JP4076385B2 - Novel microorganism and method for producing β-glucan using the same - Google Patents

Description

【００４７】
〔試験例２〕菌株のスクリーニング方法２
抗生物質シクロヘキシミドを１０μｇ／ｍｌの濃度で添加した培地を用いたこと以外は、試験例１と同様にして、菌株のスクリーニングを実施した。その結果、対象サンプルから分離・培養して生育した４０００コロニーから、第１スクリーニングにて３０菌株、第２スクリーニングにて１０菌株、第３スクリーニングにて３菌株（ＡＤＫ−７１菌株、ＡＤＫ−７７菌株、ＡＤＫ−８２菌株）が得られた。第３スクリーニングで得られた菌株について、試験例１と同様にして、βグルカン量及び生成多糖のプルランに対する純度を測定した。その結果を表２に示す。
また、第３スクリーニングの結果得られた３菌株について形態学的な観察から菌株同定をそれぞれ試みた。ＹＭ寒天培地に植菌後、２６℃、７日間培養したところ、３菌株のコロニーはいずれも、全体に光沢を生じ、中心部が黄色の輪郭を生じ、湿潤したコロニーを形成した。これらのプレートを４℃に移管して７日間保存することで、中心部の黄色は黒緑色へ変化した。ＡＤＫ−７１菌株及びＡＤＫ−７７菌株それぞれのコロニー全体は変化せず、白色〜ピンク色であった。ＡＤＫ−８２菌株はコロニー全体が黒色化した。また、これらの３菌株それぞれをＹＭ液体培地で２６℃にて３日間培養した菌体の顕微鏡観察では、３菌株いずれにおいても出芽型の分生子が認められ、酵母様の生育であった。また、これらの３菌株それぞれをＹＭ寒天培地で２６℃にて７日間培養したコロニーの菌体は、３菌株いずれにおいても、分生子がつながった鎖状に生育した菌体が観察されたが、分生子枝は認められなかった。以上から、３菌株はアウレオバシジウム プルランスと判定した。
【００４８】
【表２】

【００４９】
〔試験例３〕形態的・培養的性質
試験例１で得られた菌株の中で、ＡＤＫ−３４菌株について、ＹＭ培地（１重量％グルコース、０．５重量％ペプトン、０．３重量％酵母エキス、０．３重量％マルトエキストラクト、ｐＨ６．０）で２６℃、３日間培養した後、顕微鏡で観察した所見を示すと、細胞の大きさは短径２〜２．５μｍ、長径５〜１０μｍ、形状は卵形、表面は平滑で無色、運動性はなかった。また、菌糸はごくわずかで、幅は２．５μｍであった。不均一で表面は平滑無色であった。酵母様の出芽分生子が形成された。
【００５０】
〔試験例４〕寒天平板培養
試験例１で得られた菌株の中で、ＡＤＫ−３４菌株について、ポテトデキストロース寒天培地（栄研）で２６℃にて７日間培養した。その時の所見を以下に示す。３日間の培養で、生育は良好であり、形態は円形で、全縁がギザギザで、コロニー全体に光沢があり、表面の状態は平滑、色調は白色であった。また、Ｃｏｌｏｎｙ（コロニー）は、５日間の培養で表面が平滑で淡灰白色となり、酵母様に発育し、７日間の培養でＣｏｌｏｎｙ表面はピンク色となった。２６℃にて７日間培養したプレートを、４℃にて７日間冷蔵したところ、若干ピンク色が濃くなったが、コロニー全体に変化はなかった。
【００５１】
〔試験例５〕液体培養
試験例１で得られた菌株の中で、ＡＤＫ−３４菌株について、ＹＭ培地で培養した。その時の最適生育温度及び最適生育ｐＨを示すと、最適生育温度は２６℃であり、最適生育ｐＨは５．０〜７．０であり、生育開始時のｐＨは６．２で、培養終了後のｐＨは７．５であった。好ましい発育温度は２０〜３０℃で、最適温度は２６℃、発育可能温度は５〜４０℃であった。グルコース、フラクトース、マンノース等のヘキソース、スクロース等の二糖類並びにデンプンを分解し、いずれの炭素源でも、培養液は、粘稠になり、特有の芳香を有した。
【００５２】
試験例３〜５の結果、本発明の微生物のうち、ＡＤＫ−３４菌株は菌学的特徴からアウレオバシジウム属の菌株と同定された。
【００５３】
〔試験例６〕シクロヘキシミド耐性試験
試験例１及び２のスクリーニングで得られた菌株、並びに（財）大阪発酵研究所に保存されている、ＩＦＯ−４４６６株、ＩＦＯ−６３５３株、及びＩＦＯ−７７５７株について、各菌株のシクロヘキシミド耐性試験を実施した。即ち、各菌株をＹＭ寒天培地（ディフコ社製）にて２６℃で５日間生育させた。ＹＭ寒天培地（ディフコ社製）にシクロヘキシミドを５，１０，２０，４０，８０μｇ／ｍｌの濃度となるように添加した培地をそれぞれ調製し、上記の生育させておいた菌株を滅菌した楊子でこれらのシクロヘキシミド含有培地に植菌し、２６℃にて１０日間の培養後、生育したコロニーの有無を観察し、生育した場合はコロニーの直径を測定した。その結果を表３に示す。
【００５４】
【表３】

【００５５】
表１及び表３から明らかなように、ＩＦＯ−４４６６株、ＩＦＯ−６３５３株、及びＩＦＯ−７７５７株は、シクロヘキシミドに耐性を持たず、生産されるβグルカンのプルランに対する純度は低いものであった。一方、表１〜３から明らかなように、食品からの分離菌株の中で、シクロヘキシミドに耐性を有する菌株（ＡＤＫ−３４菌株、ＡＤＫ−７１菌株、ＡＤＫ−７７菌株、ＡＤＫ−８２菌株）は、βグルカンをプルランに対して純度よく生産し、シクロヘキシミドに耐性のない菌株の多くは、生産されたβグルカンのプルランに対する純度が低い結果となった。
【００５６】
〔試験例７〕１８Ｓ ｒＲＮＡ遺伝子の遺伝子解析
スクリーニングの結果得られたＡＤＫ−３４菌株について、１８Ｓ ｒＲＮＡ遺伝子の１７３２ｂｐの塩基配列を以下のようにして決定した。ＡＤＫ−３４菌株をポテトデキストロース培地（ディフコ社）にて振とう培養し、遠心分離、蒸留水による洗浄を３回行い、ＤＮＡ抽出用菌体を得た。この菌体からFastPrepFP120（Qbiogene）とFastDNA-kit（Qbiogene）を用いて細胞破砕し、DNeasy Plant Mini Kit（QIAGEN）によりゲノムDNA分離を行った。このゲノムＤＮＡを鋳型として、Ready-To-Go PCR Beads（Amersharm-Pharmasia Biotech）とプライマーＮＳ１及びＮＳ８を用いてＰＣＲ増幅を行った。
プライマーＮＳ１とＮＳ８の塩基配列は、NS1： (5'->3') GTAGTCATATGCTTGTCTC、NS8：(5'->3') TCCGCAGGTTCACCTACGGAを用いた。サーマルサイクラーはgeneAmp PCR System 9600(Applied Biosystems)を用いた。反応終了後、ＰＣＲ産物をQIAquickPCR purification Kit(QIAGEN)を用いて精製した。このＤＮＡフラグメントを直接シーケンシング反応に供し、塩基配列解析はABI Prism377 DNA Sequencer (Applied Biosystems) を用いて実施した。類似の塩基配列はＤＮＡデータベース（DDBJ, DNA Data Bank of Japan）からＢＬＡＳＴを利用して検索した。
その結果を配列表の配列番号１に示す。また、得られた塩基配列に基づいてデータベース検索を行い、同菌株と類縁菌との相同性を調べた結果を表４〜６に示す。決定された塩基配列（配列番号１）、相同性検索結果（表４〜６)より、ＡＤＫ−３４菌株はアウレオバシジウム プルランス（Aureobasidium pullulans）との相同性が１００％であり、完全に一致した。この結果から、本菌株はアウレオバシジウム プルランスであると判定された。
【００５７】
【表４】

