JP7452876B2 - 糸状真菌バイオマット、その製造方法及び使用方法 - Google Patents
糸状真菌バイオマット、その製造方法及び使用方法 Download PDFInfo
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- JP7452876B2 JP7452876B2 JP2021184509A JP2021184509A JP7452876B2 JP 7452876 B2 JP7452876 B2 JP 7452876B2 JP 2021184509 A JP2021184509 A JP 2021184509A JP 2021184509 A JP2021184509 A JP 2021184509A JP 7452876 B2 JP7452876 B2 JP 7452876B2
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Description
質、アミノ酸及び/又は脂質の単離及び/又は精製も提供される。これらのタンパク質、
アミノ酸及び/又は脂質は、食品、魚飼料、動物飼料、油、脂肪酸、医薬品、栄養補助食
品、殺真菌剤、除草剤、殺酵母剤、殺虫剤、バイオ潤滑剤に、及び他の価値が付加された
製品のための原料として使用することができる。
本発明は、以下の態様を含む;
[項1]
糸状菌バイオマットを製造する方法であって、
(a)有効量の少なくとも1種類の糸状菌の浮遊細胞を人工成長培地に播種するステップ
、
(b)撹乱のない状態で、所定期間、前記播種済成長培地をインキュベートするステップ
、
(c)糸状菌バイオマットを製造するステップ、及び
(d)場合により、前記糸状菌バイオマットを採取するステップを含む、上記方法;
[項2]
糸状菌バイオマットを製造する方法であって、
(a)人工成長培地に、7.5%(体積:体積)の少なくとも1種類の糸状菌の浮遊細胞
を播種するステップ、
(b)撹乱のない状態で、所定期間、前記播種済成長培地をインキュベートするステップ
、
(c)糸状菌バイオマットを製造するステップ、及び
(d)場合により、前記糸状菌バイオマットを採取するステップを含む、上記方法;
[項3]
前記少なくとも1種類の糸状菌が、MK7と命名された菌株(ATCC寄託番号PTA
-10698)、フザリウム(Fusarium)種及びクモノスカビ(Rhizopu
s)種からなる群から選択される、項1又は2に記載の方法;
[項4]
前記少なくとも1種類の糸状菌が、MK7と命名された菌株(ATCC寄託番号PTA
-10698)、フザリウム・ベネナタム(Fusarium venenatum)又
はリゾプス・オリゴスポラス(Rhizopus oligosporus)である、項
1~3のいずれか一項に記載の方法;
[項5]
前記少なくとも1種類の糸状菌が、MK7と命名された菌株(ATCC寄託番号PTA
-10698)である、項1~4のいずれか一項に記載の方法;
[項6]
前記人工培地が約18.6atmの浸透圧を有する、項1~5のいずれか一項に記載の
方法;
[項7]
前記人工培地が約0.368のイオン強度を有する、項1~6のいずれか一項に記載の
方法;
[項8]
バイオマット細胞密度が少なくとも25g/L(乾燥重量/培地(L))である少なく
とも1つの細胞層を含む、糸状菌バイオマット;
[項9]
前記糸状菌が、MK7と命名された菌株(ATCC寄託番号PTA-10698)、フ
ザリウム(Fusarium)種及びクモノスカビ(Rhizopus)種からなる群か
ら選択される、項8に記載の糸状バイオマット;
[項10]
前記糸状菌が、MK7と命名された菌株(ATCC寄託番号PTA-10698)、フ
ザリウム・ベネナタム(Fusarium venenatum)又はリゾプス・オリゴ
スポラス(Rhizopus oligosporus)である、項8又は9に記載の糸
状バイオマット;
[項11]
前記糸状菌が、MK7と命名された菌株(ATCC寄託番号PTA-10698)であ
る、項8~10のいずれか一項に記載の糸状バイオマット;
[項12]
フィラメントが、空気:バイオマット界面及び/又はバイオマット:培地界面に対して
平行に主に組織化されている、項8~11のいずれか一項に記載の糸状バイオマット;
[項13]
前記バイオマットが、少なくとも2つの構造的に異なる細胞層を含み、1つの細胞層は
人工培地及び少なくとも1つのもう一方の細胞層と接触する、項8~12のいずれか一項
に記載の糸状バイオマット;
[項14]
前記細胞層間の構造差が、層内の細胞の密度である、項8~13のいずれか一項に記載
の糸状バイオマット;
[項15]
前記1つの細胞層が、空気及び少なくとも1つのもう一方の細胞層と接触している、項
8~14のいずれか一項に記載の糸状バイオマット;
[項16]
前記バイオマットが、3つの構造的に異なる細胞層を含む、項8~15のいずれか一項
に記載の糸状バイオマット;
[項17]
前記バイオマットの引張強度が、少なくとも0.2kg/マット幅(cm)である、項
8~16のいずれか一項に記載の糸状バイオマット;
[項18]
細胞密度が少なくとも50g/l又は少なくとも75g/lである、項8~17のいず
れか一項に記載の糸状バイオマット;
[項19]
前記バイオマットは、タンパク質含有量が少なくとも40%である、項8~18のいず
れか一項に記載の糸状バイオマット;または
[項20]
前記バイオマットは、脂質含有量が少なくとも39%である、項8~19のいずれか一
項に記載の糸状バイオマット。
本明細書で使用される場合、「~を含む(comprise)」との動詞及びその活用形は、非限定的な意味で、この単語に続く項目を含むが、具体的に述べられていない項目を除外しないことを意味するように、本明細書及び特許請求の範囲で使用される。これに加え、不定冠詞「1つの(a)」又は「1つの(an)」による、ある要素への言及は、文脈上、その要素が1つ存在し、1つのみであることを明確に要求する場合を除き、その要素が1つより多く存在する可能性を除外しない。したがって、不定冠詞「1つの(a)」又は「1つの(an)」は、通常、「少なくとも1つ」を意味する。
