the present invention can be summarized as follows:
1) When Sf was cultured in the liquid medium of the present invention, the number of cells reached 50 to 70 billion per ml of the medium, which was increased about 20 times compared with the BL liquid medium.
2) In the culturing process until the maximum number of bacteria was reached under the culturing conditions of the present invention, Sf preferentially consumed Glc that was easily assimilated until 14 hours of culturing. After 19 hours when Glc was completely consumed from the medium, the concentration of lactic acid secreted and accumulated in the medium started to decrease, and lactic acid was assimilated. Thus, the culture medium pH also shifted to the alkali side, and the bacterial count reached the maximum value after 27 hours.
[0029]
  On the other hand, in the BL liquid medium, the maximum number of bacteria reached 3 billion at 19 hours when Glc of the medium was consumed. The number of bacteria did not increase even when the culture was continued thereafter, and neither the decrease in the concentration of lactic acid secreted into the medium nor the alkalinization of the medium pH was observed.
  The phenomenon similar to the characteristic found in the culture of the present invention is that, when the Glc concentration of the medium is set to a low concentration in advance, lactic acid added as a carbon source of the medium is assimilated, and the lactic acid disappears from the medium. It has been reported in Streptococcus mutans that the pH of the medium has become alkaline (Hu, G, and Sandham, H. J .: Archives of Oral Biology, 17, 729-743, 1972). From this, in the culture of the present invention, the decrease in the amount of lactic acid generated in the process of reaching the maximum number of bacteria is suggested to be a phenomenon in which microbial cells assimilated lactic acid as a new carbon source instead of Glc.
[0030]
1) The heat resistance was low in the logarithmic growth phase at 4 to 8 hours in culture (0.04% or less) and high in the stationary phase at 20 to 27 hours in culture (40% or more) under the conditions of the present invention. And when culture | cultivation was continued beyond the 27th hour which reached the maximum number of bacteria, heat resistance fell rapidly from the 30th hour onward (20% or less-several%). On the other hand, in the BL liquid medium, it was similarly low at 4 to 8 hours of culture in the logarithmic growth phase (0.04% or less), and was high in heat resistance at 19 hours after reaching the maximum number of bacteria (33%). Further, even when the culture was further continued, an almost constant heat resistance of 10 to 12% was maintained.
  When lactic acid bacteria are cultured in the medium of the present invention, high heat resistance is obtained in the vicinity of the maximum bacterial density reached in the stationary phase. One of the factors is the addition of high concentrations of peptone, which contributes to the nutrients of peptone to grow even after consuming Glc.ConversionTo do. Therefore, after reaching the maximum number of bacteria, the reason for the rapid decrease in heat resistance is presumed to be due to a decrease in nutrients of peptone from the medium (see Table 5).
[0031]
Culture conditions: One platinum loop of Sf was inoculated into 10 ml of BL liquid medium, and cultured overnight at 30 ° C. with shaking. 1.0 ml of this stationary-phase bacterial solution is inoculated into 50 ml of liquid medium (using a 500 ml Erlenmeyer flask with cotton stopper) (number of inoculated bacteria, 0.15 × 10⁸Per ml), and cultured at 30 ° C. under shaking conditions of 200 rpm. Sampling was performed at each culture time shown in Table 2, and the medium pH, the number of bacteria reached, heat resistance, lactic acid concentration, and the like at that time were analyzed. Glc was sterilized separately from other medium components and mixed with other medium components before inoculation.
  Liquid medium composition of the present invention (% by weight): 8.43% magnesium carbonate, 8.0% Glc, 12.0% high-newt SMP and 3.2% WT.
[0032]
Analysis method:
  The number of bacteria reached was determined by serially diluting 0.5 ml of the culture solution with 4.5 ml of phosphate buffer having the following composition, and 0.1 ml of the SCD agar medium “DAIGO” (manufactured by Nippon Pharmaceutical Co., Ltd.). ) Two to three were smeared. This plate was cultured at 37 ° C. for 24 to 40 hours, the number of colonies produced on the plate was measured, and the average value was converted to 1 ml of liquid medium and expressed as cfu (colony forming unit). Composition of phosphate buffer: Final solution volume adjusted to 1000 ml (pH 6.8) with 7 g of sodium chloride, 5.31 g of dipotassium phosphate, 2.65 g of monopotassium phosphate, 2.0 g of Tween 80 and deionized water.
1) The concentration of lactic acid secreted into the medium is determined based on the reagent kit for quantitative analysis of lactic acid, F-kit L-lactic acid (J.L. Measured by purchase).
2) The medium concentration of D-glucose was measured using a commercially available reagent kit for quantitative glucose analysis, F-kit D-glucose (purchased from JL International Inc.).
3) For heat resistance, after serially diluting a solution diluted with a phosphate buffer (pH 6.8) at 60 ° C for 15 minutes, 0.1 ml was smeared on an SCD agar medium and surviving after heat treatment. The number of bacteria was measured. The heat resistance was expressed in terms of the survival rate% of bacteria relative to the untreated heat.
[0033]
[Table 2]

