JP2004329077A - CULTURE PRODUCT, NEW beta-1,3-1,6 GLUCAN CONTAINED IN THE SAME AND METHOD FOR PRODUCING beta-1,3-1.6 GLUCAN USING STRAIN OF GENUS AUREOBASIDIUM - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a culture product containing β-1,3-1,6 glucan derived from a strain belonging to the genus Aureobasidium in a widely usable state in the field of foods and beverages in which three problems of (1) the yield of β-1,3-1,6 glucan in the culture product different in each culture, (2) a large amount of pellets formed in the culture product and (3) browning of the culture product by melanin pigment are solved. <P>SOLUTION: The method comprises introducing a fungus state of chlamydospore of the strain of the genus Aureobasidium. The method for culturing the strain of the genus Aureobasidium comprises stably obtaining β-1,3-1,6 glucan in high concentration in the culture product in which the chlamydospore is used in preculture. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

【００１４】
Ａｕｒｅｏｂａｓｉｄｉｕｍ属菌株がβ−１，３−１，６グルカンを生産する際に資化される炭素源を決定した。詳しくは以下のとおりである。炭素源としてサッカロース０．５重量％、ビタミンＥを添加し粉砕した中糠０．２重量％、ビタミンＣ ０．２重量％を含みｐＨ５．０〜６．０に調整した液体培地中にＦＯ−６８菌株を植菌し、２０℃で１００時間振盪培養した。培地中に生産されるβ−１，３−１，６グルカン量を前述の方法で、培地に添加した炭素源であるサッカロースとその分解物であるグルコース・フルクトースはＦキット（Ｒｏｃｈｅ製）を用いて測定し、糖成分の経時変化を追った。
【００１５】
図１に結果を示した。β−１，３−１，６グルカンの生産開始は、サッカロースがほぼ完全に分解された培養開始４０時間後であった。サッカロースの分解物であるグルコースはβ−１，３−１，６グルカンが生産されると共に減少していった。一方グルコースが存在していない７２時間以降には、サッカロースのもう一つの分解物であるフルクトースの減少は見られるがβ−１，３−１，６グルカンの生産は見られなかった。これらのことは、β−１，３−１，６グルカンの生産にグルコースが使用されていることを示していた。そこでサッカロースの代わりにグルコースを炭素源として使用しＦＯ−６８菌株を培養したところ、β−１，３−１，６グルカンが約２０時間早く生産され始めた。これらのことからＡｕｒｅｏｂａｓｉｄｉｕｍ属菌株培養にはグルコースが炭素源に適していることが明らかとなった。
【００１６】
前述したようにＡｕｒｅｏｂａｓｉｄｉｕｍ属菌株は生息環境変化に伴い増殖特性として菌糸−酵母２形性を示し、その複雑な菌形態は多糖生産能にも大きく影響している。栄養豊富な生育環境で多く見られる分芽胞子や菌糸では、それぞれ出芽と分節による増殖が優先され菌体外多糖生産は抑制され、貧栄養状態の時に見られる２重の細胞壁を持つ生体防御に優れた厚膜胞子は、休眠性細胞の性質を持ち代謝能が弱く菌体外多糖生産をほとんど行わない。一方分芽胞子から厚膜胞子への菌形態の移行時に見られる、膨張した酵母様細胞は菌体外へ活発に多糖生産を行う。Ａｕｒｅｏｂａｓｉｄｉｕｍ属菌株を用いた通気撹拌培養時において培養産物中のβ−１，３−１，６グルカン濃度は、培養終了時に見られる膨張した酵母様細胞の数だけでなく形態とも密接に関係している。つまり、細胞が大きく膨張し、内在する液胞の数が多い酵母様細胞ほどβ−１，３−１，６グルカン生産能は高くなる。
【００１７】
培養時における膨張した酵母様細胞の数と形態は、植菌する菌体の菌形態と関係しており、厚膜胞子を植菌した培養時に大きく膨張した酵母様細胞をより多く得られ、菌体外多糖生産能が最も高くなった。詳細は以下に述べる。
【００１８】
Ａｕｒｅｏｂａｓｉｄｉｕｍ属菌株を窒素源が不足した条件下で数世代植え継ぐことにより厚膜胞子を導く方法を発明した。詳しくはポテトデキストロースブロス（Ｄｉｆｃｏ社製）０．２４〜０．６重量％、グルコース１．５〜２．０重量％を含みｐＨ５．２に調整した窒素源枯渇培地にアガロース２．０重量％を加えた寒天培地にてＡｕｒｅｏｂａｓｉｄｉｕｍ属菌株を数世代生育させた後、同組成の液体培地に１〜２白金耳接種して２０℃〜３０℃で７２時間振盪培養し厚膜胞子の菌形態を誘導する方法である。Ａｕｒｅｏｂａｓｉｄｉｕｍ属菌株を窒素源が豊富な条件下で数世代植え継ぐことにより分芽胞子を導くこともできる。つまり上記の培養時に用いた窒素源枯渇培地の代わりに、ポテトデキストロースブロス（Ｄｉｆｃｏ社製）２．４重量％を溶解した培地を用いて同様の培養を行った場合には分芽胞子の菌形態を誘導することが可能である。
【００１９】
誘導したＦＯ−６８菌株の厚膜胞子と分芽胞子をそれぞれ、ビタミンＥを添加し粉砕した中糠０．１〜１．０重量％、グルコース０．５〜２．０重量％、ビタミンＣ ０．２〜０．５重量％を含みｐＨ５．０〜６．０に調整した液体培地に、培養開始時の菌数が１０^４ ｃｅｌｌ／ｍＬになるよう植菌して２０℃で９６時間振盪培養した。培養後に培養産物中の菌体数の測定、菌形態の観察、β−１，３−１，６グルカン量の測定により菌体数・菌形態と菌体外多糖生産との関係を見た。
【００２０】
菌体数はＴｈｏｍａ血球計を用い計測した。また菌形態は、細胞内部に液胞を２つ以上内在する細胞を膨張した酵母様細胞、２重の細胞壁を持つ細胞を厚膜胞子、その他の酵母型細胞を分芽胞子と分類した。β−１，３−１，６グルカン量は前述した方法にて測定した。
【００２１】
測定結果は図２に示した。誘導した厚膜胞子を用いた培養時には全菌体数の６０％以上は膨張した酵母様細胞であり、細胞自体も７〜１２ μｍと大きく、細胞内部には多くの液胞が見られ、培養産物中β−１，３−１，６グルカン量は約２．５ ｇ／Ｌと高い値であった。一方誘導した分芽胞子を用いた培養時には全菌体数の約４０％が膨張した酵母様細胞であったが、細胞自体は２〜５ μｍと小さく、細胞内部に２〜３個の液胞しか持たない細胞も見られ、培養産物中β−１，３−１，６グルカン量は約１．４ ｇ／Ｌと低い値であった。
【００２２】
さらに厚膜胞子植菌時得られた図２に示した菌体を、ビタミンＥを添加し粉砕した中糠０．１〜１．０重量％、グルコース０．５〜２．０重量％、ビタミンＣ ０．２〜０．５重量％を含みｐＨ５．０〜６．０に調整した液体培地に１／１０００量接種し、２０℃で７２時間振盪培養した際の菌体の菌形態比率と培養産物中のβ−１，３−１，６グルカン濃度を図３に示した。図から明らかなように同組成の培地に植え替えた時、３世代までほぼ同様の菌形態の比率とβ−１，３−１，６グルカン濃度が得られた。このことは、３段階の植え替えにより約１．０×１０^９倍のスケールアップを行った場合にも、一定数の大きく膨張した酵母様細胞が得られ、安定してβ−１，３−１，６グルカンが得られることを示していた。
【００２３】