【００５８】
【表５】

【００５９】
【表６】

【００６０】
〔試験例８〕ＩＴＳ−５．８Ｓ ｒＲＮＡ遺伝子
スクリーニングにより得られたＡＤＫ−３４菌株並びにＩＦＯ−６３５３株及びＩＦＯ−７７５７株について、ＩＴＳ−５．８Ｓ ｒＲＮＡ遺伝子の５６３塩基配列あるいは５６４塩基配列を決定した。各菌株をポテトデキストロース培地（ディフコ社）にて振とう培養し、遠心分離、蒸留水による洗浄を３回行い、ＤＮＡ抽出用菌体を得た。この菌体からFastPrepFP120（Qbiogene）とFastDNA-kit（Qbiogene）を用いて細胞破砕し、DNeasy Plant Mini Kit（QIAGEN）によりゲノムＤＮＡ分離を行った。このゲノムＤＮＡを鋳型として、Ready-To-Go PCR Beads（Amersharm-Pharmasia Biotech）とプライマーＩＴＳ５およびＩＴＳ４を用いてＰＣＲ増幅を行った。プライマーＩＴＳ４とＩＴＳ５の塩基配列は、ITS4： (5'->3') TCCTCCGCTTATTGATATGC、ITS5：(5'->3') GGAAGTAAAAGTCGTAACAAGG である。サーマルサイクラーはgeneAmp PCR System 9600(Applied Biosystems)を用いた。反応終了後、ＰＣＲ産物をQIAquickPCR purification Kit(QIAGEN)を用いて精製した。このＤＮＡフラグメントを直接シーケンシング反応に供し、塩基配列解析はABI Prism377 DNA Sequencer (Applied Biosystems) を用いて実施した。類似の塩基配列はＤＮＡデータベース（DDBJ, DNA Data Bank of Japan）からＢＬＡＳＴを利用して検索した。
ＡＤＫ−３４菌株の結果を配列表の配列番号２に、ＩＦＯ−６３５３株の結果を配列番号３に、ＩＦＯ−７７５７株の結果を配列番号４にそれぞれ示す。また、得られた塩基配列に基づいてデ−タベース検索を行い、同菌株と類縁菌との相同性を調べた結果を表７及び８に示す。決定された塩基配列を比較したところ、ＡＤＫ−３４菌株とＩＦＯ−６３５３株及びＩＦＯ−７７５７株とは異なり、完全一致しなかった。ＡＤＫ−３４菌株とＩＦＯ−６３５３株との相同性は９８％、ＡＤＫ−３４菌株とＩＦＯ−７７５７株との相同性は９８％であった。また、相同性検索結果（表７及び８）より、ＡＤＫ−３４菌株のＩＴＳ−５．８Ｓ領域の５６３塩基対が完全一致する菌株は、報告されていなかった。ＩＦＯ−６３５３株は、報告されているアウレオバシジウム プルランス菌株である登録番号「ＡＪ２７６０６２」と１００％（表９）、即ち完全一致し、ＩＦＯ−７７５７株は報告されているアウレオバシジウム プルランス菌株である登録番号「ＡＪ２７６０６２」と９９％で一致した（表１０）。ＡＤＫ−３４菌株は登録番号「ＡＪ２７６０６２」と９８％の一致率であった。
以上から、ＡＤＫ−３４菌株は、これまでに報告されているアウレオバシジウム プルランス菌株とはＩＴＳ−５．８Ｓ領域の塩基配列の一部が異なり、新菌株であると判定された。
【００６１】
【表７】

【００６２】
【表８】

【００６３】
【表９】

【００６４】
【表１０】

【００６５】
〔分析例１〕βグルカンの確認方法とβグルカン量の測定方法
多糖中のβグルカンの分析は、アルコールによって沈殿する全多糖量をフェノール硫酸法にて測定し、引き続き、沈殿させた多糖中のβグルカンの確認・定量を生化学工業（株）のβ−１，３−Ｄ−グルコピラノース結合を含むβグルカンの検出・測定用キットを用いて行った。以下にその手順を説明する。
先ず、測定サンプル中の全多糖量をフェノール硫酸法にて測定した。即ち、サンプル溶液３０μｌに蒸留水３０μｌを加え、ここに３００ｍＭのＮａＣｌを含むリン酸緩衝液（ｐＨ６．９）を１２０μｌ加え、さらにエタノール５４０μｌ（３倍量）を添加し、−１５℃に１０分間放置して多糖を沈殿させた。上清を除去後、１００μｌの蒸留水を添加して沈殿を溶解させた。ここに５重量％フェノール水溶液１００μｌ及び硫酸５００μｌを加えて反応させた後、該溶液の４９０ｎｍにおける吸光度を測定した。また、サンプルを加えず蒸留水１００μｌに５重量％フェノール水溶液１００μｌ及び硫酸５００μｌを加えたものをブランクとして、該ブランクの４９０ｎｍにおける吸光度を測定した。プルランの１０ｍｇ／ｍｌから２倍希釈系列を作成して標準サンプルとし、該標準サンプルを使用して検量線を作成し、該検量線と上記吸光度とから、サンプル中の全多糖量の定量を実施した。
次に、全多糖量が０．１〜１ｍｇ／ｍｌ前後の溶液を、先ず１．０ＭのＮａＯＨにて１０倍希釈し、引き続きβグルカンフリーの蒸留水にて希釈し、１０¹⁰倍まで希釈し、βグルカン希釈液を調製した。βグルカン希釈液５０μｌをチューブにとり、主反応試薬５０μｌを添加して、３７℃にて３０分間インキュベートした。続いて、亜硝酸ナトリウム溶液５０μｌ、スルファミン酸アンモニウム５０μｌ、及びＮメチル２ピロリドン溶液５０μｌを加え、反応させた後、溶液の５４５ｎｍ（対照波長６３０ｎｍ）における吸光度を測定した。また、添付のβグルカン標準品を用いて７．５〜６０ｐｇ／ｍｌのβグルカン溶液を調製し、検量線を得た。該検量線と上記吸光度とから、各βグルカン希釈液の濃度を算出し、サンプル中のβグルカン量を求めた。
【００６６】
〔分析例２〕βグルカンの分子量測定方法
βグルカンの分子量測定は、以下の通りとした。先ず、培養上清に３倍量のアルコールを加え、−２０℃に冷却して１０分間放置し、沈殿を得た。沈殿させたβグルカンを５ｍｇチューブに取り、１ｍｌの蒸留水を加えて、沸騰水中で溶解させた。該溶液を０．２２μｍのフィルターに通してＨＰＬＣ用サンプルとした。分離にはＨＰＬＣゲル濾過カラムであるＳｈｏｄｅｘのパックドカラムＫＳ−８０５（昭和電工社製）を用い、流速０．６ｍｌ／ｍｉｎ．、温度５０℃とし、検出にはＲＩ検出器を用い、水を分離溶媒として用いて実施した。分子量マーカーとしてはＳｈｏｄｅｘプルラン標準液Ｐ−８２（昭和電工社製）を用いて測定した。
【００６７】
〔分析例３〕培養液中に存在する多糖のプルランに対する純度の測定方法
プルランを特異的に分解する酵素であるプルラナーゼ（和光純薬工業社製）を用いて、培養液中に生産された多糖のプルランに対する純度を測定した。即ち、プルラナーゼによる酵素消化反応の前後で多糖量を測定し、酵素処理後の多糖量（プルラナーゼにより消化されない多糖量）と酵素処理前の多糖量とから生成多糖のプルランに対する純度を算出した。以下に多糖量の測定方法を詳述する。
先ず、プルラナーゼ酵素希釈液５０μＬにクエン酸Ｂｕｆｆｅｒ（１０ｍｍｏｌ／Ｌクエン酸−２０ｍｍｏｌ／Ｌリン酸Ｂｕｆｆｅｒ（ｐＨ６．０））２ｍＬを加えたものを酵素溶液とした。ポジティブコントロールとして、プルラン（東京化成工業社製）を１０ｍｇ／ｍＬに希釈した溶液を標準サンプル溶液として用いた。培養液あるいは標準サンプルを３０μＬずつ、２本のサンプルチューブ（１．５ｍｌ容量）に量り取り、一方に酵素溶液３０μＬを加え、一方にＰＢＳ３０μＬを加え、３７℃にて１時間反応させた。反応終了後、各サンプルに、３倍量のエタノールを加えた。良く攪拌し、−１５℃にて１０分間インキュベートした後、４℃、１５０００ｒｐｍで１０分間遠心分離を行い、上清を捨て、チューブを乾燥させ沈殿を得た。沈殿に蒸留水１００μＬを加え、撹拌し、５重量％フェノール水溶液１００μＬを加え、速やかに硫酸５００μＬを加え発色させた。冷却後、９６穴マイクロプレートに１００μＬずつ分注し、蒸留水に５重量％フェノール水溶液及び硫酸を添加した場合をブランクとして、４９０ｎｍの吸光度を測定した。また、プルランを１０ｍｇ／ｍｌ〜１μｇ／ｍｌに希釈した溶液で検量線を作成した。該検量線と上記吸光度とから、酵素消化反応前後の培養液中の多糖量をそれぞれ定量し、培養液中に存在する多糖のプルランに対する純度を算出した。なお、標準サンプルであるプルラン溶液を酵素消化した場合、１０ｍｇ／ｍｌ溶液では９０％以上の消化率が、１ｍｇ／ｍｌの濃度の溶液では９５％以上、０．１ｍｇ／ｍｌ以下では９８％以上の消化率が得られた。以上のように、プルランが生成している場合は、プルラナーゼの作用により、酵素処理後のフェノール硫酸値（吸光値）は低値となり、例えば吸光値が５０％減少している場合は、多糖のプルランに対する純度は５０％と算出した。
【００６８】
〔実施例１〕
３０ｍｌ容量の試験管にＹＭ培地（ディフコ社）５．５ｍｌを入れ、滅菌後（１２１℃、２０分間）、冷却してから、ＹＰＤ寒天培地（ディフコ社）のスラントにて保存してあるＡＤＫ−３４菌株を一白金耳、植菌し、３００ｒｐｍの振とう培養器にて２６℃で４日間培養し、種培養液を得た。別の試験管にクザペック培地（ディフコ社、シュークロース濃度３重量％）５．５ｍｌを入れ滅菌後、ＡＤＫ−３４菌株の種培養液５００μｌを添加（５％植菌）し、３００ｒｐｍの振とう培養器にて２６℃で４日間培養した。培養液は白色で顕著な粘性を有していた。培養後、培養液に等量の蒸留水を加え、１５分間、高圧蒸気殺菌してから、１００００ｒｐｍで１５分間遠心分離して多糖を含有する培養上清を得た。培養上清中のβグルカン量及び多糖のプルランに対する純度を分析例１及び分析例３の測定方法によりそれぞれ測定した。その結果、多糖のプルランに対する純度は１００％であり、βグルカン量から算出したβグルカンの対糖収率は４４％であった。なお、培養上清の４９０ｎｍによる吸光値は０．０３０であり、着色が抑制されていた。また、分析例２の測定方法によりβグルカンの分子量を測定したところ、分子量は１５万〜２００万を示した。
同様の方法でＩＦＯ−６３５３株を培養し、βグルカン量及び多糖のプルランに対する純度を測定したところ、多糖のプルランに対する純度は４０％、βグルカンの対糖収率は１１％と算出された。なお、培養上清の４９０ｎｍによる吸光値は０．１９０であり、着色が認められた。
【００６９】
〔実施例２〕
イースト窒素源基礎培地粉末（Yeast Nitrogen Base w/o Amino Acids and Ammmonium Sulfate：ディフコ社）１．７ｇを１００ｍｌの蒸留水に溶解させ、フィルター滅菌した。硝酸ナトリウム５ｇを５００ｍｌの蒸留水に溶解し、２倍濃度の硝酸ナトリウム溶液を調製した。炭素源として、シュークロース、グルコース、フラクトース、可溶性デンプン又はマルトース（和光純薬工業）を用いて、１２重量％炭素源水溶液をそれぞれ調製した。硝酸ナトリウム、炭素源水溶液及び蒸留水をオートクレーブ滅菌（１２１℃、２０ｍｉｎ．）し、滅菌済みの試験管に、イースト窒素源基礎培地１ｍｌ、硝酸ナトリウム溶液５ｍｌ、炭素源水溶液２．５ｍｌ及び蒸留水１．５ｍｌ加え、全量を１０ｍｌとし、各炭素源を添加した炭素源チェック培地を調製した。
３０ｍｌ容量の試験管に、ＹＭ培地（ディフコ社）５．５ｍｌを入れ、滅菌後（１２１℃、２０分間）、冷却してから、ＹＰＤ寒天培地（ディフコ社）のスラントにて保存してあるＡＤＫ−３４菌株を一白金耳、植菌し、３００ｒｐｍの振とう培養器にて２６℃で４日間培養し、種培養液を得た。得られた種培養液を上記炭素源チェック培地に植菌し、３００ｒｐｍの振とう培養器にて２６℃で５日間培養した。培養液は白色で顕著な粘性を有していた。培養後、培養液に等量の蒸留水を加え、１５分間、高圧蒸気殺菌してから、１００００ｒｐｍで１５分間遠心分離して多糖を含有する培養上清を得た。培養上清中のβグルカン量及び多糖のプルランに対する純度を分析例１及び分析例３の測定方法によりそれぞれ測定した。測定結果を表１１に示す。表１１から明らかなように、どの炭素源においても、多糖のプルランに対する純度は９０％以上であり、βグルカンの対糖収率は２０〜４６％と算出された。なお、培養上清の４９０ｎｍによる吸光度は０．０２６〜０．０４１であり、着色が抑制されていた。また、分析例２の測定方法によりβグルカンの分子量を測定したところ、分子量は１５万〜２００万であった。
【００７０】
【表１１】

