食品中己二烯酸快速檢測方法之開發

隨著加工食品的問世，引進許多天然及人工的添加物，於生產、加工、儲存和包裝過程中進行添加，確保食品本身的品質安全無虞。它們被應用於食品中以保持風味及改善外觀，或具有某種特定功效，以致多數的食品業者高度仰賴，因此其安全疑慮廣受矚目。加上常有業者不當濫用之情形，也引起消費者的不安。其中，防腐劑是生活中常見的食品添加物，也是近幾年食藥署稽查的重點項目之一，防腐劑中的己二烯酸 (Sorbic acid)及其鹽類，具有取得容易且價格便宜，毒性較低，其鹽類易溶於水及用途廣泛等優勢，經常造成使用不當或過量添加的情形。食用過量時會引起嘔吐、腹瀉或危害人體肝腎代謝功能。目前主要用以檢測防腐劑的方法是高效液相層析法 (HPLC)，而儀器分析的步驟繁瑣，成本高昂且須透過專業人力，因此開發可靠便捷的初篩方法，作為在第一線抽查時，達到快速篩檢之目的，具有相當潛在的必要性。為有效檢測食品中己二烯酸之含量，本研究開發基於比色定量法結合微流體紙基分析裝置的方法，利用添加氧化劑之方式使己二烯酸生成丙二醛 (Malondialdehyde)，並與硫巴比妥酸進行呈色反應，最終產生鮮明的紅色複合物作為應用依據，達到定量己二烯酸的含量。並進行比較三種氧化劑的應用優劣，分別為硫酸鐵銨〔NH4Fe(SO4)2，Ammonium iron(III) sulfate〕、硝酸鐵〔Fe(NO3)3，Iron(III) nitrate〕及重鉻酸鉀〔K2Cr2O7，Potassium dichromate〕。其中氧化劑的不同是影響呈色的主要因素，故最後選定可穩定呈色於微流體紙基晶片上的重鉻酸鉀氧化劑，並以色階 (RGB levels)分析軟體進行顏色強度分析，探討其呈色結果的最適化條件及線性關係。結果顯示，此分析法在灰階條件下，可有效定量不同濃度 (500-3000 mg/kg)之己二烯酸標準檢液，求得線性迴歸方程式為y (吸光值) = -0.0108x (濃度) + 206.57，R2=0.9955，定量極限為500 mg/kg；後續則以重鉻酸鉀進行分光光度法的探討，利用分光光度計進行全波長掃描，結果得知最大吸收波長為530 nm，並在此波長進行己二烯酸的定量分析，求得線性迴歸方程式為y (吸光值) = 0.0022 x (50-300 mg/kg) - 0.087，R2=0.9968。進一步探討常見物質及其他酸類防腐劑存在下，反應是否具有專一性，以及上述成分的共伴呈色干擾試驗，結果在專一性試驗當中發現，常見物質及其他酸類防腐劑皆不與硫巴比妥酸結合產生呈色反應，吸光值皆小於0.06；在共伴呈色試驗中，在上述物質與己二烯酸共存下，吸光值相對誤差百分比皆小於5%，證明硫巴比妥酸在此檢測方法中具有專一性，在定量己二烯酸含量時，亦不容易受其它物質影響。最後將本研究所開發之微流體紙基晶片分析法，應用於含有己二烯酸之真實食品中進行含量測定，檢測市售碳酸飲料、果醬及糕餅，其所得結果與認證實驗室測得結果比較，測定結果的相符度達八成，回收率為101%-105%，已能達到半定量之效果。經以上研究結果得知，己二烯酸與硫巴比妥酸試劑呈色反應專一且穩定，而紙基晶片的製程及分析上也達快速便捷、成本低廉、便攜等優勢，可成為廣泛應用發展的分析型工具，在食品分析領域潛在良好的應用性。