またＡｕｒｅｏｂａｓｉｄｉｕｍ属菌株は好気性菌であり、β−１，３−１，６グルカンの生産も好気的条件下にて進み、Ａｕｒｅｏｂａｓｉｄｉｕｍ属菌株を用いた培養時における培養産物中のβ−１，３−１，６グルカン濃度は、培地中に混入される空気の量に影響される。そのため、コンベンや三角フラスコを用いた浸透培養時よりも、通気攪拌培養時にβ−１，３−１，６グルカン生産能は高くなる。通気量は、培地粘度の上昇を考慮し調整する必要があるが、時間当り培地の１０〜１００倍容量の通気を行い攪拌培養した場合に、最も効率良くβ−１，３−１，６グルカンが生産される。前述した窒素源枯渇培地にて本菌を数世代培養し誘導した厚膜胞子を、米糖０．１〜１．０重量％、グルコース０．５〜２．０重量％、アスコルピン酸０．２〜０．５重量％を含みｐＨ５．０〜６．０に調整した液体培地に接種し、時間当り培地の１０〜１００倍容量の通気を行いながら、７２時間２０℃にて攪拌培養した場合には、約３．０ｇ／Ｌ以上の高濃度のβ−１，３−１，６グルカンを含む培養産物が得られた。
【００２４】
Ａｕｒｅｏｂａｓｉｄｉｕｍ属菌株培養後に得られた培養産物が持つ、多量のペレット形成とメラニン色素による褐変という２つの問題点を解決するために、本発明ではＦＯ−６８菌株を用いた。ＦＯ−６８菌株を用いた培養の場合、ペレットを含まない乳白色〜淡黄白色の一様な高粘性の液体状の培養産物を得ることができる。詳細は以下に述べる。
【００２５】
ＦＯ−６８菌株の菌体学的特徴として以下のことが上げられる。本菌は平板寒天培地・液体培地を用いた培養時には生育温度２０〜２５℃、最適生育温度２５℃、生育可能温度５℃〜４０℃、最適生育ｐＨ ５〜７を示し、生息環境に応じて他のＡｕｒｅｏｂａｓｉｄｉｕｍ属菌株と同様の増殖特性である菌糸−酵母２形性を示す。さらに菌形態は図４に示したように、増殖するための細胞で菌糸伸長の元となる分芽胞子（Ｂｌａｓｔｓｐｏｒｅ）、出芽型分芽胞子や分節型分芽胞子が成長した細胞である酵母様細胞（Ｙｅａｓｔ ｌｉｋｅ ｃｅｌｌ）、そして菌糸や酵母様細胞の分化により生じるメラニン色素合成能を持つ厚膜胞子（Ｃｈｌａｍｙｄｏｓｐｏｒｅ）の計３種類の酵母型細胞と、薄い隔壁で細胞が連結した菌糸（Ｈｙｐｈａｅ）、２重の隔壁を持ち細胞自体も肥大した膨張細胞（Ｓｗｏｌｌｅｎ ｃｅｌｌ）の計２種類の菌糸型細胞に分別することができる。
【００２６】
本菌は、栄養豊富な生育環境では分芽胞子からの菌糸伸長と出芽が活発に起こり、時間経過と共に細胞内部に液胞が見られるようになり、伸長した菌糸は２重の隔壁を持ち細胞自体も肥大した膨張細胞の状態に移行していく。一方、貧栄養状態になると膨張した菌糸の分節が観察されるようになり、他の環境要因の悪化または更なる貧栄養状態の時には、膨張細胞より更に大きな２重の細胞壁を持つ生体防御に優れた厚膜胞子が見られるようになる。
【００２７】
土壌中より単離した本菌はポテトデキストロース（日水製薬）平板寒天培地上にて２５〜３０℃で２週間培養した場合、表面を覆う粘物質による光沢と菌糸伸長による立毛とが観測される直径５〜６ ｃｍのコロニーを形成する。２週間後のコロニーは中心部に僅かなメラニン色素による褐変が見られるものの全体は白色〜淡桃色であり（図５左）、同属のＦＥＲＭ−Ｐ４２５７菌株を同条件にて２週間培養した場合に生じる全体が黒色のコロニー（図５右）とは明らかに異なる。さらに２週間培養後の本菌の平板寒天培地上にコロニーを４℃にて保存した場合、日数経過と共に褐変部分は中心部より広がり全体的に黒色となる。これらの特徴は本菌が黒色菌科のＡｕｒｅｏｂａｓｉｄｉｕｍ属の性質を持ちつつも、菌形態としてメラニン色素合成能を持つ厚膜胞子を取り難く、また厚膜胞子のメラニン色素合成能自体も低いという性質を示していた。
【００２８】
さらに通気撹拌培養時の培養産物の液色についても、平板寒天培地上のコロニーの色と同様の傾向が見られる。詳細は以下の通りである。本菌株を、米糠０．１〜１．０重量％、サッカロース０．５〜２．０重量％またはグルコース０．５〜２．０重量％、ビタミンＣ ０．２〜０．５重量％を含みｐＨ５．０〜６．０に調整した液体培地に植菌し、時間当り培地の１０〜１００倍容量の通気を行いながら、７２時間２０℃にて攪拌培養して培養産物を得た。得た培養産物を１０ ｍＭｐＨ６．０酢酸ナトリウム緩衝液にて希釈した後、４０，０００Ｇ ３０ 分間の遠心分離により不溶性固形物を除去し、得た上清の４００〜８００ ｎｍの範囲の吸光度を測定した。測定結果を図６に示す。培養産物の吸収スペクトルは、４００〜８００ ｎｍの範囲における吸光度の変化が少なく、白色に近い液色であることを示した。また本菌株の厚膜胞子を、ビタミンＥを添加し粉砕した中糠０．１〜１．０重量％、グルコース０．５〜２．０重量％、ビタミンＣ ０．２〜０．５重量％を含みｐＨ５．０〜６．０に調整した液体培地に植菌し、時間当り培地の１０〜１００倍容量の通気を行いながら、７２時間２０℃にて攪拌培養して培養産物を得た場合にも同様の傾向が見られた。
【００２９】
本菌の大半の菌糸型細胞は、幅４．０〜６．０μｍ、膨張細胞時で９．０〜１２μｍ、長さ１ ｍｍ程度であり、幅１．５〜２．５ μｍ、膨張細胞時で４．０〜５．０μｍ、長さ２ ｍｍ以上長いもので１０〜１２ ｍｍに達すＦＥＲＭ−Ｐ４２５７菌株の菌糸型細胞に対し、太く短く絡まり難い形状である。これは、通常Ａｕｒｅｏｂａｓｉｄｉｕｍ属菌株を液体培地中で培養した場合に、菌糸伸長後に分節が開始されるのに対し、本菌培養時には菌糸の分節による増殖が優先され、菌糸伸長が阻害されるからである。さらに本菌の分芽胞子は菌糸伸長よりも出芽による増殖を活発に行うため、菌形態に占める菌糸型細胞の割合自体が低い。これらの菌糸型細胞の特徴は図７に示した。実際、本菌株を、米糠０．１〜１．０重量％、サッカロース０．５〜２．０重量％またはグルコース０．５〜２．０重量％、ビタミンＣ ０．２〜０．５重量％を含みｐＨ５．０〜６．０に調整した液体培地に植菌し、時間当り培地の１０〜１００倍容量の通気を行いながら、７２時間２０℃にて攪拌培養して培養産物を得た場合には、ペレットを形成することなく一様な高粘性の液体を得ることができた。また本菌株の厚膜胞子を、ビタミンＥを添加し粉砕した中糠０．１〜１．０重量％、グルコース０．５〜２．０重量％、ビタミンＣ ０．２〜０．５重量％を含みｐＨ５．０〜６．０に調整した液体培地に植菌し、時間当り培地の１０〜１００倍容量の通気を行いながら、７２時間２０℃にて攪拌培養して培養産物を得た場合にも同様の傾向が見られた。一方以前に報告された同属のＦＥＲＭ−Ｐ４２５７菌株を同条件にて培養した場合には、培養産物中に多量のペレットが形成され、小さい物でも肉眼で確認でき大きい物は直径１ｃｍ程度の球状の形態をとっていた。
【００３０】
本菌を用い培養した場合には、培養産物中の多量のペレットとメラニン色素による褐変の２つの問題点を解決し、ペレットを含まない乳白色〜淡黄白色のβ−１，３−１，６グルカンを含む培養産物を得ることができる。さらに本発明にかかる培養方法、詳しくは厚膜胞子を、ビタミンＥを添加し粉砕した中糠０．１〜１．０重量％、グルコース０．５〜２．０重量％、ビタミンＣ ０．２〜０．５重量％を含みｐＨ５．０〜６．０に調整した液体培地に植菌後、時間当り培地の１０〜１００倍容量の通気を行いながら、７２時間２０℃にて攪拌培養する培養方法で本菌を培養した場合には、安定して高濃度のβ−１，３−１，６グルカンを含む、ペレットを含まない乳白色〜淡黄白色の培養産物を得ることができる。このＦＯ−６８培養産物は、色・物性より食品・飲料の分野で広く利用することができ、また一定量のβ−１，３−１，６グルカンを含むことから、培養産物の持つ機能性も安定する。またＦＯ−６８培養産物は生態免疫反応を調節するユニークな特性を持っており、食品分野のみならず特に医療・保健・福祉・環境など生命科学を基盤とした分野への応用が期待できる。
以下に培養産物の持つ機能性を測定した例を示す。
【００３１】
【実施例】
ＦＯ−６８培養産物のヒト末梢血単球の細胞分裂（ＤＮＡ合成能）に与える影響を見た。健常人血液からＦｉｃｏｌｌ−Ｃｏｎｒａｙ法で末梢血単核球を分離し、１％正常ヒト血清（ｈｕｍａｎ ｓｅｒｕｍ：ＨＳ）に浮遊した。次に４００倍に希釈した新しい菌株ＦＯ−６８由来の新規培養産物（以下ＦＯ−６８培養産物）を添加し、５日間、３７℃で培養した。５日後、トリチウムサイミジン（^３Ｈ−ｔｈｙｍｉｄｉｎｅ）１ μＣｉを加え、その取り込み量を液体シンチレーションカウンターで測定し、培養産物の末梢血単核球の細胞分裂能（ＤＮＡ合成能）に与える影響を調べた。その結果、表２に示すように、添加群は無添加群に比べて有意に末梢血単核球のＤＮＡ合成能を亢進させた。
【００３２】
【表２】