【００７１】
〔実施例３〕
ＡＤＫ−３４株をＹＭ培地に植菌し、２６にて３日間培養して、３００ｍｌの種培養液を得た。フルゾーン翼を搭載した５Ｌジャーファーメンター（丸菱バイオエンジ社）に、クザペック培地３Ｌ及びシュークロース３００ｇを入れ、滅菌・冷却後、１００ｍｌの種培養液を植菌し、２６℃にて７２時間の培養を実施し、培養液３Ｌを得た。培養液を８０℃にて３０分間加熱して、殺菌した後、等量の蒸留水を添加し、よく混合してから、８０００ｒｐｍで１５分間遠心分離して、培養希釈液を得た。培養希釈液をさらに蒸留水で５倍希釈し、吸光度を測定したところ、４９０ｎｍにおいて０．０２３であった。培養希釈液１００ｍｌに等量のエタノールを添加し、得られた沈殿を分離し、エタノールで洗浄し、そのまま５０ｍｌの蒸留水に溶解させた。この操作を再度繰り返し、透析膜（分子量３０００カット）に入れ１０倍量の蒸留水で透析を実施した。最終的に１００ｍｌの多糖溶液を得た。このときのβグルカン量は２５ｍｇ／ｍｌ、多糖のプルランに対する純度は９５％、βグルカンの分子量は１５万〜２００万と算出された。
【００７２】
【発明の効果】
本発明によれば、βグルカンを効率よく高い生産速度で製造するのに有用な新規な微生物、及び該微生物を用いたβグルカンの製造方法を提供することができる。
【００７３】
【配列表】