[0034]
Test Example 2 “Characteristics of Liquid Culture of the Present Invention, Part 2: Specificity of Inorganic Salts on Increase”
In the liquid culture conditions of the present invention, the specific effect on the increase in Sf when adding a high concentration of inorganic salt was examined, and the results obtained are shown in Table 3.
[0035]
  As a result, the following points were found as features related to inorganic salts:
1) When the WT is added to the medium at a concentration of 3.2%, Glc and Hynewt R as high as 10% each, 120 × 10 × 10 without adding an inorganic salt⁸Bacteria counts as high as cfu / ml were obtained. In the conventional batch culture method, this number of bacteria is the upper limit that can be reached, and even if an additive is added, the number of bacteria could not be further increased. The present inventors examined the increase effect by adding a higher concentration of inorganic salt to this medium condition. As a result, as shown in Table 3, it was found that when calcium carbonate was added at a high concentration of 6.00% or magnesium carbonate as high as 5.06%, the concentration further increased. As other inorganic salts, the number of bacteria was slightly increased as compared with the condition in which potassium bicarbonate was not added, but the number of bacteria reached was lower than that in the condition without any addition of calcium chloride or the like.
[0036]
2) What is characteristic is that when calcium sulfate (gypsum), magnesium carbonate, or calcium carbonate having poor solubility is added, the heat resistance is about 50%, almost the same as when no heat resistance is added. . On the other hand, when a water-soluble inorganic salt such as sodium chloride was added, the heat resistance decreased. When these sparingly soluble inorganic salts are added, it is thought that heat resistance is prevented from being reduced because of their poor effect on the cells due to their sparingly soluble properties. It was also observed that the heat resistance is lowered if is too high.
[0037]
3) The concentration of lactic acid in the medium was high when both conditions were maintained, that the medium pH remained high after culturing and that the number of bacteria was high. Among them, the addition of sparingly soluble magnesium carbonate resulted in the highest lactic acid concentration, and both heavy and light had the same effect. On the other hand, even in the same magnesium salt as magnesium carbonate, water-soluble magnesium chloride and magnesium sulfate had lower lactic acid concentration as well as the number of bacteria reached compared to the case where no inorganic salt was added. Incidentally, the concentration of lactic acid per number of bacteria (mg / 100 million) is as follows: sodium chloride (0.70), magnesium chloride (0.32), magnesium carbonate (0.14), calcium carbonate (0.11), magnesium sulfate ( 0.093) in order, and it was not recognized that lactic acid production was promoted even when magnesium chloride and magnesium sulfate were added to the basic composition of the liquid medium of the present invention.
[0038]
Culture conditions: 10 ml of BL liquid medium was inoculated with one platinum loop of Sf, and cultured overnight at 30 ° C. with shaking. 0.1 ml of this stationary-phase bacterial solution is inoculated into 10 ml of liquid medium (50 ml test tube with cotton plug) (the number of inoculated bacteria is 0.25 × 10⁸Cultivated at 30 ° C. for 21 hours. The shaking speed was set at 300 times / min. Glucose was sterilized separately from the other ingredients and mixed with the remaining ingredients before inoculation.
  Liquid medium composition (% by weight): 0.6M inorganic salts, 10.0% Glc, 10.0 High New® and 3.2% WT.
[0039]
[Table 3]