【００３３】
ＦＯ−６８培養産物の白血病細胞株に与える影響を見た。各種白血病細胞株２×１０^５個にＦＯ−６８培養産物を添加し、３日間、３７℃で培養した。培養後、血球計算盤を用いて生細胞数を計測し、白血病細胞株の増殖を検討した。その結果表３に示すように、添加群は無添加群に比べて有意に白血病細胞株の増殖を抑制した。
【００３４】
【表３】

【００３５】
ＦＯ−６８培養産物のサイトカイン産生に与える影響を見た。単球系細胞株（Ｕ９３７）に３０倍希釈したＦＯ−６８培養産物を、添加し、２４時間、３７℃で培養した。また、健常人末梢血単核球に４００倍希釈のＦＯ−６８培養産物を添加し、３７℃で８時間および２４時間培養した。培養後、培養上清中のサイトカインの中でＩＬ（インターロイキン）−１β、ＩＬ−２、ＩＬ−４、ＩＬ−６、ＩＬ−８、ＩＦＮ（インターフェロン）−γの量を酵素抗体法（ｅｎｚｙｍｅ ｉｍｍｕｎｏａｓｓａｙ：ＥＩＡ）で調べた。その結果表４および表５に示すように添加群は無添加群に比べて有意にＩＬ−８の産生を誘導した。ＩＬ−８は主に免疫担当細胞の走化性を誘導するサイトカインで、好中球、単球、肺胞マクロファージ、血管内皮細胞などから産生される。また、最近は血管新生を促す因子としても注目され、免疫担当細胞の走化や集積に関与しているものと考えられる。
【００３６】
【表４】