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a microorganism useful for obtaining β-glucan, which is a polysaccharide effective as a dietary fiber, antitumor agent, immune enhancer, and the like, and a method for producing β-glucan using the microorganism.
[0002]
[Prior art]
β-glucan is a polysaccharide that is non-digestible by human digestive enzymes, as represented by cellulose, and thus acts as a dietary fiber, that is, suppresses cholesterol lowering action, intestinal regulation action, and blood sugar level increase. It is a material that can be expected to work and is useful for preventing lifestyle-related diseases. β-glucan is defined as a polysaccharide that mainly holds β-bonds of glucose in the molecule, but β-bonding mode of glucose, types of sugars, or functions such as phosphate, sulfate, and carboxyl groups Various molecular species exist in nature due to differences in the degree of modification of constituent sugars by groups. Some of these so-called β-glucans have a function of stimulating the immune system and activating the defense mechanism of the living body. In recent years, research on influenza prevention or cancer treatment / aging prevention using immune modifiers (BRM) that activate the immune system including some β-glucans has attracted attention. It has become necessary to provide effective β-glucan, or to provide a cheaper and more stable method for producing such β-glucan material.
[0003]
Β-glucans that activate the immune system include plant cell wall components (Japanese Patent Publication No. Sho 62-6692 and Japanese Patent Laid-Open Publication No. 2001-323001), fruit bodies of basidiomycetes (mushrooms) and mycelium (K. Sasaki et al., Carbohydrate Res., Vol. 47, 99-104 (1976), Japanese Patent Laid-Open No. 05-345725), cell wall components of microbial cells, β-glucan secreted and produced outside the cells, etc. It has been known.
[0004]
It is generally well known that cell wall components of microbial cells contain β-glucan and have an action to enhance immune activity. However, it is particularly safe and highly useful as a food. Yeast cells (JP 54-138115, JP 09-103266), lactic acid bacteria (JP 03-229702, JP 10-167972), Aureobasidium Fungus bodies (Japanese Patent Publication No. 06-92441) and the like are known.
[0005]
Moreover, as a microorganism that secretes and produces β-glucan having a function of enhancing immune activity to the outside of the microbial cell, as described in JP-A No. 03-2202, Macrophomopsis genus or Alkaligenes genus Curdlan produced (Science of dietary fiber, p108, Asakura Shoten, 1997), Aureobasidium pullulans (Agaric. Biol. Chem. 47 (6), 1167-1172 (1983), JP-A-6- No. 340701) is known.
[0006]
Β-glucan contained in fruit bodies and mycelium of basidiomycetes (mushrooms) has high immunopotentiating activity, and some are used as pharmaceuticals, such as lentinan extracted from shiitake fruit bodies. However, β-glucan contained in the fruiting bodies and mycelium of basidiomycetes (mushrooms) generally varies greatly depending on the growth and cultivation conditions, and it is necessary to separate β-glucan by extraction operation. As a result, β-glucan obtained as a result covers a wide variety of molecular species, and the quality is unstable, such as a mixture of high and low molecular weight substances.
[0007]
Thus, in basidiomycetes (mushrooms), it is a problem at present to stably produce a certain β-glucan having high activity. On the other hand, the cell wall of microbial cells contains a large amount of β-glucan, which is interesting as a source of β-glucan. However, since the cell wall of the microbial cell itself contains components other than β-glucan and is insoluble in water, extraction operation is required in the same way as basidiomycete to obtain highly effective water-soluble β-glucan. Therefore, it can be said that the establishment of a method for stably extracting β-glucan of a certain quality is an issue.
[0008]
On the other hand, fermentative production of β-glucan using a microorganism that secretes and produces water-soluble β-glucan with high immune enhancing activity outside the cell body can obtain uniform and water-soluble highly active β-glucan, This is a very effective method. Based on this idea, a method for producing β-glucan using a microorganism belonging to the genus Aureobasidium, which is known to secrete and produce highly active β-glucan outside the bacterial body, has been proposed.
[0009]
However, it is known that microorganisms belonging to the genus Aureobasidium secrete and produce pullulan, which is an α-glucan, when cultured using a carbon source used for general microorganism culture such as sucrose ( JP-B 51-36360, JP-B 51-42199), and it was difficult to produce β-glucan with high purity (Japanese Patent Laid-Open No. 06-340701). Microorganisms belonging to the genus Aureobasidium are called black yeasts by common names, and are produced because the cells or the culture solution are colored black by the melanin pigment produced by the cells, and the resulting β-glucan is also colored. Β-glucan has been significantly impaired in quality. In order to improve this point, a method for producing a mutant such as pullulan using the mutant by obtaining a mutant that does not recognize coloration by mutagen treatment has been proposed (Japanese Patent Publication No. 4-18835). ). However, a strain that does not completely produce melanin and efficiently secretes and produces high-purity β-glucan outside the bacterial body (does not secrete and produce pullulan outside the bacterial body) has not yet been known.
[0010]
In addition, various culture methods for suppressing the production of pullulan, which is listed as an impurity, in the β-glucan production process, that is, the culture process, have been studied. In JP-A-06-340701 and JP-A-07-51080, pH adjustment or There has been proposed a method capable of suppressing the production of pullulan and producing β-glucan with high purity by using a special saccharide as a carbon source. In this case, the operation is complicated because special culture conditions are set, and the cost of the medium used for culture is high because a special carbon source is used, which causes problems in producing β-glucan. Yes.
[0011]
[Problems to be solved by the invention]
From the above, in order to produce β-glucan using microorganisms belonging to the genus Aureobasidium, is it possible to produce impurities such as pullulan even when cultivated with an inexpensive saccharide generally used for microbial culture as a carbon source? Alternatively, a highly active and high-quality β-glucan that is suppressed and that the production of β-glucan is substantially inhibited from producing or inhibiting the production of melanin pigment, and the produced β-glucan is not colored. Therefore, there is a need for a strain that efficiently secretes and produces sucrose.
[0012]
Therefore, an object of the present invention is to produce a novel microorganism useful for efficiently producing a high-activity and high-quality β-glucan from an inexpensive sugar such as sucrose at a high production rate, and production of β-glucan using the microorganism. It is an object of the present invention to provide a method and β-glucan that is secreted and produced outside the cell by the microorganism.
[0013]
[Means for Solving the Problems]
In order to solve the above-mentioned problems, the present inventors have searched for a microorganism having the ability to efficiently secrete and produce β-glucan having a high purity from sucrose outside the cell body. As a result, a novel microorganism has been found that secretes and produces high-quality β-glucan outside the cell with high efficiency. As a result of further studies, it was found that a strain belonging to the genus Aureobasidium that is resistant to the antibiotic cycloheximide efficiently secretes and produces high-purity β-glucan outside the cells.
[0014]
  The present invention has been made based on the above findings, and the sequence of 1732 bases of the 18S rRNA gene is represented by the nucleotide sequence shown in SEQ ID NO: 1 in the Sequence Listing.ColumnIn addition, it has resistance to cycloheximide, an antibiotic, and has the ability to secrete and produce β-glucan outside the cellAureobasidium pulllance ( Aureobasidium pulullans ) ADK-34 (FERM P-18932) strainIt provides microorganisms.
  Further, the present invention relates to a nucleotide sequence represented by SEQ ID NO: 2 in the Sequence Listing, wherein the sequence of 563 bases of the ITS-5.8S rRNA gene.ColumnAnd has the ability to secrete and produce β-glucan outside the cellAureobasidium pulllance ( Aureobasidium pulullans ) ADK-34 (FERM P-18932) strain,Preferably, the present invention further provides a microorganism having resistance to the antibiotic cycloheximide.
  The present invention also provides the above-mentioned microorganism having the ability to secrete and produce β-glucan having at least β-1,3-D-glucopyranose bond in its structure..
  The present invention also provides a method for producing β-glucan, characterized in that the microorganism is cultured (preferably cultured in a culture solution containing saccharide as a carbon source) and β-glucan is secreted and produced outside the cell. To do.
  Further, the present invention is secreted and produced outside the cells by culturing Aureobasidium pullulans (Areobasidium pullulans) ADK-34 (FERM P-18932) strain,The absorbance at 490 nm of the culture diluted solution obtained by appropriately diluting the culture solution and removing the cells by centrifugation (10000 × g, 10 min.) Is the same as that at 490 nm of the culture diluted solution of IFO-6353 strain obtained by culturing in the same manner. In the condition where the absorbance is 0.1 or more, it is 0.099 or less,A β-glucan having at least a β-1,3-D-glucopyranose bond in its structure is provided.
[0015]
DETAILED DESCRIPTION OF THE INVENTION
  In the microorganism of the present invention, the 1732 base sequence of the 18S rRNA gene has the base sequence shown in SEQ ID NO: 1 in the sequence listing.ColumnA microorganism having resistance to cycloheximide, which is an antibiotic produced by Streptomyces griseus, and the like, and having the ability to secrete and produce β-glucan outside the cell, and ITS -The sequence of 563 bases of the 5.8S rRNA gene is represented by SEQ ID NO: 2 in the sequence listing.ColumnAnd a microorganism that has the ability to secrete and produce β-glucan outside the cell, and is preferably resistant to cycloheximide, an antibiotic produced by Streptomyces griseus, etc..
[0017]
  The inventors have isolated the microorganisms of the present invention from the environment as detailed in the examples. SeparateFineAmong organisms, microorganisms belonging to the genus Aureobasidium were observed. Among them, one strain having high purity and capable of efficiently secreting and producing non-coloring β-glucan outside the cells was named ADK-34 strain. This strain will be displayed at the National Institute of Advanced Industrial Science and Technology Patent Organism Depositary as a microbiological label (label for identification given by the depositor) “Aureobasidium pullulans ADK-34” and the deposit number “FERM P-18932”. It has been deposited.
[0018]
In addition, for drug resistance, since the concentration showing resistance may vary depending on the condition of the strain, the type of medium, and the culture period, in the present invention, “having resistance to cycloheximide” means IFO-4466 strain, It refers to having resistance to cycloheximide as compared to these strains using IFO-6353 strain and IFO-7757 strain as a standard. That is, when a strain is inoculated into a solid medium (agar plate) containing cycloheximide and cultured at 26 ° C. for 10 days, the solid medium having a cycloheximide concentration in which growth of these IFO strains is not observed is observed. This refers to the case where colony formation due to bacterial cell growth of 0.1 mm or more, preferably 0.3 mm or more, more preferably 0.5 mm or more is observed. Usually, these IFO strains do not grow when the concentration of cycloheximide in the solid medium is 20 μg / ml or more.
[0019]
Further, in the present invention, non-coloring property or coloration suppressed, strain or culture solution or β-glucan is a culture obtained by appropriately diluting the culture solution and removing the cells by centrifugation (10000 × g, 10 min.). In the case of the strain of the microorganism of the present invention, the absorbance of the diluted solution was measured at 490 nm and the absorbance at 490 nm of the culture diluted solution of IFO-6353 strain obtained by culturing in the same manner was 0.1 or more. 099 or less, preferably 0.060 or less, more preferably 0.050 or less.
Further, in the present invention, high purity means the phenol sulfate value before enzyme treatment of the phenol sulfate value after the enzyme treatment when performing the pullulanase enzyme treatment in the purity measurement for the pullulan of the produced polysaccharide shown in Analysis Example 3 described later. The ratio to is 75% or more, preferably 85% or more, and more preferably 90% or more.
[0020]
The microorganism of the present invention is useful for producing β-glucan with high purity and high efficiency outside the cells. The β-glucan produced by the microorganism of the present invention is preferably a β-glucan having at least a β-1,3-D-glucopyranose bond in its structure.
[0021]
A preferred embodiment of the method for producing β-glucan using the microorganism of the present invention will be described below.
In the method for producing β-glucan using the microorganism of the present invention, the strain of the microorganism of the present invention is allowed to act on a medium in which the microorganism can grow to produce β-glucan in a medium outside the cells. In addition, the cultured cells obtained by culturing the strain of the microorganism of the present invention are isolated, and the cultured cells are allowed to act on a solution or medium containing a saccharide that is a substrate for β-glucan, so that β Glucan may be produced. However, since there may be cases where secreted β-glucan prepared for secretion outside the cell body is encapsulated in the cell body, β-glucan prepared and accumulated in the cell body for secretion in this way is also the present invention. Is a β-glucan that is secreted outside the cell.
[0022]
In the method for producing β-glucan using the microorganism of the present invention, the medium for culturing the strain of the microorganism of the present invention is a nutrient source (carbon source, nitrogen source, etc.) that can be normally used by microorganisms belonging to the genus Aureobasidium. An ordinary medium containing an inorganic salt) and further containing an organic nutrient can be used if necessary, but a culture solution containing saccharide as a carbon source is preferable. As these media, various synthetic media, semi-synthetic media, natural media and the like can be used.
[0023]
As the carbon source, saccharides are preferable, and as the saccharides, monosaccharides such as glucose, fructose, mannose, galactose, xylose, arabinose, disaccharides such as sucrose, maltose, lactose, trehalose, fructo-oligosaccharides, xylooligosaccharides, etc. And oligosaccharides such as dextrin and starch, and these can be used alone or in combination. Among these, as the main carbon source, it is preferable to use hexasaccharides such as glucose and fructose, disaccharides such as sucrose and lactose, starches and dextrins, and polysaccharides such as hydrolysates of these carbohydrates. In addition, beet juice, sugarcane juice, fruit juices including citrus fruits, or those obtained by adding sugar to these juices can also be used. In addition, alcohols such as glycerol and ethylene glycol, sugar alcohols such as mannitol, sorbitol and erythritol, and other carbon sources such as organic acids can be used as appropriate. These carbon sources may be added at any time during the culture. For example, saccharides such as sucrose are preferably added to the medium in an amount of 3 to 500 g / l, more preferably 5 to 300 g / l, and most preferably 10 to 200 g. By appropriately feeding so as to be in a concentration range of / l, the production rate and production amount of β-glucan can be relatively increased.
[0024]
Examples of the nitrogen source include peptone, meat extract, soybean powder, casein, amino acid, malt extract, corn steep liquor, casein decomposition product, yeast extract, organic nitrogen source such as urea, sodium nitrate, ammonium sulfate, ammonia gas, aqueous ammonia, etc. These inorganic nitrogen sources can be used alone or in combination.
[0025]
Examples of the inorganic salts include sodium chloride, potassium chloride, calcium carbonate, magnesium sulfate, sodium phosphate, potassium phosphate, cobalt chloride and the like, heavy metal salts, etc., and vitamins as necessary. Can also be used. In addition, when foaming occurs during culture, various known antifoaming agents can be appropriately added to the medium.
[0026]
The culture conditions for the strain of the microorganism of the present invention are not particularly limited, and can be appropriately selected within a range in which the strain can grow well. Usually, it is good to culture for about 2 to 8 days at pH 5.0 to 8.5 and 20 ° C. to 35 ° C., but these culture conditions can be appropriately changed according to the type and characteristics of the microorganism strain used, external conditions, etc. The optimum condition can be selected. In addition, the inoculation amount of the strain to the medium is preferably 1 platinum ear in the case of flask culture, and 1 to 10% (v / v) of the seed culture solution in the case of scale-up. However, this does not apply as long as culture is possible.
[0027]
Cultivation of the microorganism strain of the present invention is carried out under aerobic conditions such as by aeration stirring and shaking. The culture time is preferably carried out until the target concentration of β-glucan is reached, and is usually 2 to 5 days. Alternatively, β-glucan may be continuously produced by continuously adding sugars and medium components that are β-glucan substrates. Saccharose such as sucrose, which is a substrate of β-glucan, can be added to the culture solution as a powdered sugar or as a liquid sugar dissolved in water at a high concentration. 1 to 500 g is preferred. When the addition amount is more than 500 g / l, the production rate is remarkably slow, which is not preferable. After the addition of the substrate, the substrate is preferably treated at 25 to 35 ° C. for 1 to 7 days, particularly about 2 to 5 days by shaking or aeration and stirring, and the reaction is allowed to proceed under aerobic conditions. Β-glucan can be produced from a sugar such as sucrose.
[0028]
In addition, a cultured microbial cell obtained by culturing the microorganism strain of the present invention can be isolated, and β-glucan can be produced using the cultured microbial cell or an extract of the cultured microbial cell as a catalyst. In this case, a saccharide solution or a medium as a substrate may be added to the suspension of cultured cells, cultured cell preparations or treated cultured cells. Examples of the cultured cell preparation include cell disrupted liquid obtained by homogenizing the cultured cell, and the cultured cell processed product is obtained by subjecting the cultured cell or cell disrupted solution to ion exchange in an alginate gel. Examples include immobilized cells immobilized on resin, ceramic, chitosan, and the like. Examples of the solution that can be used for preparing suspensions of these cultured cells, cultured cell preparations, or treated cultured cells include the above-mentioned medium, Tris-acetic acid, Tris-hydrochloric acid, sodium succinate, and sodium citrate. In addition, a buffer solution such as sodium phosphate or potassium phosphate may be used alone or in combination. The pH of the buffer is preferably 3.5 to 9.0, more preferably 5.0 to 8.0, and most preferably 5.2 to 7.8. The amount of cultured cells in the suspension is not particularly limited, but is preferably about 0.1 to 10% in terms of wet volume ratio.
[0029]
Addition of the saccharide or medium as a substrate to the suspension can be carried out at any concentration and any volume, but the amount of substrate added per time is preferably within a range where the activity of the cells can be maintained. For example, 0.1 to 500 g per 1 liter of suspension may be added once or several times, or may be continuously added at about 0.5 to 500 g / day. Β-glucan can be produced by incubating a solution in which cells and a substrate are allowed to act at an arbitrary time and temperature, and this incubation is the same as the above-described culture of the microorganism strain of the present invention. It is preferable to implement under conditions.
[0030]
After culturing as described above, β-glucan secreted and produced outside the cells is separated and collected from the culture solution according to a conventional method. Specifically, various known purification means are selected, such as separating and removing solids such as bacterial cells from the culture solution by centrifugation, filtration, etc., removing impurities and salts with activated carbon, ion exchange resin, etc. Can be done in combination. Furthermore, for example, adsorption / elution to hydrophobic resin, solvent precipitation using ethanol, methanol, ethyl acetate, n-butanol, etc., column method using silica gel, etc., or thin layer chromatography, for fractionation using reverse phase column Separation and purification can be performed by using high performance liquid chromatography alone or in combination as appropriate, and optionally using it repeatedly.
[0031]
In addition, before and after separating β-glucan secreted and produced outside the cells by the above method, the cells may be sterilized. The sterilization temperature is not particularly limited as long as it is a temperature at which the cells die, but is preferably 50 ° C. or higher, more preferably 60 ° C., and most preferably 80 ° C. or higher. Moreover, the secretory β-glucan prepared in the cells can be extracted with hot water at a higher temperature, for example, at 90 ° C. or higher or 121 ° C. under pressure. The sterilization time and hot water extraction time can be set arbitrarily, but preferably 10 minutes or more and 8 hours or less, more preferably 15 minutes or more and 6 hours or less, and most preferably 30 minutes or more and 2 hours or less. Mixing is suppressed and β-glucan is not deteriorated, which is preferable.
[0033]
  Further, when β-glucan is produced using Aureobasidium pulullans ADK-34 (FERM P-18932) strain, β-glucan having at least β-1,3-D-glucopyranose bond in its structure is obtained. It is possibleThe
[0034]
The production method of β-glucan using the microorganism of the present invention significantly suppresses pullulan production as compared to the culture using the strain of Aureobasidium pullulans known so far or the microorganism belonging to the genus Aureobasidium. Therefore, there is an advantage that β-glucan can be easily separated from the culture and the purification operation for obtaining high-purity β-glucan can be simplified. In addition, there are fewer impurities compared to cell walls of microorganisms belonging to the genus Aureobasidium, yeast, lactic acid bacteria, basidiomycetes and extracted β-glucan from plants, and at the same time the isolation and purification operations can be simplified. Since coloring of glucan is remarkably suppressed, it is easy to stably obtain β-glucan having high quality and constant quality.
[0035]
The β-glucan obtained in the present invention has no color and is of high quality, and can be used for various applications as it is or by adding it to other products.
Examples of uses of β-glucan obtained in the present invention include foods, food additives, cosmetics, toiletry products, chemical products, and pharmaceuticals.
[0036]
Specific examples of foods are listed below.
Examples of oils and fats include margarine, shortening, mayonnaise, cream, salad oil, deep oil, whipped cream, foaming emulsified fat, dressing, fat spread, custard cream, dip cream and the like. The β-glucan obtained in the present invention is excellent in compatibility with fats and oils, and is preferably used together with other raw materials after being added to the fats and oils to obtain a fat-and-oil composition containing β-glucan.
[0037]
Furthermore, examples of cereal-related products include foods based on wheat flour, foods based on rice, processed rice products, processed wheat products, processed corn products, processed soybean products, etc. Bakery products such as pies and danish, hot cakes, donuts, pizzas, tempura, etc., premixes of them, biscuits, cookies, snacks and other confectionery, raw noodles, dry noodles, instant noodles, cup noodles, udon, soba, Chinese Examples include noodles such as noodles, rice noodles, and pasta, rice products such as cooked rice, rice bran, aseptic cooked rice, retort cooked rice, Kamisin flour, rice bran powder, dumplings, rice crackers, and hail.
[0038]
Furthermore, examples of the confectionery include Western confectionery such as chocolate, candy drop, candy, chewing gum, baked confectionery, cake, bun, or Japanese confectionery.
Further, processed meat products include ham / sausage, hamburger and the like, and processed fishery products include chikuwa, kamaboko, fried fish, fish sausage and the like.
Furthermore, examples of dairy products include butter, cheese, ice cream, yogurt and the like.
[0039]
Further beverages include alcoholic beverages such as beer, liquor, Japanese sake, whiskey, brandy, Western liquor, shochu, distilled liquor, sparkling liquor, wine, fruit liquor, coffee, tea, Japanese tea, oolong tea, Chinese tea, cocoa, carbonated beverages. , Drinks such as nutrition drinks, sports drinks, coffee drinks, carbonated drinks, lactic acid bacteria drinks, fruit juices and fruit drinks.
Furthermore, examples of seasonings and sauces include roux such as spices, sauce, sauces for grilled meat, dressings, sauces, miso, soy sauce, curry, and hayashi. Examples of soups include soups such as corn soup, potato soup, and pumpkin soup. Other examples include jam, peanut butter and sprinkles.
[0040]
Furthermore, as long-term preservation foods, marine products, livestock meat, fruits, vegetables, mushrooms, corn beef, jam, tomatoes, etc. canned or bottled, frozen foods, etc. Also, curry, stew, meat sauce, mabo tofu, meat vegetable mixed Examples include retort foods such as boiled, soup and cooked rice. Examples of powdered foods include powdered instant foods such as beverages, soups, and miso soups.
Further examples include health foods, medicinal foods, baby foods such as baby foods, sick foods such as liquid foods, elderly foods, diet foods, and supplements.
Furthermore, a microwave oven heating food, a microwave oven cooking food, etc. are mentioned.
[0041]
Food additives include emulsifiers, thickeners, thickening stabilizers, quality improvers, antioxidants, stabilizers, preservatives, flavorings, sweeteners, colorants, bleaching agents, acidulants, gum bases, seasonings, Examples include bitters, nutrient enhancers, spices, and manufacturing agents.
[0042]
Cosmetics and toiletry products include skin cosmetics, hair cosmetics, medicated cosmetics, oral preparations, etc., specifically, lotion, emulsion, skin milk, cream, ointment, lotion, calamine lotion, sunscreen agent , Suntan agent, after shave lotion, pre-shave lotion, makeup base, pack, cleansing, facial cleanser, anti-acne cosmetic, essence and other basic cosmetics; foundation, white powder, eye shadow, eyeliner, eyebrow, teak, lipstick Makeup cosmetics such as nail color; shampoo, rinse, conditioner, hair color, hair tonic, set agent, hair conditioner, hair restorer, hair nourishing agent, body powder, deodorant, hair remover, soap, body shampoo, hand soap, perfume , Toothpaste, oral care , Bath salts, and the like.
[0043]
Examples of chemical products include surfactants, emulsifiers, thickeners, viscosity modifiers, and the like.
Examples of the drug include drugs that have a cholesterol-lowering action, an intestinal regulating action, and an action that suppresses an increase in blood glucose level, and that prevent lifestyle-related diseases, drugs that have an immune enhancing action, and the like.
[0044]
【Example】
EXAMPLES The present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples. In Test Examples 1 and 2, the screening method for the microorganism strain of the present invention is shown, and in Test Examples 3 to 7, the mycological properties of the ADK-34 strain are shown. Moreover, Examples 1-3 show the Example of the manufacturing method of the beta glucan of this invention using ADK-34 strain.
[0045]
[Test Example 1] Strain screening method 1
In Japan, microorganisms that adhere to and grow on the surface of commercially available foods that are usually consumed without cooking, centering on traditional preserved foods, were widely isolated and screened for strains that produce β-glucan. The screening method is described in detail below.
First, the food as the target sample was placed in a sterilized petri dish, and 10 ml of sterilized PBS was added thereto, and the sample surface was repeatedly washed with PBS using a sterilized dropper to obtain a washing liquid in which the surface of the sample was washed. The obtained washing solution was diluted 10 to 100 times with sterilized PBS, 200 μl of this was added to the plate, spread with a congeal rod, and cultured at room temperature for 2 weeks. The plate used was YM medium (manufactured by Difco) and 20 ml of a medium supplemented with chloramphenicol 100 μg / ml and agar 1.5% by weight, respectively, and solidified. Of the approximately 20,000 grown colonies, those that were milky in the early stage of growth and gradually formed a glossy and wet colony throughout the colony, and the entire colony was glossy, with a slightly yellow / brown center or yellow / brown That formed a wet colony, formed a milky white to pink colony, formed a colony that gradually became greenish and fluffy, and exhibited the above four traits From the fungus. In addition, although the whole colony turned pink, the swelled colony at the upper part and the expansion to the outer peripheral part were not recognized, and the glossy colony was excluded. The strains that have been fished are subjected to the isolation of single bacteria, cultured again for 7 days, and then those with budding-type conidia observed by microscopy and those with yeast-like growth are observed, and conidia branches are observed. Were excluded and used as the first screening result.
Next, the strain obtained by the first screening was subjected to liquid culture. That is, a medium in which sucrose was added to YM medium to 5% by weight was prepared, and cultured at 26 ° C. for 4 days using a 24-well microplate to obtain a culture solution. The grown strain was uniformly dispersed throughout the culture solution, and a strain that gave a viscous culture solution was left, which was used as the second screening result.
Next, the bacterial strain obtained from the second screening result was isolated, inoculated into a YM liquid medium, and cultured at 26 ° C. for 96 hours. An equal amount of distilled water was added to the obtained culture solution, and after sterilization at 121 ° C. for 20 minutes, the culture solution was centrifuged (8,000 rpm) to obtain a culture supernatant. Add 2 volumes of ethanol to 30 μl of the culture supernatant, obtain a precipitate by centrifugation (1000 rpm, 10 min), add 100 μl of distilled water, measure the total polysaccharide content by the phenol-sulfuric acid method, A value higher than the value obtained by the operation was judged as positive. Positive results were taken as the third screening result.
From about 20,000 colonies that were separated and cultured from the target sample, 180 strains were obtained in the first screening, 50 strains were obtained in the second screening, and 14 strains were obtained in the third screening. The following analysis of the amount of β-glucan in the culture supernatant of the strain obtained in the third screening and the three strains obtained from the Osaka Fermentation Research Institute, namely IFO-4466 strain, IFO-6353 strain and IFO-7757 strain While measuring by the measuring method shown in Example 1, the purity with respect to pullulan of the produced polysaccharide in the culture broth was measured by the measuring method shown in Analysis Example 3 below. The results are shown in Table 1. It was concluded that the polysaccharide produced by the ADK-34 strain was not digested by pullulanase, the amount of the polysaccharide almost coincided with the quantitative value of β-glucan, and β-glucan was produced with high purity.
[0046]
[Table 1]

