- 學位論文
食品中苯甲酸鈉紙基快速檢測試片之開發
Development of paper-based rapid detection method of Sodium Benzoate in food
摘要
食品防腐劑是一類食品添加劑，旨在阻止食品腐敗和變質，從而延長其保存期限。苯甲酸(Benzoic Acid)有抑制真菌、細菌、黴菌生長的作用，抑菌性能最強且價格低廉，為食品加工業者最喜用的防腐劑之一。苯甲酸對微生物有強烈的毒性，但其鈉鹽的毒性則相對較低，安全的每日最大攝取量為每公斤體重每日5毫克以下。苯甲酸及其鹽類被廣泛應用於乾酪、乳酪、奶油、脫水水果、蘿蔔乾、醬油；或是魚貝類乾製品、豆皮豆乾與醃漬蔬菜等，國內容許用量限制在0.6-1.0 g/kg以下。 目前檢測食品中苯甲酸濃度的方法主要為高效能液相層析法(High performance liquid chromatograph, HPLC)，然而因成本較高、樣品製備步驟耗時及操作複雜，不利於市售產品的快速篩檢，導致對於更快速及更經濟的初篩方法之需求日益增加。本研究以Janovsky Reaction之原理，開發出一套簡易、方便且完善的快速檢測系統。苯甲酸經硝化後衍生成3,5-二硝基苯甲酸(3,5-Dinitrobenzoic Acid)，以異丁醇萃取後取1毫升於試管，加入2毫升丙酮及1毫升0.1 N氫氧化鈉溶液便能產生具有紫色之Janovsky錯化合物，首先利用分光光度法進行定量，發現在波長555 nm處有最大吸光值，依不同濃度(0-5000 ppm)可得到迴歸方程式y (吸光值) = 0.0002 x (濃度) + 0.0145, R2 = 0.9995，檢測之定量極限(LOQ)估計可達50ppm。將此呈色方法進行食品成分及常用防腐劑之專一性測定及共伴吸光反應，結果皆發現無明顯干擾。 利用化學反應呈色的檢測方法，因其操作簡單、價格低廉、消耗微量試劑及樣品、便於攜帶等優點，紙基分析方法成為近年來蓬勃發展的分析工具。為求更方便、快速地檢測食品中之苯甲酸鈉，本研究利用分光光度法之原理開發紙基分析檢測試片 (Microfluidic paper-based analytical devices, μPADs)。利用Janovsky Reaction之原理，在濾紙的親水反應區中滴入2.4 µL 5N氫氧化鈉溶液、1.2 µL丙酮及1.8 µL樣品溶液，將濾紙移入烘箱以120℃烘烤60秒使其反應呈現黃色，以桌上型多功能印表機掃描，再使用Photoshop軟體讀取紅色 (R)、綠色 (G)、藍色 (B) 值，即可快速檢測苯甲酸鈉之含量。結果顯示，R+G+B值依不同濃度(0-5000 ppm)可得到迴歸方程式y (R+G+B值) = -0.029 x (濃度) + 736.5, R² = 0.9959，檢測之定量極限(LOQ)估計可達500ppm。將此呈色方法進行食品成分及常用防腐劑之專一性測定及共伴吸光反應，結果同樣發現無明顯干擾。 以本研究所開發之分光光度法快速檢測系統及紙基檢測試片，應用於真實食品樣品中苯甲酸鈉含量檢測，以外部添加法分別添加100, 250, 500, 1000, 2000 ppm的苯甲酸鈉於醃嫩薑中，對於分光光度法其與農水產品檢驗中心相符度可達83-100%，回收率可達82-100%，對於紙基檢測試片之相符度可達83-99%，500ppm以上回收率可達82-94%。由於其便利性，快速性和低成本，紙基檢測試片已被證明是具潛力的工具對於快速篩選食品中苯甲酸鈉。爾後可望發展更好地分析能力，使消費者和衛生單位受益。
並列摘要
Food preservatives are a group of food additives aiming to detent food spoilage and deterioration, and hence to extent its shelf life. Benzoic Acid inhibits the growth of fungi, bacteria and molds. Due to its strongest antibacterial property and low price, Benzoic Acid and its salts are the most frequently abused preservatives in Taiwan. It has a strong toxicity to microorganisms, but the toxicity of its sodium salt is relatively low, and the acceptable daily intake (ADI) is below 5 mg / kg of body weight per day. They are widely used in cheese, cream, butter, dehydrated fruit, dried radish, soy sauce; dried fish and shellfish products, dried bean curd, and pickled vegetables, etc., the domestic allowable doses are limited to 0.6-1.0 g / kg or less (as acid). At present, the method for detecting the concentrations of Benzoic Acid in foods is mainly by High Performance Liquid Chromatograph (HPLC). However, due to its high cost, time-consuming sample preparation, and complicated operation, it is disadvantageous in fast screening for market products. The need for initial fast screening method is gradually increasing. This study developed a simple, convenient, and complete rapid detection system based on Janovsky Reaction. Benzoic Acid is derivatized into 3,5-Dinitrobenzoic Acid after nitration. Then extract by the Isobutanol and take 1 mL to a test tube. Adding 2 mL Acetone and 1 mL 0.1 N NaOH solution can produce Janovsky compound with purple color with maximum absorbance occuured at 555 nm, and then use spectrophotometry to quantify. The regression equation for different concentrations (0-5000 ppm) is y (absorbance) = 0.0002 x (in ppm) + 0.0145, R2 = 0.9995. The Limit of Quantitation (LOQ) of the assay is estimated to be up to 50 ppm. The specificity as well as synergy (co-effect) test, by food compositions and other preservatives, found no significant interference on Sodium Benzoate Janovsky reaction. With its low price, low consumption of chemical reagents and sample, and easy portability, the paper-based analysis method has become a booming analytical tool in recent years. In order for more convenient and quick detection of Sodium Benzoate in foods, the principle of spectrophotometry was used to develop Microfluidic paper-based analytical devices (μPADs). In this study, 2.4 µL 5N Sodium Hydroxide solution, 1.2 µL Acetone, and 1.8 µL sample solution were dropped into the hydrophilic reaction zone of the filter paper. The filter paper was then transfered to an oven and baked at 120° C for 60 seconds to produce the yellow color. Scanned by the desktop multi-function laser printer, and the red (R), green (G), and blue (B) values are read by Photoshop software to quickly detect Sodium Benzoate. The regression equation for different concentrations (0-5000 ppm) is y (R+G+B value) = -0.029 x (in ppm) + 736.5, R2 = 0.9959. The Limit of Quantitation (LOQ) of the assay is estimated to be up to 500 ppm. The specificity as well as synergy (co-effect) test, by food compositions and other preservatives, found no significant interference on this developed μPADs for the determination of Sodium Benzoate in foods. The spectrophotometric rapid detection system and μPADs developed in this study were applied to the detection of Sodium Benzoate in real food samples. 250, 500, 1000, and 2000 ppm of Sodium Benzoate were added to pickled ginger by external addition. For spectroscopic test method developed in this study, it’s found that conformance range between 83-100%. The recovery of external addition achieved 82-100%. For paper-based method, it’s found that conformance range between 83-99%. The recovery of external addition achieved 82-94% above 500 ppm. Due to its convenience, rapidity, and low cost, µPADs has been proven to be a potentially capable tool for rapid screening of Sodium Benzoate in foods. More efforts are needed to stretch this exploration for better analyzing capability, to benefit consumers as well as the Authority.