[0040]
Test Example 3 “Characteristics of Liquid Culture of the Present Invention, Part 3: Specificity of Supplemental Medium Nutrients for Glc and Peptone Depends on Species”
Regarding the specificity of supplemental nutrients in the liquid culture conditions of the present invention, from the results shown in Table 4,
The following features were found:
1) It was found that the specificity of supplemental nutrients on the increase depends on the bacterial species used. In addition to WT, the components that showed an effect with Sf were AK, LFK, embryo medium, yeast powder or yeast extract. On the other hand, the components that showed an effect on L. casei NRIC1917 were limited to AK and germ medium. When AK was added, both Sf and L. casei strains had almost the same number of reached bacteria.
[0041]
2) Addition of yeast powder acted to suppress the increase of L. casei NRIC1917 strain, and the yeast extract was not as effective as Sf. The inhibitory action by adding yeast powder was expected to occur even when the optimal component AK coexists. This was not as effective as AK when WT was added. WT is composed of yeast powder 37% and LFK 10% in addition to AK 50%, and it is speculated that the AK increasing effect was suppressed by the contained yeast powder.
[0042]
3) The growth of Sf was accelerated by the addition of LFK compared to the germ medium, but the growth of L. casei NRIC1917 strain was the same number of cells reached in LFK as in the germ medium. LFK is a sputum obtained by solid-cultivating Sf in a germ medium, and secretes a growth promoting substance for the same strain in the solid culture, and this substance has a property of not exerting a growth promoting action on a heterogeneous L. casei strain. It was guessed.
[0043]
Culture conditions: 10 ml of BL liquid medium was inoculated with one platinum loop each of Sf and Lactobacillus casei NRIC1917 strain, and cultured overnight at 30 ° C. with shaking. 0.1 ml of these stationary phase bacterial solutions were inoculated into 10 ml of liquid medium (50 ml test tube with cotton stopper), and Sf was cultured at 30 ° C. for 20 hours and L. casei NRIC1917 strain was shaken for 27 hours (shaking speed 300 times / Min). Glucose was sterilized separately from other medium components and mixed with other components immediately before inoculation. The germ medium used as a supplemental nutrient is a medium of AK or LFK obtained by solid culture of Aspergillus oryzae and lactic acid bacteria, and is a mixture of defatted rice germ and defatted wheat germ.
  Liquid medium (% by weight): 5% calcium carbonate, 10% Glc and 5% hynewt S were added as common components, and 3.2% supplemental nutrients were added. In addition, supplemental nutrients were also added, and the medium pH after autoclaving was as follows: no addition, 7.53; WT, 7.09; AK, 7.06; LFK, 7.29; germ medium, 7 .47; Yeast powder, 7.12; Yeast extract, 6.64.
[0044]
[Table 4]

[0045]
Test Example 4 “Characteristics of Liquid Culture of the Present Invention, Part 4: Increase is better aerobic than anaerobic culture conditions”
With respect to the effects of aerobic and anaerobic shaking cultures on growth, the results shown in Table 5 revealed the following characteristics:
1) The number of bacteria increased in the aerobic culture condition compared to the anaerobic condition. However, 270x10 even under anaerobic conditions⁸The number of bacteria reached as high as cfu / ml suggests that even anaerobic lactic acid bacteria that are difficult to grow in the presence of oxygen can be increased by the culture conditions of the present invention regardless of their genus or species.
[0046]
2) In order to increase Sf by liquid culture, (1) magnesium carbonate and calcium carbonate mixture, (2) Glc, (3) high-newt S, regardless of whether the culture conditions are aerobic or anaerobic And (4) 4 elements composed of WT were found to be essential requirements for liquid medium composition. By the way, the medium lactic acid concentration increased as the number of bacteria reached was higher and the medium pH was close to alkaline.
[0047]
3) In order to ensure high heat resistance, it was necessary to add both Glc and hynewt as medium components at high concentrations, and it was found that the addition of peptone was particularly important.
[0048]
Culture conditions: 10 ml of BL liquid medium was inoculated with one platinum loop of Sf, and cultured overnight at 30 ° C. with shaking. 0.1 ml of this stationary phase bacterial solution is inoculated into 10 ml of liquid medium (50 ml test tube with cotton plug) (number of inoculated bacteria, 0.35 × 10 5⁸cfu / ml), cotton plugs were used for aerobic culture conditions, while carbon dioxide was sealed and rubber plugs were used for anaerobic culture conditions. Both were cultured with shaking at 30 ° C. for 20 hours (shaking speed: 300 times / min). Glucose was sterilized together with other medium components.
[0049]
[Table 5]