[0047]
[Test Example 2] Strain screening method 2
Screening of strains was carried out in the same manner as in Test Example 1 except that a medium supplemented with the antibiotic cycloheximide at a concentration of 10 μg / ml was used. As a result, from 4000 colonies grown after separation and culture from the target sample, 30 strains in the first screening, 10 strains in the second screening, 3 strains in the third screening (ADK-71 strain, ADK-77 strain) , ADK-82 strain). For the strain obtained in the third screening, the amount of β-glucan and the purity of the produced polysaccharide with respect to pullulan were measured in the same manner as in Test Example 1. The results are shown in Table 2.
Moreover, strain identification was tried from the morphological observation about three strains obtained as a result of the 3rd screening, respectively. After inoculating on a YM agar medium and culturing at 26 ° C. for 7 days, all three colonies were glossy and had a yellow outline at the center, forming a wet colony. When these plates were transferred to 4 ° C. and stored for 7 days, the central yellow color changed to black-green. The entire colonies of the ADK-71 strain and the ADK-77 strain were not changed, and were white to pink. The entire ADK-82 strain was blackened. In addition, in the microscopic observation of the cells obtained by culturing each of these three strains in a YM liquid medium at 26 ° C. for 3 days, budding type conidia were observed in all three strains, and the growth was yeast-like. In addition, the colony cells obtained by culturing each of these three strains on a YM agar medium at 26 ° C. for 7 days were observed to grow in a chain with conidia connected in all three strains. No conidia were found. From the above, three strains were determined to be Aureobasidium pullulans.
[0048]
[Table 2]

