JP5670998B2 - エンバク中のアベナンスラミド濃度を増加させる方法 - Google Patents
エンバク中のアベナンスラミド濃度を増加させる方法 Download PDFInfo
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Description
エンバク材料の質的及び量的なアベナンスラミド組成物の決定を以下に記載するように行った。エンバク試料、すなわち種子を、恒量まで37℃で、オーブン中で乾燥させ(約48時間)、分析まで−20℃で真空パックビニール袋中に貯蔵した。抽出前に市販のコーヒーミルを使用して種子を挽いた。抽出及び定量分析は、一般的に、2反復を使用して行った。
還流酸性化80%エタノール(エタノール:水:氷酢酸、80:19.9:0.1(v/v/v))75mlに、挽いたエンバク試料10gを、抗酸化剤としての亜ジチオン酸ナトリウム5mgと共に、激しく攪拌しながら添加した。混合物を熱から離し、攪拌しながら室温で20分間冷却させた。次いで、全内容物を、フリットディスクを備えた目盛り付きガラスクロマトグラフィーカラムにデカントした。重力によって懸濁液を沈降させ、透明な上澄みを有する軽く圧縮された抽出物ベッド(ベッド容積=VbmL)を形成した。上澄みを重力流によって回収し、抽出ベッドを「浸出抽出」によって3×Vbの酸性化80%エタノールで溶出し、透明な黄緑色溶離液を得た。
この抽出物から親油性成分を除去するため、Octyl Sepharose(商標) CL 4-Bクロマトグラフィービーズを添加し(抽出物g当たり0.5ml)、混合物を回転蒸発によって真空下40℃で濃縮乾固した。酸化を防ぐために、蒸留水を混合物に添加して、確実に乾燥中にアベナンスラミドが沈殿するようにした。乾燥混合物を酸性化50%エタノール(エタノール:水:氷酢酸、50:49.9:0.1(v/v/v))に再懸濁し、前もって重力圧縮し、酸性化50%エタノール(例えば、25mlの試料10gについて、最終ベッド容積Vb=30mL)にあらかじめ平衡化したOctyl Sepharose(商標)CL 4-Bを含有する目盛り付きガラスクロマトグラフィーカラムに定量的に移した。次いで、カラムを3×Vbの酸性化50%エタノールで溶出した。合わせた溶離液を回転蒸発によって真空下40℃で濃縮して本質的に脂質を含まない抽出物を得た。
第1の方法では、精製アベナンスラミド分画を50%エタノール(エタノール:水、50:50(v/v))5mlに溶解し、0.45μmフィルターを通して濾過し、HPLCにかけた。試料(10μl)は、Rheodyne(商標)インジェクターを使用して、C18ガードカラムを備えたCERA Column Cooler 250を使用して25℃に維持したC18逆相カラム(ODS Hypersil(商標)C18、5μm、4.6mm×250mm)に注入した。Thermo Separation Products(TSP)Spectra System P4000ポンプを使用してHPLC分析を行い、TSP SpectraSystem(商標)UV3000スペクトルスキャン検出器及びChromQuestソフトウェアを使用して330nmで監視した。流速は1分当たり0.8mlに維持した。HPLC用溶媒は、A:メタノール、B:水及びC:5%酢酸とした。溶媒の勾配(容積%)は、40A:55B:5Cからなり、40分で50A:45B:5Cに直線的に増加し、15分で80A:15B:5Cに直線的に増加し、次いで3分で100Aに達し、3分間維持した。溶媒の勾配は、3分で元の状態に戻し、4分間平衡化した。全ての主要アベナンスラミドのピークを、相対的保持時間及びUVスペクトル(240〜380nmを監視)の真正標準(authentic standards)との比較によって同定した。全ての微量アベナンスラミドは、HPLC−質量分析のみによって同定した。
微量アベナンスラミドのピークを、Surveyor(商標)HPLC-UVダイオードアレイ検出器システムを備えたThermo Finnigan LCQ Advantage(商標)質量分析計を使用して、HPLC−MS−MSによって同定した(HPLC条件:Hypersil(商標)ODS、120Å、5μ、250×4.6mmカラム)。HPLC UVモニタリングは330nmで行った。1分当たり0.8mLの流速で、第1の方法で上記したのと同じ溶媒系を使用した。溶媒の勾配(容積%)は、40A:55B:5Cからなり、80分で60A:35B:5Cに直線的に増加し、5分で80A:15B:5Aに直線的に増加し、次いで3分で100Aに達し、3分間維持した。溶媒の勾配は、3分で元の状態に戻し、3分間平衡化した。MS−MS条件:エレクトロスプレーイオン化(ESI,electro-spray ionization、ネガティブモード)、電源電圧:4.5Kボルト、キャピラリー電圧:−10ボルト、キャピラリー温度:300℃、シースガス流量:80%満、補助ガス流量:20%最大(分割流無し)。
