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Analysing peroxidase activity by 3,3'',5,5''-tetramethylbenzidine and its application.
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|摘要:||稻米為台灣重要的作物，新鮮度為消費者購買稻米時一重要的指標，但稻穀會隨著貯藏而逐漸老化。前人研究已指出穀粒酸鹼值與過氧化酶dianisidine 呈色法皆能顯示稻米新鮮程度。本試驗發展出另一套間接推估過氧化酵素活性之檢測法可辨別不同年期與貯藏條件之稻米，亦以穀粒酸鹼值檢測法與過氧化酶dianisidine 呈色法同時進行檢測。以 3,3’,5,5’-tetramethylbenzidine（TMB）做為過氧化酶反應基質，並將糙米或白米置入 TMB 反應溶液中，其呈色產物為藍色，在酸性環境下則轉變為黃色（OD450nm），並使用光電比色之方式量化溶液呈色結果。以 96 well 之血清盤為反應槽一槽置入一粒米，將反應溶液吸出後以全波長免疫分析移讀取吸光值。TMB反應溶液之黃色呈色程度不同顯示其米樣過氧化酶活性亦不同，可應用於判斷不同年期之糙米或白米米樣。此方法具有以 log(OD450 nm)之值表示樣品過氧化酶活性高低，在操作上可以快速、簡便的推估米樣的過氧化酶活性。|
Rice is one of most important staple in Taiwan. Freshness is an important palatability characteristic of rice grains. However, this characteristic deteriorates rapidly during dry storage. Previous study showed that soluble acidity, a colorimeter system to monitor peroxidase activity reacted with dianisidine could be used as a reliable indexes to reflect the freshness of rice grains. In the present study, we develop another indirect method to examine the freshness based on peroxidase activity changes of rice samples harvested from different crop seasons or stored under conditions known to accelerate deterioration. In addition, a statistical procedure to analyze the freshness measurements (e.g. acidity, or peroxidase activity) of a batch of rice grains was tested. Bulk samples of intact brown or polish rice grains were soaked in a 3,3',5,5'-tetramethylbenzidine (TMB) reaction solution, which colored products is blue, and it changes into yellow(OD450 nm) in acid condition, and optical absorbance of incubating solution was determined colorimetrically. Alternatively, intact brown or polish rice grains were incubated in a similar solution in 96-well microplates with one grain per well, and the optical absorbance of incubation solution were measured by a microplate reader. Marked differences in the development of yellow color in incubation solution and in vitro peroxidase activity were also found among the brown or polish rice grains sampled from different season. The changes in the color of the incubation solutions measured by either of the above methods paralleled the changes in grain activities of peroxidase assayed using an in vitro method. The proposed method can differentiate the brown or polish rice grains harvested in different seasons. The advantages of this testing method using log(OD450 nm) over the conventional enzyme activity assay are the rapidity and simplicity with which log(OD450 nm) can estimate the in vitro determination of peroxidease activity of tested rice grains.
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