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食品中のアクリルアミド迅速定量法の開発と低減化に向けた減少機構の解析

吉岡, 俊暁 YOSHIOKA, Toshiaki ヨシオカ, トシアキ 九州大学

2020.03.23

概要

第一章 緒論
アクリルアミド (AA) は,反応性の高いアクリル基を持つ揮発性の高極性化合物である.AA およびその酸化物であるグリシダミドは,生体内で神経系タンパク質あるいは DNA への結合によって毒性を示すことが報告されており,神経毒性を持つ遺伝毒性発癌物質と考えられている.2002年にスウェーデンの研究グループが,様々な加熱食品に AA が含まれていることを発表し世界中で大きな話題となった.食品中の AA は,アスパラギンと還元糖を含む食品の加熱中にメイラード反応副産物として生成すると考えられている.遺伝毒性発癌物質は毒性発現の閾値が設定できないため,食品中の AA はできるだけ低減することが求められる.EU では AA 低減のため各食品に目標値が設定されており,食品事業者が AA 含量の観測および低減化に向けた取り組みを行っている.日本では農林水産省が食品中の AA 低減の指針を示しており,食品事業者が低減化に向けた研究を行っている.また,食材や調理法は国ごとに異なるため,様々な食品の AA 量の把握は重要である.これらの理由から,現在数百検体におよぶ食品サンプルの AA の実態調査や低減研究に活用可能な簡便,迅速な AA 定量分析法が求められている.また,食品中の AA 低減法として,1) 農学的手法,2) 生物学的手法,3) 物理学的手法,4) 化学的手法,5) 食品中の成分との反応による AA 低減法が考案されている.特に,揚げ物・焼き物の AA 低減は,3) 物理学的手法である加熱温度・加熱時間の最適化,あるいは 4) 化学的手法である食品添加物の使用によって一定の効果が得られている.食品の中でもコーヒー飲料は AA の摂取量に対する寄与率が高いため,コーヒー飲料の AA 低減は重要な課題である.しかし,コーヒー飲料では,原料生豆の焙煎工程時に AA が生成しており,工程上加熱の最適化や食品添加物の使用は難しい.一方,コーヒー飲料保存中に何らかの物質との付加反応による AA の減少が示唆されているが,その全貌は明らかになっていない.コーヒー飲料中のAA との反応物質が特定できれば,コーヒー飲料での AA 低減の戦略を立案できる可能性がある.

本研究では,食品中の AA 迅速高感度定量分析法の開発およびコーヒー飲料中の AA 減衰機構を解明することで AA 低減化に向けた新たな戦略を立案することを目的とした.

第二章 SFC/MS/MS を用いた AA の高感度迅速定量法の開発
近年の AA の定量分析は,主に超高速液体クロマトグラフィー/ タンデム質量分析 (UHPLC/MS/MS) によって実施されてきた.しかし,UHPLC/MS/MS を用いて食品中の低濃度の AA (5 ng g‒1 以下) を検出・定量するためには,煩雑な前処理が必要であり,感度およびスループットの点で課題が残っていた.そこで,様々な食品中の AA 含量を迅速かつ高感度に観測する手法を開発するために,超臨界流体クロマトグラフィー/タンデム質量分析 (SFC/MS/MS) に着目した.

SFC の移動相に用いられる超臨界二酸化炭素は,低粘性・高拡散性という理想的な移動相特性から, UHPLC と比較して高流速領域でも高い分離効率を示す場合がある.また,SFC をエレクトロスプレーイオン化質量分析に接続することでUHPLC を接続する場合と比べて高感度に検出できる事例がいくつか報告されている.そこで,本章では,AA の高感度定量分析および迅速前処理を組み合わせた新規の AA 迅速高感度定量分析法を開発するために,①SFC/MS/MS による AA の高感度検出,②マトリックス効果低減による AA の感度改善,③抽出液濃縮時の AA の揮発防止による感度向上,④サンプル前処理工程の迅速化,および⑤抽出液の異物除去を行うメンブレンフィルターからの AA 溶出防止による定量精度向上,という 5 つの課題解決に取り組んだ.まず,SFC/MS/MSを用いて,AA の分離に最適なカラムの選定および各種分析条件の検討を行った.最適化した SFC/MS/MS 分析法と従来の UHPLC/MS/MS 法の AA 分析における感度を同一の質量分析計を用いて比較した結果,開発した SFC/MS/MS 分析法は従来法と比べて約 10 倍の高感度化を達成した.続いて,当該 SFC/MS/MS 分析法に適した迅速前処理法の検討を行った.具体的には,従来の AA測定のための簡易前処理法である QuEChERS 法をさらに簡略化させることで,迅速な前処理法の開発に取り組んだ.QuEChERS 法で得られた抽出液に対して精製・濃縮操作を伴わずに簡便な希釈および遠心操作のみで前処理を行う手法とした.その結果,①-⑤の全ての課題を解決し,特に AA 含有量の少ない飲料類においても AA の定量が可能であった.また,本分析法による 1 サンプルあたりの分析所要時間は約 35 分であり,LC 法 (75 分) と比べて処理時間を大幅に短縮できた.さらに,本分析法は,希釈や遠心分離といった短時間での同時処理が実施可能な手段を用いるため,多検体分析を必要とする AA の実態調査,低減研究に対して非常に有効な手法になると考えられる.

第三章 コーヒー飲料中の AA 減少機構の解析
コーヒー飲料中の AA は保存期間中に何らかの物質と反応することで減少していると示唆されているが,その詳細は不明である.そこで,本章では安定同位体標識および超高速液体クロマトグラフィー/高分解能質量分析 (UHPLC/HRMS/MS) で得られた精密質量データをもとに AA 付加体の探索を行った.UHPLC/HRMS/MS を用いて,AA と 13C3-AA を添加したミルクコーヒー抽出液のノンターゲット分析を行った.続いて,ミルクコーヒー中に含まれる 5000 種以上の化合物ピークの中から,AA と 13C3-AA の精密質量差 (Δm/z = 3.0102) および保持時間の一致度をもとに AA 付加体候補化合物の絞込みを行った.最終的に,得られた AA 付加体候補化合物の標準品を合成し比較することで,AA 付加体の構造決定を行った.その結果,ミルクコーヒー液中の 3HP と Py,およびミルクコーヒータンパク質中の Lys と Cys の 4 種が,AA 付加体を形成していることが明らかとなった.また,ミルクコーヒー保存期間中の AA 減少量に対する AA 付加体の合計生成率は 75.3% (3HP-AA, 5.1%; Py-AA, 0.6%; Lys-AA, 51.7%; Cys-AA, 17.9%) であり,ミルクコーヒー中で AA が減少する要因の大部分を突き止めた.特にタンパク質中のLys とCys がAA 低減に寄与しており,内在性のタンパク質と AA の反応はコーヒー飲料の新しい AA 低減法となりうる.また,タンパク質は様々な食品に存在するため,コーヒー飲料以外の食品に対してもその適用が期待できる.

第四章 総括
本研究では,様々な食品中の AA を迅速かつ高感度に定量できる SFC/MS/MS 分析法を開発した.当該分析法は,多検体分析を必要とする AA の実態調査や低減研究に非常に有効である.また,ミルクコーヒー保存中のAA の減少は 4 種のAA 付加体の生成が原因であり,その合計生成量は 75.3%であった.特に,タンパク質中の Lys と Cys が AA 減少に大きく寄与しており,タンパク質と AAとの反応性を利用したコーヒー飲料の新しい AA 低減法に応用できる可能性が期待できる.

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