糸状菌バイオマットを生産するために、人工培地が使用される。人工培地は、天然に見出されるものと比較して、細胞周期時間を増加させ(すなわち、成長速度を増加させ)、細胞密度を増加させるために必要な栄養素を与える。人工培地は、窒素(N)、リン(P)、カルシウム(Ca)、マグネシウム(Mg)、炭素(C)、カリウム(K)、硫黄(S)といった多量栄養素を少なくとも含む。炭素源を補うために、微量栄養素、例えば、鉄(Fe)、ホウ素(B)、クロム(Cr)、銅(Cu)、セレン(Se)、マンガン(Mn)、モリブデン(Mo)、バナジウム(V)、亜鉛(Zn)も、培地に添加することができる。炭素源、例えば、リグノセルロース系原料、スイートホエイ及び/又は酸ホエイは、典型的には、さらなる微量栄養素が必要とされないように、十分な微量栄養素を与える。
本発明は、単離された真菌種及び/又は菌株の純粋な培養物、又は2種類の真菌種及び/又は菌株の純粋な共培養物を使用するか、又は3種類以上の真菌種及び/又は菌株の実質的に純粋な培養物から構成される。多数の単離された糸状真菌種及び/又は菌株、例えば、フシスポリウム(Fusisporium)、プソイドフザリウム(Psedofusarium)、ジベレラ(Gibberella)、スポロトリチェラ(Sporotrichella)、アスペルギルス(Aspergillus)ペニシリウム(Penicillium)、トリコデルマ(Triocoderma)、ピチア spp.(Pichia spp)、ケカビ.sp(Mucorales sp.)内の種(例えば、リゾプス sp.(Rhizopus sp.))の種及び/又は菌株、及びこれらの組み合わせを使用することができる。生物学的に純粋な培養物/共培養物/実質的に純粋な培養物は、MK7と命名される単離された糸状好酸性真菌株(ATCC Accession Deposit No.PTA-10698として寄託されている)、又はその活性な突然変異体も含んでいてもよい。遺伝的に改変された糸状菌の生物学的に純粋な培養物も使用することができる。純粋な真菌種及び/又は菌株及び/又はその子孫は、典型的には、分生子、小分生子、大分生子、分生子殻(pycnidia)、厚膜胞子、菌糸、菌糸の一部、菌糸体、又はこれらの組み合わせの形態である。
(a)容器中、真菌種又は菌株及び/又はその子孫のうち1つ以上を、糖、グリセロール、リグノセルロース系原料、炭素を含有する農業、工業及び都市廃棄物、炭水化物、酵母抽出物、カサミノ酸、酸ホエイ、スイートホエイ及び/又はこれらの組み合わせからなる群から選択される炭素源を含む人工培地に播種することであって、人工培地が、表面発酵によって、上述の単離された真菌株の成長を補助することができる、播種すること、
(b)上述の単離された真菌株を上述の人工培地中で成長させ、糸状菌バイオマットを生産すること、
(c)上述の糸状菌バイオマットを採取すること、並びに
(d)場合により、上述の糸状菌バイオマットからの産物を単離し、精製し、及び/又は生産することを含む。
本開示は、人工培地に、所望の糸状真菌種及び/又は菌株の浮遊細胞の懸濁物を播種することによって、表面発酵を開始する。播種材料リアクターからの播種材料培養物を、所望の期間内に成熟したバイオマットを産生する濃度で、人工培地に加える。理論的には、この培地に、1個の細胞を播種することができた。しかし、このような播種は、成熟したバイオマットを発達させるためには、非常に厳しい滅菌条件及び著しく長い期間を必要とする。典型的には、成長培地1リットル当たり、0.5~1.0gの細胞を播種すると、3~6日でバイオマットが産生されるだろう。例えば、使用する培地の7.5%(体積対体積)で、約10g/Lの細胞を含有する播種材料を加えると、3~6日でバイオマットが産生されるだろう。人工培地にバブリング又は他の手段によって外部から酸素が導入されない場合、周囲条件又はほぼ周囲条件から十分な酸素を集めることができる。
本発明で使用される好酸性真菌種及び/又は菌株は、以下の識別する特性を少なくとも有する、リグノセルロースを分解する糸状真菌株及び/又はその子孫である。
(a)単離された菌株は、好酸性であり、約0.68~約8.5の範囲のpHで成長することができ、
(b)人工培地から、好気性条件、微好気性条件、嫌気性条件、又はこれらの組み合わせで、表面発酵によって、タンパク質と脂質を含む糸状バイオマットを生成する。ここで、人工培地の炭素源には、炭水化物、リグノセルロース系原料、炭素含有廃棄物(例えば酸ホエイ)、又はこれらの組み合わせが含まれる。
(c)タンパク質、脂質アミノ酸、酵素、核酸(ヌクレオチド)、炭水化物、繊維(例えばβグルカン)、ポリケチド、アルカロイド、顔料及び抗生物質などを生産する能力。例としては、限定されないが、エステル、グルタミン酸、アスパラギン酸、アミラーゼ、プロテアーゼ、セルラーゼ、キシラナーゼ、リパーゼ、ペルオキシダーゼ、マンガンペルオキシダーゼ、核酸/ヌクレオチド:DNA/RNA、プリン、ピリミジン、オレイン酸、パルミトレイン酸、β-グルカン、キチン、β-カロテン、グリコシド、フェノール類、18ページ、段落[80]に記載される炭素源及び藻類原料からのテルペノイド、種々の嫌気性条件、好気性微好気性条件及び/又はこれらの任意の組み合わせでの、バイオ燃料の生産中に作られる廃棄物(例えば、処理した藻類バイオマス、グリセロール)からのものが挙げられる。
(d)配列番号1に対して少なくとも98%の同一性を共有する18S rRNA及びITS領域DNA配列を含む。
好酸性フザリウム種に関する情報、同定し、単離し、培養する方法は、Nelsonら(Taxonomy,Biology,and Clinical Aspects of Fusarium Species,1994,Clinical Microbiology Reviews,7(4):479-504)、Toussoun及びNelson(1976,Fusarium),Booth(Fusarium:laboratory guide to the identification of the major species,1977,Commonwealth Mycological Institute,ISBN 0851983839,9780851983837)及びLeslieら(The Fusarium laboratory manual,2006,Wiley-Blackwell,ISBN 0813819199,9780813819198)に記載されており、それぞれ、その全体が本明細書に援用により組み込まれる。