[0050]
Test Example 5 “Characteristics of Liquid Culture of the Present Invention, Part 5: Shaking during culture is essential for increase. Concentration ranges of various medium components when calcium carbonate alone is added.”
  The essential factors affecting the increase in the number of bacteria under the culture conditions of the present invention were analyzed, and the results of the test examples shown in Table 6-2 revealed the following:
[0051]
1) When static culture is performed using the medium composition of the present invention, 100 × 10⁸The number of bacteria was so low that it did not reach cfu / ml. Incidentally, under the conditions deviating from the medium composition of the present invention, there were also cases where the stationary culture had a higher number of reached bacteria than the shaking culture, as in medium Nos. 22 and 23.
[0052]
It has been reported that NADH oxidase activity is significantly higher when aerobically cultured by shaking lactic acid bacteria (Leuconostoc mesenteroides) than when it is statically cultured, and this enzyme acts as a terminal electron acceptor for oxygen. Therefore, it has been reported that supplementing the oxygen concentration of the liquid medium to the medium by shaking facilitates the production of ATP by consuming oxygen, resulting in an increase in the cell concentration (Con, A. et al .: Journal of General Microbiology, 132, 1789-1796, 1986). Therefore, the essential factors for shaking in the present invention are (1) it is easy for water-insoluble calcium carbonate to ensure uniformity in the medium by shaking the medium, and (2) an increase in NADH oxidase activity. It is done.
[0053]
2) With the medium composition of the present invention, a high bacterial density was reached by shaking culture. No.15, 17 and 20 showed cases where the lactic acid concentration in the medium was higher than that in the shaking culture despite the low bacterial density in the stationary culture. The culture pH is shifted to the acidic side in stationary culture compared to shaking culture, and the production of organic acids such as lactic acid per cell is higher in static culture, and the anaerobic atmosphere in static culture It was speculated that the production of lactic acid was activated thanks to its high level.
[0054]
3) The optimum amount of WT added was 3.2%, and when a higher concentration was added, the number of reached bacteria decreased.
4) The number of bacteria reached by Sf increased as the concentration of calcium carbonate added increased.
5) Along with calcium carbonate and Glc, also increased using polypeptone as peptone and yeast extract as supplemental nutrient.
6) When galactose is added instead of Glc in the calcium carbonate addition system, the number of bacteria reached is 200 × 10⁸Although it reached an increase state similar to cfu / ml and Glc, the medium pH after shaking culture was slightly shifted to the alkali side from the medium pH before culture. When calculated backward from the lactic acid concentration in the medium, the pH should be shifted to the acidic side by about 0.2, so it was thought that the alkaline substance was secreted during the culture.
[0055]
Culture conditions: One platinum loop of Sf was inoculated into 10 ml of BL liquid medium, and cultured overnight at 30 ° C. with shaking. 0.1 ml of this stationary-phase bacterial solution was inoculated into 10 ml (50 ml test tube) of a liquid medium having the composition shown in Table 6-1.⁸cfu / ml). The test tube was cotton plugged and cultured aerobically at 30 ° C. for 20 hours, under the conditions of shaking (shaking speed 300 times / min) and standing. Under both culture conditions, the number of reached bacteria was measured immediately after culturing. D-glucose and galactose (Gal) were sterilized separately from other media components and mixed prior to inoculation. Furthermore, a Briggs modified medium was used as a known liquid medium [medium composition: 2.0% Glc, 1.5% polypeptone, 0.6% yeast extract, 0.5% sodium chloride, 0.1% Rheodor TW120. 0.05% soluble starch, 0.05% L-cysteine hydrochloride, 0.2% light calcium carbonate, 20% tomato juice, 7.5% liver extract]
[0056]
[Table 6-1]

[0057]
[Table 6-2]

[0058]
Test Example 6 “Characteristics of Liquid Culture of the Present Invention, Part 5: Effective Concentration Range of Various Medium Components in a Magnesium Carbonate Single Addition System”
Further analysis of essential factors affecting the increase in the number of bacteria under the culture conditions of the present invention was carried out. From the test results shown in Tables 6 and 7, the concentration ranges of the four medium components necessary for the increase were set as follows. :
(1) Magnesium carbonate and calcium carbonate had an increase effect when added in an amount of 2% or more. When these salts were added at a concentration higher than 13%, it was difficult to handle due to precipitation at the bottom of the culture tank, foaming during culture medium sterilization, or culture.
(2) Any monosaccharide may be used as long as the strain to be used can be assimilated. In the case of Glc, the effect of increasing was observed by addition of 4% or more. In addition, when Glc was added at a concentration higher than 15%, the browning of the medium became large, and it was difficult to treat lyophilized powder.
(3) The effect of increasing the high-newt was observed by adding 3% or more. In addition, it was not desirable to add a high concentration of high newt of 13% or more because the medium cost increased.
(4) The effect of increasing WT was observed when 0.5% or more was added, and the optimum concentration was 3.2%. In addition, when the addition amount was increased from 5%, insoluble residue increased and handling became difficult. In this case, there was a problem that the cost of producing koji extract increased, although there was no problem if koji extract was used.
[0059]
Culture conditions: One platinum loop of Sf was inoculated into 10 ml of BL liquid medium, and cultured overnight at 30 ° C. with shaking. 0.5 ml of this stationary-phase bacterial solution was inoculated into 50 ml of a medium having the composition shown in Table 7 (500 ml Erlenmeyer flask with cotton plug) (inoculum count 0.38 × 10⁸cfu / ml) at 30 ° C. for 19 hours at 200 rpm. Glc was sterilized separately from the other components.
[0060]
[Table 7]