[0049]
[Test Example 3] Morphological and culture properties
Among the strains obtained in Test Example 1, for the ADK-34 strain, YM medium (1 wt% glucose, 0.5 wt% peptone, 0.3 wt% yeast extract, 0.3 wt% malto extract, After observation at pH 6.0) at 26 ° C. for 3 days, the microscopic observation shows that the cells are 2 to 2.5 μm in the minor axis, 5 to 10 μm in the major axis, the shape is oval, and the surface is smooth. Colorless, no motility. In addition, the mycelium was very small and the width was 2.5 μm. The surface was uneven and the surface was smooth and colorless. Yeast-like budding conidia were formed.
[0050]
[Test Example 4] Agar plate culture
Among the strains obtained in Test Example 1, the ADK-34 strain was cultured on potato dextrose agar medium (Eiken) at 26 ° C. for 7 days. The findings at that time are shown below. After 3 days of culture, the growth was good, the shape was circular, the edges were jagged, the entire colony was shiny, the surface was smooth, and the color was white. Moreover, Colony (colony) became smooth and light grayish white after 5 days of culture, grew like yeast, and the surface of Colony became pink after 7 days of culture. When the plate cultured at 26 ° C. for 7 days was refrigerated at 4 ° C. for 7 days, the pink color was slightly increased, but the entire colony was not changed.
[0051]
[Test Example 5] Liquid culture
Among the strains obtained in Test Example 1, ADK-34 strain was cultured in YM medium. When the optimum growth temperature and optimum growth pH at that time are shown, the optimum growth temperature is 26 ° C., the optimum growth pH is 5.0 to 7.0, and the pH at the start of growth is 6.2. The pH of was 7.5. The preferable growth temperature was 20 to 30 ° C., the optimum temperature was 26 ° C., and the growth possible temperature was 5 to 40 ° C. It decomposed hexoses such as glucose, fructose and mannose, disaccharides such as sucrose, and starch, and the culture solution became viscous and had a characteristic aroma with any carbon source.
[0052]
As a result of Test Examples 3 to 5, among the microorganisms of the present invention, the ADK-34 strain was identified as a strain of the genus Aureobasidium from the mycological characteristics.
[0053]
[Test Example 6] Cycloheximide resistance test
Cycloheximide resistance test of each strain for strains obtained by screening in Test Examples 1 and 2, and IFO-4466 strain, IFO-6353 strain, and IFO-7757 strain preserved in Osaka Fermentation Research Institute Carried out. That is, each strain was grown on a YM agar medium (Difco) at 26 ° C. for 5 days. Medium prepared by adding cycloheximide to YM agar medium (manufactured by Difco) to a concentration of 5, 10, 20, 40, and 80 μg / ml was prepared, and these strains that had been grown were sterilized. Were inoculated into a cycloheximide-containing medium, cultured at 26 ° C. for 10 days, and then observed for the presence of grown colonies. When grown, the diameter of the colonies was measured. The results are shown in Table 3.
[0054]
[Table 3]

