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Title: Allergenicity of Peanut Proteins is Retained Following Enzymatic Hydrolysis

item GUO, R - Duke University Medical Center
item SHI, X - North Carolina State University
item White, Brittany
item Sanders, Timothy
item BURKS, A - Duke University Medical Center
item Davis, Jack
item KULIS, M - Duke University Medical Center

Submitted to: American Academy of Allergy Asthma and Immunology
Publication Type: Abstract Only
Publication Acceptance Date: 1/30/2012
Publication Date: N/A
Citation: N/A

Interpretive Summary:

Technical Abstract: Rationale: Hydrolysis of peanut proteins by food-grade enzymes may reduce allergenicity and could lead to safer forms of immunotherapy. Methods: Light roasted peanut flour extracts were digested with pepsin (37°C, pH 2), Alcalase (60°C pH 8), or Flavourzyme (50°C, pH 7) up to 1 hr, or sequentially with Alcalase and Flavourzyme for 30 min each. Hydrolysates were analyzed by size-exclusion chromatography (SEC), SDS-PAGE, and Western blotting with human peanut-allergic plasma. Basophil degranulation assays were conducted on whole blood from peanut-allergic individuals and monitored by CD63 up-regulation, while T cell proliferation was investigated by a CFSE-incorporation assay. Results: Hydrolysis with pepsin, Alcalase or Flavourzyme resulted in distinct SDS-PAGE and SEC patterns and the degree of hydrolysis (DH) after 1 hr was approximately 15, 22 and 27%, respectively. DH for sequential hydrolysates was approximately 37%. All peptides were less than ~ 20 kDa and proportions of lower MW peptides increased with increasing DH. Western blotting demonstrated that IgE-binding fragments existed for each of the hydrolysates although the sizes were much smaller than the non-hydrolyzed controls. No appreciable effect was seen with any of the 3 hydrolysates in the basophil assays (n=5 subjects) compared to non-hydrolyzed controls. T cell proliferation assays (n=5 subjects) indicated that hydrolysates retained ~50% of the CD4+ T cell stimulation effect as compared to non-hydrolyzed controls. Allergenicity data for sequential hydrolysates and fractions collected from SEC will also be presented. Conclusions: Hydrolysis of peanut proteins yields low MW peptides that retain IgE cross-linking properties as evidenced by basophil degranulation assays.