摘要:食品過敏是食安重要議題之一,食品過敏反應可能引發消化道發炎、皮膚起疹紅腫,或呼吸不適等症狀,嚴重者甚至全身性過敏休克而危害健康。在法規上,世界各國多要求詳盡標示食品中的過敏原,因此也需要建立分析食品中過敏原的技術,支持食品過敏原的檢驗及有效管理。目前免疫化學分析方法在分析食品過敏原的應用最廣,但是常有食品基質干擾、偽陰性、偽陽性、定量精確性不足、偵測對象受限、無法鑑定待測物等缺點。以質譜儀為基礎的分析方法,具有可用於鑑定、高專一性、高靈敏性、高通量等優勢,然而以質譜方法分析過敏原這類蛋白高分子成份,在鑑定及定量上較分析小分子化合物困難,目前仍面臨樣品前處理方法困難、不易取得同位素標定之內標準品、質譜參數複雜等技術障礙,尚待開發解決方案。
本計畫之目標是建立低成本高準確之食品過敏原分析平台,擬以植物性食品為對象,解決蛋白質含量低、細胞破碎困難、具較多干擾物質等樣品製備上等等問題,另一方面,二甲基穩定同位素標記法,為近年胜肽標記常用之方法,具有標記時間短、無副反應、高標記效率、成本低廉等無可取代之優點,我們將藉此技術,開發低成本的同位素標定內標準品製備程序,同時配合三重四極矩質譜儀,發展高準確性的過敏原鑑定及絕對定量方法。實驗上,第一年度擬以黃豆 (Glycine max) 作為代表作物,建立種子類食品過敏原分析平台;第二年度擬以奇異果 (Actinidia deliciosa) 作為代表作物,建立蔬果類食品過敏原分析平台;第三年度擬以牛肝菌 (Boletus edulis) 作為代表作物,建立厚壁植物及真菌類食品過敏原分析平台。此計畫與分析平台的完成,預期能夠創新及建立食品過敏原之分析模式與範本,此分析技術並將能方便應用於其他牛乳、漁產、甲殼類、禽蛋等動物性過敏原,以及食品中其他蛋白質高分子成份之定量分析,加強我國食品安全管理,並有助於提升全國人民之食品安全。
Abstract: Food allergy, one of the major issues in food safety management, which could affect health by eliciting localized sensitization, and anaphylaxis shock in some severe cases. In most countries, authorities demand food allergen labeling. In order to assist authorities inspect and manage food allergen, development of technique for analyzing food allergen is needed. Currently immunochemistry-based methods are widely applied in food allergen determination. However, they possess some disadvantages such as matrix interference, false-negative result, false-positive result, insufficient precision, limited target allergen, and inability to identify target. In contrast, mass spectrometry-based methods possess advantages like ability to identify target, high specificity, high sensitivity, and high throughput; however, mass spectrometry-based methods face challenges like difficulty in sample preparation, difficulty in obtaining internal standard and lack of cost efficient labeling method when analyzing high molecular weight compounds, such as protein.
In this project, our goal is to establish a cost-efficient and high accurate platform for food allergen analysis, specifically for plant-food allergen, which is difficult to prepare sample due to low protein content, difficulty in cell disruption and high interfering content. On the other hand, stable isotope dimethyl labeling method is a popular method for labeling peptides in recent year, which has unreplaceable advantages like short labeling time, no side reaction, high labeling efficiency, and low cost. We plan to apply this technique in the development of a low cost internal standard preparation procedure, and combine it with the usage of triple quadrupole mass spectrometry to establish a cost-efficient and high accurate allergen identification and absolute quantification method. In the first year, we plan to use soybean (Glycine max) as representative to establish allergen analysis platform for seeds crops. In the second year, we plan to use kiwifruit (Actinidia deliciosa) as representative to establish allergen analysis platform for vegetables and fruits. In the third year, we plan to use king bolete (Boletus edulis) as representative to establish allergen analysis platform for pachydermatous plants and fungus. With the completion of this project, we will establish pattern and example of plant-food allergen analysis platform, which can be more easily applied to quantification analysis of animal allergen such as milk, seafood, crustaceans, eggs or other high molecular weight protein content in food to enhance management of food safety in our country and to help improve food safety for the whole nation.