[0061]
Test Example 7 “Characteristics of Liquid Culture of the Present Invention, Part 5: Specificity of Carbohydrates on Increase”
  Sugars that can be utilised by Sf to produce lactic acid were selected (BERGEY's Manual of Systemic Bacteriology, Volume 2, Williams & Wilkins, 1064, 1986), and the range of carbohydrates affecting the increase was analyzed. As a result, galactose, which was effective for the increase under calcium carbonate addition conditions, became ineffective, and all saccharides increased, except that glycerin and mannitol were ineffective. Among them, hexoses such as Glc showed the highest effect, and disaccharides showed high maltose effect.
[0062]
Test method
  One platinum loop was inoculated into 50 ml of BL liquid medium and Sf was aerobically cultured for 8 hours under shaking at 30 ° C. and 120 rpm. The number of bacteria reached is 32x10⁸cfu / ml, and the medium pH at that time was 4.69. 0.1 ml of the bacterial solution in the early stationary phase was inoculated into 10 ml of the main culture medium, and cultured at 30 ° C. for 18 hours with shaking (300 times / min). The medium for main culture was supplemented with 8.0% of the following saccharides, but all were sterilized and mixed with other ingredients immediately before inoculation. The common ingredients (% by weight) were as follows: 4.22% Magnesium carbonate, 6.0% high-newt SMP, and 3.2% WT (50 ml test tube with cotton plug)
[0063]
[Table 8]

[0064]
Test Example 8 “Characteristics of Liquid Culture of the Present Invention, Part 6: Effects of Simultaneous and Separate Sterilization of Maltose on Growth of Sf, Growth Phase, and Addition / No Addition of Magnesium Carbonate”
  The effects of simultaneous sterilization / separate sterilization of maltose, growth phase, and addition / non-addition of magnesium carbonate on the increase in Sf were examined, and the results are shown in Table 9. It was found that the medium sterilized with maltose had a higher medium pH after sterilization than the medium sterilized at the same time, and the separately sterilized medium was more likely to increase as the pH changed to alkali. Furthermore, when the browning substance (melanoidin) is produced during sterilization, the amount of lactic acid produced is expected to decrease as the absolute amount of sugar that the bacteria can assimilate decreases.
  The number of bacteria reached under the culture conditions with magnesium carbonate added does not affect the growth phase of the inoculum, and the medium with maltose separately sterilized is higher than the simultaneous sterilized medium. It was significantly higher than the co-sterilized medium. On the other hand, under the culture conditions without adding magnesium carbonate, the number of reached bacteria was slightly higher in the separate sterilized medium than in the co-sterilized medium, but conversely, the lactic acid concentration in the separate sterilized medium was co-sterilized. It decreased to about 50% from the medium.
[0065]
Test method
  One volume of platinum was inoculated into 10 ml of BL liquid medium and Sf was aerobically shaken at 30 ° C. for 6.5 hours to prepare cells in the logarithmic growth phase. The number of bacteria reached is 3.6 x 10⁸cfu / ml, and the medium pH at that time was 6.23. 0.1 ml of the bacterial solution in the logarithmic growth phase was inoculated into 10 ml of the main culture medium, followed by shaking culture at 30 ° C. for 18 hours. Similarly, Sf was inoculated into a BL liquid medium, and aerobically shaken at 30 ° C. for 21.5 hours to prepare stationary cells. The number of bacteria reached is 14.5x10⁸cfu / ml, and the medium pH at that time was 4.32. 0.1 ml of this stationary phase bacterial solution was inoculated into 10 ml of the main culture medium, followed by shaking culture at 30 ° C. for 20 hours. Further, the above-mentioned stationary-phase bacterial solution was diluted 10-fold with phosphate buffer (pH 6.8) and allowed to stand at room temperature for 3 hours, and then 0.1 ml of this bacterial solution was inoculated into 10 ml of the main culture medium. The shaking culture was carried out at 17 ° C. for 17 hours. The culture medium was added with maltose monohydrate (8.0% (w / w)), but in the case of separate sterilization, the separately sterilized material was mixed with other components immediately before inoculation and co-sterilized. In this case, sterilize together with other components, and other common components (% by weight) are as follows: 4.22% magnesium carbonate (addition and non-addition are shown in Table 9), 6.0 % High Neutral SMP, and 3.2% WT (50 ml test tube with cotton plug). Shaking speed, 300 rpm.
[0066]
[Table 9]

[0067]
Test Example 9 “Characteristics of Liquid Culture of the Present Invention, Part 7: Hydrotalcite Concentration Necessary for Increasing Sf”
  Under the liquid culture conditions of the present invention, the lower limit of the effective concentration of synthetic hydrotalcite was examined, and the results are shown in Table 10. Synthetic hydrotalcite showed an effect of increase at an addition concentration of 1.0% or more, and the medium lactic acid concentration was also increased accordingly.
[0068]
Test method
    Sf was inoculated into 10 ml of BL liquid medium with one platinum loop and aerobically cultured under shaking conditions of 30 ° C. and 300 rpm for 21.5 hours to prepare stationary cells. The number of bacteria reached is 14.5x10⁸cfu / ml, and the medium pH at that time was 4.32. 0.1 ml of this stationary-phase bacterial solution was inoculated into 10 ml of the main culture liquid medium, and cultured at 30 ° C. for 20 hours with shaking (speed, 300 rpm). Common components (% by weight) of the liquid medium for main culture are as follows: 6.0% high-newt SMP, 8.0% Glc, 3.2% WT and synthetic hydrotalcite (50 ml test tube with cotton plug) Glc was sterilized separately and mixed with other ingredients immediately before inoculation.
[0069]
[Table 10]

