Different experimental approaches to highlight the relationship between processing parameters and protein/peptide integrity in lupin proteins

DIFFERENT EXPERIMENTAL APPROACHES TO HIGHLIGHT THE RELATIONSHIP BETWEEN PROCESSING PARAMETERS AND PROTEIN/PEPTIDE INTEGRITY IN LUPIN PROTEINS Sirtori Elena1, Resta Donatella2, Brambilla Francesca1, Arnoldi Anna2 1Laboratory of Food Chemistry and Mass Spectrometry, Department of Endocrinology, Pathophysiology and Applied Biology, University of Milan, Italy. 2HPF-Nutraceutics, Milan, Italy. elena.sirtori@unimi.it During the last decade functional foods have gained attention from the Food Industry because of their nutraceutical properties and role in the maintenance of good health. Lupin is developing as functional ingredients since it has been shown experimentally to have hypocholesterolemic and hypotensive properties. Since it is known that industrial processing causes protein denaturation and, consequently, a certain degree of protein insolubilisation, with deleterious effects on the nutritive value and nutraceutical properties of proteins, it appeared necessary to estimate the integrity of the lupin proteins subjected to different technological treatments and how much they are altered by processing. The present investigation was designed to evaluate the effects of several processing conditions on a lupin protein isolate (LPI) from Lupinus angustifolius. The first part of the study was dedicated to the identification of the main spots in the 2-dimentional map of L. angustifolius seed protein, combining the canonical proteomic approach with the “de novo peptide sequencing". With the aim of mimic industrial processing of functional foods, the LPI was thermal and mechanical treated. Heated was done in an oven from 100 °C to 200 °C for different times of exposure, and mechanical stress was applied using an Ultraturrax and an homogeniser in order to simulate different mixing and extrusion devices. The protein degradation was studied via differential scanning calorimetry, 2D-electrophoresis, and mass spectrometry, which enabled the fingerprint of the available peptides after processing. The results showed a decreasing denaturation enthalpy of the LPI (decreasing content of native protein) with increased strength of the treatments, even if some intense spots were still present on 2D-gels. The identity of these spots, confirmed via HPLC-Chip-Ion Trap and HPLC-Chip-qTOF analyses, indicated that 11S and 2S proteins were the most resistant, but that also some peptides belong to 7S family were still present. In conclusion, these preliminary observations indicate that, despite the thermal processes at various temperatures and conditions applied to lupin proteins, several peptides with putative biological activity remain intact and stable. The information obtained could help to better exploit the functional properties of these proteins as food ingredients.