- 學位論文
以傅立葉轉換紅外光光譜檢測白蝦中過敏原之方法建立
Establishment of a method for the detection of allergens in whiteleg shrimp by Fourier transform infrared spectroscopy
摘要
白蝦 (Litopenaeus vannamei) 屬於甲殼類動物 (Crustacea) 之一，其富含蛋白質、不飽和脂肪酸、維生素及礦物質等，通常被認為是營養的良好來源。然而於國內外甲殼類被列為食物重要過敏原之一，會引發人體一連串過敏反應，包含蕁麻疹、氣喘、腹瀉等，嚴重的話會導致休克甚至死亡。白蝦中主要過敏原為原肌球蛋白 (Tropomyosin, TM) 以及精胺酸激酶 (Arginine kinase, AK)，前者具熱穩定性，後者為蝦中重要生化反應酵素之一。目前，有許多檢測過敏原之方法，像是針對過敏原蛋白之免疫測定法，如酵素連結免疫吸附分析法 (Enzyme-linked immunosorbent assay, ELISA)，或是針對過敏原DNA片段之核酸檢測法，如聚合酶連鎖反應 (Polymerase chain reaction, PCR)，上述過敏原檢測法都相對耗時且成本高，亦造成誤判結果，然而，傅立葉轉換紅外光光譜法 (Fourier transform infrared spectroscopy, FTIR) 之優點包含分析速度快且樣品製備簡單，常與化學計量學結合使用來優化分析結果，因此，本研究目的為建立傅立葉轉換紅外光光譜快速檢測白蝦中兩種過敏原之定量方法。本研究先以純化TM與AK為目標，分別利用不同的蛋白質分離方法進行純化，TM以等電點沉澱、硫酸銨沉澱以及加熱沉澱方式，而AK以硫酸銨沉澱與離子交換層析方法，接著經超過濾濃縮，再以十二烷基硫酸鈉聚丙烯醯胺凝膠電泳 (Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, SDS-PAGE) 與西方墨點法進行確認，最後凍乾製成粉末並低溫保存備用。接著將粉末於0-10000 ppm之範圍內，配製成101個濃度之標準溶液，並利用衰減式全反射 (Attenuated total reflectance, ATR) 搭配FTIR於4000-650 cm-1下，條件設定以解析度4 cm-1掃描64次，所收集到的光譜圖結合化學計量學中的偏最小平方迴歸 (Partial least-squares regression, PLSR) 來建立與評估定量模型。根據結果顯示，TM與AK之最適定量模型條件皆於1800-1000 cm-1波數範圍下之原始圖譜，兩者之驗證組之均方根誤差與校正組交叉驗證之均方根誤差相對低，且兩組驗證之R2值都接近於1，也可應用於市售蝦類產品中過敏原之定量分析。
並列摘要
Whiteleg shrimp (Litopenaeus vannamei) is one of the crustaceans (Crustacea). It is rich in protein, unsaturated fatty acids, vitamins, and minerals, etc., and is generally considered to be a good source of nutrition. However, crustaceans are listed as one of the important food allergens at home and abroad, which can trigger a series of allergic reactions in the human body, including urticaria, asthma, diarrhea, etc., and severe cases can lead to shock or even death. The main allergens in whiteleg shrimp are Tropomyosin (TM) and Arginine kinase (AK), the former is heat-stable, and the latter is one of the important biochemical reaction enzymes in shrimp. At present, there are many methods for detecting allergens, such as immunoassays for allergen proteins, such as enzyme-linked immunosorbent assay (ELISA), or nucleic acid detection methods for allergen DNA fragments, such as polymerase chain reaction (PCR), the above allergen detection methods are relatively time-consuming and costly, and also cause misjudged results. However, the advantages of Fourier transform infrared spectroscopy (FTIR) include the analysis speed is fast and sample preparation is simple, and it is often used in combination with chemometrics to optimize the analysis results. Therefore, the purpose of this study is to establish a quantitative method for the rapid detection of two allergens in whiteleg shrimp by Fourier transform infrared spectroscopy. In this study, the purification of TM and AK was first carried out by using different protein separation methods. TM was purified by isoelectric point precipitation, ammonium sulfate precipitation, and heating precipitation, while AK was purified by ammonium sulfate precipitation and ion exchange chromatography. Concentrated by ultrafiltration, then confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot, and finally freeze-dried to make powder and stored at low temperature spare. Then the powder was prepared into standard solutions with 101 concentrations in the range of 0-10000 ppm, and the attenuated total reflectance (ATR) was combined with FTIR at 4000-650 cm-1, and the conditions were set to analyze. The collected spectra were scanned 64 times at a resolution of 4 cm-1 and combined with partial least-squares regression (PLSR) in chemometrics to establish and evaluate quantitative models. According to the results, the most suitable quantitative model conditions of TM and AK are the original spectra in the wavenumber range of 1800-1000 cm-1. The root mean square error of the validation group, and the root mean square error of the cross-validation group of the calibration group are relatively low. , and the R2 values of the two sets of verification are close to 1. It can also be applied to the quantitative analysis of allergens in commercially available shrimp products.