【００３７】
【表５】

【００３８】
ＦＯ−６８培養産物のマウス脾臓細胞のＤＮＡ合成能に与える影響を見た。Ｃ５７ＢＬ／６Ｎマウス由来の脾臓細胞を型のごとく調整した。次に１００倍に希釈したＦＯ−６８培養産物を添加し、２日間、３７℃で培養した。２日後、トリチウムサイミジン１ μＣｉを加え、その取り込み量を液体シンチレーションカウンターで測定し、脾臓細胞の分裂（ＤＮＡ合成能）を調べた。その結果、表６に示すように、添加群は無添加群に比べて脾臓細胞のＤＮＡ合成能を亢進させた。
【００３９】
【表６】

【００４０】
ＦＯ−６８培養産物のマウス脾臓細胞からのＩＬ−１２産生に与える影響を見た。Ｃ５７ＢＬ／６Ｎマウス由来の脾臓細胞を型のごとく調整した。次に１００倍に希釈したＦＯ−６８培養産物を添加し、２〜３日間、３７℃で培養した。培養後、培養液を回収し、培養液中のＩＬ−１２の量をＥＩＡ法で測定した。その結果、表７に示すように、添加群は無添加群に比べて脾臓細胞からのＩＬ−１２産生を有意に亢進させた。ＩＬ−１２は抗腫瘍活性を示すＮＫ（ナチュラルキラー）細胞の活性化因子で、静止状態のＮＫおよびＴ細胞からのＩＦＮ−γ（インターフェロン）の産生を強く誘導し、ＮＫおよびＴ細胞の細胞傷害活性を高める重要なサイトカインである。本結果からＦＯ−６８培養産物は免疫系の活性化のみならず、抗腫瘍活性や抗微生物活性に優れた培養産物であることが判明した。
【００４１】
【表７】

【００４２】
ＦＯ−６８培養産物のマウス腹腔細胞のＮＫ活性への影響を見た。ＦＯ−６８培養産物をＣ５７ＢＬ／６Ｎマウスの腹腔内に１日おきに５回注射した後、アイソトープであるクロム（^５１Ｃｒ）でラベルしたＹＡＣ−１細胞（マウス白血病細胞株）を標的細胞としたクロムリリーズ法で、腹腔細胞のＮＫ活性を測定した。その結果、表８に示すように、投与群は未投与群に比べてＮＫ活性を誘導した。本結果からＦＯ−６８培養産物は抗腫瘍活性を高めることが判明した。
【００４３】
【表８】

【００４４】
前述したように、本発明にかかる新規培養方法で本菌を培養した時に生産される培養産物は、細胞のＤＮＡ合成能の増加・サイトカイン産生の誘導・ＮＫ活性の獲得など生態免疫反応を調節するユニークな特性を持っており、抗腫瘍活性や抗微生物活性などを高める。β−１，３−１，６グルカンの機能性はその構造により異なり、ＦＯ−６８培養産物中に含まれるβ−１，３−１，６グルカンは、糖の構造解析の結果から高い分岐率を持つ新規β−１，３−１，６グルカンであった。今回得た高い分岐率を持つ新規β−１，３−１，６グルカンは、その持つ生態免疫反応から特に医療・保健・福祉・環境・食品など生命科学を基盤とした分野への応用が期待できる。糖の構造解析の詳細は以下に述べる。
【００４５】
窒素枯渇培地で誘導した本菌の厚膜胞子を、ビタミンＥを添加し粉砕した中糠０．１〜１．０重量％、グルコース０．５〜２．０重量％、ビタミンＣ ０．２〜０．５重量％を含みｐＨ５．０〜６．０に調整した液体培地に植菌後、時間当り培地の１０〜１００倍容量の通気を行いながら、７２時間２０℃にて攪拌培養した。こうして得られた培養産物を蒸留水にて１０倍希釈した後、４０，０００Ｇ ３０ 分間の遠心分離により不溶性固形物を除去した。得られた上清に１／５容量のクロロホルム：１‐ブタノール＝５：１の混合液を加えて３０分間激しく撹拌した後、遠心分離して得た上清液から分液ロートを用いて水層を分取した。この操作をさらに２回繰り返して得た水層に、終濃度７０ ％までエタノールを加えて多糖を沈殿させた後、遠心分離にて上清を取り除いた。こうして得た沈殿を２０ｍＭクエン酸‐リン酸緩衝液（ｐＨ ６．０）で再溶解した溶液に、同緩衝液で１０００倍希釈したプルラナーゼ懸濁液（Ｗａｋｏ社製）を１／３容量加えて３０ ℃にて２４時間反応させた後、分画分子量１２，０００〜１４，０００のα−セルロース膜中に入れて２００倍量の蒸留水に対して３ 回透析した。透析膜内液に終濃度８０ ％までエタノールを加えて多糖を沈殿させた後、遠心分離を行い、得た沈殿を常温減圧乾燥して菌体外分泌多糖の試料とした。
【００４６】
菌体外分泌多糖のメチル化を以下に記載する方法にて行った。試料１００ ｍｇにジメチルスルホキシド１０ ｍＬを加えた後、窒素流入化にて５５〜６０℃で１時間撹拌して溶解した。室温に冷却後、メチルスルフォニルカルバニオン３．０ ｍＬを加えて室温で５時間撹拌した後、ヨウ化メチル１．０ ｍＬを加えて２０℃以下に冷却しながら１時間撹拌した。このメチルスルフォニルカルバニオンとヨウ化メチルを加え撹拌する操作を３回繰り返した。反応液に精製水５０ｍＬを加えた後、クロロホルム２０ｍＬを加えて分液ロートにて５分間激しく撹拌してメチル化糖を抽出する操作を５回繰り返した。このクロロホルム抽出液に無水硫酸ナトリウムを加えて１時間脱水した後、４０℃で減圧乾固した物をメチル化糖とした。メチル化は赤外吸収分析により確認した。
【００４７】
段落［００４５］で得られたメチル化糖のアルジトールアセテート法は以下に記載した。メチル化糖を７２％Ｈ_２ＳＯ_４３．０ ｍＬに溶解した後、精製水１８ ｍＬを加えて１００℃で４時間加水分解した。室温まで冷却した溶液を炭酸バリウムで中和した後、遠心分離を行い、硫酸バリウムを除去した上清を５ ｍＬまで減圧濃縮した。濃縮液に水素化ホウ素ナトリウム（ＮａＢＨ_４）３０ ｍｇを加えて激しく撹拌した後、２時間放置して還元した。過剰なＮａＢＨ_４を分解するために気体が発生しなくなるまで５％酢酸水溶液を滴下した後、陽イオン交換樹脂アンバーライトＩＲ １２０Ｂを１５ ｍＬ加えて室温で３時間放置した。吸引濾過にて樹脂を取り除いた濾液を減圧乾固した後、１％塩酸酸性メタノール１０ ｍＬを加えて４０℃で減圧乾固する操作を３回繰り返した。次に、無水酢酸とピリジンを各々２．０ ｍＬ加えて１１０℃で３時間アセチル化した後、室温まで冷却後、精製水１０ ｍＬを加えて減圧乾固する操作を３回繰り返ことで未反応の無水酢酸とピリジンを除去した。さらに、トルエン１０ ｍＬを加えて減圧乾固する操作を３回繰り返した後、クロロホルム１０ ｍＬを加えて３時間放置し、アルジトールアセテート誘導体を抽出した。この抽出液の吸引濾過後の濾液は、減圧乾固してクロロホルム５ ｍＬに溶解する操作を２回行った後、ガスクロマトグラム質量分析の試料に用いた。
【００４８】
ガスクロマトグラムの結果を図８に、またマスフラグメントの結果を図９，１０，１１，１２と表９に示す。図８は本菌体外多糖の部分メチル化アルジトールアセテート誘導体のガスクロマトグラム（ＦＯ−６８の糖鎖構造の６Ｐ上段図、Ｆｉｇ．１ ＦＯ−６８ Ｖ．Ｃの部分メチル化アルジトールアセテート誘導体のガスクロマトグラムを使用）を示し、また図９は本菌体外多糖のマスフラグメント（ＦＯ−６８の糖鎖構造の６Ｐ下段図、Ｆｉｇ．２ ＦＯ−６８ Ｖ．Ｃのマスフラグメントの内、Ｐｅａｋ１の図を使用）を示し、図１０は本菌体外多糖のマスフラグメント（ＦＯ−６８の糖鎖構造の７Ｐ上段図、Ｆｉｇ．２ ＦＯ−６８ Ｖ．Ｃのマスフラグメントの内、Ｐｅａｋ２の図を使用）を示し、図１１は本菌体外多糖のマスフラグメント（ＦＯ−６８の糖鎖構造の７Ｐ下段図、Ｆｉｇ．２ ＦＯ−６８ Ｖ．Ｃのマスフラグメントの内、Ｐｅａｋ３の図を使用）を示し、図１２は本菌体外多糖のマスフラグメント（ＦＯ−６８の糖鎖構造の８Ｐ上段図、Ｆｉｇ．２ ＦＯ−６８ Ｖ．Ｃのマスフラグメントの内、Ｐｅａｋ４の図を使用）を示す。Ｐｅａｋ１は表９に示したように２，３，４，６−Ｍｅ_４−ＧＰであり、１位の炭素でのみ結合した非還元末端基のグルコースを表している。Ｐｅａｋ４は表９に示したようにＧ１結合（ｐｅａｋ１）を多く持ち、図８に示したようにＰｅａｋ１とほぼ同等の強度であることから、２，４−Ｍｅ_２−Ｇと考えられ１，３，６位の炭素に結合を持つグルコースと推定した。Ｐｅａｋ３は表９に示したように２，４，６−Ｍｅ_３−Ｇであり、１，３位の炭素に結合を持つグルコースであった。Ｐｅａｋ２は、糖であるフラグメントが見られたが、これはガスクロマトグラムの分離能によりＰｅａｋ３フラグメントが混合したためであり、Ｐｅａｋ２は非糖であった。以上の結果は菌体外多糖が、僅かな非糖部位を持つものの、主に１，３，６結合の分岐構造に非還元末端が結合した１，３−１，６グルカンでことを示していた。ガスクロマトグラムのピーク面積により各グルコースのモル比を求めた。その結果、２，３，４，６−Ｍｅ_４−Ｇ：２，４−Ｍｅ_２−Ｇ：２，４，６−Ｍｅ_３−Ｇ＝１６：３：１３であった。
【００４９】
【表９】

