利用分子內螢光能量轉移現象選擇性檢測色胺酸並應用於醬油品質之評估

Abstract

本研究開發一套選擇性檢測色胺酸 (tryptophan) 含量的螢光流動注射分析系統。檢測原理利用色胺酸與鄰苯二甲醛 (o-phthalaldehyde, OPA) 結合後，在280 nm激發時能產生分子內螢光共振能量轉移的特性，使吸收波長向右偏移至445 nm，有別於其他的胺基酸其吸收波長約落於340 nm處，因此即能選擇性地量測胺基酸溶液中的色胺酸含量。由於胺基與鄰苯二甲醛結合後所形成的異吲哚官能基，在280 nm與300 nm激發 (445nm接收) 時，會有微量且近乎相同強度的螢光生成反應，故可將280 nm與300 nm激發的螢光強度相減，即可得到僅因螢光共振能量轉移的螢光量值，以其定量色胺酸的含量。研究中，搭配流動注射分析系統，以提高螢光生成反應的再現性與精準度。樣品注射後，反應時間約5分鐘。系統檢測範圍為0.01 - 10 μM，檢測極限為10 nM。檢量線之相關係數＞0.9893，CV值於檢測濃度大於0.5 uM時小於6.5% (n=3)。 在醬油製造過程中，若以非傳統釀造的酸水解方式分解原料的蛋白質，色胺酸極容易會被氧化，藉由色胺酸於醬油產品中的含量，即可分辨純釀造醬油、混和醬油與化學醬油。此外，為了避免一些特殊醬油，如加高湯稀釋的和風醬油，被視為非純釀造醬油，以色胺酸與總胺基酸態氮量的比值作為評估醬油品質的標準。最後再以毛細管電泳法驗證本研究的螢光流動注射分析系統所檢測的醬油品質結果。

Tryptophan was detected with a fluorometric flow-injection manifold after reacting with mM order of fluorogenic o-phthalaldehyde (OPA) / thiol reagent (pH 10.0) in the carrier stream (0.63 mL/min). Based on the intra-molecular fluorescence resonance energy transfer (FRET) between the indole moiety of tryptophan and the isoindole moiety derivatized by reacting with OPA, the difference in fluorescence intensity obtained at 280 nm and 300 nm excitation was used to detect tryptophan content with satisfactory precision (CV < 6.5% for concentration higher than 0.5 μM), linearity (0.01 - 10 μM, R2 = 0.9893) and sensitivity. Since tryptophan will be oxidized during manufacturing non-fermented soy sauce by acid-hydrolysis procedure, the above method was used to discriminate purely fermented soy sauces from adulterated and chemically-treated soy sauces in less than 5 min. The ratio of tryptophan to total amino acid content served as the index for judgment, and the results were validated by capillary electrophoresis.