Release of Major Peanut Allergens from Their Matrix under Various pH and Simulated Saliva Conditions-Ara h2 and Ara h6 Are Readily Bio-Accessible

Abstract

The oral mucosa is the first immune tissue that encounters allergens upon ingestion of food. We hypothesized that the bio-accessibility of allergens at this stage may be a key determinant for sensitization. Light roasted peanut flour was suspended at various pH in buffers mimicking saliva. Protein concentrations and allergens profiles were determined in the supernatants. Peanut protein solubility was poor in the pH range between 3 and 6, while at a low pH (1.5) and at moderately high pHs (>8), it increased. In the pH range of saliva, between 6.5 and 8.5, the allergens Ara h2 and Ara h6 were readily released, whereas Ara h1 and Ara h3 were poorly released. Increasing the pH from 6.5 to 8.5 slightly increased the release of Ara h1 and Ara h3, but the recovery remained low (approximately 20%) compared to that of Ara h2 and Ara h6 (approximately 100% and 65%, respectively). This remarkable difference in the extraction kinetics suggests that Ara h2 and Ara h6 are the first allergens an individual is exposed to upon ingestion of peanut-containing food. We conclude that the peanut allergens Ara h2 and Ara h6 are quickly bio-accessible in the mouth, potentially explaining their extraordinary allergenicity.

Keywords: Arachis hypogaea; allergen; bio-accessibility; peanut; saliva.

Figure 1

Protein release from light roasted peanut flour at various pHs. Extraction was conducted for 90 min at room temperature. Protein concentrations are shown, as measured with the BCA method (mean values of triplicate experiments; the bars show SD).

Figure 2

Protein profiles of extracts of light roasted peanut flour prepared at various pHs. Extraction was conducted for 90 min. Protein profiles determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), under reducing conditions, on 10–20% gradient gels). The line at the bottom shows the extraction pHs (M refers to the marker proteins, indicated in the left margin in kDa). The arrows at the right point to major allergens. The rectangle in the lane corresponding to pH = 6.0 indicates the Ara h2 doublet (top) and Ara h6 band (bottom).

Figure 3

Protein profiles of light roasted peanut flour extracted with artificial saliva (pH = 7.8). Extraction time up to 20 min. Protein profiles were determined by SDS-PAGE, under reducing conditions, on 10% gels. The molecular weight marker is indicated on left in kDa, numbers at the bottom indicate extraction time (minutes). Arrows on the right margin point to major allergens.

Figure 4

Protein profiles of light roasted peanut flour released at various pHs mimicking saliva. Extraction time 20 min. Protein profiles determined by SDS-PAGE, under reducing conditions, on 10% gels. The molecular weight marker is indicated on the left in kDa. Numbers at the bottom indicate the pH of the extraction buffer. Arrows on the right margin point to major allergens.

Figure 5

Release of individual peanut allergens from their matrix of light roasted peanut flour dissolved at various pHs mimicking saliva. Panel A: Protein concentrations in the supernatants, in mg/mL. Panel B: Protein concentrations in the supernatants expressed as percentage of the theoretical maximum value. Mean values of two experiments are shown (SDs were typically between 0 and 8% of the mean (except in one case were the SD was 13% of the values; for Ara h6 at pH = 7.9). SDs are not shown here to improve readability.

Figure 6

Protein profiles of light roasted peanut flour pellets after extraction at various pHs mimicking saliva. Extraction time 20 min. Protein profiles determined by SDS-PAGE, under reducing conditions, on 10% gels. The marker is indicated on the left in kDa. The numbers at the bottom indicate the pH of the extraction buffer. Arrows on the right margin point to major allergens (note that the area indicted by Ara h2 and Ara h6 is empty indicating no residual Ara h2 and Ara h6 in the pellets.