[0070]
Test Example 10 “Sf augmentation liquid culture using koji extract and production of freeze-dried bacterial powder”
1. Liquid culture
In the pre-culture, 10 ml of GPY liquid medium (50 ml test tube with cotton plug) composed of 1.0% Glc, 1.0% polypeptone and 1.0% yeast extract was inoculated in a platinum loop. The culture was allowed to stand at 37 ° C. for 24 hours. The WT extract was suspended in deionized water at 10% and boiled for 30 minutes, and then centrifuged at 9000 rpm for 10 minutes to recover the supernatant fraction, which was used as the WT extract (10%). The medium composition (% by weight) of the present invention is 4.2% light magnesium carbonate, 8.0% Glc, 6.0% high-newt SMP, and 3.2% WT extract. However, Glc was sterilized in an autoclave separately from other components and mixed with other components before inoculation (medium solution amount, 200 ml and 100 ml). As a comparative example, BL liquid medium was used (medium liquid volume, 186 ml and 96 ml).
  In the main culture, the Sf bacterial solution precultured in 200 ml (2 L-Sakaguchi flask with cotton stopper) and 100 ml (1 L-Sakaguchi flask with cotton stopper), respectively, of the medium of the present invention and the comparative example was 0.38 × 10.⁸The inoculum was inoculated so that the number of bacteria was cfu / ml, and cultured with shaking at 30 ° C. for 24 hours. The shaking speed was 150 reciprocations per minute.
[0071]
  As a result, the number of bacteria reached in the culture medium of the present invention is 405 × 10 6 when the medium volume is 200 ml.⁸In the case of cfu / ml and medium volume of 100 ml, the number of reached bacteria is 465 × 10⁸cfu / ml. The total number of bacteria obtained by the liquid culture of the present invention is 127,500 × 10⁸cfu, which was 22.2 times higher than when cultured in BL medium used as a comparative example. The number of bacteria reached in the BL liquid medium is 17 × 10 when the medium volume is 186 ml.⁸In the case of cfu / ml and a medium volume of 96 ml, the number of reached bacteria is 27 × 10⁸cfu / ml and the total number of bacteria obtained by culture is 5,754 × 10⁸cfu.
[0072]
2. Production of freeze-dried powder
  Preparation of the lyophilized powder is as follows: After adjusting the pH of each culture solution to pH 5.0 with 1N hydrochloric acid to dissolve poorly soluble magnesium carbonate, it is centrifuged at 9000 rpm for 5 minutes to precipitate the cells. Minutes were collected. The cells were suspended in a phosphate buffer (pH 6.8), centrifuged and collected twice, and washed. Thereafter, the cells were suspended with a stabilizer (composition: 10% scum milk and 1% sodium glutamate). In the case of the culture of the culture medium of the present invention, 8 ml of the suspension in 50 ml of the suspension of the stabilizer was further diluted 2-fold with the stabilizer and lyophilized to obtain 1.95 g of a white lyophilized powder. 12,900x10⁸The number of bacteria was cfu / g, and its heat resistance was 26.0%. Furthermore, as a result of freeze-drying the remaining amount (42 ml), 6.49 g of a white freeze-dried powder was obtained, which was 23,400 × 10⁸The number of bacteria was cfu / g, and the heat resistance was 17.2%. Therefore, the bacterial yield after centrifuging and freeze-drying using 200 ml and 100 ml of medium was 138%. The total bacterial count of the bacterial powder obtained by freeze-drying the liquid culture of the present invention is 177,021 × 10⁸It was cfu, and the number of bacteria was 26.4 times higher than that of the comparative example. In the case of a culture of BL medium, 6.2 ml of 9.2 ml of the stabilizer suspension was lyophilized, resulting in 0.96 g of a white lyophilized powder, 5,180 × 10 5⁸The number of bacteria was cfu / g, and its heat resistance was 23.3%. The remaining 3 ml was further diluted about 3.3 times with a stabilizer and lyophilized to obtain 1.10 g of a white lyophilized powder.⁸The number of bacteria was cfu / g, and its heat resistance was 30.9%. Therefore, 200 ml medium and 100 ml medium are pooled, the number yield of bacteria through all the steps is 116%, and the total number of bacteria obtained by freeze-drying the BL liquid culture is 6,700 × 10 6⁸cfu.
[0073]
Test Example 11 “Increased liquid culture of Sf using jar fermenter”
One platinum loop of Sf was inoculated into 50 ml of BL liquid medium, and cultured with shaking at 130 rpm at 30 ° C. overnight.
10 ml of this liquid type was inoculated into 1000 ml of the liquid medium of the present invention, and predetermined culture was performed. The obtained results are shown in Table 11. Under the condition where stirring was not performed, the number of bacteria reached was about half, and the culture was performed in a state where a precipitate was present at the bottom of the jar. The number of reached bacteria was the same in both the atmosphere and carbon dioxide aeration when cultured with stirring in the liquid medium composition of the present invention. On the other hand, the concentration of lactic acid in the medium was significantly higher under anaerobic stirring culture conditions in which carbon dioxide gas was aerated than in aerobic stirring culture conditions in which air was aerated.
[0074]
  Composition of liquid medium (% by weight) of the present invention: 4.22% magnesium carbonate, 8.0% Glc, 6.0% high-newt SMP, 3.2% WT (Glc was sterilized together with other medium components. The medium pH after sterilization was 8.07.)
  Jar Fermenter: Jar Fermenter MBF manufactured by Tokyo Science Instruments Co., Ltd., 2.5 L culture condition: 30 ° C., 24 hours culture. Stirring speed, 500 rpm. Aeration rate, 1vvm
[0075]
[Table 11]

