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