上述のように、高いC:N比を有する人工培地中で培養すると、藻類及び他の脂質を産生する生物と比較して、高い脂質含有量を有し、より望ましい脂質プロファイルを有する糸状菌バイオマットが産生される。脂質は、単離された糸状バイオマスから抽出することができる。ある場合には、脂質は、主に脂肪酸アシル基を有するトリアシルグリセリドである。ある場合では、脂肪酸は、本質的に不飽和脂肪酸及び/又は飽和脂肪酸である。不飽和脂肪酸としては、オレイン酸(18:1)、α-リノレン酸(18:3)、エイコセン酸(20:1)、及びこれらの組み合わせが挙げられる。飽和脂肪酸としては、パルミチン酸(16:0)、ステアリン酸(18:0)、アラキジン酸(20:0)、ベヘン酸(22:0)、及びこれらの組み合わせが挙げられる。産生し得る他の種類の脂質としては、限定されないが、ステロール(例えばエルゴステロール、D2前駆体中のビタミン)、ジアシルグリセリド、カロテノイド、飽和脂肪(例えば、酪酸、ヘキサン酸、オクタン酸、デカン酸、ドデカン酸、トリデカン酸、テトラデカン酸、ペンタデカン酸、ヘキサデカン酸、ヘプタデカン酸、オクタデカン酸、ノナデカン酸、エイコサン酸、ドコサン酸、テトラコサン酸)、モノ不飽和脂肪(例えば、テトラデセン酸、ペンタデセン酸、ヘキサデセン酸、ヘプタデセン酸、オクタデセン酸、エイコセン酸、ドコセン酸、cis-テトラコセン酸)、多価不飽和脂肪(例えば、ヘキサデカジエン酸、リノール酸、リノレン酸、α-リノレン酸、γ-リノレン酸、パリナリン酸、エイコサジエン酸、アラキドン酸、チモノドン酸、ブラシン酸、クルパノドン酸、ドコサヘキサエン酸)が挙げられる。
フザリウム・オキシスポラム f.sp.リコペルシシ(Fusarium oxysporum f.sp.lycopersici)株4287のゲノムは、最近配列決定されており、リグニン、ヘミセルロース及びセルロースの分解に関与する様々な遺伝子を有することが示されている。さらに、これらの物質の分解に関与する酵素(例えば、セルラーゼ、キシラナーゼ、リグニナーゼ、グルクロニダーゼ、アラビノフラノシダーゼ、アラビノガラクタナーゼ、フェルラ酸エステラーゼ、リパーゼ、ペクチナーゼ、グルコマンナーゼ、アミラーゼ、ラミナリナーゼ、キシログルカナーゼ、ガラクタナーゼ、グルコアミラーゼ、ペクチン酸リアーゼ、キチナーゼ、exo-[3-D-グルコサミニダーゼ、セロビオースデヒドロゲナーゼ、アセチルキシランエステラーゼ、キシロシダーゼ、a-L-アラビノフラノシダーゼ、フェルロイルエステラーゼ、エンドグルカナーゼ、[3-グルコシダーゼ、Mn-ペルオキシダーゼ及びラカーゼ)は、F.oxysporum strain F3(Xirosら(2009)Enhanced ethanol production from brewer’s spent grain by a Fusarium oxysporum consolidated system.Biotechnol Biofuels 10:4)で広く研究されている。結果的に、好酸性糸状真菌種及び/又は菌株、例えば、フザリウム(Fusarium)種、及びMK7と命名された好酸菌性糸状菌株は、リグノセルロース材料及び廃液(例えば酸ホエイ)などの複合炭素源を加水分解するために完全に装備されることが期待される)。これらの好酸性糸状真菌種及び/又は菌株は、他の糸状菌株(pH>2、Starkey、1973)と比較して、かなり低いpH(0.7~7.5)で成長することができ、リグニン、ヘミセルロース及びセルロースの分解のための酵素は、低いpHでもっと高い活性を有するだろうということも期待される。酸性条件下で高活性を有する酵素は、低pHで実施されるプロセスにおいて特に有用である。
しばしば、微生物に基づく生産は、汚染を防ぐために費用と時間のかかる方法の使用を必要とする。上述のように、糸状好酸性真菌種及び/又は菌株は、他の生物による汚染に対して非常に耐性である。例えば、フザリウム属のメンバーが強力な抗生物質、殺虫剤及び植物毒性を有する毒素(例えば、フモニシン)を生成することが知られている。同様に、糸状好酸性MK7真菌株は、糸状菌バイオマットの生産に使用される場合、外部抗生物質をほとんど必要としないか、又は全く必要としない。いくつかのフザリウム種は、9種類の異なる毒素を産生し得る。その生成は、異なる宿主植物との関係に依存する(Marasasら、1984,Toxigenic Fusarium species,identity and mycotoxicology,ISBN 0271003480)。毒素の産生も、発酵に使用される培地によって異なる。フザリウム種によって産生され、分泌される毒素としては、限定されないが、ビカベリン、エンニアチン、フザリン酸、リコマラスミン、モニリホルミン、オキシスポロン、トリコテセン、ゼエレロン(zearelones)、種々のナフトキノン及びアントラキノン(例えば、ノナケチドナフタザリンキノン、ビカベリン及びノルビカベリン、ヘプタケチド、ネクタリアフロン、5-0-メチルジャバニシン、及びアンヒドロフサルビン(anhydrofusarubin)ラクトール)が挙げられる。
例1:自然環境におけるMK7株
自然発生株MK7は、自然界の藻類、古細菌及び細菌と常に関連しており、平均密度が0.5g乾燥バイオマス/井戸水(L)未満であることを特徴とする(図1)。これに加え、MK7は、「ストリーマー」として自然界に存在する。Purcellらは、「ストリーマー」を以下のように定義している。「ストリーマーは、付着点から流水中に突出している糸状及び他の細胞形態を有する水中凝集物である」(Purcellら(2007)FEMS Microbiology Ecology7 60:456-466)。MK7株は、全ストリーマーバイオマスの割合として10%未満である。さらに、MK7株バイオマスは、本質的に、大きな分生子細胞としてバイオマスの30%を超えることを特徴とし、本開示の方法の概要によって作られる表面発酵バイオマット中には決して見出されない。
