Purpose: To critically assess the enzymatic approach used by the Ahmedna group to reduce and/or eliminate the allergens contained in whole peanuts or peanut derivatives.
Methods: Peanuts (Spanish and Runner cultivars) were sanitized and then treated to enzymatic digestion using a panel of endopeptidases under optimized conditions. In some instances, enzymes were applied to homemade peanut butter or to extracted peanut proteins directly. Following treatment, nuts were rinsed, heated overnight at 70°C and then ground into a homogenous paste. Proteins were then isolated using a standard extraction protocol. Assessment of enzyme activity against the major peanut allergens, Ara h1, Ara h2 and Ara h3, was performed using a combination of SDS-PAGE electrophoresis, western blotting, and ELISA techniques. Assessment of immunogenicity was performed using pooled sera obtained from peanut-allergic patients.
Results: Protein gel analysis of the allergen profile of whole nuts following enzymatic treatment with trypsin and α-chymotrypsin demonstrated only a modest reduction of Ara h1, Ara h2 and Ara h3 bands. Furthermore, assessment of the immunoreactivity of these bands and their subsequent cleavage products using ELISA and western blot analysis revealed the overt presence of immunoreactive peptides.
Significance: Enzymatic digestion of allergens contained in whole nut matrix of peanuts using trypsin or α-chymotrypsin is incomplete at best, and results in the generation of peptides that fully retain immunogenic epitopes capable of eliciting allergic responses. These results indicate that additional approaches are required that are aimed at reducing the immunogenic potential of peanut allergens.