Submitted to: Journal of Agricultural and Food Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/26/2024
Publication Date: 10/7/2024
Citation: Hao, M., Wang, Q., Wang, J., Che, H., Guo, X., Zhang, Y. 2024. Identification, purification, and characterization of Kunitz-type inhibitors from white-fleshed pitaya (Selenicereus undatus) and red-fleshed pitaya (Selenicereus costaricensis) seeds as novel food allergens. Journal of Agricultural and Food Chemistry. 72(41):22836-22847. https://doi.org/10.1021/acs.jafc.4c04328.
DOI: https://doi.org/10.1021/acs.jafc.4c04328

Interpretive Summary: The prevalence of food allergies is increasing, and the foods known as allergen sources are also expanding. More protein families have been found to have family members capable of eliciting allergic reactions. But what makes a food protein a food allergen remains elusive. More information is required to provide a better understanding of the allergenicity of food allergens, and uncovering and characterizing new food allergens is needed. Identifying novel food allergens is also necessary for scientists and physicians to develop better diagnoses and prognoses. This study reported the isolation and recombinant production of the Kunitz-type protease inhibitor from two kinds of dragon fruits. These inhibitors were identified as food allergens with patient sera, and their thermal stability, denaturation, and refolding were investigated with circular dichroism and fluorescence spectroscopies. The results can be used to obtain valuable reagents for studying the properties of food allergens and developing methods for allergen mitigation.

Technical Abstract: The consumption of pitaya fruits is on the rise because of their nutritional value and health benefits. However, pitaya fruits are also known to be capable of resulting in food allergies. In this study, two allergens are purified chromatographically and identified as Kunitz-type protease inhibitors by LC-MS/MS with chymotrypsin proteolysis and Biomolecular technologies. These two natural allergens in pitaya seeds are glycoproteins with apparent molecular masses of ~21.60 kDa. Their thermal stability and reversibility of their unfolding were determined by CD and fluorescence spectroscopies. Recombinant allergens produced in E.coil with the pCold' vector were well-folded and predominantly ß-sheet proteins, the same as the known structures of Kunitz-type protease inhibitors. They also displayed the ability to be recognized by patients’ serum IgE, comparable to their natural counterparts. IgE reactivity analysis with ten sera from patients primarily sensitized to pitaya reveals that Kunitz-type protease inhibitors are major pitaya allergens. These findings expand the understanding of the allergen sources and the knowledge required to understand the allergenicity of proteins from different species.