以下の手順で使用されるMK7-1液体培地は、表1Aに列挙される成分を、22~30℃の脱イオン水(18.2MΩ)に添加した後、13N HClを用い、元より低いpHであるpH2.8に調整することによって調製された。pHは、Oakton Instruments 150型のpH計とプローブ(オレンジバーグ、NY)を用いて測定した。次いで、培地を20分間沸騰させ、使用前に室温(約23℃)まで冷却した。液体培地を添加する直前に、Oakton Instruments 150のpH計とプローブを用いてpHを再び調べ、必要に応じてpH2.8に調整し直す。
トレイに播種するために使用される培養物を、MK7-1液体培地中、深部発酵条件下で10Lバイオリアクター中で成長させた。他の大きさのバイオリアクターに修正可能であり、10Lの大きさのリアクターの選択は、限定するものと解釈すべきではないことを注記しておくべきである。10Lリアクターは、直径10.16cmの透明なPVC管の長さ1.3mの部分から構成されており、底部にPVCエンドキャップを有している。3mmオリフィスを備えるプラスチック通気口/接続具を底部のエンドキャップと管に接続し、空気を供給するために通気口に接続した。プラスチックのサンプリング口/バルブを、底部エンドキャップの底から15cmの透明なPVC壁の側面に取り付けた。透明なPVCリアクターの上部を滅菌ガーゼで覆い、リアクターから気体を逃がすことができるように、3mmの穴を有するPVCエンドキャップをゆるく取り付けることによって、所定位置に保持した。組み立てられたバイオリアクターを図2に示す。
糸状好酸性MK7真菌株を、浅いトレイリアクターで成長させた。この革新的な考えの教示から、異なるトレイの大きさに修正可能であることを注記しておくべきである。この例では、ポリエチレントレイの内寸は、幅が41.27cm、長さが61.28cmであり、高さが2.54cmの側壁を有する(マットの成長に利用可能な全表面積=0.253m2、図5、Winco、アイダホフォールズ、ID)。トレイは、汚染の可能性を最小限に抑えるために、使用前に屑片及び化学物質をきれいに取り除き、滅菌しておくことが望ましい。その結果、使用前に、トレイを石鹸及び温かい飲料水道水(50~70℃)で十分に洗浄し、温かい水道水で1分間十分に洗浄し、全ての石鹸残留物が除去されたことを確認した。その後、トレイの全ての表面が70%イソプロパノール/30%脱イオン水(18.2MΩ)溶液で濡れるまで、トレイ全表面に噴霧し、上述のアルコール混合物に浸した紙タオルを用い、手袋を付けた手でトレイを拭いた。その後、漏れたり乾燥したりせずにトレイが液体培地(以下に記載)を受け入れ、保持することができるように、トレイをラックシステム内に配置した。
バイオマットの成長及び生産性に対するトレイサイズの影響を試験するために、0.02m2Pyrex(登録商標)ガラストレイ、0.25m2ポリプロピレントレイ、1.77m2プラスチックライナー付きトレイ中、7.5%グリセロールを含むMK7-1培地上でMK7株糸状菌バイオマットを成長させた。表面積に対する液体培地の体積の比率は、全ての処理について6L/m2であった。成長速度は、トレイサイズによって最小限しか影響を受けず、6日後に、線形の成長速度が観察された(図8)。乾燥バイオマスの生産性は、それぞれ0.02、0.25、1.77m2のトレイで、1.32、1.54及び1.57/m2/hであった。
糸状好酸性MK7真菌株の成長特性(すなわち、成長速度、細胞密度、基質変換効率、マット形成)の成長特性は、成長培地の選択の関数として顕著に変動し、培養物は、SSF/深部発酵条件又はSSSF/表面発酵条件で成長する。糸状好酸性MK7真菌株を、イエローストーン国立公園の微生物の自然環境中に見出される化学物質を模倣するために最初に設計されたが、他の糸状真菌にとって有益であることがわかっている栄養源に適合するように窒素、リン、カルシウム及びマグネシウムの濃度を高めた、従来の(2009年5月から2012年11月まで)MK7A培地上で栽培した(表2、MK7A)。MK7-1培地は、糸状好酸性MK7真菌株の成長特性を、特に、表面発酵によるマット形成に関して向上させ、改善するために開発された(表2)。具体的には、リン、カルシウム、マグネシウム、窒素を増やし、さらなる窒素源(尿素)を加えた。カルシウム、マグネシウムを増やし、尿素を加えると、具体的には、成長速度が増加し、マット形成が向上した。
MK7株の糸状菌バイオマットは、MK7A培地及びMK7-1培地を用い、炭素源(原料)としてスクロース又はグリセロールを用いた表面発酵条件下で生産された。MK7A培地及びMK7-1培地から生産される糸状菌バイオマットを評価するために、5種類の異なる培地配合物を調製した。(1)MK7A培地中、4%のスクロース、(2)MK7-1培地中、4%のスクロース、(3)MK7A培地中、4%のグリセロール、(4)MK7-1培地中、10%のグリセロール、(5)MK7-1培地中、12.5%のグリセロール。5種類全ての培地配合物のpHを、濃HClを適切に添加して2.7に調整し、その後、10分間沸騰させた。室温(約23℃)まで冷却した後、7.5%(体積/体積)の対数成長期にあるMK7株播種材料を各培地に添加した。pHを2.7に再び調整し、播種した培地を250mLずつに小分けし、これを消毒した0.023m2のPyrex(登録商標)ガラストレイに添加した。次いで、トレイをトレイラックシステムに入れ、培地と播種材料の混合物を23℃±1℃でインキュベートした。
バイオマットの構造は、透過型光学顕微鏡によって決定した。ここで、7.5%グリセロールを含むMK7-1培地上で5日間成長させたMK7株からバイオマットを生産した。バイオマットを採取し、凍結させ(-20℃)、1cm×1cmの正方形試験片に切除した後、クリオモルド(VWR 25608-916、ラドナー、PA)中の10%ゼラチン(Sigma G2500-3000 Bloomゼラチン、Sigma-Aldrich、セントルイス、MO)で包み込んだ。液体窒素浴の蒸気相にクリオモルドをさらすことによって、ゼラチン/組織サンプルをすばやく凍結させた後、-20℃に一晩置いた。