個々のアベナンスラミドは、外部真正アベナンスラミドA標準に対する330nmのピーク面積を決定することによって定量化し、アベナンスラミドA重量当量として表した。総アベナンスラミドは、アベナンスラミドA重量当量として計算した個々のアベナンスラミド量を合計することによって計算し、乾燥重量基準で百万分率(ppm)アベナンスラミドA当量として表した。
新しく収穫した休眠、殻無し、覆い無し(bald)播種エンバク育種系統(VAO-48)の種子試料及び非休眠、殻無し、覆い無し播種エンバク育種系統(VAO-2、品種AC GEHLとして現在登録されている)の種子試料を以下に記載のように製麦した。
休眠、殻無し、覆い無し(VAO-48) 1%
非休眠、殻無し、覆い無し(VAO-22) 75%
休眠エンバクは、貯蔵の条件(例えば、酸素濃度、温度、水分含量)に応じて、時間と共にその二次休眠から覚醒することが知られている。特に、低酸素濃度は、休眠を延長する、及び/又は発芽を阻害する。サブゼロ温度(例えば、−20℃)で貯蔵した種子は、長期間その遺伝的に伝達する形質の大半を保持することができることも、エンバク育種家及び種子胚芽プラズマ保存技術分野の当業者に知られている。しかしながら、製麦などの大規模工程に実用的であるためには、種子を休眠状態に維持するための高価な低温貯蔵に頼らず、休眠エンバクの一定供給が、一年中定期的に利用可能であるべきである。そのため、休眠エンバクの休眠を延長及び増強し、非休眠エンバクの二次休眠を誘発することが好ましい。
二次休眠を増強及び誘発する他の方法が存在する。エンバクの休眠を増強及び誘発する非限定的な代表的な方法は、30〜70℃の乾熱で2週間までの種々の期間エンバクを処理することである。この手順は通常、2段階で行う。第1段階は、約30℃の温度で数日間、種子の水分含量を約3%まで下げることを含み、その後、第2段階は約70℃で1週間までである。このような処理はまた、種子伝染性糸状菌及び細菌胞子を減少させるのに有効であることも示されてきた。この体制下で種子は損傷せず、いくつかの場合には、発芽率が事実上増加する。
伝統的な製麦では、発芽及び後の発酵のための種子のデンプン貯蔵物質の遊離糖への同時の加水分解を最大化するように最適温度が選択される。これらの目的のため、最適温度は通常、約25℃未満である。温度のアベナンスラミド蓄積への影響を研究するため、4種の異なる温度で、暗中で4日間偽製麦を行った。比較のため、散乱光下、室温でも偽製麦を行った。これらの研究のため、加熱前処理及び嫌気的浸麦によって休眠状態にした非休眠、殻無しエンバク品種を使用した。
偽製麦工程中のアベナンスラミド蓄積の時間経過を研究するために、非休眠、殻無しエンバク品種を使用して、種々の期間で工程を行い、蓄積したアベナンスラミド濃度を決定した。
嫌気的浸麦段階中のCa+2イオンの存在が、その後の製麦段階中のアベナンスラミドの蓄積に何らかの影響を有するかどうかを調査するために、浸麦水に一連の増加するCa+2濃度を使用し、製麦後にアベナンスラミド含量を決定した。
非休眠、殻無し、無毛のエンバク系統VAO-22及び浸麦水中でより高濃度のCa+2を実施例7で使用した。
前述のように、偽製麦は、大部分は未製麦穀粒と本質的に同じであり、それゆえ、従来の乾燥摩耗製粉、例えばSatake製粉を通してふすま及び脱ふすま製品への加工に容易に使用可能なエンバク穀粒を生じる。
0〜7(重量)%ふすま分画
7〜15(重量)%ふすま分画
15〜23(重量)%ふすま分画
23(重量)%を超える脱ふすま挽割
0〜10.7(重量)%ふすま分画
10.7〜20.6(重量)%ふすま分画
20.6(重量)%を超える脱ふすま挽割
偽製麦ふすまの外層中のアベナンスラミド濃度は、全粒穀粒中の濃度に対して高いが、このふすまの収率(製麦穀粒の7〜10%から)は低い。穀粒の特徴的な細長い形状及び深く区切られた中央のしわの形態のために、ふすま及び胚乳の明確な分離が限定され、胚乳組織の混入が最小限のふすまの収率は、少ない脱ふすま及び低いふすま分画製粉収率に制限される。しかしながら、殻無し、無毛、非休眠系統VAO-22育種系統は、短い穀粒及び比較的浅いしわを有し、全体形状及び形態がコムギ穀粒に似ており、Satake摩耗製粉にはるかにより適したものになっている。
0〜10.1(重量)%ふすま分画
10.1〜20.1(重量)%ふすま分画
20.1(重量)%を超える脱ふすま挽割
殻無し、無毛エンバク品種を使用する経済的利点にもかかわらず、最終製品中の殻の包含が正当化され得る技術の潜在的用途、例えば、それだけに限定されないが、殻の除去が経済的であると判明していない、飼料用途、非食品用産業用分画用途が存在する。
Claims (19)
