加工食品中花生蛋白夹心酶联免疫吸附测定的前处理方法探究与改进

Exploration and improvement of the pretreatment method for peanut proteins sandwich enzyme-linked immunosorbent assay in processed food

中文关键词:  前处理  提取探究  花生  过敏  夹心酶联免疫吸附测定法  加工食品  可靠检测

英文关键词:pretreatment  extracting exploration  peanut  allergy  sandwich enzyme-linked immunosorbent assay  processed food  reliable detection

中文摘要:

      目的 构建一种可配合花生蛋白夹心酶联免疫吸附测定(enzyme-linked immunosorbent assay, ELISA)试剂盒实现对于加工食品中花生成分可靠检测的高质量样品前处理方法。方法 对9种不同pH的提取缓冲液提取未处理、湿热处理、干热处理花生蛋白效果进行比较分析以确定最优提取缓冲液类型。而后, 将吐温-20、二硫苏糖醇(dithiothreitol, DTT)、十二烷基硫酸钠(sodium dodecyl sulfate, SDS)以单独或组合添加的方式加入上一步获得的最优提取缓冲液中, 提取3组花生蛋白, 并对提取效果与夹心ELISA试剂盒检测花生蛋白回收率进行比较分析以确定最优添加剂配方。结果 最终确定添加0.2% SDS和0.1%吐温-20的碳酸盐缓冲液(50 mmol/L, pH 9.6)为最优提取缓冲液。向碳酸盐缓冲液中加入SDS、吐温-20较好地提升了花生蛋白的提取率和回收率, 将湿热处理花生蛋白回收率提高了2倍以上。结论 本研究实现了对商业加工食品中花生成分的可靠检测, 为推动过敏原信息在食品标签的准确标注提供了技术支撑, 也为相关研究提供了参考。

英文摘要:

      Objective To develop a high-quality sample pretreatment method applicable to the peanut proteins sandwich enzyme-linked immunosorbent assay (ELISA) test kit for reliable detection of peanut in commercially processed food. Methods The extraction effects of proteins from untreated, wet heat-treated, and dry heat-treated peanut extracted by 9 extraction buffers of different pH were compared and analyzed in order to determine the optimal type of extraction buffer. Afterward, 0.1% Tween-20, 10 mmol/L dithiothreitol (DTT), and 0.2% sodium dodecyl sulfate (SDS) as the additives were added separately or in combination into the optimal extraction buffer obtained in the previous step to extract proteins from 3 groups of peanut. The extraction effects and the recoveries of peanut proteins detected by the sandwich ELISA test kit were compared and analyzed to determine the optimal additive formulation. Results Carbonate buffer (50 mmol/L, pH 9.6) with 0.2% SDS and 0.1% Tween-20 was ultimately determined to be the optimal extraction buffer. Adding SDS and Tween-20 into carbonate buffer effectively improved the extraction efficiency and the recovery of the peanut proteins. The recovery of the proteins from wet heat-treated peanut was improved by more than 2 times. Conclusion In this study, we developed a high-quality sample pretreatment method applied to the detection of the peanut proteins sandwich ELISA test kit, providing technical support for its reliable detection of peanut in commercially processed food, and promoting the accurate labeling of allergen information in the food labels. It also provides a reference for the related researches.