Separation and Enrichment of Antioxidant Peptides from Whey Protein Isolate Hydrolysate by Aqueous Two-Phase Extraction and Aqueous Two-Phase Flotation

At present, peptides are separated by molecular exclusion chromatography and liquid chromatography. A separation method is needed in any case, which can be scaled up for industrial scale. In this study, aqueous two-phase extraction (ATPE) and aqueous two-phase flotation (ATPF) were applied to separate and enrich antioxidant peptides from trypsin hydrolysates of whey protein isolates (WPI). The best experimental conditions were investigated, and the results were evaluated using the 2,2'-Azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS) free radical scavenging activity of the peptides-per-unit concentration and the recovery rate (gamma) of peptides in the top phase of both ATPE and ATPF. Under optimal conditions, the gamma and ABTS free radical scavenging activity per unit concentration in top phase of ATPE could reach 38.75% and 12.94%, respectively, and in ATPF could reach 11.71% and 29.18%, respectively. The purified peptides were characterized by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and reversed-phase high-performance liquid chromatography (RP-HPLC). PeptideCutter and PeptideMass were applied to analyze and calculate the peptide sequencing. KILDKVGINYWLAHK, VGINYWLAHKALCSEK, and TPEVDDEALEKFDKALK sequences having antioxidant activity were detected in the top phase of ATPE, and VGINYWLAHKALCSEK, KILLDKVGINYWLAHK, ILLDKVGINYVVLAHK, IIAEKTKIPAVFK, KIIAEKTKIPAVFK, and VYVEELKPTPEGDLEILLQK sequences having antioxidant activity were detected in the top phase of ATPF. In conclusion, antioxidant peptides were successfully separated from the WPI hydrolysate by ATPE and ATPF; compared with ATPE, ATPF has superior specificity in separating antioxidant peptides.