食品中軟體動物免疫測定法之開發

• Main Authors: 蔡佳霖, TSAI, CHIA-LIN
• Other Authors: 食品安全碩士學位學程, 陳奕廷
• Format: Thesis
• Language: Chinese
• Published: 2021
• Subjects: 食品過敏;軟體動物;熱穩定蛋白質;免疫測定法; food allergy;mollusk;thermal stable protein;immunoassay; Crassostrea gigas
• Online Access: http://libir.tmu.edu.tw/handle/987654321/61323; http://libir.tmu.edu.tw/bitstream/987654321/61323/2/index.html

碩士 指導教授：陳奕廷 委員：陳玉華 委員：侯又禎 軟體動物及其加工製品為《過敏原標示之建議事項》公告建議標示的食品過敏原，然而目前仍無軟體動物免疫測定法開發的技術。免疫測定法以特定蛋白質作為檢測標的物，普遍用於食品產業的快篩檢測。軟體動物測定法的開發首先需要確定標的蛋白質，並開發免疫測定方法。本研究分析與鑑定軟體動物肌肉中熱穩定蛋白質，探討合適的標的蛋白質。收集常見食用軟體動物以十二烷基硫酸鈉聚丙烯醯胺凝膠電泳分析軟體動物肌肉熱穩定蛋白質，並搭配液相層析串聯四極桿飛行時間質譜儀鑑定共同的熱穩定蛋白質。實驗結果顯示，35-40 kDa蛋白質為17種常見食用的軟體動物肌肉中共同且大量存在之熱穩定蛋白質。真鎖管、九孔螺、扇貝肌肉中的35-40 kDa蛋白質在蒸煮、水煮、烘烤、油炸條件中皆能維持熱穩定性，經鑑定結果得知，35-40 kDa蛋白質為原肌球蛋白（tropomyosin）。因此，本研究以tropomyosin作為免疫測定法開發的標的蛋白質。以tropomyosin開發的抗體所建構的sandwich ELISA能檢測所有測試的軟體動物肌肉加熱樣本（23物種），且對甲殼類、魚類、陸生動物、兩棲類樣本無非專一性反應。檢測真鎖管、九孔螺、太平洋牡蠣的肌肉加熱樣本的偵測極限皆為0.5 ppm。此外，對於常見的熱風乾燥、鹽漬、烘焙、煉製品、罐頭、煙燻的市售軟體動物加工食品皆能成功檢測。未來此sandwich ELISA檢測方法能轉為快篩試劑的形式，供軟體動物過敏患者將可利用此快篩試劑即時檢測飲食中潛在軟體動物之成分。本研究中開發的免疫測定法可供大量篩檢，落實食品安全法規與提升軟體動物過敏患者之食品安全。 Mollusk and its products are in the list of suggested food allergens according to “Regulation of Food Allergen Labelling”. At present, there is no immunoassay available for the detection of mollusk. Immunoassay detects specific protein as analyte and is commonly used in the food industries. The first step of the development of an immunoassay for the detection of mollusks is the determination of a marker protein. This study characterized thermal stable proteins in mollusk muscle and identified a suitable marker protein. Common edible mollusk species were collected and analyzed by SDS-PAGE and liquid chromatography-quadrupole-time of flight mass spectrometer for the identification of the main thermal stable proteins in mollusk muscle tissue. The results indicated that 35-40 kDa protein was the most abundant thermal stable muscle protein among 17 mollusk species. 35-40 kDa protein of Loligo edulis, Haliotis diversicolor and Patinopecten yessoensis kept its integrity under the food processing methods including steaming, boiling, baking and frying. 35-40 kDa protein was identified as tropomyosin. These results suggested that tropomyosin is a suitable marker protein for the development of an immunoassay for the detection of mollusk. The sandwich ELISA developed by mollusk-specific antibodies is able to detect cooked samples of all 23 mollusk species tested without non-specific cross-reaction with crustacean, fish, land animals and amphibian. The limit of detection of the optimized sandwich ELISA was 0.5 ppm for all three cooked mollusk samples (Loligo edulis, Haliotis diversicolor and Crassostrea gigas). In addition, the sandwich ELISA was also capable of detecting common processed mollusk products, including hot-air drying, salting, baked, surimi, canned and smoked products. In the future, the sandwich ELISA assay can be converted to lateral-flow assay. Mollusk allergy patients can use this lateral-flow assay to detection mollusk in foods and food industrials can also perform high throughput screening by this lateral-flow assay. The developed mollusk-specific immunoassay provides significant benefit for food allergen labeling law and improves food safety for mollusk allergy patients.