扁桃仁蛋白饮料中扁桃仁蛋白高特异性的定量方法

Highly Specific Quantitative Estimation of Almond Kernel Protein in Almond Protein Beverag

摘要:

建立基于竞争酶联免疫分析（ELISA）的扁桃仁蛋白饮料中扁桃仁蛋白定量方法。使用双向电泳比较了扁桃仁和杏仁的蛋白质组上的差异，并且测序了杏仁prunin蛋白的氨基酸序列。鉴定，提取，纯化了扁桃仁分子量27 ku且pI 5.5~8.0的prunin蛋白亚基片段并且将其作为抗原制备特异性抗扁桃仁prunin蛋白单克隆抗体。使用单克隆抗体建立了检测扁桃仁蛋白的竞争酶联免疫分析方法。基于扁桃仁可溶全蛋白作为标准品，该方法的IC50为10.3 μg/mL，线性范围为2.0~100 μg/mL (R2=0.991)。该方法特异性良好，与杏仁和其他可食种子蛋白无交叉反应。样品前处理使用含有0.1%二硫苏糖醇（DTT）的7M尿素作为提取液。检测植物蛋白饮料样品的检出限为300 μg/mL，相对标准偏差<10%。使用巴氏杀菌，超高温瞬时灭菌和高压灭菌的蛋白平均回收率分别为97%，93%和84%。

Abstract:

A new method was established to quantitatively estimate almond kernel protein in almond protein beverage using competitive enzyme-linked immunosorbent assay (ELISA). Subsequently, two-dimensional electrophoresis (2-DE) was used to compare the proteomic differences between almond kernel and apricot kernel. Amino acid sequences of apricot pruning protein were determined and a Pru-1 subunit fragment with a mass of 27 kDa and pI (isoelectric point) of 5.5 to 8.0 was identified, extracted, purified, and used as an antigen to prepare a specific monoclonal antibody. This was used to establish the highly specific ELISA method to detect almond kernel protein. With almond soluble protein as standard, the half-maximal inhibitory concentration (IC50) of this new method was found to be 10.3 μg/mL and linear detection range was from 2.0 to 100 μg/mL (R2 = 0.991). The method exhibited good specificity and had no cross-reactivity with the proteins of test apricot kernels and other edible plant seeds. In the pre-treatment procedure, 7 M urea with 0.5% dithiothreitol (DTT) was used as sample extraction solution. The limit of detection for plant protein beverage was 300 μg/mL, with relative standard deviation lessthan10%. The average recoveries of pasteurization, ultra high-temperature sterilization, and autoclave sterilization were 97%, 93%, and 84%, respectively.