Identification of 30 KDa Bombyx mori allergen by immunoblotting and MALDI-TOF/TOF mass spectrometry

Edible insects can be a source of sustainable protein with low environmental impact. Among them, the silkworm (Bombyx mori) pupae (SWP) has been included in the EFSA (European Food Safety Authority) list of insect species with a interesting food application. Insects are a traditionally food in East and Southeast Asia and every year in China 1000 people have allergic reaction after SWP ingestion. In the Europe the introduction of insect into the diet will take place after study of specific risk assessment, including the evaluation of the allergenic potential. The aim of this work was to evaluate the allergenicity of SWP by immunoblotting performed with the sera of two patient sensitized during manipulation of insect flour and to identify the immuno-reactive proteins with mass spectrometry approach. Total proteins extract were separated by SDS-PAGE and transferred into nitrocellulose membrane. The membranes were incubated o.n. with the two sensitized patient sera. The immune-complex was detected with anti-human IgE AP Conjugate antibody. The reactive band was cut from the gel, destained and digested by trypsin (o.n., at 37°C) and the resulting peptides were analyzed by MALDI-TOF/TOF. On the three more relevant piques were performed the Post Source Decay(PSD) analysis. The mass spectra were analyzed by Flex Analysis software and the m/z list were used by Protein Prospector search software (MS-Fit and MS-Tag)to query UniProt data base. The Immunoblotting results show only one reactive band for both the sensitized patients. The identification show different protein belonging of 30Kda Lipoprotein family, one of the most abundant protein in SWP hemolymph. This is a defense protein and it's already known to be a food allergen. This preliminary study about SWP allergenicity could improve the state-of-the-art about edible insect safety from the allergenic point of view. It's one of the first evaluation of SWP allergenic potential using the sera of two Italian patient sensitized during manipulation of insect flour; a rare case of allergy to insect proteins in Europe.