食品中過敏原牛乳β-乳球蛋白之質譜檢驗方法   全文下載

Mass Spectrometry Method of β-Lactoglobulin from Milk in Foods

食品過敏原是指會引起人體過敏反應的蛋白質，為加強食品過敏原的標示資訊，參考國際規範及國人食品過敏之臨床調查資料，訂定「食品過敏原標示規定」，其中牛奶為強制標示項目之一。常見的牛奶過敏原蛋白檢測方式有兩種，一為核酸分析方法，惟僅能針對牛之物種做檢測；二為酵素連結免疫吸附法（Enzyme-linked Immunosorbent Assay, ELISA）檢測套組，然對於複雜基質或高度加工的市售產品容易有偽陰性或偽陽性的結果。為能提高檢測食品過敏原蛋白正確性，本研究利用液相層析串聯質譜儀的高準確、高靈敏及高穩定性等優點開發食品中牛乳過敏原的定性方法。市售牛乳經萃取及淨化後，以液相層析串聯四極桿飛行時間式質譜儀（Liquid Chromatograph-Quadrupole Time-of-Flight Mass Spectrometer, LC/Q-TOF MS）進行總離子層析分析（Total ion chromatogram），找出具有專一性的過敏原「牛乳β-乳球蛋白」胜肽，續以液相層析串聯四極桿質譜儀（QTRAP Liquid Chromatograph-Mass Spectrometer, QTRAP LC/MS/MS）進行方法確效試驗及市售產品方法適用性評估。結果顯示選定專一胜肽離子對之滯留時間（Retention time）差小於0.1分鐘；離子對訊雜比（Signal/noise ratio）大於10；離子比（Ion ratio）±30%內；同日間變異係數小於8.50%；異日間變異係數小於12.42%，皆符合確效規範，偵測極限為2 ppm。產品適用性評估結果顯示17件市售產品皆準確檢出是否含有牛乳β-乳球蛋白。因此本研究開發的牛乳β-乳球蛋白之質譜定性分析方法可運用於市售產品之檢測，為國人健康把關。

Food allergens refer to proteins that trigger human allergic reactions. Regulations Governing Food Allergen Labeling, referencing international standards and clinical survey data on food allergies among the populace, was established to strengthen the labeling information of food allergens. Milk is one of the mandatory labeling items. Currently, there are two common methods for detecting milk allergenic proteins. One is nucleic acid analysis, which is only applicable to the detection of bovine species. The other is Enzyme-linked Immunosorbent Assay (ELISA) detection kits, but they may produce false negative or false positive results in complex matrices or highly processed commercial products. In order to increase the accuracy of allergenic proteins detection, this study developed a qualitative method for detecting cow’s milk allergens by using liquid chromatography-tandem mass spectrometry. After extraction, cow’s milk underwent full spectrum analysis using liquid chromatograph-quadrupole time-of-flight mass spectrometer (LC/Q-TOF MS) to identify the specific allergenic peptide,“β-lactoglobulin.”Further method validation and applicability evaluation for commercial products were performed by QTRAP Liquid Chromatograph-Mass Spectrometer (QTRAP LC/MS/MS). The results demonstrated that the selected specific peptide ion pairs exhibited a retention time difference less than 0.1 minute, a signal-to- noise ratio greater than 10, ion ratios within±30%, intra-day variability with a coefficient of variation less than 8.50%, and inter-day variability with a coefficient of variation less than 12.42%. All of these data meet validation criteria with a detection limit of 2 ppm. Results of product applicability assessment showed accurate detection ofβ-lactoglobulin in 17 commercially available products. Therefore, the mass spectrometry based qualitative analysis method forβ-lactoglobulin detection developed in this study can be used to the detection of commercially available products to maintain food safety.