【００５０】
段落［００４５］に記載した箱守法でメチル化した菌体外多糖を重水素化クロロホルムに溶解した後、テトラメチルシラン（ＴＭＳ）を内部標準として、フーリエ変換式核磁気共鳴吸収装置にて^１３Ｃ核磁気共鳴吸収スペクトルを測定した。その結果を図１３に示す。図１３は本菌体外多糖の^１３ＣＮＭＲスペクトル（ＦＯ−６８の糖鎖構造の１Ｐ最下段図、ＦＯ−６８ の^１３ＣＮＭＲスペクトルの図を使用）を示す。
７０〜８０ ｐｐｍの吸収は重クロロホルムの３重線であり、矢印で示した１００ ｐｐｍ位の僅かに見られるピークと９０〜１００ ｐｐｍ付近にα−アノマーの吸収が見られないことから、菌体外多糖に存在する（１−３）結合と（１−６）結合は共にβ配向していることが分かった。
【００５１】
以上の分析結果から構成糖であるグルコースの結合様式は下図であり、１→６結合で１つのグルコースを持つグルコースが１→３結合で連続している分岐率が高い構造を持つβ−１，３−１，６グルカンであった。

【００５２】
【発明の効果】
Ａｕｒｅｏｂａｓｉｄｉｕｍ属には特定の条件下で培養した場合、生理活性を持つ水溶性のβ−１，３−１，６グルカンを菌体外に分泌する菌株は１菌株報告されていたものの、β−１，３−１，６グルカン生産に適した培養方法は確立されていなかった。そのため一定した収量のβ−１，３−１，６グルカンを得ることは難しく、Ａｕｒｅｏｂａｓｉｄｉｕｍ属菌株由来の培養産物の持つ機能性は培養毎で異なっていた。そこでβ−１，３−１，６グルカン生産に最適な培地組成と、β−１，３−１，６グルカン生産能の高い膨張した酵母様細胞を誘導する方法から、Ａｕｒｅｏｂａｓｉｄｉｕｍ属菌株の新規培養法を発明した。つまり、窒素枯渇培地で誘導した厚膜胞子を、ビタミンＥを添加し粉砕した中糠０．１〜１．０重量％、グルコース０．５〜２．０重量％、ビタミンＣ ０．２〜０．５重量％を含みｐＨ５．０〜６．０に調整した液体培地に植菌し、数多くの液胞を持つ大きい膨張した酵母様細胞を誘導しつつ、時間当り培地の１０〜１００倍容量の通気を行いながら、７２時間２０℃にて撹拌培養する方法である。この培養方法により、Ａｕｒｅｏｂａｓｉｄｉｕｍ属菌株を用いて安定して高濃度のβ−１，３−１，６グルカンが得られるようになった。
【００５３】
またＡｕｒｅｏｂａｓｉｄｉｕｍ属は不完全菌に分類される菌であり、液体培養時には不完全菌の特徴である糸状菌体が絡まった多量のペレットを形成した。加えてメラニン色素合成能が非常に高く、培養産物にはメラニン色素による褐変が見られた。これらの大量に含まれるペレットと褐変による変色により、Ａｕｒｅｏｂａｓｉｄｉｕｍ属菌株由来の培養産物の利用分野も限られていた。しかし本発明で用いた新規Ａｕｒｅｏｂａｓｉｄｉｕｍ属菌株ＦＯ−６８は、メラニン色素合成能を持つ厚膜胞子を形成し難く、厚膜胞子のメラニン色素合成能自体も低く、太く短く絡まり難い形状の菌糸型細胞を形成し、菌形態に占める菌糸型細胞の割合自体低い性質を持つ。そのためＦＯ−６８菌株を培養して得られる培養産物は、ペレットを含まず乳白色〜淡黄白色の一様な高粘性の液体状であり、食品・飲料に広く利用される形態でβ−１，３−１，６グルカンを提供することを可能にした。このことは、β−１，３−１，６グルカンの日常的な摂取を助け、ガン・高血圧・糖尿病等の生活習慣病の予防という、予防医学の観点からも有益である。
【００５４】
さらに前述した本発明にかかる新規培養方法でＦＯ−６８菌株を培養した時に生産されるβ−１，３−１，６グルカンの構造を解析した結果、報告の無い分岐率が高い構造を持つ新規β−１，３−１，６グルカンであった。この新規β−１，３−１，６グルカンを含む培養産物は、細胞のＤＮＡ合成能の増加・サイトカイン産生の誘導・ＮＫ活性の獲得など生態免疫反応を調節するユニークな特性を持っており、抗腫瘍活性や抗微生物活性などを高める。今回得た高い分岐率を持つ新規β−１，３−１，６グルカンは、その持つ生態免疫反応から特に医療・保健・福祉・環境・食品など生命科学を基盤とした分野への応用が期待できる。
【図面の簡単な説明】
【図１】Ａｕｒｅｏｂａｓｉｄｉｕｍ属菌株培養時の培地中糖成分の経時変化を示す図表である。
【図２】培養終了時における菌形態と菌体外β−１，３−１，６グルカンの関連図表である。
【図３】厚膜胞子植菌時に誘導した菌形態比率の継続性と菌体外β−１，３−１，６グルカンの関連図表である。
【図４】Ａｕｒｅｏｂａｓｉｄｉｕｍ属ＦＯ−６８菌株の様々な菌形態を示す図面代用写真である。
【図５】平板寒天培地上のコロニー色を示す図面代用写真である。
【図６】ＦＯ−６８菌株を培養して得た培養産物の液色を示す図表である。
【図７】液体培地中で観察されるＡｕｒｅｏｂａｓｉｄｉｕｍ属の菌糸型細胞の特徴を示す図面代用写真である。
【図８】本菌体外多糖の部分メチル化アルジトールアセテート誘導体のガスクロマトグラムを示す図表である。
【図９】本菌体外多糖のマスフラグメントを示す図表である。
【図１０】本菌体外多糖のマスフラグメントを示す図表である。
【図１１】本菌体外多糖のマスフラグメントを示す図表である。
【図１２】本菌体外多糖のマスフラグメントを示す図表である。
【図１３】本菌体外多糖の^１３ＣＮＭＲスペクトルを示す図表である。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a culture product produced by the FO-68 strain [(Accession number) FERM P-19327], a novel β-1,3-1,6-glucan contained therein, and a β-protein using a strain of the genus Aureobasidium. The present invention relates to a 1,3-1,6-glucan production method.
[0002]
[Prior art]
[Patent Document 1] Japanese Patent Publication No. 3-48201
[Patent Document 2] JP-A-2002-204687
[0003]
Daily ingestion of β-1,3-1,6-glucan is effective in preventing lifestyle-related diseases such as cancer, hypertension, and diabetes because it induces immunostimulation. Therefore, many functional foods, quasi-drugs, and pharmaceuticals have been produced using β-1,3-1,6-glucan contained in basidiomycetes such as Agaricaceae, Agaricus mushroom and Agaricus mushroom.
In addition, a health food using β-1,3-1,6-glucan produced outside the cells by a strain of the genus Aureobasidium (Aureobasidium sp. FERM-P No. 4257, ATCC No. 20524) belonging to the same genus as the present invention, Food additives are also manufactured.
[0004]
[Problems to be solved by the invention]
From the viewpoint of preventive medicine, it is preferable to ingest the food periodically to make the most of the functionality of the food, and the immunostimulatory activity of β-1,3-1,6-glucan is also the same. For this reason, β-1,3-1,6-glucan is also widely used in foods and beverages, and it is preferable that it can be exchanged daily. However, β-1,3-1,6-glucan is currently limited by its physical properties and price. Only used in form.
[0005]
For example, β-1,3-1,6-glucan contained in basidiomycetes is a polysaccharide classified as a structural polysaccharide, and is tightly bound to proteins and other polysaccharides to constitute a cell wall. Most of them are neutral polysaccharides without charge. For this reason, the extraction efficiency is poor, and when extracted, it often does not show water solubility or has an unpleasant odor. In addition, depending on the extraction method, the binding mode of the monosaccharide is changed, and the original polysaccharide structure is lost. Based on these physical properties, basidiomycetes use β-1,3-1,6-glucan in health foods that can be eaten in the form of mushrooms, dried products, or powdered forms thereof, or expensive drugs that have undergone mild extraction and advanced purification processes. Limited to quasi-drugs and pharmaceuticals. That is, unless β-1,3-1,6 glucan is water-soluble and can be easily obtained without going through an extraction process, it cannot be widely used in the field of foods and beverages.
[0006]
On the other hand, there are strains of the genus Aureobasidium that secrete, when cultured under specific conditions, water-soluble β-1,3-1,6-glucan having physiological activity outside the cells. Β-1,3-1,6-glucan contained in the culture product obtained by culturing the cells is not bound to proteins or other polysaccharides, and is soluble in water. Can be used in various fields without going through the extraction process. However, the genus Aureobasidium is usually a bacterium that produces pullulan in which maltotriose or maltotetraose is linked by α-1,6 bonds outside the cell, and β-1,3-1,6 glucan is extracellularly produced. Currently, only one kind of strain to be produced has been reported, and a culture method suitable for β-1,3-1,6-glucan production has not been established. Therefore, it is difficult to obtain a constant yield of β-1,3-1,6-glucan, and the functionality of the culture product varies from culture to culture. The genus Aureobasidium is a bacterium classified as an incomplete bacterium, and forms a large amount of pellets entangled with filamentous fungi characteristic of the incomplete bacterium during liquid culture. In addition, β-1,3-1,6-glucan is a very viscous substance, and a pellet formed from a culture product that is a highly concentrated solution of β-1,3-1,6-glucan is centrifuged or filtered. It is difficult to remove by the method and the like, and remains as an impurity in the culture product. Furthermore, Aureobasidium genus strains have a very high melanin pigment synthesizing ability, also called black yeast, and the culture product shows browning due to melanin pigment, shows dark yellow to brown when antioxidant is added, and shows black when not added, Secondary products that can be processed are limited. Due to the heterogeneity of the β-1,3-1,6-glucan content, the large amount of pellets, and the discoloration due to browning, the field of application of culture products derived from Aureobasidium strains has been limited.
[0007]
Therefore, in the present invention, (1) the yield of β-1,3-1,6-glucan in a culture product that differs from one culture to another, (2) a large amount of pellets formed in the culture product, and (3) the production of a melanin-based culture product The present invention solves the three problems of browning and provides a culture product containing β-1,3-1,6-glucan derived from a strain of the genus Aureobasidium in a form that can be widely used in the food and beverage fields.
[0008]
[Means for Solving the Problems]
In order to achieve the above-mentioned object, the present invention has invented a novel culture method capable of stably obtaining a high concentration of β-1,3-1,6-glucan in consideration of the influence of bacterial form on polysaccharide production ability. . Using the newly isolated Aureobasidium strain FO-68 [(Accession number) FERM P-19327], the two problems of a large amount of pellets formed in the culture product and browning due to melanin pigment were solved. Furthermore, β-1,3-1,6-glucan having a unique structure was obtained by culturing Aureobasidium strain FO-68 by a novel culture method.
The scientific properties and taxonomic position of Aureobasidium strain FO-68 are as follows.
(Scientific properties)
This bacterium produces a highly viscous high molecular polysaccharide. This substance easily aggregates with ethanol and can be easily recovered. The present polysaccharide β-type, the main chain is a 1.3 bond, and is a polysaccharide having a branch from positions 3 and 6. In addition, it is easily aggregated by aluminum ions or the like. This substance is effective as a food additive and functional food with immunity, and is effective as a feed for growth promotion and wastewater treatment.
(Taxonomic position)
When cultured on a potato dextrose plate agar culture at 25 to 30 ° C. for 2 weeks, a colony having a diameter of 5 to 6 cm is formed in which glossiness due to a viscous substance covering the surface and piloerection due to hyphal elongation are observed. Browning due to a slight melanin pigment is observed at the center of the colony, but the whole is white to pale pink and changes to black-brown over time. The cells were three types of yeast-type cells, blastospores (Blastspore), yeast-like cells (Yeast like cells), and chlamydospores capable of synthesizing melanin pigment, and mycelia (Hyphae) in which the cells were connected to the cells were enlarged. It can be classified into two types of swollen cells. Decomposes hexoma such as glucose, fructose and galactose, sucrose and starch. The culture becomes noticeably viscous.
Due to mycological properties, it is closely related to imperfect fungi and Aureobasidium pullulans.
[0009]
BEST MODE FOR CARRYING OUT THE INVENTION
Aureobasidium strains exhibit hyphae-yeast dimorphism as growth characteristics with changes in habitat, and their morphology can be classified into three types of yeast cells and two types of hyphae cells. Among them, cells that actively produce β-1,3-1,6-glucan outside the cells are swollen yeast-like cells having many vacuoles, and the number of cells and the internal vacuoles Is related to the morphology of the cells to be inoculated.
[0010]
Therefore, a novel culture method most suitable for producing β-1,3-1,6-glucan of a strain of the genus Aureobasidium was invented in consideration of the influence of the bacterial form on the polysaccharide-producing ability. In other words, chlamydospores induced by a nitrogen-depleted medium were added with vitamin E and pulverized to obtain 0.1-1.0% by weight of bran, 0.5-2.0% by weight of glucose, and 0.2-0.0% by weight of vitamin C. This is a culture method of inoculating a liquid medium containing 5% by weight and adjusted to pH 5.0 to 6.0, followed by agitating culture at 20 ° C. for 72 hours while aerating 10 to 100 times the volume of the medium per hour. . Details are described below.
[0011]
The amount of β-1,3-1,6-glucan produced extracellularly by culturing Aureobasidium strain was measured by the following method. After diluting the culture product 10 times with distilled water, and removing the insoluble solids by centrifugation at 40,000 G for 30 minutes, ethanol was added to the final concentration of 70% to precipitate the polysaccharide. The supernatant was removed by centrifugation. A 1/3 volume of a pullulanase suspension (manufactured by Wako) diluted 1000-fold with the same buffer was re-dissolved in a 20 mM citrate-phosphate buffer (pH 6.0). The mixture was subjected to an enzyme reaction at 30 ° C. for 24 hours to obtain a β-1,3-1,6-glucan measurement sample. This sample was placed in an α-cellulose membrane having a molecular weight cutoff of 12,000 to 14,000, dialyzed three times against 200 times the amount of distilled water, and then ethanol was added to a final concentration of 80% to the dialysis membrane solution. Then, the supernatant was removed by centrifugation, and the total amount of sugar contained in the obtained precipitate was measured by the phenol sulfate method to obtain the amount of β-1,3-1,6-glucan.
[0012]
Rice bran was used as a carbon source for culturing Aureobasidium strains. Rice bran is rich in carbon, nitrogen, lipids, vitamins and minerals, and has a high nutritional value by itself, so that it was most suitable for culturing Aureobasidium strains. Rice bran can be broadly classified into three types, red bran, medium bran, and white bran, depending on the degree of rice milling. The more starch is transferred from red bran to white bran, the greater the starch quality and, conversely, the less protein and lipid. In a liquid medium containing 0.2% by weight of rice bran, 0.5% by weight of glucose, and 0.2% by weight of vitamin C and adjusted to pH 5.0 to 6.0, FO- newly isolated as a strain of the genus Aureobasidium was added. After inoculating 68 strains and shaking culture at 20 ° C. for 72 hours, the amount of β-1,3-1,6-glucan produced in each medium was measured. Table 1 shows the results. Red bran and middle bran show the values when using bran at the time of production and pulverization, respectively, and white bran show the value using bran at the time of production because of the properties of bran, which are ground at the time of production. From the results, the highest β-1,3-1,6-glucan concentration was found in the medium using Nakanuka. This is because not only proteins as nitrogen sources but also starch and lipids play an important role in the production of β-1,3-1,6-glucan by Aureobasidium strains. When the rice bran is finely crushed to 0.5 to 2.0 mm, assimilation by bacteria is more likely to proceed, and the number of bacteria increases and β-1,3-1,6-glucan increases in a short time. Concentrations were seen. In order to further prevent the oxidation of lipids, when vitamin E (D1-α-tocophenol) is added at the time of pulverizing the rice bran, the β-1,3 is as high as or higher than that when the rice bran immediately after the pulverization is used alone. A -1,6 glucan concentration was obtained. In other words, in the culture of the genus Aureobasidium, ground bran is suitable as a nitrogen source. By adding and grinding vitamin E, the oxidation of lipids is suppressed, and more stable production of β-1,3-1,6-glucan is achieved. Made it possible.
[0013]
[Table 1]