MK7-3培地で5日間成長させたMK7バイオマットの引張強度を評価した。ここでは、幅25.4cm、長さ46cm、厚さ3.5mmのマットを使用した。マットの含水率は約85%であり、乾燥重量の約15%に相当する。総乾燥重量は、70g/0.25m2トレイ又は280g/m2であった。このマットの密度は0.08g/cm3であった。
粗グリセリンを炭素源及び栄養源(原料)として使用し、緻密なMK7株バイオマットを8日間で製造した。粗グリセリンは、バイオディーゼル生産の副産物であり、W-2 Fuels(製品コードGL32000、バッチ4300、カタログ番号56-81-5、エイドリアン、MI)から得た。粗グリセリンは、75~85%のグリセリン、2~10%の水、2~5%の塩、1~2%の脂肪、油又はエステル、1%未満のメタノールで構成されていた。
連結されたMK7株の糸状菌バイオマットは、炭素源及び栄養源(原料)として乾燥小麦蒸留可溶物(ds)を用いて7日間で生産された。小麦dsは、タンパク質31.5%、油8.6%、デンプン2.8%、砂糖13.5%、繊維2.7%、灰分8.5%、カルシウム0.19%、マグネシウム0.29%、カリウム1.7%、リン0.78%、硫酸3.5%から構成されていた。2種類の成長培地処理を調製した。処理1は、水中5%のds乾燥重量を使用し、処理2は、1/2強度のMK7-1塩培地中5%ds乾燥重量を使用した。混合物のpHを3.4に調整した。混合物に、7.5%(体積:体積)の例3に記載したように調製したMK7株播種材料を播種し、その培地175mlをアルコール滅菌した12.7×12.7cmのプラスチックトレイに添加した。糸状菌バイオマットを室温(約23℃)で7日間インキュベートした後に採取した。唯一の炭素源及び栄養源としての5%ds上で成長させたバイオマット(処理1、MK7-1塩を含まない)は、厚さが平均2.7mmであり(n=3トレイ)、明確な層化を示さず、平均乾燥重量は0.83gであり、平均密度は0.019g/cm3であり、変換効率は約10%であった。気中菌糸は観察されず、マットは、全体的に液体で飽和されていた。すなわち、マットの上部表面は、液体の表面にあった。
緻密なMK7株の糸状菌バイオマットは、唯一の炭素源及び栄養源(原料)としてコーンスティープリカーを用い、わずか4日間で生産された。さらに、デンプンを5%添加したコーンスティープリカーも、緻密な糸状菌バイオマットを生成することができることが示された。コーンスティープリカーは、トウモロコシ湿式粉砕の粘性のある副生成物であり、アミノ酸、ビタミン、及びミネラルの組成を有し、微生物発酵の補助剤として適している。この例で使用したコーンスティープリカーは、Santa Cruz Biotechnology,Inc.(ダラス、TX;ロット番号B0116)から購入した。これらの実験は、MK7-1栄養素の代替物としてのコーンスティープリカーの使用を示した。処理は、唯一の炭素源及び栄養源として10%及び20%のコーンスティープリカー、(それぞれ)5%デンプンを含む10%及び20%のコーンスティープリカーを含んでおり、デンプンは、糸状菌バイオマットの成長のためのさらなる炭素を与える。
成長実験は、唯一の炭素源及び栄養源として牛の肥育池水を用いて実施した。水の初期溶存有機炭素含有量は4g/Lであり、初期総溶存窒素含有量は0.8g/Lであった。
緻密なMK7株バイオマットは、Acid Whey Surrogate Medium(AWS)から7日間で製造された。AWS培地の組成は、Tsakaliら(2010)に記載されている酸ホエイの典型的な組成に基づいていた。この例で使用したTsakali培地及びAWS培地の組成を表4に記載する。
主な炭素源及び栄養源として酸ホエイを用い、MK7株バイオマットを6日間成長させた。バイオマットは、種々の処理を行った125mLの原料酸ホエイを使用し、滅菌済みの12.7×12.7cm(0.016m2)のポリプロピレントレイで生産された。この処理は、pHを調整し、選択した栄養素を加え、及び/又は競合する微生物の存在を最小限にするために加熱した後、成長速度とバイオマスの生産性を評価するために行われる。
嫌気性消化物を唯一の炭素源及び栄養源(原料)として使用し、緻密なMK7株バイオマットを7日間で製造した。嫌気性消化物は、酸素制限条件下で、リグノセルロース富化バイオマス(例えば、トウモロコシ茎葉、小麦わら、牛肥料)の微生物発酵後に残るリグニンに富む固形残留物である。嫌気性消化物は、微生物によるさらなる分解に抵抗性があると考えられており、この理由から、土壌改質として一般に使用されているか、燃焼され、電気の生成のために蒸気発生器に動力を供給している。
リゾプス・オリゴスポラス(Rhizopus oligosporus)及びフザリウム・ベネナタム(Fusarium venenatum)の成長を、MK7株について例1、2及び3に記載の表面発酵技術を用いて評価した。
SSF手順
ここでいう固体状態発酵(SSF)とは、含水量が低い、通常50%未満の固形分で起こる微生物発酵プロセスを意味する。
SSSF播種材料は例3に記載されており、手順は以下の例に記載されている(すなわち、液体層の上に浮遊するサトウダイコンパルプ及び他のリグノセルロース系材料の変換)。具体的には、本明細書で言及するSSSFは、固形基質が液体表面より下に沈められたときに起きる発酵を意味し、糸状菌バイオマットは、沈められた固体由来の炭素及び栄養素を用い、液体表面上で成長する。産生された糸状菌バイオマスは凝集性であり、高密度であり、原料を含まない(図14C)。
MK7株バイオマスは、SSF法及びSSSF法を用い、主な炭素源としてサトウダイコンパルプを用いて生産された。サトウダイコンパルプは、処理工場でサトウダイコンパルプから砂糖を除去した後に残る、サトウダイコンの野菜部分である。サトウダイコンパルプは、ビリングス、モンタナのWestern Sugar Cooperative製造工場から得て、約24%の乾燥物質、9.1%の粗タンパク質、0.6%の粗脂質、23.1%の粗繊維、4%の灰分、0.56%のカルシウムで構成されていた。