[0076]
Test Example 12 “Enhanced liquid culture of E. faecalis JCM5803 strain & production of freeze-dried bacterial powder”
1. Liquid culture
  One ml of Enterococcus faecalis JCM5803 strain was inoculated into 10 ml of BL liquid medium (test tube with a silico stopper), and cultured with shaking at 30 ° C. overnight. The number of bacteria reached is 39.5x10⁸cfu / ml. 0.5 ml of the bacterial solution was inoculated into 50 ml of a liquid medium having two compositions shown in Table 9 (500 ml Erlenmeyer flask with cotton plug) and cultured at 30 ° C. and a shaking speed of 130 rpm for 24 hours. Glc was sterilized by autoclave together with other components. The analysis results after culture are shown in Table 12. In both types of the medium of the present invention, the number of reached bacteria is 400 × 10 6⁸cfu / ml and 325x10⁸The number of bacteria was increased to cfu / ml. Incidentally, in the BL liquid medium, the number of bacteria reached in the 10 ml liquid medium used for the preparation of the liquid species was reduced to about 30% in the main culture using 50 ml of the medium. When this strain was subjected to aerobic shaking culture in a BL liquid medium, it was presumed that growth was likely to be inhibited when the amount of oxygen supplied was larger than Sf used in the test examples.
[0077]
[Table 12]

[0078]
2. Freeze drying
  40.0 ml of the above-mentioned two types of culture solutions were adjusted to pH 5.0 with 1N hydrochloric acid to solubilize insoluble carbonate, and then centrifuged at 9000 rpm for 5 minutes to collect the cells, and phosphate buffer (pH 6. 8) Suspended in 30 ml. Again, the cells were collected by centrifugation at 9000 rpm for 5 minutes to obtain washed cells. The cells were suspended in 15 ml of a stabilizer (composition: 10% skimmed milk & 1% sodium glutamate; Koch sterilized for 30 minutes), transferred to a 100 ml eggplant flask, and freeze-dried overnight. The obtained freeze-dried powder was evaluated and the results are shown in Table 13. The two types of the culture media of the present invention were 5.48 times higher in the magnesium carbonate-containing medium and 2.00 times higher in the calcium carbonate-containing medium than the BL liquid medium. .
[0079]
[Table 13]

[0080]
Test Example 13. “Liquid culture of Enterococcus faecium IFO3128 and production of freeze-dried bacterial powder”
1. Liquid culture
The inoculum is filled with 10 ml of BL liquid medium in a test tube with a silico stopper and sterilized by autoclaving for 20 minutes. Prepared by culturing. The number of bacteria in each culture was measured by diluting the bacterial solution 10-fold with phosphate buffer (pH 6.8), and then 0.1 ml of the solution was smeared on a BL agar medium and aerobically cultured at 37 ° C. And forming colonies. The obtained results are shown in Table 14.
[0081]
[Table 14]

[0082]
0.5 ml of the above inoculum was inoculated into 50 ml of a liquid medium placed in a 500 ml Erlenmeyer flask equipped with a silico stopper, and cultured at 30 ° C. under shaking conditions at 120 rpm for 20 hours. The obtained culture broth was measured for the number of bacteria under the same conditions as the inoculum, and the results are shown in Table 15. The number of bacteria increased in the liquid medium of the present invention compared to the comparative example, and in particular, magnesium carbonate was added. The prepared medium was increased in comparison with the medium supplemented with calcium carbonate.
[0083]
Composition A. BL liquid medium (comparative example)
Composition B. 3.2% WT Extract, 6.0% High Neutral SMP, 8% Glc, 4.22% Magnesium Carbonate
Composition C. 3.2% WT extract, 5.0% high-newt SMP, 10% Glc, 5.0% calcium carbonate
* Preparation of WT extract: WT was suspended in deionized water to 10 wt%, boiled for 30 minutes, centrifuged at 9000 rpm for 10 minutes, and the supernatant fraction was collected to obtain 10% WT extract. .
[0084]
[Table 15]

