Separation of bioactive peptides by electrodialysis with ultrafiltration membranes: membrane characteristics, ex-situ and in-situ digestion and their impact on peptide migration

Dissertation presented to Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa for obtaining the master degree in Membrane Engineering

Trypsic hydrolysis of whey protein isolate was performed simultaneously with (in-situ) and before (ex-situ) fractionation by electrodialysis with ultrafiltration membrane (EDUF) to obtain bioactive peptides. Two ultrafiltration membrane (UFM) materials, PES and PVDF, were used for a 120 minute EDUF fractionation of the hydrolysate. The two membranes showed similar zeta potential measurements at the pH of operation, 7.8, but had significantly different conductivity which decreased significantly after use in EDUF. Peptide migration to anionic (A-RC) and cationic (C+RC) peptide recovery compartment was strongly dependent on the electrical conductivity of the UFMs than their types or the digestion strategy used. For UFMs with close values of conductivity, peptide migration to the A-RC was observed to be higher with in-situ digestion while peptide migration to the C+RC was higher in an ex-situ digestion. When the two membrane types, PES and PVDF, have closer values of conductivities, PVDF was observed to exhibit more migration than PES. Peptide migration to the C+RC varied from 16.56 ± 5.36 μg/mL to 103.10 ± 2.76 μg/mL for PVDF membrane with significantly different conductivities: 2.73 ± 0.32 mS/cm and 5.47 ± 0.56 mS/cm, respectively. Peptide migration to the A-RC varied from 4.41 ± 0.86 μg/mL to 49.65 ± 6.13 μg/ml for PVDF membrane with significantly different conductivities: 3.163 ± 0.12 mS/cm and 5.23 ± 0.04 mS/cm, respectively. HPLC-MS studies showed 23 major peaks that were generated on whey protein isolate digestion by trypsin. 9 of these peaks migrated to the A-RC while 3 migrated to the C+RC. Among the anionic peptides 3 peptides are known to have hypocholesterolemic effect, 1 is antibacterial and 1 is antihypertensive. Among the cationic peptides 1 is antihypertensive. EDUF appears to be a powerful new technique that can fractionate bioactive peptides in terms of mass and charge.

The EM3E Master is an Education Programme supported by the European Commission, the European Membrane Society (EMS), the European Membrane House (EMH), and a large international network of industrial companies, research centres and universities