Boiling and Frying Peanuts Decreases Soluble Peanut (Arachis Hypogaea) Allergens Ara h 1 and Ara h 2 But Does Not Generate Hypoallergenic Peanuts

Abstract

Peanut allergy continues to be a problem in most developed countries of the world. We sought a processing method that would alter allergenic peanut proteins, such that allergen recognition by IgE from allergic individuals would be significantly reduced or eliminated. Such a method would render accidental exposures to trace amounts of peanuts safer. A combination of boiling and frying decreased recovery of Ara h 1 and Ara h 2 at their expected MWs. In contrast, treatment with high pressures under varying temperatures had no effect on protein extraction profiles. Antibodies specific for Ara h 1, Ara h 2, and Ara h 6 bound proteins extracted from raw samples but not in boiled/fried samples. However, pre-incubation of serum with boiled/fried extract removed most raw peanut-reactive IgE from solution, including IgE directed to Ara h 1 and 2. Thus, this method of processing is unlikely to generate a peanut product tolerated by peanut allergic patients. Importantly, variability in individual patients' IgE repertoires may mean that some patients' IgE would bind fewer polypeptides in the sequentially processed seed.

Fig 1. Comparison of chloroform/methanol defatted (df) and non-defatted (ndf), buffer D extracted raw and boiled/fried peanut samples.

50μg protein (as determined using the Bradford assay on extracts prior to centrifugation) was loaded per lane on a 14% reducing SDS-PAGE. Lane labeled “spun” was loaded with non-defatted samples that had been centrifuged for one minute at 15,000 rpm in a benchtop eppendorf centrifuge. Arrows indicate differentially detected proteins.

Fig 2. 12% reducing SDS-PAGE of all 38 defatted, buffer D extracted peanut extracts.

A raw peanut extract (Raw) from defatted peanut was included on each gel as an internal control. Lanes are labeled with extract number (Table 1). Each lane was loaded with 65μg of protein.

Fig 3. Dot blot of all 38 defatted, buffer D extracted peanut samples (1μg/dot).

(A) Dot blot layout. Samples were blotted with serum from an atopic (mold sensitive), non-food allergic patient with presumably no peanut-specific IgE (B) and a peanut allergic individual (C).

Fig 4. Immunoblots of defatted, buffer D extracted peanut samples (numbers match Table 1) blotted with serum from peanut allergic individuals.

Serum was from individuals with a broad IgE repertoire (A, B), individuals with a more limited IgE repertoire (C, D), or an atopic (mold sensitive), non-food allergic patient with presumably no peanut-specific IgE (E). Extracts were run on 14% SDS-PAGE, 50μg per lane, and transferred to 0.22μm NC before blotting.

Fig 5. Immunoblots of defatted, buffer D extracted peanut samples (numbers match Table 1) blotted with polyclonal galline anti-Ara antibodies.

A) anti-Ara h 1, B) anti-Ara h 2, C) anti-Ara h 6 and D) anti-Ara h 3. Extracts were run on 14% reducing SDS-PAGE, 50μg per lane, and transferred to 0.22μm NC before blotting.

Fig 6. Inhibition immunoblots.

A) Inhibition immunoblot of raw peanut (sample 38) with sera from peanut allergic individuals presorbed with boiled/fried peanut (sample 37). B) Inhibition immunoblot of boiled/fried peanut (sample 37) with sera from peanut allergic individuals presorbed with purified Ara h 2 protein (h2) or not presorbed (0). C) Inhibition immunoblot of raw peanut (sample 38) with sera 4 presorbed with purified Ara h 2 (5μg or 10μg) or Ara h 6 (5μg or 10μg). “P” indicates a sample was presorbed with pollen extract.