[0014]
The carbon source which was assimilated when the Aureobasidium strain produced β-1,3-1,6-glucan was determined. The details are as follows. FO- in a liquid medium containing 0.5% by weight of saccharose as a carbon source, 0.2% by weight of crushed bran with added vitamin E and 0.2% by weight of vitamin C and adjusted to pH 5.0 to 6.0. 68 strains were inoculated and cultured with shaking at 20 ° C. for 100 hours. The amount of β-1,3-1,6-glucan produced in the medium was determined by the above-mentioned method using the F kit (Roche) for saccharose as a carbon source added to the medium and glucose fructose as a decomposition product thereof. And the change over time of the sugar component was followed.
[0015]
FIG. 1 shows the results. The production of β-1,3-1,6-glucan was started 40 hours after the start of the culture when saccharose was almost completely degraded. Glucose, which is a decomposition product of saccharose, decreased as β-1,3-1,6-glucan was produced. On the other hand, after 72 hours in the absence of glucose, fructose, another degradation product of saccharose, was reduced, but production of β-1,3-1,6-glucan was not observed. These facts indicated that glucose was used for production of β-1,3-1,6-glucan. Then, when FO-68 strain was cultured using glucose as a carbon source instead of saccharose, β-1,3-1,6-glucan started to be produced about 20 hours earlier. These facts revealed that glucose is suitable as a carbon source for cultivation of Aureobasidium strains.
[0016]
As described above, the strain of the genus Aureobasidium exhibits hypha-yeast dimorphism as a growth characteristic as the habitat changes, and its complicated morphology greatly affects the polysaccharide-producing ability. In spores and hyphae that are often found in nutrient-rich growth environments, germination and segmental growth are prioritized, and extracellular polysaccharide production is suppressed. Superior chlamydospores have the properties of dormant cells, have poor metabolic capacity, and hardly produce extracellular polysaccharides. On the other hand, the swollen yeast-like cells, which are observed during the transfer of bacterial form from spores to chlamydospores, actively produce polysaccharide outside the cells. During aeration and agitation culture using Aureobasidium strains, the concentration of β-1,3-1,6-glucan in the culture product is closely related not only to the number of swollen yeast-like cells observed at the end of the culture but also to the morphology. I have. In other words, yeast-like cells in which the cells are greatly expanded and the number of internal vacuoles is large, the β-1,3-1,6-glucan-producing ability becomes high.
[0017]
The number and morphology of expanded yeast-like cells during cultivation are related to the morphology of the cells to be inoculated, and more yeast-like cells that have greatly expanded during inoculation with chlamydospores can be obtained. The ability to produce exopolysaccharide was highest. Details are described below.
[0018]
The present inventors have invented a method for deriving chlamydospores by subculturing Aureobasidium genus strains for several generations under a condition where the nitrogen source is insufficient. Specifically, 2.0% by weight of agarose was added to a nitrogen source depleted medium containing potato dextrose broth (manufactured by Difco) 0.24 to 0.6% by weight and glucose 1.5 to 2.0% by weight and adjusted to pH 5.2. After growing Aureobasidium strains for several generations on the added agar medium, 1 to 2 loops are inoculated into a liquid medium of the same composition, and cultured with shaking at 20 ° C to 30 ° C for 72 hours to induce the morphology of chlamydospores. How to The spores can also be derived by subculturing Aureobasidium strains for several generations under conditions rich in nitrogen sources. That is, when the same culture is performed using a medium in which 2.4% by weight of potato dextrose broth (manufactured by Difco) is dissolved instead of the nitrogen source depleted medium used in the above culture, the bacterial morphology of the spore spores Can be induced.
[0019]
The chlamydospores and spore spores of the induced FO-68 strain were added with vitamin E and crushed, respectively, to 0.1 to 1.0% by weight of Nakanobu, 0.5 to 2.0% by weight of glucose, and Vitamin C0. In a liquid medium containing 0.2 to 0.5% by weight and adjusted to pH 5.0 to 6.0, ⁴ The cells were inoculated to a cell / mL and cultured with shaking at 20 ° C. for 96 hours. After the cultivation, the number of cells in the culture product was measured, the morphology of the cells was observed, and the amount of β-1,3-1,6-glucan was measured.
[0020]
The number of cells was measured using a Thomas hemocytometer. The bacterial morphology was classified into yeast-like cells obtained by expanding cells having two or more vacuoles inside the cells, cells having double cell walls as chlamydospores, and other yeast cells as spore spores. The amount of β-1,3-1,6-glucan was measured by the method described above.
[0021]
The measurement results are shown in FIG. At the time of culture using induced chlamydospores, more than 60% of the total number of cells are swollen yeast-like cells, the cells themselves are as large as 7 to 12 μm, and many vacuoles are seen inside the cells. The amount of β-1,3-1,6-glucan in the product was as high as about 2.5 g / L. On the other hand, at the time of cultivation using the induced spores, about 40% of the total number of cells was swollen yeast-like cells, but the cells themselves were as small as 2 to 5 μm, and 2-3 vacuoles were present inside the cells. Some cells had only β-1,3-1,6-glucan in the culture product, which was a low value of about 1.4 g / L.
[0022]
Further, the bacterial cells shown in FIG. 2 obtained at the time of inoculation of chlamydospores were added with vitamin E and pulverized, and 0.1 to 1.0% by weight of bran, 0.5 to 2.0% by weight of glucose, C. Inoculation of 1/1000 amount to a liquid medium containing 0.2 to 0.5% by weight and adjusted to pH 5.0 to 6.0, and cultivation with shaking at 20 ° C. for 72 hours and cultivation of bacterial cells. The β-1,3-1,6-glucan concentration in the product is shown in FIG. As is clear from the figure, when the medium was replaced with a medium having the same composition, almost the same ratio of bacterial forms and β-1,3-1,6-glucan concentrations were obtained up to three generations. This is approximately 1.0 × 10 ⁹ Even when the scale was doubled, a certain number of greatly expanded yeast-like cells were obtained, indicating that β-1,3-1,6-glucan was stably obtained.
[0023]
The Aureobasidium genus strain is an aerobic bacterium, and the production of β-1,3-1,6-glucan proceeds under aerobic conditions, and β-1 in the culture product at the time of culture using the Aureobasidium genus strain. The 3-1 and 6 glucan concentration is affected by the amount of air mixed into the medium. Therefore, the β-1,3-1,6-glucan-producing ability becomes higher during aeration and agitation culture than during permeation culture using a convenon or an Erlenmeyer flask. The amount of aeration needs to be adjusted in consideration of an increase in the viscosity of the medium, but when aeration is performed with aeration of 10 to 100 times the volume of the medium per hour and stirring culture is performed, β-1,3-1,6-glucan is most efficiently used. Is produced. The chorion spores induced by culturing this bacterium for several generations in the above-mentioned nitrogen source depleted medium were converted to 0.1 to 1.0% by weight of rice sugar, 0.5 to 2.0% by weight of glucose, and 0.2 of ascorbic acid When inoculated into a liquid medium containing 0.5% by weight and adjusted to pH 5.0 to 6.0, and cultured with stirring at 20 ° C. for 72 hours while aerating 10 to 100 times the volume of the medium per hour, As a result, a culture product containing a high concentration of β-1,3-1,6-glucan of about 3.0 g / L or more was obtained.
[0024]
In order to solve two problems of the culture product obtained after culturing the Aureobasidium genus strain, that is, formation of a large amount of pellets and browning due to melanin pigment, FO-68 strain was used in the present invention. In the case of culturing using the FO-68 strain, a milky white to pale yellowish white uniform high-viscosity liquid culture product containing no pellet can be obtained. Details are described below.
[0025]
The following are the somatic characteristics of the FO-68 strain. This bacterium exhibits a growth temperature of 20 to 25 ° C, an optimum growth temperature of 25 ° C, a cultivable temperature of 5 ° C to 40 ° C, and an optimum growth pH of 5 to 7 at the time of culturing using a plate agar medium or a liquid medium, depending on the habitat. It shows hyphal-yeast dimorphism, which is a growth characteristic similar to other Aureobasidium strains. Further, as shown in FIG. 4, the morphology of the cells is, as shown in FIG. 4, yeast-like cells, which are blastocyspores (Blastspores) which are the cells to proliferate and cause germ-line elongation, and cells in which budding-type spores and segmental-type spores grow. A total of three types of yeast cells, a yeast like cell, a chlamydospore capable of synthesizing melanin pigment generated by the differentiation of hyphae and yeast-like cells, and a hyphae in which cells are connected by a thin septum (Hyphae). Swollen cells, which have double partition walls and have enlarged cells themselves, can be classified into a total of two types of hyphal cells.
[0026]
In this nutrient-rich growth environment, hyphae elongate and germinate actively from the spores, and vacuoles can be seen inside the cells over time. It itself shifts to a state of enlarged swelling cells. On the other hand, in the case of oligotrophic condition, segments of expanded hyphae are observed, and in the case of deterioration of other environmental factors or further oligotrophic condition, superior biological defense with a double cell wall larger than expanded cells. Thick spores can be seen.
[0027]
When the fungus isolated from the soil is cultured on a potato dextrose (Nissui Pharmaceutical) flat plate agar medium at 25 to 30 ° C. for 2 weeks, gloss due to a viscous substance covering the surface and piloerection due to hyphal elongation are observed. Form colonies 5-6 cm in diameter. The colony after 2 weeks shows a slight browning due to melanin pigment in the center, but the whole is white to pale pink (Fig. 5 left). When the FERM-P4257 strain of the same genus is cultured under the same conditions for 2 weeks The resulting whole is clearly different from the black colonies (FIG. 5, right). When the colonies are stored at 4 ° C. on the plate agar medium of the present bacterium after further culturing for 2 weeks, the browned portion spreads from the center and becomes black as a whole with the passage of days. These characteristics are that this bacterium has the property of Aureobasidium belonging to the genus Aspergillus, but it is difficult to remove chlamydospores having melanin pigment synthesizing ability as a bacterial form, and the melanin pigment synthesizing ability of chlamydospores itself is low. Was shown.
[0028]
Furthermore, the same tendency as the color of the colonies on the plate agar medium is observed in the liquid color of the culture product during the aeration and stirring culture. Details are as follows. The strain contains 0.1-1.0% by weight of rice bran, 0.5-2.0% by weight of saccharose or 0.5-2.0% by weight of glucose, and 0.2-0.5% by weight of vitamin C. The cells were inoculated into a liquid medium adjusted to pH 5.0 to 6.0, and cultured with stirring at 20 ° C. for 72 hours while aerating 10 to 100 times the volume of the medium per hour to obtain a culture product. After diluting the obtained culture product with 10 mM pH 6.0 sodium acetate buffer, insoluble solids were removed by centrifugation at 40,000 G for 30 minutes, and the absorbance of the obtained supernatant was measured in the range of 400 to 800 nm. did. FIG. 6 shows the measurement results. The absorption spectrum of the culture product showed little change in absorbance in the range of 400 to 800 nm, indicating a liquid color close to white. The chlamydospores of this strain were crushed by adding vitamin E to 0.1-1.0% by weight of bran, 0.5-2.0% by weight of glucose, and 0.2-0.5% by weight of vitamin C. When a culture product is obtained by inoculating a liquid medium adjusted to pH 5.0 to 6.0 and containing aeration and incubating at 20 ° C. for 72 hours with aeration at 10 to 100 times the volume of the medium per hour. A similar trend was observed.
[0029]
Most mycelial cells of this bacterium have a width of 4.0 to 6.0 μm, about 9.0 to 12 μm when expanded, and about 1 mm in length, and a width of 1.5 to 2.5 μm when expanded. It is thick, short and hard to entangle with hyphal cells of the FERM-P4257 strain, which is 4.0-5.0 μm long and 2-12 mm long and reaches 10-12 mm. This is because, usually, when a strain of the genus Aureobasidium is cultured in a liquid medium, segmentation is started after hyphal elongation, whereas during cultivation of this fungus, growth by hyphal segments is prioritized, and hyphal elongation is inhibited. is there. Furthermore, the germ spores of this bacterium are more actively proliferated by budding than hyphal elongation, and the ratio of mycelial cells in the morphology is low. The characteristics of these hyphal cells are shown in FIG. In fact, this strain is characterized by rice bran 0.1-1.0% by weight, saccharose 0.5-2.0% by weight or glucose 0.5-2.0% by weight, vitamin C 0.2-0.5% by weight When a culture product is obtained by inoculating a liquid medium adjusted to pH 5.0 to 6.0 and containing aeration and incubating at 20 ° C. for 72 hours with aeration at 10 to 100 times the volume of the medium per hour. A uniform high-viscosity liquid could be obtained without forming pellets. The chlamydospores of this strain were crushed by adding vitamin E to 0.1-1.0% by weight of bran, 0.5-2.0% by weight of glucose, and 0.2-0.5% by weight of vitamin C. When a culture product is obtained by inoculating a liquid medium adjusted to pH 5.0 to 6.0 and containing aeration and incubating at 20 ° C. for 72 hours with aeration at 10 to 100 times the volume of the medium per hour. A similar trend was observed. On the other hand, when a previously reported strain of the same genus, FERM-P4257, was cultured under the same conditions, a large amount of pellets were formed in the culture product, and even small ones could be visually confirmed and large ones had a spherical shape of about 1 cm in diameter. Was in the form.
[0030]
When cultivation is performed using this bacterium, two problems of a large amount of pellets in the culture product and browning due to melanin pigment are solved, and milky white to pale yellowish white β-1,3-1,6 containing no pellets. A culture product containing glucan can be obtained. Further, the culture method according to the present invention, specifically, chlamydospores obtained by adding and crushing vitamin E to 0.1-1.0% by weight of medium bran, 0.5-2.0% by weight of glucose, and 0.2% of vitamin C After inoculating a liquid medium containing 0.5% by weight and adjusting the pH to 5.0 to 6.0, culturing with stirring at 20 ° C. for 72 hours while aerating 10 to 100 times the volume of the medium per hour. When this bacterium is cultured by the method, a milky white to pale yellow-white culture product containing a high concentration of β-1,3-1,6-glucan and containing no pellet can be stably obtained. This FO-68 culture product can be widely used in the field of foods and beverages rather than colors and physical properties, and contains a certain amount of β-1,3-1,6-glucan. Is also stable. Further, the FO-68 culture product has a unique property of regulating an ecological immune response, and can be expected to be applied not only to the food field but also to fields based on life science such as medical care, health care, welfare, and the environment.
An example in which the functionality of a culture product is measured is described below.
[0031]
【Example】
The effect of the FO-68 culture product on the cell division (DNA synthesis ability) of human peripheral blood monocytes was examined. Peripheral blood mononuclear cells were separated from healthy human blood by the Ficoll-Conray method and suspended in 1% normal human serum (HS). Next, a new culture product derived from a new strain FO-68 diluted 400 times (hereinafter referred to as a FO-68 culture product) was added, and cultured at 37 ° C for 5 days. Five days later, tritium thymidine ( ³ 1 μCi of H-thymidine was added, and the amount of the uptake was measured with a liquid scintillation counter, and the effect of the culture product on the cell division ability (DNA synthesis ability) of peripheral blood mononuclear cells was examined. As a result, as shown in Table 2, the addition group significantly enhanced the DNA synthesis ability of peripheral blood mononuclear cells as compared with the non-addition group.
[0032]
[Table 2]

