Processing Can Alter the Properties of Peanut Extract Preparations (1)
Several different enzyme-linked immunosorbent assay (ELISA) methods are being used to detect either the common peanut allergens, such as Ara h 1, or total peanut protein, all of which require that these proteins be extractable from a food source.
Ara h 1 (63 kDa), Ara h 2 doublet bands (19 and 21 kDa), and Ara h 3 (40 kDa) acidic subunits are indicated with arrows. The levels of Ara h 2 in each sample show much less visible change in comparison with Ara h 1, in either the soluble or the insoluble fractions. In addition, as the length of exposure to heat increases, high-molecular-weight-smearing of the proteins in the lanes containing the insoluble fractions is seen.
The Ara h 1 monomer can be seen as a single band in lane 1, which contains pure Ara h 1, and in lane 2, which contains the soluble fraction of raw peanut. In the soluble portion of boiled, fried, and roasted samples, the relative Ara h 1 levels decrease with increased heat treatment. However, not only does the relative level of Ara h 1 increase in the insoluble fraction of each sample, it is also clear from specific bands and smears recognized by the antibody that Ara h 1 is present in the higher molecular weight aggregates or oligomers.
Soluble Ara h 2 is reduced by heat treatment, particularly in the roasted and boiled samples, but to a much lesser extent than that seen with Ara h 1, with the exception of the peanut sample boiled for 45 min. In this boiled peanut sample, the Ara h 2 protein is almost completely insoluble and barely detectable with the anti-raw Ara h 2 antibody in the soluble fraction. The anti-raw Ara h 2 antibody recognizes the Ara h 2 in the various forms of processed peanut and in both the supernatant and in the pellet fractions as well.
The IgE binding to Ara h 1 in both fried and roasted soluble portions decreases as do the levels of Ara h 1 with increased heating time. In the soluble fractions (S) of boiled (45 min) and roasted (dark roast, 50 min) peanuts, even though it seems like a reduction in the level of extractable Ara h 2, IgE binding to Ara h 2 is not different in the boiled sample and is higher in the dark roast peanut than in the other soluble samples.
While denaturation of most proteins at temperatures above 80 °C results in the loss of almost all secondary and tertiary structure, heating of purified Ara h 1 leads to a more structured secondary conformation of the protein, with an increased content of extended β-sheet structures leading to the formation of large protein complexes or aggregates.