| Insect contamination of grain products is one of the main factors that causes serious decrease in quality of processed products. We have developed a rapid detection method for insect parts in wheat flour, based on special peptides of insects, as these peptides gave a consistent response for a very broad range of insects. Five of the most common insects of stored products, Cryptolestes ferrugineus (Stephens), Rhyzopertha dominica (F.), Sitophilus oryzae (Linnaeus), Tribolium castaneum (Herbst), and Tribolium confusum (Jacquelin du Val), were investigated in this experiment; T. castaneum is the predominant insect in flour. In this research, an Agilent 1100 reversed phase high performance liquid chromatograph (RP-HPLC) was used for peptide purification and fractionation. Matrix-assisted laser desorption/ionization (MALDI) was used to read the RP-HPLC fractions, and the spectra were analyzed by an in-house software developed by the Time-of-Flight Lab in the Department of Physics and Astronomy at the University of Manitoba. Initially, three protein purification buffers, 50% acetonitrile; sterilized water; and the buffer of 10 mM imidazole pH 7.0, 1 mM phenylthiourea, 1% Triton X, and protease inhibitor cocktail mixture (PTU buffer), were compared. Later, T. castaneum (red flour beetle) protein identification and peptide spectra mapping were done using the protein fingerprinting technique. Peptide spectra mapping method, which could set up biomarkers for each type of insect, was proven to be a more effective and practical way to detect insect contamination. This peptide mapping technique could obtain 55 stable biomarkers of T. castaneum, when T. castaneum was 0.1% in wheat flour by mass. This level needs to be lowered further during future research. |