[0055]
As is clear from Tables 1 and 3, the IFO-4466, IFO-6353, and IFO-7757 strains were not resistant to cycloheximide, and the purity of β-glucan produced with respect to pullulan was low. . On the other hand, as apparent from Tables 1 to 3, among the isolates from food, the strains having resistance to cycloheximide (ADK-34 strain, ADK-71 strain, ADK-77 strain, ADK-82 strain) are: β-glucan was produced with high purity against pullulan, and many of the strains not resistant to cycloheximide resulted in low purity of β-glucan produced against pullulan.
[0056]
[Test Example 7] Genetic analysis of 18S rRNA gene
About the ADK-34 strain obtained as a result of the screening, the base sequence of 1732 bp of 18S rRNA gene was determined as follows. The ADK-34 strain was cultured with shaking in a potato dextrose medium (Difco), and centrifuged and washed with distilled water three times to obtain a cell for DNA extraction. Cells were disrupted from the cells using FastPrepFP120 (Qbiogene) and FastDNA-kit (Qbiogene), and genomic DNA was isolated using DNeasy Plant Mini Kit (QIAGEN). Using this genomic DNA as a template, PCR amplification was performed using Ready-To-Go PCR Beads (Amersharm-Pharmasia Biotech) and primers NS1 and NS8.
NS1: (5 ′-> 3 ′) GTAGTCATATGCTTGTCTC, NS8: (5 ′-> 3 ′) TCCGCAGGTTCACCTACGGA were used as the base sequences of the primers NS1 and NS8. As the thermal cycler, geneAmp PCR System 9600 (Applied Biosystems) was used. After completion of the reaction, the PCR product was purified using QIAquick PCR purification Kit (QIAGEN). This DNA fragment was directly subjected to sequencing reaction, and the base sequence analysis was performed using ABI Prism377 DNA Sequencer (Applied Biosystems). Similar base sequences were searched from DNA databases (DDBJ, DNA Data Bank of Japan) using BLAST.
The result is shown in SEQ ID NO: 1 in the sequence listing. Moreover, the database search was performed based on the obtained base sequence, and the results of examining the homology between the same strain and related bacteria are shown in Tables 4-6. From the determined base sequence (SEQ ID NO: 1) and homology search results (Tables 4 to 6), the ADK-34 strain had 100% homology with Aureobasidium pullulans and was completely consistent. . From this result, this strain was determined to be Aureobasidium pullulans.
[0057]
[Table 4]

[0058]
[Table 5]

[0059]
[Table 6]

[0060]
[Test Example 8] ITS-5.8S rRNA gene
For the ADK-34 strain, IFO-6353 strain, and IFO-7757 strain obtained by screening, the 563-base sequence or 564-base sequence of the ITS-5.8S rRNA gene was determined. Each strain was cultured with shaking in a potato dextrose medium (Difco), and centrifuged and washed with distilled water three times to obtain bacterial cells for DNA extraction. Cells were disrupted from the cells using FastPrepFP120 (Qbiogene) and FastDNA-kit (Qbiogene), and genomic DNA was isolated using DNeasy Plant Mini Kit (QIAGEN). Using this genomic DNA as a template, PCR amplification was performed using Ready-To-Go PCR Beads (Amersharm-Pharmasia Biotech) and primers ITS5 and ITS4. The base sequences of the primers ITS4 and ITS5 are ITS4: (5 ′-> 3 ′) TCCTCCGCTTATTGATATGC, ITS5: (5 ′-> 3 ′) GGAAGTAAAAGTCGTAACAAGG. As the thermal cycler, geneAmp PCR System 9600 (Applied Biosystems) was used. After completion of the reaction, the PCR product was purified using QIAquick PCR purification Kit (QIAGEN). This DNA fragment was directly subjected to sequencing reaction, and the base sequence analysis was performed using ABI Prism377 DNA Sequencer (Applied Biosystems). Similar base sequences were searched from DNA databases (DDBJ, DNA Data Bank of Japan) using BLAST.
The results of ADK-34 strain are shown in SEQ ID NO: 2 in the sequence listing, the results of IFO-6353 strain are shown in SEQ ID NO: 3, and the results of IFO-7757 strain are shown in SEQ ID NO: 4, respectively. In addition, Tables 7 and 8 show the results of conducting a database search based on the obtained base sequence and examining the homology between the strain and related bacteria. When the determined base sequences were compared, the ADK-34 strain, the IFO-6353 strain, and the IFO-7757 strain were not completely matched. The homology between the ADK-34 strain and the IFO-6353 strain was 98%, and the homology between the ADK-34 strain and the IFO-7757 strain was 98%. In addition, from the homology search results (Tables 7 and 8), no strains in which the 563 base pairs of the ITS-5.8S region of the ADK-34 strain completely matched were reported. The IFO-6353 strain is 100% (Table 9), that is, completely consistent with the reported Aureobasidium pullulans strain registration number “AJ276062”, and the IFO-7757 strain is a reported Aureobasidium pullulans strain. It coincided with a certain registration number “AJ276062” at 99% (Table 10). The ADK-34 strain had a registration rate of 98% with the registration number “AJ276062”.
From the above, the ADK-34 strain was determined to be a new strain, with a part of the base sequence of the ITS-5.8S region being different from the Aureobasidium pullulans strains reported so far.
[0061]
[Table 7]

[0062]
[Table 8]

[0063]
[Table 9]

[0064]
[Table 10]

