High-pressure and temperature autoclaving of peanuts reduces the proportion of intact allergenic proteins

High-pressure and temperature autoclaving of peanuts reduces the proportion of intact allergenic proteins Casey G. Cohena, Wei Zhaoa, Liane Beaudetteb, Duncan Lejtenyib, Bertrand J. Jean-Claudea, Bruce D. Mazera,bTo the Editor,Peanut allergy is extremely common, affecting approximately 1.5% of children in North America, Australia and the UK1. Peanut does not appear to denature under normal cooking conditions. Structural biology analyses have focused on the three-dimensional structure of major peanut protein allergens such as Ara h 1 and Ara h 2. This class of proteins is rich in disulfide bridges, which explains their resistance to denaturation at high temperature2. Analysis of whole autoclaved roasted peanuts demonstrated a decrease of IgE-binding capacity of peanut allergens and in wheal size by skin prick test, as well as the unfolding of proteins and reduction in overall secondary structure3. The objective of this study was to evaluate the effects of thermal processing, particularly roasting and autoclaving, on the allergenicity of major peanut protein allergens Ara h2 and Ara h8.Western blot analyses were performed on protein extracts of each processing condition. In all autoclaved extracts, no distinct bands were observed for Ara h 2 or Ara h 8, while high levels of band intensity were observed for the raw and roasted extracts (Figure 1A). For both Ara h 2 and Ara h 8, raw and roasted protein extracts showed high levels of detection by ELISA (Figure 1B). However, for the autoclaved extract, detection was reduced by approximately 50% for Ara h 2, while no Ara h 8 was detected in the autoclaved extracts, independent of extract concentration (Figure 1B). Furthermore, IgE binding decreased significantly upon autoclaving peanut (Figure 1C, p < 0.0001), as shown by ELISA using the sera of 4 highly allergic patients (ImmunoCAP >50 IU).Nine peanut-allergic individuals were subjected to skin prick tests (SPT) with a panel of protein extracts derived from raw, roasted and autoclaved peanuts and the resulting wheal diameters were measured (Table I). Within the peanut-allergic group, a statistically significant reduction in mean wheal diameter was observed using the autoclaved extract when compared to raw (p < 0.01). Additional information was revealed when the allergic group was further stratified into two groups: (1) patients who have previously experienced systemic symptoms or anaphylaxis to peanut or have demonstrated high likelihood based on past SPT and serum-IgE tests, and (2) those who have experienced only oral symptoms upon peanut consumption. A striking decrease in wheal size was observed when using autoclaved extracts compared to raw in the group that experiences oral symptoms. In contrast, this was not observed in the group at risk for anaphylaxis.Our discovery of complete and partial degradation of Ara h 8 and Ara h 2, respectively, under autoclaving may have significant clinical implications. Currently, whole protein extracts created from raw or roasted peanuts are routinely used in SPTs for the diagnosis of peanut allergy in the clinic. Sensitivity to Ara h 2 has proven to be one of the best predictors of anaphylaxis in allergic patients4, while isolated Ara h 8 sensitization correlates with oral symptoms or tolerance to peanut in almost all cases5. Our results indicate that the use of an autoclaved peanut extract, in addition to the current whole protein extract, has the potential to serve as an improved diagnostic tool (patent applied6), distinguishing between two subsets of peanut-allergic patients: (1) those at risk for anaphylaxis, and primarily have Ara h 2-specific IgE, versus (2) those who will only experience oral symptoms to peanut, and predominantly present Ara h 8-specific IgE. As depicted in Figure 1D, patients with a positive SPT result using both the whole peanut extract (raw or roasted) and the autoclaved peanut extract will be classified as at risk for anaphylaxis (subset 1). Importantly, patients with a positive SPT result using the whole peanut extract, but a negative SPT result using the autoclaved extract, will experience only oral symptoms upon peanut consumption (subset 2). Patients tolerant to peanut will experience a negative SPT result with both extracts.Altogether, the data reported in this study suggest that high-pressure and temperature autoclaving leads to a significant denaturation of allergenic peanut proteins. This discovery is being further developed into an improved diagnostic test for peanut-allergic patient stratification. Further studies are required to optimize a degree of complete reduction of intact allergens by autoclaving.