Perner Sebastian P, Heupel Linda, Zimmermann Lisa, Peters Yasmin, Vongehr Kai U, El-Bedewy Hesham, Siebeneicher Susanne, Weiß Thomas, Hektor Thomas, Lindemann Bernd, Loos-Theisen Simone, Schneider Klaus
University of Applied Sciences Fresenius, Institute for Biomolecular Research, Limburger Straße 2, 65510 Idstein, Germany.
Hochschule Geisenheim University, Institute for Food Safety, Rüdesheimer Straße 28, 65366 Geisenheim, Germany.
J AOAC Int. 2019 Sep 1;102(5):1271-1279. doi: 10.5740/jaoacint.19-0055. Epub 2019 Mar 19.
Western society is facing an increase in the number of food-allergic individuals, with rising incidence in the past years. Therefore, allergen-free food and accurate and reliable analysis of allergen contamination are essential for the protection of consumers. Yet, there is limited understanding on the effect of food processing on allergenicity and on the ability of available methods to detect trace contamination in processed food. Available studies addressing this have relied on sample processing on a laboratory scale. In this study, industry-like processing under precisely defined conditions (ranging from 110 to 150°C roasting temperatures) was employed to better understand the limitations of state-of-the-art methods for detecting traces of hazelnut and almond in processed food. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis indicated an overall reduction in extracted proteins from roasted nut samples, and with matrix-assisted laser desorption ionization time-of-flight Cor a 9 and Prunin, were identified as majorly expressed proteins for hazelnut and almond, respectively. A commercial ELISA kit detected nut traces only up to a 130°C roasting temperature. Untargeted MS (Orbitrap) analysis was able to detect traces of nuts roasted up to 150°C while also confirming Cor a 9 and Prunin as the major expressed proteins for hazelnut and almond, respectively. Preparing cookie dough spiked with roasted nut samples, a complex food matrix was simulated. Analysis by ELISA showed the same limitations encountered for pure nuts samples, hardly detecting traces of nuts roasted above 130°C. Targeted MS (linear ion trap) using multiple reaction monitoring methods for one proteotypic peptide for Cor a 9 and Prunin, respectively, enabled a detection of nut traces up to 150°C. The results indicated that a reduced extractability because of temperature-related effects (e.g., protein denaturation, cross-linking, poor solubility) caused the significant differences between the ELISA and MS analysis. Overall, the results of this study may form the basis to improve allergen detection after roasting through improved extraction methods and refined ELISA formats.
西方社会中食物过敏个体的数量呈上升趋势，在过去几年里发病率不断攀升。因此，无过敏原食品以及对过敏原污染进行准确可靠的分析对于保护消费者至关重要。然而，人们对于食品加工对致敏性的影响以及现有方法检测加工食品中痕量污染物的能力了解有限。针对这一问题的现有研究依赖于实验室规模的样品处理。在本研究中，采用在精确限定条件下（烘焙温度范围为110至150°C）类似工业生产的加工方式，以更好地了解检测加工食品中痕量榛子和杏仁的现有方法的局限性。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分析表明，烘焙坚果样品中提取的蛋白质总体减少，并且通过基质辅助激光解吸电离飞行时间质谱分别鉴定出Cor a 9和李属蛋白为榛子和杏仁中主要表达的蛋白质。一种商业酶联免疫吸附测定试剂盒仅能检测到烘焙温度达130°C时的坚果痕量。非靶向质谱（轨道阱）分析能够检测出烘焙至150°C的坚果痕量，同时也分别确认了Cor a 9和李属蛋白为榛子和杏仁中主要表达的蛋白质。通过制备添加烘焙坚果样品的饼干面团，模拟了复杂的食品基质。酶联免疫吸附测定分析显示，纯坚果样品存在同样的局限性，几乎无法检测出烘焙温度高于130°C时的坚果痕量。分别针对Cor a 9和李属蛋白的一种蛋白型肽，采用多反应监测方法的靶向质谱（线性离子阱）能够检测出烘焙至150°C时的坚果痕量。结果表明，由于温度相关效应（如蛋白质变性、交联、溶解性差）导致的提取率降低，造成了酶联免疫吸附测定和质谱分析之间的显著差异。总体而言，本研究结果可为通过改进提取方法和优化酶联免疫吸附测定形式来提高烘焙后过敏原检测水平奠定基础。
