Different experimental approaches to highlight the relationship between industrial processing and protein/peptide integrity in bioactive pea proteins

DIFFERENT EXPERIMENTAL APPROACHES TO HIGHLIGHT THE RELATIONSHIP BETWEEN INDUSTRIAL PROCESSING AND PROTEIN/PEPTIDE INTEGRITY IN BIOACTIVE PEA PROTEINS Sirtori1 E., Resta2 D., Isak1 I., Arnoldi1 A. 1Department of Endocrinology, Pathophysiology and Applied Biology, University of Milan, 20133 Milan, Italy. 2HPF-Nutraceutics, 20133 Milan, Italy. E-mail: elena.sirtori@unimi.it Background: During the last decade food industry has increased its attention on functional foods, defined as any food or food ingredient able to provide an health benefit beyond the nutritional properties. The nutraceutical properties depend on the presence of bioactive components such as proteins or peptides with an in-vivo biological effect. Several literature data reported many examples of food bioactive proteins characterized by different biological activities such as hypotensive, hypocholesterolemic and hypoglycemic activity. Recent studies carried out in the European Craft Project “Bioprofibre” have highlighted specific biological properties of proteins from Pisum sativum, in agreement with previous literature results. Since data regarding the consequences of industrial processing on the quality of pea protein are scarce, the present investigation was designed to evaluate the effects of mild and harsh thermal and mechanical treatments on a industrial pea protein isolate. In fact, industrial processing is known to cause protein denaturation with either deleterious or beneficial effects on the nutritive value and nutraceutical properties of proteins. Methods: The total protein extract (TPE) from pea seeds (cv Attika) and an industrial pea protein isolate, (PPI, Pisane®) were examined. PPI was treated thermally in an oven from 65°C to 200°C for different exposure time, and mechanically (ultraturrax, high pressure homogenizer). After each treatment, the soluble proteins were extracted and the degradation behavior of the major storage proteins was studied. Differential Scanning Calorimetry (DSC) and proteomic techniques, such as 2D-electrophoresis and mass spectrometry, were used to investigate the effects of different processing conditions on the protein profile and to assess the availability of intact/resistant peptides after treatments. Results: After a prolonged heating treatment no native proteins were present and the proteins solubility was greatly decreased, even if some spots with high intensity were still present on 2D-gels. The identity of these gel spots was confirmed via HPLC-Chip-MS/MS showed that the vicilin 30 kDa fragment and legumin acidic subunit were the most resistant. The tryptic peptides derived from them were still available after 30 min dry heating at 120°C. The results of DSC analysis, used to determine the content of native/degraded proteins, showed a decreasing denaturation enthalpy with increasing of thermal and mechanical treatments. On the other hand, the mechanical stress applied did not show any relevant difference in the DSC thermograms and in 2D-gels, meaning that these kind of mechanical treatments did not affect the native structures of the protein fractions. Conclusions: This study could help in understanding the relationship between the thermal and mechanical effects on protein integrity, by using 2D-gels, Differential scanning calorimetry (DSC) and gel filtration chromatography. The results indicate that after heating treatments pea protein isolate partially or completely lost their protein integrity, however the HPLC-Chip-MS/MS shotgun proteomics analysis was enabled, especially after harsh treatments, to determine the presence of specific and intact available peptides with putative biological activity.