Establishment of an analytical platform for the absolute quantification of kiwifruit (Actinidia deliciosa) allergens using dimethyl labeling coupled with mass spectrometry

• Main Authors: YI-Chen Shih, 施奕辰
• Other Authors: Fuu Sheu
• Format: Others
• Language: zh-TW
• Published: 2018
• Online Access: http://ndltd.ncl.edu.tw/handle/p6edt5

碩士 === 國立臺灣大學 === 園藝暨景觀學系 === 107 === Food allergy management is one of the main issues in food safety. For efficient management of allergy, an effective analytical method is required. In the past, antibody-based Immuno-assay ELISA was the most popular analytical method for allergen determination, however it has some disadvantages in the use of antibody. Recently, mass spectrometry-based methods gains popularity because it can overcome the disadvantages of antibody-based methods, also possesses advantages such as high throughput, and ability to provide molecular evident etc. Quantification of plant allergens, especially fruit allergens, which have low protein contents, is difficult to achieve, compared to other food allergens. Kiwi fruit (Actinidia deliciosa) is one of the popular fruits in Taiwan. It ranks the second highest among the imported fresh fruits. Kiwi fruit is also an allergenic fruit, of which allergic popularity is rated sixth among food allergens in Europe and tenth in Taiwan. Many governments suggest allergenic labeling on food label. In our study, we choose kiwi fruit as an example to develop a MS-based quantification method for fruit allergen. Our results consisted of three major parts. In the first part, we have developed and optimized sample preparation methods for mass spectrometry analysis of fruit allergen using kiwifruit as a model sample. We have developed two fruit optimized phenol extraction methods, including optimized phenol extraction method (OPE) and micro phenol extraction method (MPE). Both methods had yielded a high extraction efficiency. OPE had a higher protein extraction weight, while MPE had some other advantages such as low cost, fast, simple and was well compatible with further mass spectrometry sample preparation steps. Meanwhile we have optimized trypsin in-solution digestion method and confirmed efficiency as well as isotopic effect of stable isotope dimethyl labeling method. We have also combined different methods of sample preparation steps into one continuous, low intra-method conversion loss process. In the second part, we have established the method of mass spectrometry for allergen quantification of kiwifruits. First, we used the results in the test of Q-ToF to decide the target allergens (Act d 1, Act d 5, and Act d 11) and representative peptides (AD1, AD5, and AD11). Then, we decided the quantitative and qualitative daughter ions of representative peptides on QqQ MS while establishing and optimizing parameters of LC and MS. After validation of our methods, AD1H and AD5H we found to have satisified validation characteristics, including range, accuracy, repeatability, instrumental quantification limit, instrumental detection limit, specificity, and linearity. We have successfully developed and validated mass spectrometry-based allergen quantification method for kiwifruits under the MRM mode. In the third part, we have compared eight common kiwi fruit products to study the influence of different process methods on kiwi allergens. We have determined the allergen contents of Act d 1 and Act d 5 in all samples, which indicated that our method was robust and could be applied to some other common fruit products. We found that allergen contents were significantly reduced under long time heat processing. In conclusion, we have developed two fruit optimized phenol extraction methods, while MPE was combined with rest of the sample preparation methods into one continuous MS sample preparation process. In addition, we have developed a MS-based allergen quantification method for kiwifruits and its derivative products. Our study not only could be an example of analytical method development of food allergen, but also could be applied to improve food safety.