緻密なMK7株バイオマットは、ホモジナイズされたブロッコリー又はホモジナイズされたニンジンを原料として使用して、6日間で生産された。ブロッコリーとニンジンは、ボーズマン、モンタナ州のCostco Wholesaleから購入した。100グラムの各原料を、市販のフードプロセッサーで、金属ブレードを用い、高速で個別に5分間ホモジナイズし、9000mlの水道水を用い、2リットルのビーカーに入れた。中程度の塩を以下のように加えて混合物を形成した。
都市有機廃棄物に対する酸及び塩基の前処理の影響は、原料から糸状菌バイオマットへの変換率の関数として評価した。ケンタッキー・ブルーグラスの切り抜きとトネリコの木の葉は、含水量が8%未満になるまで60℃で別々に乾燥させた。各原料は、市販のブレンダーで細かい粉末に粉砕した。
・100ml水道水中、pH2.5で(33%HClで調整)、10gの草/葉(乾燥重量で50:50)の3個組を、10分間沸騰させることによって前処理した。
・100ml水道水中、pH2.5で(33%HClで調整)、10mMのMnSO4を含み、10gの草/葉(乾燥重量で50:50)の3個組を、10分間沸騰させることによって前処理した。
・100mlのMK7-1培地中、pH2.5で(33%HClで調整)、10gの草/葉(乾燥重量で50:50)の3個組を、10分間沸騰させることによって前処理した。
・100mlのMK7-1培地中、pH2.5で(33%HClで調整)、10gの草/葉(乾燥重量で50:50)の3個組を、10mMのMnSO4と共に10分間沸騰させることによって前処理した。
・100ml水道水中、pH10.75で(1%NaOHで調整)、10gの草/葉(乾燥重量で50:50)の3個組を、10分間沸騰させることによって前処理した。HClを用い、最終pHを2.5に調整した。
・100ml水道水中、pH10.75で(1%NaOHで調整)、10mMのMnSO4を含み、10gの草/葉(乾燥重量で50:50)の3個組を、10分間沸騰させることによって前処理した。HClを用い、最終pHを2.5に調整した。
・100mlのMK7-1培地中、pH10.75で(1%NaOHで調整)、10gの草/葉(乾燥重量で50:50)の3個組を、10分間沸騰させることによって前処理した。HClを用い、最終pHを2.5に調整した。
・100mlのMK7-1培地中、pH10.75で(1%NaOHで調整)、10gの草/葉(乾燥重量で50:50)の3個組を、10mMのMnSO4と共に10分間沸騰させることによって前処理した。HClを用い、最終pHを2.5に調整した。
・100mlの水道水中、10gの草/葉(乾燥重量で50:50)を3個組。最終pH5.5。
・100mlの水道水、10mMのMnSO4中、10gの草/葉(乾燥重量で50:50)を3個組。最終pH5.5。
・100mlのMK7-1培地中、10gの草/葉(乾燥重量で50:50)を3個組。最終pH5.5。
・100mlのMK7-1培地、10mMのMnSO4中、10gの草/葉(乾燥重量で50:50)を3個組。最終pH5.5。
サンプルを12.7×12.7cmのトレイに入れ、カバーし、次いで7日間インキュベートした。結果を図15に示す。それぞれの場合に、前処理を適用すると、得られた変換率が増えた。すなわち、酸又は塩基による前処理を適用することによって、また、マンガンの添加によって、得られた糸状菌バイオマットに対する原料の変換量が増えた。
緻密なMK株及び糸状菌バイオマットは、炭素源及び栄養源(原料)としてデンプンを用い、わずか4日間で生産された。これらの具体的な実験で使用されたデンプンは、Argo Food Companies,Inc(メンフィス、TN)によって製造された100% Argo Corn Starchであり、ボーズマン、MHのAlbertsonスーパーマーケットから購入した。
緻密なMK7株の糸状菌バイオマットは、炭素源及び栄養源(原料)としてジャガイモ処理廃棄物を用い、7日間で生産された。ジャガイモ処理廃棄物は、一般に、ジャガイモの処理中に生成し、洗浄、皮むき、切断操作(すなわち、フライドポテト、ポテトキューブ、フレークなど)からの廃液を含む。ジャガイモ処理廃液は、ひとかたまりの複数のジャガイモ処理廃液のピリンで構成される廃棄堆積物も含み、覆いのない自然環境にさらされる。この例では、複数の様々なジャガイモの処理廃棄物で構成されるジャガイモ処理廃液を、2016年9月21日にホワイトホール、モンタナ州のBauch Farmsから入手し、MK7株バイオマットを成長させるための炭素源及び栄養源として48時間以内に使用した。
栄養分析は、SSSF方法論に対し、SSF法から得られたバイオマットと比較し、Eurofinsによって実施された。SSFサンプルは、Michigan Biotechnology Instituteのアンモニア繊維拡張(AFEX)で前処理したトウモロコシ茎葉上で培養したMK7株から得た。150gのAFEXを500mlの水道水に添加し、濃HCLでpHを3.5に調整した後、121℃でオートクレーブ処理した。得られた混合物に、例18に従って25mlのMK7株播種材料を播種した。スラリーを23×23cmのPyrex(登録商標)ガラストレイに移し、室温で11日間インキュベートした。統合したMK7株バイオマスとトウモロコシ茎葉を採取し、60℃で48時間乾燥させた。Eurofins USA(デモインズ、アイオワ州)では、総タンパク質、総繊維、総炭水化物、灰分及び総脂肪についてサンプルを分析した。