[0033]
The effect of the FO-68 culture product on the leukemia cell line was observed. Various leukemia cell lines 2 × 10 ⁵ The FO-68 culture product was added to the cells, and the cells were cultured at 37 ° C for 3 days. After the culture, the number of viable cells was counted using a hemocytometer, and the proliferation of the leukemia cell line was examined. As a result, as shown in Table 3, the addition group significantly suppressed the growth of the leukemia cell line as compared with the non-addition group.
[0034]
[Table 3]

[0035]
The effect of the FO-68 culture product on cytokine production was observed. A 30-fold diluted FO-68 culture product was added to a monocyte cell line (U937), and the cells were cultured for 24 hours at 37 ° C. In addition, a 400-fold diluted FO-68 culture product was added to healthy human peripheral blood mononuclear cells, and cultured at 37 ° C for 8 hours and 24 hours. After the culture, the amounts of IL (interleukin) -1β, IL-2, IL-4, IL-6, IL-8, and IFN (interferon) -γ among the cytokines in the culture supernatant were determined by the enzyme antibody method (enzyme). (immunoassay: EIA). As a result, as shown in Tables 4 and 5, the added group induced IL-8 production significantly more than the non-added group. IL-8 is a cytokine that mainly induces chemotaxis of immunocompetent cells, and is produced from neutrophils, monocytes, alveolar macrophages, vascular endothelial cells, and the like. Recently, it has also been noted as a factor that promotes angiogenesis, and is considered to be involved in chemotaxis and accumulation of immunocompetent cells.
[0036]
[Table 4]

[0037]
[Table 5]

[0038]
The effect of the FO-68 culture product on the DNA synthesis ability of mouse spleen cells was examined. Spleen cells from C57BL / 6N mice were adjusted as per type. Next, the FO-68 culture product diluted 100-fold was added, and the cells were cultured at 37 ° C for 2 days. Two days later, 1 μCi of tritiated thymidine was added, and the amount of the uptake was measured with a liquid scintillation counter to examine the division of spleen cells (DNA synthesis ability). As a result, as shown in Table 6, the addition group enhanced the DNA synthesis ability of spleen cells as compared with the non-addition group.
[0039]
[Table 6]

[0040]
The effect of the FO-68 culture product on IL-12 production from mouse spleen cells was examined. Spleen cells from C57BL / 6N mice were adjusted as per type. Next, the FO-68 culture product diluted 100-fold was added, and the cells were cultured at 37 ° C for 2 to 3 days. After the culture, the culture solution was collected, and the amount of IL-12 in the culture solution was measured by the EIA method. As a result, as shown in Table 7, the addition group significantly enhanced IL-12 production from spleen cells as compared with the non-addition group. IL-12 is an activator of NK (natural killer) cells exhibiting antitumor activity, strongly induces the production of IFN-γ (interferon) from quiescent NK and T cells, and induces cytotoxicity of NK and T cells. It is an important cytokine that enhances activity. From these results, it was found that the FO-68 culture product was a culture product having excellent antitumor activity and antimicrobial activity as well as activation of the immune system.
[0041]
[Table 7]

[0042]
The effect of the FO-68 culture product on the NK activity of mouse peritoneal cells was observed. After injecting the FO-68 culture product intraperitoneally five times every other day into C57BL / 6N mice, the isotope chromium ( ⁵¹ NK activity of peritoneal cells was measured by the chromium release method using YAC-1 cells (mouse leukemia cell line) labeled with Cr) as target cells. As a result, as shown in Table 8, the administration group induced NK activity as compared with the non-administration group. From this result, it was found that the FO-68 culture product enhanced the antitumor activity.
[0043]
[Table 8]

[0044]
As described above, a culture product produced when the present bacterium is cultured by the novel culture method according to the present invention regulates an ecological immune response such as an increase in DNA synthesis ability of cells, induction of cytokine production, and acquisition of NK activity. It has unique properties and enhances antitumor activity and antimicrobial activity. The function of β-1,3-1,6-glucan differs depending on its structure, and β-1,3-1,6-glucan contained in the FO-68 culture product has a high branching ratio based on the results of sugar structural analysis. Is a novel β-1,3-1,6 glucan having The new β-1,3-1,6-glucan with high branching rate obtained this time is expected to be applied to life science-based fields such as medical care, health, welfare, environment, and food, especially from its ecological immune response. it can. Details of the structural analysis of the sugar are described below.
[0045]
The chlamydospores of this bacterium induced in a nitrogen-depleted medium were added with vitamin E and pulverized into crushed rice bran 0.1-1.0% by weight, glucose 0.5-2.0% by weight, vitamin C 0.2- After inoculation into a liquid medium containing 0.5% by weight and adjusted to pH 5.0 to 6.0, the cells were cultured with stirring at 20 ° C. for 72 hours while aerating 10 to 100 times the volume of the medium per hour. The culture product thus obtained was diluted 10-fold with distilled water, and then insoluble solids were removed by centrifugation at 40,000 G for 30 minutes. To the obtained supernatant, 1/5 volume of a mixed solution of chloroform: 1-butanol = 5: 1 was added, and the mixture was vigorously stirred for 30 minutes. Then, the supernatant obtained by centrifugation was subjected to water separation using a separating funnel. The layers were separated. This operation was repeated twice more, and ethanol was added to the aqueous layer to a final concentration of 70% to precipitate the polysaccharide, and the supernatant was removed by centrifugation. To a solution obtained by re-dissolving the thus-obtained precipitate with a 20 mM citrate-phosphate buffer (pH 6.0) was added 1/3 volume of a pullulanase suspension (manufactured by Wako) diluted 1000-fold with the same buffer. After reacting at 30 ° C. for 24 hours, the mixture was placed in an α-cellulose membrane having a molecular weight cutoff of 12,000 to 14,000 and dialyzed three times against 200 times the amount of distilled water. Ethanol was added to the final concentration in the dialysis membrane to 80% to precipitate the polysaccharide, followed by centrifugation, and the resulting precipitate was dried at room temperature under reduced pressure to obtain a sample of extracellular secretory polysaccharide.
[0046]
Methylation of the extracellular secretory polysaccharide was performed by the method described below. After adding 10 mL of dimethyl sulfoxide to 100 mg of the sample, the mixture was dissolved by stirring at 55 to 60 ° C. for 1 hour under nitrogen flow. After cooling to room temperature, 3.0 mL of methylsulfonylcarbanion was added, and the mixture was stirred at room temperature for 5 hours. Then, 1.0 mL of methyl iodide was added, and the mixture was stirred for 1 hour while cooling to 20 ° C or lower. This operation of adding methylsulfonyl carbanion and methyl iodide and stirring was repeated three times. The operation of adding 50 mL of purified water to the reaction solution, adding 20 mL of chloroform, and vigorously stirring for 5 minutes with a separating funnel to extract methylated sugar was repeated 5 times. Anhydrous sodium sulfate was added to this chloroform extract, dehydrated for 1 hour, and dried at 40 ° C. under reduced pressure to obtain methylated sugar. Methylation was confirmed by infrared absorption analysis.
[0047]
The alditol acetate method of the methylated sugar obtained in paragraph [0045] is described below. 72% H methylated sugar ₂ SO ₄ After dissolving in 3.0 mL, 18 mL of purified water was added and hydrolyzed at 100 ° C. for 4 hours. After the solution cooled to room temperature was neutralized with barium carbonate, the solution was centrifuged, and the supernatant from which barium sulfate had been removed was concentrated under reduced pressure to 5 mL. Add sodium borohydride (NaBH) to the concentrate ₄ ) 30 mg was added and stirred vigorously, and then left for 2 hours to reduce. Excess NaBH ₄ A 5% aqueous solution of acetic acid was added dropwise until no gas was generated in order to decompose, and then 15 mL of a cation exchange resin Amberlite IR 120B was added, and the mixture was allowed to stand at room temperature for 3 hours. The filtrate from which the resin had been removed by suction filtration was dried to dryness under reduced pressure, and the operation of adding 10 mL of 1% hydrochloric acid-methanol to dryness at 40 ° C. under reduced pressure was repeated three times. Next, 2.0 mL each of acetic anhydride and pyridine were added, and the mixture was acetylated at 110 ° C. for 3 hours. After cooling to room temperature, 10 mL of purified water was added, and the operation of drying under reduced pressure was repeated three times. Acetic anhydride and pyridine in the reaction were removed. Further, the operation of adding 10 mL of toluene and drying under reduced pressure was repeated three times, then, 10 mL of chloroform was added, and the mixture was allowed to stand for 3 hours to extract an alditol acetate derivative. The filtrate obtained by suction filtration of the extract was dried under reduced pressure and dissolved twice in 5 mL of chloroform, and then used twice as a sample for gas chromatography mass spectrometry.
[0048]
The results of the gas chromatogram are shown in FIG. 8, and the results of the mass fragment are shown in FIGS. 9, 10, 11, 12 and Table 9. FIG. 8 is a gas chromatogram of the partially methylated alditol acetate derivative of the present extracellular polysaccharide (6P top view of the sugar chain structure of FO-68; FIG. 8 shows the partially methylated alditol acetate derivative of FIG. 1 FO-68 VC). FIG. 9 shows the mass fragment of the present extracellular polysaccharide (6P lower diagram of the sugar chain structure of FO-68; FIG. 9 shows the mass fragment of Peak 1 among the mass fragments of FIG. 2 FO-68 VC). FIG. 10 shows the mass fragment of the present extracellular polysaccharide (7P upper diagram of the sugar chain structure of FO-68; FIG. 10 shows the diagram of Peak 2 among the mass fragments of FIG. 2 FO-68 VC). FIG. 11 shows the mass fragment of the present exopolysaccharide (7P lower diagram of the sugar chain structure of FO-68, FIG. 2 FO-68 VC, among the mass fragments of Pea). FIG. 12 shows the mass fragment of the present extracellular polysaccharide (8P upper diagram of the sugar chain structure of FO-68, Fig. 2 FO-68 VC, of the mass fragment of Peak4). (Using figure). Peak 1 was 2,3,4,6-Me as shown in Table 9. ₄ -GP, which represents glucose at the non-reducing end group bonded only at the 1-position carbon. Peak4 has many G1 bonds (peak1) as shown in Table 9 and has almost the same strength as Peak1 as shown in FIG. ₂ It was presumed to be -G and glucose having a bond at the 1,3,6-position carbon. Peak 3 was 2,4,6-Me as shown in Table 9. ₃ -G, and glucose having a bond at the 1,3-position carbon. Peak2 was a sugar fragment, which was due to the mixing of Peak3 fragments due to the resolution of the gas chromatogram, and Peak2 was non-sugar. The above results indicate that although the exopolysaccharide has a slight non-sugar site, it is mainly a 1,3-1,6 glucan in which the non-reducing end is bonded to a 1,3,6-bonded branched structure. Was. The molar ratio of each glucose was determined from the peak area of the gas chromatogram. As a result, 2,3,4,6-Me ₄ -G: 2,4-Me ₂ -G: 2,4,6-Me ₃ -G = 16: 3: 13.
[0049]
[Table 9]

