A new paradigm to search for allergenic proteins in novel foods by integrating proteomics analysis and in silico sequence homology prediction: Focus on spirulina and chlorella microalgae

Since novel nutrient sources with high protein content, such as yeast, fungi, bacteria, algae, and insects, are increasingly introduced in the consumer market, safety evaluation studies on their potentially allergenic proteins are required. A pipeline for in silico establishing the sequence-based homology between proteins of spirulina (Arthrospira platensis) and chlorella (Chlorella vulgaris) micro-algae and those included in the AllergenOnline (AO) database (AllergenOnline.org) is described. The extracted proteins were first identified through tryptic peptides analysis by reversed-phase liquid chromatography and high resolution/accuracy Fourier-transform tandem mass spectrometry (RPLC-ESI-FTMS/MS), followed by a quest on the UniProt database. The AO database was subsequently interrogated to assess sequence similarity between identified microalgal proteins and known allergens, based on criteria established by the World Health Organization (WHO) and Food and Agriculture Organization (FAO). A direct search for microalgal proteins already included in allergen databases was also performed using the Allergome database. Six proteins exhibiting a significant homology with food allergens were identified in spirulina extracts. Five of them, i.e., two thioredoxins (D4ZSU6, K1VP15), a superoxide dismutase (C3V3P3), a glyceraldehyde-3-phosphate dehydrogenase (K1W168), and a triosephosphate isomerase (D5A635), resulted from the search on AO. The sixth protein, C-phycocyanin beta subunit (P72508), was directly obtained after examining the Allergome database. Two proteins exhibiting significant sequence homology with food allergens were retrieved in chlorella extracts, viz. calmodulin (A0A2P6TFR8), which is related to troponin c (D7F1Q2), and fructose-bisphosphate aldolase (A0A2P6TDD0). Specific serum screenings based on immunochemical tests should be undertaken to confirm or rule out the allergenicity of the identified proteins.