糸状好酸性MK7真菌株バイオマットは、MK7-1培地において例2及び3に記載の方法を使用し、トレイリアクター中で産生された。6つのトレイからの糸状バイオマスを合わせた後、60℃で45分間、50℃で72時間乾燥させた。この糸状バイオマス400gを、栄養分析のために、デモイン、アイオワ州のEurofins Scientific Inc.のNutritional Analysis Centerに送った。アミノ酸は、Association of Official Agricultural Chemists(AOAC)Official Methods of Analysisで公開された国際的に認められた、以下のような方法を用いて分析した。トリプトファンについてはAOAC 988.15、シスチン及びメチオニンについてはAOAC 994.12改変法、アラニン、アルギニン、アスパラギン酸、グルタミン酸、グリシン、ヒスチジン、イソロイシン、ロイシン、フェニルアラニン、プロリン、セリン、トレオニン、総リシン、チロシン及びバリンについてはAOAC 982.30改変法。Eurofinsによって報告された糸状好酸性MK7真菌株サンプルのアミノ酸組成を、魚飼料について使用したフザリウム・ベネナタム(Fusarium venenatum)のアミノ酸組成と比較する(Alriksson,B.ら、(2014)Fish feed from wood.Cellulose Chemistry and Technology 48:9-10(2014),Quorn(Nutritional Profile of Quorn Mycoprotein,2009),egg albumin(Food and Agriculture Organization of the United Nations.The Amino Acid Content of Foods and Biological Data on Proteins,Nutritional Study #24.Rome(1970).UNIPUB,Inc.,4611-F Assembly Drive,Lanham,MD 20706)and Rhizopus oligosporus(Graham,D.C.,Steinkraus,K.H. & Hackler,L.R.(1976)Factors affecting production of mold mycelium and protein in synthetic media.Appl Environ Microbiol 32:381-387)in Table 8。総タンパク質含有量は、4.5%の含水量のバイオマスの41.5%として測定された。注目すべきことに、糸状好酸性MK7真菌株は、4種類の他のタンパク質源の全てと比較して、より高い濃度の必須アミノ酸を含むことが示され、糸状好酸性MK7真菌株は、食品及び飼料にとって非常に望ましいタンパク質源となった。
培地調製:125g/Lのグリセロール(The Chemistry Store-Kosher Food Grade Glycerol>99.7%、ASIN:B00KN1LRWQ、インターネット上で入手可能)(562.5g)と、C:N比を40:1に変更するNH4NO3及び尿素窒素濃度(C源中の炭素のモル数:窒素化合物中のNのモル数)を用い、4.5リットルのMK7-1培地を調製した。
異なる条件下で成長させたMK7株の5つのサンプルを、マイコトキシンの存在についてアッセイした。サンプル1のバイオマスは、C:N比が30:1であったことを除いて、例1に記載した播種材料を生成するために使用したのと同じ条件下で、10Lのバイオリアクターで生産された。バイオマスサンプルを、例1に記載した高減圧濾過装置を用い、0.2umのフィルターに通して濾過することによって集めた。
Claims (55)
- 糸状真菌バイオマスであって、前記バイオマスが:
マットの形態であり、
単一の真菌株または真菌種であり、
生存能力の不活性化、酵素の不活性化、またはそれらの組み合わせにより処理され、
少なくとも0.05kg/マット幅(cm)の引張強度を有し、かつ
気中菌糸を含む、
糸状真菌バイオマス。 - 前記マットが気中菌糸層及び人工培地と接触する底部層を含む互いに接触する少なくとも2つの構造的に異なる細胞層を含み、ここで前記気中菌糸層が前記底部層より密度が低い、請求項1に記載の糸状真菌バイオマス。
- 前記マットが前記気中菌糸層及び前記底部層の間に遷移層をさらに含む、請求項2に記載の糸状真菌バイオマス。
- 前記底部層が、第一の方向に整列したフィラメントを含み、かつ前記気中菌糸層が第一の方向に主に垂直に配向する気中菌糸を含む、請求項2に記載の糸状真菌バイオマス。
- 上部気中菌糸層の厚さに対する底部層の厚さの比が少なくとも0.41である、請求項2に記載の糸状真菌バイオマス。
- 前記マットが1mm~30mmの厚さである、請求項1に記載の糸状真菌バイオマス。
- 前記マットが少なくとも40%のタンパク質含有量を有する、請求項1に記載の糸状真菌バイオマス。
- 前記マットが少なくとも39%の脂質含有量を有する、請求項1に記載の糸状真菌バイオマス。
- 前記マットが、引き裂かれることなく扱って持ち上げて移動させるのに十分な引張強度及び構造一体性を有する、請求項1に記載の糸状真菌バイオマス。
- 前記マットの引張強度が少なくとも0.2kg/マット幅(cm)である、請求項1に記載の糸状真菌バイオマス。
- 細胞外多糖をさらに含む、請求項1に記載の糸状真菌バイオマス。
- 少なくとも1つの細胞層を含む糸状真菌バイオマットであって、前記バイオマットが少なくとも25g乾燥重量/l培地の細胞密度及び1mm~30mmの厚さを有し、かつ不活性化されている、糸状真菌バイオマット。
- 前記バイオマットの引張強度が少なくとも0.05kg/マット幅(cm)である、請求項12に記載のバイオマット。
- 前記バイオマットの引張強度が少なくとも0.2kg/マット幅(cm)である、請求項13に記載のバイオマット。
- 請求項1に記載の糸状真菌バイオマスの製造方法であって:
(a)人工成長培地へ少なくとも1つの糸状真菌の有効量の生物学的に純粋な培養物を播種するステップ;および
(b)播種した糸状真菌を前記人工成長培地の表面で糸状真菌バイオマットを産生するようインキュベートするステップ、を含む、製造方法。 - (c)糸状真菌バイオマットを採取するステップをさらに含む、請求項15に記載の方法。
- 前記人工成長培地が、液体人工成長培地である、請求項15に記載の方法。
- ステップ(a)が少なくとも1つの糸状真菌の浮遊細胞を人工成長培地に播種するステップを含む、請求項15に記載の方法。
- 前記人工成長培地が、18.6atmの浸透圧を有する、請求項15に記載の方法。
- 前記人工成長培地が、0.368のイオン強度を有する、請求項15に記載の方法。