[0050]
The exopolysaccharide methylated by the Hakomori method described in paragraph [0045] is dissolved in deuterated chloroform, and then tetramethylsilane (TMS) is used as an internal standard in a Fourier transform nuclear magnetic resonance absorption apparatus. ^Thirteen The C nuclear magnetic resonance absorption spectrum was measured. The result is shown in FIG. FIG. 13 shows the exopolysaccharide ^Thirteen CNMR spectrum (1P bottom view of sugar chain structure of FO-68, FO-68 ^Thirteen CNMR spectrum figure).
The absorption at 70 to 80 ppm is a triplet of deuterated chloroform, and a slight peak at about 100 ppm indicated by an arrow and no absorption of α-anomers around 90 to 100 ppm are observed. It was found that both the (1-3) bond and the (1-6) bond present in the outer polysaccharide are β-oriented.
[0051]
From the above analysis results, the binding mode of glucose, which is a constituent sugar, is as shown in the figure below. Β-1, which has a high branching rate in which glucose having one glucose at 1 → 6 bonds is continuous at 1 → 3 bonds, 3-1 and 6 glucan.

[0052]
【The invention's effect】
In the genus Aureobasidium, one strain secreting extracellular water-soluble β-1,3-1,6-glucan having physiological activity when cultured under specific conditions was reported. A culture method suitable for the production of 3,3-1,6-glucan has not been established. Therefore, it was difficult to obtain a constant yield of β-1,3-1,6-glucan, and the functionality of culture products derived from Aureobasidium strains was different for each culture. Therefore, a new culture of Aureobasidium genus strain is performed by using a medium composition optimal for producing β-1,3-1,6-glucan and a method of inducing expanded yeast-like cells having high β-1,3-1,6-glucan producing ability. Invented the law. That is, chlamydospores induced in a nitrogen-depleted medium were crushed with vitamin E by adding 0.1-1.0% by weight of bran, 0.5-2.0% by weight of glucose, and 0.2-0% by weight of vitamin C. Inoculated in a liquid medium containing 0.5% by weight and adjusted to pH 5.0 to 6.0 to induce large swollen yeast-like cells having a large number of vacuoles, while inducing 10 to 100 times the volume of the medium per hour. This is a method of culturing with stirring at 20 ° C. for 72 hours while performing aeration. By this culturing method, a high concentration of β-1,3-1,6-glucan can be obtained stably using Aureobasidium strains.
[0053]
The genus Aureobasidium is a bacterium classified as an incomplete bacterium, and formed a large amount of pellets entangled with filamentous fungi characteristic of the incomplete bacterium during liquid culture. In addition, the melanin pigment synthesis ability was very high, and the culture product showed browning due to the melanin pigment. Due to these large amounts of pellets and discoloration due to browning, the field of application of culture products derived from Aureobasidium strains has been limited. However, the novel Aureobasidium genus strain FO-68 used in the present invention is difficult to form chlamydospores having melanin pigment synthesizing ability, the melanin pigment synthesizing ability of chlamydospores itself is low, and the mycelium-type cells are thick, short and hard to entangle. And the ratio of mycelial cells in the bacterial morphology itself is low. Therefore, the culture product obtained by culturing the FO-68 strain is a milky white to pale yellowish white, uniform, highly viscous liquid containing no pellet, and β-1, 3-1 and 6 glucans can be provided. This helps daily intake of β-1,3-1,6-glucan and is also beneficial from the viewpoint of preventive medicine, that is, prevention of lifestyle-related diseases such as cancer, hypertension, and diabetes.
[0054]
Furthermore, as a result of analyzing the structure of β-1,3-1,6-glucan produced when the FO-68 strain is cultured by the above-described novel culture method according to the present invention, a novel structure having a high unbranched branching rate has been reported. β-1,3-1,6 glucan. Culture products containing this novel β-1,3-1,6-glucan have unique properties that regulate ecological immune responses, such as increasing cell DNA synthesis ability, inducing cytokine production, and acquiring NK activity. Enhances antitumor activity and antimicrobial activity. The new β-1,3-1,6-glucan with high branching rate obtained this time is expected to be applied to life science-based fields such as medical care, health, welfare, environment, and food, especially from its ecological immune response. it can.
[Brief description of the drawings]
BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is a chart showing a change over time of a sugar component in a culture medium during cultivation of an Aureobasidium strain.
FIG. 2 is a table showing the relationship between bacterial morphology and extracellular β-1,3-1,6-glucan at the end of culture.
FIG. 3 is a table showing the relationship between the continuity of the bacterial form ratio induced during inoculation of chlamydospores and extracellular β-1,3-1,6-glucan.
FIG. 4 is a photograph as a substitute for a drawing, showing various bacterial forms of Aureobasidium genus FO-68.
FIG. 5 is a photograph as a substitute of a drawing, showing the color of colonies on a plate agar medium.
FIG. 6 is a table showing the liquid colors of culture products obtained by culturing the FO-68 strain.
FIG. 7 is a photograph substituted for a drawing, which shows the characteristics of mycelial cells of the genus Aureobasidium observed in a liquid medium.
FIG. 8 is a table showing a gas chromatogram of a partially methylated alditol acetate derivative of the present exopolysaccharide.
FIG. 9 is a chart showing mass fragments of the present exopolysaccharide.
FIG. 10 is a chart showing mass fragments of the present exopolysaccharide.
FIG. 11 is a chart showing mass fragments of the present exopolysaccharide.
FIG. 12 is a chart showing mass fragments of the present exopolysaccharide.
FIG. 13 shows that the exopolysaccharide ^Thirteen It is a chart which shows a CNMR spectrum.

Claims (5)

A method for inducing the morphology of chlamydospores of Aureobasidium strains. A method for culturing a strain of the genus Aureobasidium capable of stably obtaining β-1,3-1,6-glucan at a high concentration in a culture product obtained by using the chlamydospores derived in the method of claim 1 for preculture. A viscous white culture product containing no pellet obtained by culturing the novel isolate FERM P-19327. A viscous white culture product obtained by culturing the novel isolate FERM P-19327 by the method of claim 2 and containing no pellet containing a fixed amount of β-1,3-1,6-glucan. A highly branched extracellular water-soluble β-1,3-1,6-glucan obtained by culturing the newly obtained strain FERM P-19327 by the method of claim 2.

Images