D1.31 - Identification of Process-Modified Allergens in Tiger Nut Milk (Horchata) as the Cause of IgE-Mediated Anaphylaxis: Molecular Evidence of Aldolase and α/β-Hydrolase Proteins

Tiger nut (Cyperus esculentus) is not commonly regarded as a major allergenic source, yet its processed derivative, tiger nut milk (horchata), may induce immediate IgE-mediated reactions.

We report a patient with anaphylaxis after horchata ingestion and negative prick-by-prick to fresh tiger nut. 

Horchata and tiger nut (hydrosoluble and liposoluble fractions) were extracted using standardized PBS-based extraction and lipid phase purification. SDS-PAGE and IgE–Western blot were performed under reducing conditions, and proteomic identification (LC-MS/MS) was conducted for the 25 kDa and 40 kDa hydrosoluble horchata bands. An ALEX² inhibition assay using hydrosoluble horchata extract evaluated cross-reactivity with profile-related allergens.

Clear differences were observed between horchata and fresh tiger nut protein profiles, with the hydrosoluble fractions showing a richer band pattern than the liposoluble portions. IgE–Western blot of hydrosoluble horchata revealed two IgE-reactive proteins of approximately 40 kDa and 25 kDa, while the liposoluble fraction demonstrated a ~13 kDa band. In tiger nut, IgE recognition was limited to a ~13 kDa band in the hydrosoluble extract and a ~15 kDa band in the liposoluble extract. LC-MS/MS identified the 40 kDa horchata band as fructose-bisphosphate aldolase (UniProt A0AAV8C2B4), a known allergen in crab, several molds, and Artemisia, where it is associated with pollen–food syndrome.

In addition, the 25 kDa band was identified as an α/β-hydrolase superfamily protein (UniProt A0AAV8FE51). The inhibition study demonstrated decreased IgE binding to several profilins (Cuc m 2, Hev b 8, Mer a 1, Phl p 12 and Pho d 2) and walnut pollen (Jug r), indicating that the ~13 kDa tiger nut band could correspond to a profilin—likely present in horchata at levels below Western blot detection but sufficient for immunological inhibition. Together, these results demonstrate that the patient’s IgE profile is directed primarily against processing-modified proteins in horchata rather than native tiger nut proteins.

This case shows that tiger nut milk can induce IgE-mediated anaphylaxis through allergens that differ from those present in the raw tuber. Processing appears to modify or expose epitopes in aldolase and α/β-hydrolase proteins, generating clinically relevant allergens absent in fresh tiger nut.