關鍵字

己二烯酸；硫巴比妥酸；丙二醛；重鉻酸鉀；微流體紙基裝置

並列摘要

With the advent of processed foods, more additives have been introduced, of both natural and artificial origins. These additions can be made during production, processing, storage, and packaging, to ensure the quality and safety of the food itself. They are added to food to preserve flavor or improve its taste and appearance, or modify certain function. As a result, most food manufacturers could easily become highly dependent on these additives, thus their safety concerns have attracted attention, and the cases of misuse (abuse) also caused consumer anxiety. Preservatives have been widely used among food additives, and also a major compound inspected by Taiwan Food and Drug Administration (TFDA) in recent years. Sorbic acid and its salts in preservatives are easy to obtain and inexpensive, with low toxicity, and their salts are easily soluble in water, so have a wide range of uses. They often cause improper use or excessive additions, and excessive consumption may cause vomiting, diarrhea or endanger the liver and kidney metabolic functions. At present, the primary method used to analyze preservatives is high-performance liquid chromatography (HPLC), and the steps of instrumental analysis are time-consuming, costly, and require professional manpower. Therefore, reliable and convenient preliminary screening methods are developed as a first-line spot check. In order to achieve the purpose of rapid screening, it is potentially necessary to develop those techniques. In order to effectively detect the content of sorbic acid in foods, this study developed a method based on colorimetric quantitative method combined with a microfluidic paper-based analysis device, using an oxidizing agent to convert sorbic acid to malondialdehyde, and subquencially reacts with thiobarbituric acid for colorimetry. The reaction will eventually produce a bright red complex compound as the basis for application, to achieve a quantitative determination of sorbic acid. And compare the application advantages and disadvantages of the three oxidants: ammonium iron (III) sulfate [NH4Fe (SO4)2], iron (III) nitrate [Fe(NO3)3], and potassium dichromate [K2Cr2O7]. Among them, the difference of oxidants is the main factor affecting the color rendering. Therefore, the potassium dichromate oxidizing agent that can stabilize the color rendering on the microfluidic paper-based device is finally selected, and the color intensity analysis is carried out with RGB levels analysis software to explore the optimal conditions and linear relationship of color rendering results. The results show that this analysis method can effectively quantify the standard test solution of sorbic acid at different concentrations (500-3000 mg/kg) under gray-scale conditions. The linear regression equation is y (absorbance value) = -0.0108x (concentration) + 206.57, R2 = 0.9955, the limit of quantification is 500 mg/kg. The spectrophotometric method was applied with potassium dichromate, and the full wavelength scan was carried out with a spectrophotometer. As a result, the maximum absorption wavelength occurred at 530 nm, and quantitative analysis of sorbic acid was performed at this wavelength. The linear regression equation was y (absorbance value) = 0.0022 x (50-300 mg/kg)-0.087, R2 = 0.9968. Further exploring whether the reaction is specific in the presence of common food ingredients and other acid preservatives, as well as the color interference (synergistic effects) by the above ingredients. The result in the specific test showed that common food ingredients and other acid preservatives did not react with thiobarbituric acid to produce a color reaction, while the absorbance values were all less than 0.06; in the synergistic effect test, in the presence of above compounds with sorbic acid, the relative error of the absorbance value is less than 5%, which proves that thiobarbituric acid has specificity in this detection method, and is not easily affected by others when quantifying the content of sorbic acid in foods. Finally, the microfluidic paper-based analysis method developed by this research was applied to the determination of sorbic acid content in real food, samples include commercially available carbonated drinks, jams and pastrys. The results obtained are compared with the results measured by certified laboratory, which showed the consistency of the measurement results is more than 80%, and the recovery rate is 101%-105%, being able to achieve a semi-quantitative outcome. According to the above research results, the color reaction of sorbic acid and thiobarbituric acid reagent is specific and stable, and the process and analysis of paper-based device are also fast, convenient, low-cost, and portable. It has become an analytical tool for widespread application and development. Potentially wide applicability in the field of food analysis could be expected.