- 前記人工成長培地が、グリセロール、酸ホエイ、デンプン、コーンスティープリカー、ジャガイモ廃棄物、サトウダイコン廃棄物、デキストロース、グルコース、マルトース、ガラクトース、マンノース、コーンシロップ、野菜スクラップ、牛の飼料用水からなる群から選択される炭素源を含む、請求項15に記載の方法。
- 前記人工成長培地が、農業廃棄物、医療用有機廃棄物、都市有機廃棄物、食品加工廃棄物、バイオ燃料製造廃棄物、工業廃棄物、リグノセルロースを含有する廃棄物、乳製品廃棄物、屠殺場廃棄物及びこれらの組み合わせからなる群から選択される廃棄物を含む炭素源を含む、請求項15に記載の方法。
- 前記人工成長培地が、単糖類、二糖類及びこれらの組み合わせからなる群から選択される炭素源を含む、請求項15に記載の方法。
- 前記人工成長培地が、グリセロール、糖、乾燥小麦蒸留可溶物、コーンスティープリカー、牛の肥育池水、酸ホエイ、植物廃棄物、コーンシロップ及びこれらの組み合わせからなる群から選択される炭素源を含む、請求項15に記載の方法。
- 人工成長培地が、硝酸アンモニウム、尿素、リン酸二水素カリウム、塩化カルシウム、硫酸マグネシウム、及び酵母抽出物を含む、請求項15に記載の方法。
- 人工成長培地が、硫酸第二鉄、硫酸亜鉛、塩化マンガン、塩化コバルト、硫酸銅及びホウ酸をさらに含む、請求項15に記載の方法。
- 7.5%(体積:体積)の浮遊細胞が前記人工成長培地に播種される、請求項15に記載の方法。
- 前記人工成長培地が窒素源を含む、請求項15に記載の方法。
- 前記窒素源が硝酸塩、アンモニウム塩、タンパク質、ペプチド、尿素、窒素を含む廃液、及びこれらの組み合わせからなる群から選択される、請求項28に記載の方法。
- 前記人工成長培地が炭素及び窒素を含み、C:Nの比が7.5:1以下である、請求項15に記載の方法。
- 前記人工成長培地が、炭素及び窒素を含み、C:Nの比が7.5:1を超える、請求項15に記載の方法。
- 真菌菌糸体の成長方法であって:
複数の容器を提供するステップ、ここで各容器は培地及び真菌を含有するキャビティを定義する;
閉じたインキュベーション室に前記容器を置くステップ;
真菌菌糸体バイオマスを産生するのに十分なだけ制御された環境条件で閉じたインキュベーション室を維持するステップ;
インキュベーション室に空気流を作るステップ;
各容器において液体培地及び真菌をインキュベートするステップ;および
各容器において液体培地及び真菌の上に空気流を通過させるステップ;を含み、
ここで、インキュベートするステップが真菌が培地を消化し、各容器において真菌菌糸体バイオマスを生成するのに十分な時間である、方法。 - 真菌菌糸体バイオマスが単一の真菌株または真菌種のものである、請求項32に記載の方法。
- 空気流が前記容器の横方向に閉じたインキュベーション室へ作られる、請求項32に記載の方法。
- 空気流が前記容器の水平方向へ作られる、請求項34に記載の方法。
- 前記容器がインキュベーション室内で垂直方向に間隔を空けた複数の列に積み重ねられている、請求項32に記載の方法。
- 制御された環境条件が相対湿度、二酸化炭素含有量、および温度を含む、請求項32に記載の方法。
- 制御された環境条件が、相対湿度が90%-100%で維持されること、温度が少なくとも30°Cで維持されること、及びこれらの組み合わせからなる群から選択される、請求項37に記載の方法。
- 空気流が前記容器の横方向に閉じたインキュベーション室へ作られる、請求項32に記載の方法。
- 空気流が前記容器の水平方向へ作られる、請求項32に記載の方法。
- 空気流がインキュベートのステップの間に調整される、請求項32に記載の方法。
- 真菌菌糸体が気中菌糸を含む、請求項32に記載の方法。
- 各容器において液体培地及び真菌の上に空気流を通過させるステップが、各容器において液体培地及び真菌の表面を空気流を通過させることを含む、請求項32に記載の方法。
- 真菌菌糸体バイオマスが真菌菌糸体バイオマスのバイオマットである、請求項32に記載の方法。
- 真菌菌糸体バイオマスが単一の真菌株または真菌種のものである、請求項44に記載の方法。
- 真菌菌糸体バイオマスが細胞外多糖類をさらに含む、請求項32に記載の方法。
- 各容器においての成長中の菌糸体に空気流を通過させることをさらに含む、請求項32に記載の方法。
- 前記容器が蓋を備えていない、請求項32に記載の方法。
- 人工培地及び単離された真菌株及び/又はその子孫を使用して有用な製品を生産する方法であって、
(a)容器中の、真菌種又は菌株及び/又はその子孫のうち1つ以上を、糖、グリセロール、リグノセルロース系原料、炭素を含有する農業、工業及び都市廃棄物、炭水化物、酵母抽出物、カサミノ酸、酸ホエイ、スイートホエイ及び/又はこれらの組み合わせからなる群から選択される炭素源を有する人工培地に播種することであって、前記人工培地が、表面発酵によって、前記単離された真菌株の成長を補助することができる、播種すること、
(b)前記単離された真菌株を前記人工培地中で成長させ、1つ以上の糸状真菌バイオマットを生産すること、
(c)1つ以上の前記糸状真菌バイオマットを採取すること、
ここで、1つ以上の前記糸状真菌バイオマットが、
生存能力の不活性化、酵素の不活性化、またはそれらの組み合わせにより処理され、
少なくとも0.05kg/マット幅(cm)の引張強度を有し、かつ
気中菌糸を含む、採取すること、
を含む、方法。 - (d)前記糸状真菌バイオマットからの産物を単離し、精製し、及び/又は生産することをさらに含む、請求項49に記載の方法。
- 前記有用な製品が、タンパク質が豊富なバイオマス、バイオマット及び/又は糸状真菌バイオマット、食品、魚飼料製品、動物飼料製品、バイオプラスチック及び/又はこれらの前駆体に使用するためのタンパク質バイオマットである、請求項49に記載の方法。
- 請求項1に記載の糸状真菌バイオマスを含む食品。
- 前記食品が、ヒト食品、魚飼料、および動物飼料からなる群から選択される、請求項52に記載の食品。
- 前記糸状真菌バイオマスが、全ての必須アミノ酸を含む、請求項52に記載の食品。
- 前記糸状真菌バイオマスが、20%分岐鎖アミノ酸を含むタンパク質をさらに含む、請求項52に記載の食品。
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