- 休眠中の栽培種のエンバク(Avena sativa)を提供するステップと、
前記休眠中の栽培種のエンバクを、製麦(malting)手順を伴うが、発芽はしていないエンバクをもたらす処理である偽製麦(false malting)に供するステップと
を含む、アベナンスラミド濃度が増加した休眠中の栽培種のエンバクを生産する方法。 - 栽培種のエンバクが非休眠性であり、休眠中の栽培種のエンバクを提供するステップが、前記休眠を誘発させるステップをさらに含む、請求項1に記載の方法。
- 栽培種のエンバクが休眠性であり、休眠中の栽培種のエンバクを提供するステップが、前記休眠を増強させるステップをさらに含む、請求項1に記載の方法。
- 休眠を誘発又は増強するために、栽培種のエンバクを嫌気的に浸麦するステップをさらに含む、請求項1〜3のいずれかに記載の方法。
- 偽製麦が、栽培種のエンバクを4〜40℃の温度で96〜120時間インキュベートするステップを含む、請求項1〜4のいずれかに記載の方法。
- 嫌気的浸麦が、栽培種のエンバクを4〜40℃で12〜18時間、水に浸漬するステップを含む、請求項4又は5に記載の方法。
- 嫌気的浸麦が、カルシウムイオンを含む水中である、請求項4〜6のいずれかに記載の方法。
- 休眠を誘発するために偽製麦前に栽培種のエンバクを乾熱処理するステップをさらに含む、請求項1〜7のいずれかに記載の方法。
- 栽培種のエンバクが休眠性の、殻無しの栽培種のエンバクである、請求項1又は3に記載の方法。
- 栽培種のエンバク中の増加したアベナンスラミド濃度が、乾燥量基準で、750ppmより高い、請求項1〜9のいずれかに記載の方法。
- 栽培種のエンバクが30〜40℃で48〜72時間乾熱処理され、その後70℃で144〜168時間さらに乾熱処理される、請求項8〜10のいずれかに記載の方法。
- 栽培種のエンバクが被覆されており、休眠性の栽培種のエンバクであり、前記栽培種のエンバクの殻を除去するステップをさらに含む、請求項1又は3に記載の方法。
- 栽培種のエンバクが、乾燥量基準で3〜10%の最終水分含量まで乾燥される、請求項8〜11のいずれかに記載の方法。
- 栽培種のエンバクが、35%の水分含量まで嫌気的に浸麦される、請求項4〜13のいずれかに記載の方法。
- 請求項1〜14のいずれかに記載の方法に従って生産される栽培種のエンバク。
- 食品製品を製造するための、請求項1〜14のいずれかに記載の方法に従って生産された栽培種のエンバクの使用。
- 外側のふすま成分及び残留脱ふすま挽割を回収するために摩耗製粉を使用して、請求項1〜14のいずれかに記載の方法に従って生産された栽培種のエンバクから得られるエンバクふすま及び脱ふすまエンバク挽割。
- 外側のふすま成分が、摩耗製粉前の栽培種のエンバクの初期重量の3〜30%を含む、請求項17に記載のエンバクふすま及び脱ふすまエンバク挽割。
- 脱ふすま挽割が、摩耗製粉前の栽培種のエンバクの初期重量の97〜70%を含む、請求項17又は18に記載のエンバクふすま及び脱ふすまエンバク挽割。
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US10334869B2 (en) | 2019-07-02 |
WO2010108277A1 (en) | 2010-09-30 |
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AU2010228083B2 (en) | 2014-05-22 |
CN102369289A (zh) | 2012-03-07 |
DK2411527T3 (en) | 2019-01-07 |
AU2010228083A1 (en) | 2011-11-03 |
EP2411527A1 (en) | 2012-02-01 |
US20120082740A1 (en) | 2012-04-05 |
CN102369289B (zh) | 2015-02-04 |
RU2011141719A (ru) | 2013-05-10 |
HK1165833A1 (en) | 2012-10-12 |
EP2411527A4 (en) | 2015-02-11 |
ES2700455T3 (es) | 2019-02-15 |
CA2756554C (en) | 2016-04-26 |
CA2756554A1 (en) | 2010-09-30 |
EP2411527B1 (en) | 2018-11-07 |
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JP2012521746A (ja) | 2012-09-20 |
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