[0085]
2. Freeze drying
  40.0 ml of the culture medium with medium composition B and C was adjusted to pH 5.0 with 1N hydrochloric acid to solubilize the hardly soluble carbonate, and centrifuged at 9000 rpm for 5 minutes to collect the bacterial cells. It was suspended in 30 ml of buffer (pH 6.8). Again, the cells were collected by centrifugation at 9000 rpm for 5 minutes to obtain washed cells. The washed cells were suspended in 10 ml of a stabilizer (composition: 10% skim milk & 1% sodium glutamate; Koch sterilized for 30 minutes), and the entire amount was placed in a 100 ml eggplant flask and freeze-dried overnight. Table 16 shows the weight and the number of bacteria of the obtained freeze-dried powder. In any of the strains, when the bacterial solution obtained under the liquid culture conditions of the present invention was lyophilized, a lyophilized powder having a significantly higher number of bacteria was obtained.
[0086]
[Table 16]

[0087]
Test Example 14. “Enhanced liquid culture of lactobacilli and production of freeze-dried bacterial powder”
1. Liquid culture
One platinum loop of Lactobacillus casei NRIC1917 strain was inoculated into 10 ml of BL liquid medium (50 ml test tube with a silico stopper), and cultured overnight at 30 ° C. (shaking speed, 300 rpm). The number of bacteria reached is 31x10⁸cfu / ml. 0.5 ml of the stationary phase bacterial solution was added to 50 ml of the liquid medium of the present invention (medium composition (% by weight): 5.0% calcium carbonate, 10.0% Glc, 5.0% high-newt SMP, and 3.2%). WT; 500 ml Erlenmeyer flask with cotton stopper) and cultured at 30 ° C. and shaking speed 130 rpm for 30 hours. Glc was sterilized by autoclave together with other components. The number of bacteria reached after culture is 152x10⁸cfu / mlThe lactic acid concentration in the medium was 38.9 mg / ml.
[0088]
2. Freeze drying
  40.0 ml of the above culture solution was adjusted to pH 5.0 with 1N hydrochloric acid to dissolve hardly soluble calcium carbonate, and then centrifuged at 9000 rpm for 5 minutes to recover the cells. The cells were suspended in 30.0 ml of phosphate buffer (pH 6.8), centrifuged at 9000 rpm for 5 minutes, and the cells were collected and washed. The washed cells were suspended in 15.0 ml of a stabilizer (composition: 10% skimmed milk and 1% sodium glutamate; processed for 30 minutes by Koch sterilization), transferred to a 100 ml eggplant flask, and freeze-dried overnight. . The obtained freeze-dried bacterial powder has a weight of 3.15 g and a bacterial count of 675 × 10 6.⁸cfu / g.
[0089]
【The invention's effect】
(1) Magnesium carbonate, calcium carbonate or hydrotalcite alone or an inorganic salt mixture containing these (2) monosaccharides or disaccharides, (3) peptone, (4) straw, cocoon extract, germ medium, yeast, or It has been found that a lactic acid bacterium culture having an increased number of bacteria can be obtained by using yeast extract alone or a mixture containing these in a liquid medium and culturing lactic acid bacteria with stirring.

Claims (5)

(1) magnesium carbonate,
(2) monosaccharide or disaccharide,
(3) Peptone and
(4) Enterococcus faecalis or Enterococcus faecium characterized by stirring and culturing a mixture containing one or more selected from sputum, persimmon extract, germ medium, yeast, and yeast extract in a liquid medium Culture method. (1) 2-13% (w / w) magnesium carbonate,
(2) 5-15% (w / w) monosaccharides,
(3) 5-13% (w / w) peptone, and
(4) 2-5% (w / w) of 1 type or 2 types chosen from a cocoon, a cocoon extract, a germ medium, yeast, or a yeast extract,
2. The method of culturing Enterococcus faecalis or Enterococcus faecium according to claim 1, wherein a mixture comprising 2. The method of culturing Enterococcus faecalis or Enterococcus faecium according to claim 1, wherein the bacterium or cocoon extract-producing bacterium is a filamentous fungus. The method of culturing Enterococcus faecalis or Enterococcus faecium according to claim 3, wherein the filamentous fungus is Aspergillus oryzae. After culture | cultivating by the method of Claim 1, after adding carbonate to a culture medium and adjusting a medium pH to 4-6 and melt | dissolving carbonate, a microbial cell is isolate | separated from a culture medium and collect | recovered, and freeze-drying A process for producing Enterococcus faecalis or Enterococcus faecium powder, characterized in that: