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Structure-viscosity relationship of exopolysaccharide produced by Lactobacillus fermentum MTCC 25067

MENGI Bharat 帯広畜産大学

2021.02.08

概要

インド産伝統的発酵乳「ダヒ」から単離された乳酸菌Lactobacillus fermentumMTCC25067株が生産する菌体外多糖(HePS)はキサンタンガ厶と同等の高い粘性を示す。近年、同菌株の培養液が示す高粘性は複数の当該HePS分子が会合し超分子ネットワークを形成することに起因すると示された。予備実験として生菌数、pH値、培養上清中の残存グルコース濃度、培養液の粘性を培養144時間にわたり測定した。その結果、生菌数は培養開始後16時間から24時間で最高値を示したのち漸減した。pH値と残存グルコース濃度は培養開始後24時間で大幅に減少したのち一定値を示した。同菌株の培養液が示す粘性は培養開始後48時間で最大となり、その後、定常期の間、少なくとも培養開始後144時間までは低下した。生菌数、pH値、残存グルコース濃度は3反復行った実験間での誤差は非常に小さい値であったのに対し、培養液粘性に関しては培養開始後24時間から96時間にかけて大きく変動した。そこで本博士論文は、同HePSが示す構造粘性相関と、その物理化学的性質の培養期間中における経時変化を明らかにすることを目的とした。まず、同菌株の48時間培養上清と144時間培養上清から精製したHePS(以下それぞれHePS48hおよびHePS144hと略す)の特徴を比較した。HePS48hおよびHePS144hの分子量分布パターンをゲル濾過クロマトグラフィーとフェノール硫酸法による糖濃度測定により見積もったところ、HePS48hおよびHePS144hはほぼ同様のピークパターンを示したがHePS144bにのみ第3のピークが観察されたことから、HePS144hがより多くの低分子画分を有することが示された。次に、600-MHz 1D ¹H-NMRを用いてHePS分子の側鎖構造を解析した。3位置換D-Glcpのピーク面積に対する比として計算したところ、末端D-Glcp、3位置換D-Glcp、2,3位置換D-Glcp、6位置換D-Galpのピーク面積比はHePS48hとHePS144hでほぼ同様であった。従って、HePS48hおよびHePS144hの側鎖構造に相違はないと結論した。次に、レオメーターを用いてHePS48hおよびHePS144hの1%水溶液の25°Cにおける0.1/s to 100/sの範囲における定常ずり粘度を求めたところ、ずり速度の上昇に伴いずり応力が低下する非ニュートン流体に典型的なパターンを示した。そのような特徴を示す物質として大きな回転半径を有する直鎖状高分子が挙げられる。一方、低ずり速度における定常ずり粘度はHePS144h水溶液と比較してHePS48h水溶液は有意に高い値を示した。従って、HePS_水溶液の粘性低下はHePS分子の切断による低分子化が一因であると考えられた。さらに、原子間力顕微鏡(AFM)を用いたHePS48hおよびHePS144hの微細構造観察の結果、両HePS共にネットワーク構造が観察されたが、HePS48hとは対照的にHePS144hは細く短かいHePS繊維が大きな割合を占めていることが示された。従って、これもHePS144h水溶液の低ずり速度における定常ずり粘度低下の一因であると示唆された。二次代謝産物や培地成分などバクテリア周囲に存在する物質と菌体との相互作用も培養液の物性に大きな影饗を与えることが推定される。そこで菌体を含む培地成分とHePS分子との相互作用が粘性に与える影響を調べるため、0.02%のHePS48hまたはHePS144hをMRS培地に溶解し粘性を測定した。その結果、両HePS溶液ともニュートン流体の挙動を示し、菌体培養液と比較して有意に低い粘性を与えた。また、AFMにより培養液そのものを用いて菌体の形状解析を実施したところ、48時間培養では菌体周辺部に明瞭なHePS超分子ネットワークが観察されたが144時間培養ではこれが消失していることが示された。さらに、144時間培養液中で形成されたHePSネットワークには網の目の交点が他の部分よりも1.9倍高い構造が観察され、利湯は不明であるが48時間培養液中で同様の構造は観察されなかった。これらの発見により、長時間培養後、死菌体の細胞壁の脆弱化により菌体表層とHePS超分子ネットワーク間の相互作用が低下し、その結果として培養液の粘性が低下したとする仮説を得た。また、同菌株のコロニーの走査型電子顕微鏡(SEM)の結果、菌体周辺に幾層にも重なった明瞭なHePS超分子ネットワークの存在を認めたことから、超分子ネットワーク形成は当該HePS分子が有する性質であることが支持された。以上から、正確な培養条件の制御とHePS分解因子の探索が効果的に培養液粘性の低下を防ぐ方法であると示された。また、産業利用のためには当該HePSと食品成分との相互作用を明らかにするべきである。さらに、当該HePS生産に関連する遺伝子の慟きを明らかにする必要がある。本研究では、乳酸菌の遺伝子破壊株作製に汎用される二手法、すなわち抗生物質耐性遺伝子をマーカーとして用いるプラスミドと温度感受性ベクターpORI28プラスミドを使用し、当該菌株の糖転移酵素遺伝子LF25067_00121(GenBank ID: AP017973.1)遺伝子破壊株作製用プラスミドを構築し、同菌株の野生型を宿主として用いたHePS生合成関連遺伝子破壊株を取得するため方法を最適化した。その結果、電気穿孔によるコンピテントセルへのプラスミド導入は、菌体の十分な洗浄によるHePS除去が重要であることが示された。以上、本研究で得られた知見を適用することで、培餐上清や発酵食品の粘性を操作するために必要な当該HePS分子と菌体表層との相互作用の理解に関して重要な知見を得ることが今後期待される。

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