Direct-Affinity Reverse Extraction (DARE) screening for bioactive food-derived peptides
Special Symposium - Innovations in Food Technology (LMC Congress)
Innovations in Food Technology - Poster Session (LMC/Food - P1)
Keywords: bioactive components
Foods of the future should provide not only appropriate nutrients and sufficient energy for human function but also contribute to the well-being and health of consumers. Bioactive milk-derived peptides will occur at highest concentrations in the gastrointestinal (GI) tract, where innate immune responses mediated by Toll-like receptors (TLR) are particularly important for the host defence against microbial infection.
In order to rapidly and efficiently characterize novel foods for presence of potentially bioactive components, we propose to identify bioactive peptides by Direct Affinity Reverse Extraction (DARE), arguing that for a peptide to have a biological effect it must bind to a relevant target protein with high affinity. This novel method utilizes immobilized target biomolecules to extract peptides with high binding affinity from complex hydrolysates containing thousands of different peptides. We use biomolecules of importance for the innate immune response as targets directly immobilized on MALDI-TOF plates for subsequent identification by accurate mass analysis and sequencing of bound peptides.
We have developed strategies for immobilising biotin- or polyhistidine-tagged biomolecules as targets for the DARE technology. Milk-derived peptides have been generated simulating a gastrointestinal digest. Subsequent incubation of the target biomolecules with milk-derived peptides results in binding of putative bioactive peptides with high affinity for the target biomolecule, and we have subsequently identified these peptides directly by MS. The activity of these putative bioactive peptides will be tested by using a murine intestinal epithelial cell line (MICC12 cells) transfected with the luciferase reporter gene under control of NF-B, and thus we can simply measure bioactivity of these peptides in the absence or presence of natural ligands that stimulate the innate immune response.
Presented Wednesday 19, 13:30 to 15:00, in session Innovations in Food Technology - Poster Session (LMC/Food - P1).
