Peanut Allergens Attached with p-Aminobenzamidine Are More Resistant to Digestion than Native Allergens

Despite being known as resistant proteins, peanut allergens (Ara h 1 and Ara h 2) can be digested and cause allergic reactions. Making the allergens more resistant to digestion may aid in non-absorption and excretion of the allergens. Our objectives were to make Ara h 1 and Ara h 2 more resistant to digestion and test them in a model system using trypsin as the digestive enzyme. The resistant allergens were prepared by covalently attaching p-aminobenzamidine (pABA), a protease inhibitor, to peanut allergens in an extract or on a PVDF membrane using glutaraldehyde, and were then tested for resistance to trypsin digestion. SDS-PAGE and Western blot were performed to determine the allergenic capacity of the modified allergens. A control was prepared using glycine instead. Results showed that Ara h 2, when covalently attached with pABA, was more resistant to trypin digestion than the native allergen. Similarly, Ara h 1, prepared on a PVDF membrane and treated with pABA, displayed a resistance to trypsin digestion. Treatment of the allergens with glycine (a control) instead of pABA showed that the modified allergens were as digestible as native allergens. Blot assays showed that the pABA-treated allergens exhibited a lower allergenic capacity than native allergens. It was concluded that pABA, when attached to peanut allergen Ara h 1 or Ara h 2, inhibited digestion of the allergen by trypsin and reduced their allergenic capacity as well.

Selection of specific nanobodies against peanut allergen through unbiased immunization strategy and the developed immuno-assay

Peanut allergy is considered to be a major health issue with global effects.To date,no effective curative approach has been applied for the therapy of the anaphylaxis resulting from the peanut allergens.The accurate and effective detection methods for the surveillance of allergens in food are still the primary strategies to avoid allergic diseases.In this study,nanobodies(Nbs)derived from the Heavy-Chain only Antibodies(HCAbs)were selected against the general peanut protein extract through the unbiased strategy to facilitate the development of the sandwich ELISA for the detection and surveillance of peanut allergen contamination.The target antigen of the selected Nb was identified as peanut allergen Ara h 3,and a cross-reaction was observed with the member of Gly 1 from the Ara h 3 family.The applicability of the self-paired Nb P43 on the establishment of the immuno-assay was verified.A sandwich ELISA against peanut allergen was developed,which reached a linear range of 0.2-10.6μg/mL,and a limit of detection of 53.13 ng/mL.

Effects of different cooking methods on peanut allergenicity

Peanut allergy is a type of serious food allergy worldwide.In this study,peanuts were treated with two different cooking methods.The effect of cooking on digestion stability was evaluated by simulated gastrointestinal digestions.The effects of cooking on the allergenicity were assessed by immunoblotting,passive cutaneous anaphylaxis (PCA) mice model and rat basophilic leukemia (RBL)-2H3 cell model.Results showed that raw peanuts were more stable to digestion than boiled and fried peanuts.SDS-PAGE showed that the quantities of allergens Ara h 1 (64 kDa),Ara h 2 (20 kDa),and Ara h 3 (38 kDa) in the boiled and fried peanuts were all reduced.Meanwhile,immunoblotting revealed the IgE binding capacities of boiled and fried peanuts were decreased significantly,especially the fried peanuts.In the PCA mice model,the vascular permeability of allergic mice,as well as the release of histamine,were both alleviated in mice gavaged with boiled and fried peanuts compared with raw peanuts.Lastly,the release of β-hexosaminidase of RBL-2H3 cells treated with boiled and fried peanuts was significantly decreased compared with the raw peanuts.In addition,the analyses of circular dichroism (CD) spectra showed that the α-helical content of peanut proteins was decreased after boiling and frying,indicating the structure stability of peanut allergens decreased.The analyses of ultraviolet (UV) spectra showed that aromatic amino acid residues were exposed,causing the change of the tertiary structure of peanut allergens after boiling and frying.In conclusion,the allergenicity of peanuts was significantly reduced after boiling and frying,especially frying.