[0065]
[Analysis Example 1] Confirmation method of β-glucan and measurement method of β-glucan amount
For analysis of β-glucan in polysaccharide, the total amount of polysaccharide precipitated by alcohol is measured by the phenol-sulfuric acid method, and then β-glucan in the precipitated polysaccharide is confirmed and quantified by β-1 of Seikagaku Corporation. This was carried out using a β-glucan detection / measurement kit containing a 3-D-glucopyranose bond. The procedure will be described below.
First, the total polysaccharide amount in the measurement sample was measured by the phenol sulfuric acid method. That is, 30 μl of distilled water is added to 30 μl of the sample solution, 120 μl of phosphate buffer (pH 6.9) containing 300 mM NaCl is added thereto, 540 μl of ethanol (3 times volume) is further added, and the mixture is added to −15 ° C. for 10 minutes. The polysaccharide was left to stand. After removing the supernatant, 100 μl of distilled water was added to dissolve the precipitate. After 100 μl of 5 wt% phenol aqueous solution and 500 μl sulfuric acid were added and reacted, the absorbance at 490 nm of the solution was measured. Further, the absorbance at 490 nm of the blank was measured using a blank obtained by adding 100 μl of a 5 wt% phenol aqueous solution and 500 μl of sulfuric acid to 100 μl of distilled water without adding a sample. Create a 2-fold dilution series from pullulan 10 mg / ml as a standard sample, create a standard curve using the standard sample, and quantitate the total amount of polysaccharides in the sample from the standard curve and the absorbance above did.
Next, a solution having a total polysaccharide amount of about 0.1 to 1 mg / ml is first diluted 10-fold with 1.0 M NaOH, and subsequently diluted with β-glucan-free distilled water.^TenIt diluted to 1 time, and the beta glucan dilution liquid was prepared. 50 μl of β-glucan diluted solution was taken in a tube, 50 μl of the main reaction reagent was added, and incubated at 37 ° C. for 30 minutes. Subsequently, 50 μl of sodium nitrite solution, 50 μl of ammonium sulfamate, and 50 μl of N methyl 2-pyrrolidone solution were added and reacted, and then the absorbance of the solution at 545 nm (control wavelength: 630 nm) was measured. A 7.5 to 60 pg / ml β-glucan solution was prepared using the attached β-glucan standard, and a calibration curve was obtained. From the calibration curve and the absorbance, the concentration of each β-glucan diluted solution was calculated to determine the amount of β-glucan in the sample.
[0066]
[Analysis Example 2] Method for measuring molecular weight of β-glucan
The molecular weight of β-glucan was measured as follows. First, 3 times the amount of alcohol was added to the culture supernatant, cooled to −20 ° C., and allowed to stand for 10 minutes to obtain a precipitate. Precipitated β-glucan was taken in a 5 mg tube, 1 ml of distilled water was added and dissolved in boiling water. The solution was passed through a 0.22 μm filter to obtain a sample for HPLC. For separation, Shodex packed column KS-805 (manufactured by Showa Denko KK), which is an HPLC gel filtration column, was used at a flow rate of 0.6 ml / min. The temperature was 50 ° C., an RI detector was used for detection, and water was used as a separation solvent. The molecular weight marker was measured using Shodex pullulan standard solution P-82 (manufactured by Showa Denko KK).
[0067]
[Analysis Example 3] Method for measuring the purity of a polysaccharide present in a culture solution with respect to pullulan
Using a pullulanase (manufactured by Wako Pure Chemical Industries, Ltd.), an enzyme that specifically degrades pullulan, the purity of the polysaccharide produced in the culture broth was measured. That is, the amount of polysaccharide was measured before and after the enzyme digestion reaction with pullulanase, and the purity of the produced polysaccharide with respect to pullulan was calculated from the amount of polysaccharide after enzyme treatment (the amount of polysaccharide not digested by pullulanase) and the amount of polysaccharide before enzyme treatment. The method for measuring the amount of polysaccharide will be described in detail below.
First, an enzyme solution was prepared by adding 2 mL of citrate buffer (10 mmol / L citrate-20 mmol / L phosphate buffer (pH 6.0)) to 50 μL of pullulanase enzyme diluted solution. As a positive control, a solution obtained by diluting pullulan (manufactured by Tokyo Chemical Industry Co., Ltd.) to 10 mg / mL was used as a standard sample solution. 30 μL of the culture solution or standard sample was weighed into two sample tubes (1.5 ml capacity), 30 μL of enzyme solution was added to one side, 30 μL of PBS was added to one side, and reacted at 37 ° C. for 1 hour. After completion of the reaction, 3 times the amount of ethanol was added to each sample. The mixture was well stirred and incubated at −15 ° C. for 10 minutes, followed by centrifugation at 4 ° C. and 15000 rpm for 10 minutes. The supernatant was discarded and the tube was dried to obtain a precipitate. 100 μL of distilled water was added to the precipitate, stirred, 100 μL of a 5 wt% phenol aqueous solution was added, and 500 μL of sulfuric acid was quickly added to cause color development. After cooling, 100 μL each was dispensed into a 96-well microplate, and the absorbance at 490 nm was measured using a 5 wt% aqueous phenol solution and sulfuric acid added to distilled water as a blank. A calibration curve was prepared with a solution obtained by diluting pullulan to 10 mg / ml to 1 μg / ml. From the calibration curve and the absorbance described above, the amount of polysaccharide in the culture solution before and after the enzyme digestion reaction was quantified, and the purity of the polysaccharide present in the culture solution with respect to pullulan was calculated. When the pullulan solution as a standard sample was enzymatically digested, the digestibility of 90% or more was 10 mg / ml solution, 95% or more for a 1 mg / ml concentration solution, and 98% or more for 0.1 mg / ml or less. Digestibility was obtained. As described above, when pullulan is generated, the phenol sulfate value (absorption value) after the enzyme treatment becomes low due to the action of pullulanase. For example, when the absorbance value is reduced by 50%, The purity with respect to pullulan was calculated as 50%.
[0068]
[Example 1]
Place YM medium (Difco) 5.5 ml into a 30 ml test tube, sterilize (121 ° C., 20 minutes), cool, and store in YPD agar medium (Difco) slant. 34 strains were inoculated into one platinum loop and cultured at 26 ° C. for 4 days in a 300 rpm shaking incubator to obtain a seed culture solution. Put 5.5 ml of kuzapek medium (Difco, sucrose concentration 3% by weight) in another test tube, sterilize, add 500 μl of seed culture solution of ADK-34 strain (5% inoculation), and shake culture at 300 rpm. Incubator was cultured at 26 ° C. for 4 days. The culture medium was white and had a significant viscosity. After culturing, an equal volume of distilled water was added to the culture solution, pasteurized with high-pressure steam for 15 minutes, and then centrifuged at 10,000 rpm for 15 minutes to obtain a culture supernatant containing polysaccharide. The amount of β-glucan in the culture supernatant and the purity of the polysaccharide with respect to pullulan were measured by the measurement methods of Analysis Example 1 and Analysis Example 3, respectively. As a result, the purity of the polysaccharide with respect to pullulan was 100%, and the yield of β-glucan to sugar calculated from the amount of β-glucan was 44%. The absorbance value at 490 nm of the culture supernatant was 0.030, and coloring was suppressed. Further, when the molecular weight of β-glucan was measured by the measurement method of Analysis Example 2, the molecular weight was 150,000 to 2,000,000.
The IFO-6353 strain was cultured in the same manner, and the amount of β-glucan and the purity of the polysaccharide with respect to pullulan were measured. As a result, the purity of polysaccharide with respect to pullulan was calculated to be 40%, and the yield of β-glucan with respect to sugar was calculated to be 11%. The absorbance value at 490 nm of the culture supernatant was 0.190, and coloring was observed.
[0069]
[Example 2]
Yeast Nitrogen Base w / o Amino Acids and Ammmonium Sulfate (1.7 g) was dissolved in 100 ml of distilled water and sterilized by filtration. 5 g of sodium nitrate was dissolved in 500 ml of distilled water to prepare a 2-fold concentration sodium nitrate solution. As the carbon source, sucrose, glucose, fructose, soluble starch or maltose (Wako Pure Chemical Industries, Ltd.) was used to prepare 12 wt% carbon source aqueous solutions, respectively. Sodium nitrate, an aqueous carbon source solution and distilled water were sterilized by autoclave (121 ° C., 20 min.). In a sterilized test tube, 1 ml of yeast nitrogen source basal medium, 5 ml of sodium nitrate solution, 2.5 ml of aqueous carbon source solution and distilled water 1 .5 ml was added to make a total volume of 10 ml, and a carbon source check medium added with each carbon source was prepared.
ADK containing 5.5 ml of YM medium (Difco) in a 30 ml test tube, sterilized (121 ° C., 20 minutes), cooled, and stored in a slant of YPD agar medium (Difco) -34 strains were inoculated into one platinum loop and cultured at 26 ° C. for 4 days in a 300 rpm shaking incubator to obtain a seed culture solution. The obtained seed culture solution was inoculated into the carbon source check medium and cultured at 26 ° C. for 5 days in a 300 rpm shaking incubator. The culture medium was white and had a significant viscosity. After culturing, an equal volume of distilled water was added to the culture solution, pasteurized with high-pressure steam for 15 minutes, and then centrifuged at 10,000 rpm for 15 minutes to obtain a culture supernatant containing polysaccharide. The amount of β-glucan in the culture supernatant and the purity of the polysaccharide with respect to pullulan were measured by the measurement methods of Analysis Example 1 and Analysis Example 3, respectively. Table 11 shows the measurement results. As is clear from Table 11, the purity of the polysaccharide with respect to pullulan was 90% or higher and the yield of β-glucan with respect to sugar was calculated to be 20 to 46% in any carbon source. The absorbance at 490 nm of the culture supernatant was 0.026 to 0.041, and coloring was suppressed. Further, when the molecular weight of β-glucan was measured by the measurement method of Analysis Example 2, the molecular weight was 150,000 to 2,000,000.
[0070]
[Table 11]

[0071]
Example 3
ADK-34 strain was inoculated into YM medium and cultured at 26 for 3 days to obtain 300 ml of seed culture solution. Into a 5L jar fermenter (Maruhishi Bio-Engineering) equipped with a full zone wing, put 3L of Kuzapek medium and 300g of sucrose, inoculate 100 ml of seed culture solution after sterilization and cooling, and incubate at 26 ° C for 72 hours. Culture was performed to obtain 3 L of a culture solution. The culture solution was heated at 80 ° C. for 30 minutes to sterilize, and then an equal amount of distilled water was added and mixed well, followed by centrifugation at 8000 rpm for 15 minutes to obtain a culture dilution. The culture dilution was further diluted 5-fold with distilled water, and the absorbance was measured and found to be 0.023 at 490 nm. An equal amount of ethanol was added to 100 ml of the culture dilution, and the resulting precipitate was separated, washed with ethanol, and directly dissolved in 50 ml of distilled water. This operation was repeated again and put into a dialysis membrane (molecular weight 3000 cut), and dialyzed with 10 times the amount of distilled water. Finally, 100 ml polysaccharide solution was obtained. At this time, the amount of β-glucan was calculated to be 25 mg / ml, the purity of the polysaccharide with respect to pullulan was 95%, and the molecular weight of β-glucan was calculated to be 150,000 to 2,000,000.
[0072]
【The invention's effect】
According to the present invention, it is possible to provide a novel microorganism useful for efficiently producing β-glucan at a high production rate, and a method for producing β-glucan using the microorganism.
[0073]
[Sequence Listing]

Claims (7)

1732 bases of the sequence of 18S rRNA gene comprises a nucleotide sequence shown in SEQ ID NO: 1, further comprising a resistance to cycloheximide (cycloheximide) is an antibiotic, and secrete β-glucan extracellularly Aureobasidium pullulans having the ability to produce (Aureobasidium pulullans) ADK-34 ( FERM P-18932) microorganism is strain. ITS-5.8S rRNA gene 563 base sequence comprises a nucleotide sequence shown in SEQ ID NO: 2, and Aureobasidium pullulans having the ability to produce and secrete the β-glucan extracellularly (Aureobasidium pulullans ) A microorganism that is an ADK-34 (FERM P-18932) strain .   The microorganism according to claim 2, which has resistance to cycloheximide which is an antibiotic.   The microorganism according to any one of claims 1 to 3, which has the ability to secrete and produce β-glucan having at least a β-1,3-D-glucopyranose bond in its structure outside the cell. A method for producing β-glucan, wherein the microorganism according to any one of claims 1 to 4 is cultured, and β-glucan is secreted and produced outside the cells. The method for producing β-glucan according to claim 5 , wherein the microorganism is cultured using a culture solution containing saccharide as a carbon source. Aureobasidium pulullans (Aureobasidium pulullans) ADK-34 (FERM P-18932) strain is cultured and secreted and produced outside the cells.
The absorbance at 490 nm of the culture diluted solution obtained by appropriately diluting the culture solution and removing the cells by centrifugation (10000 × g, 10 min.) Is the same as that at 490 nm of the culture diluted solution of IFO-6353 strain obtained by culturing in the same manner. In the condition where the absorbance is 0.1 or more, it is 0.099 or less,
Β-glucan having at least a β-1,3-D-glucopyranose bond in its structure.