Separation of Antioxidant Peptides from Pepsin Hydrolysate of Whey Protein Isolate by ATPS of EOPO Co-polymer (UCON)/Phosphate

Abstract

An aqueous two-phase system (ATPS) consisting of poly(ethylene glycol-ran-propylene glycol) monobutyl ether (UCON)/phosphate was developed for the separation of the antioxidant peptides from pepsin hydrolysate of Whey Protein Isolate (WPI). The efficiency of the separation was evaluated based on the DPPH radical scavenging activity, ABTS radical scavenging activity and ferric reducing antioxidant power (FRAP) of the separated peptides. The effects of some parameters on the partition of antioxidant peptides were investigated. An efficient separation of antioxidant peptides was achieved using ATPS with pH of 4.0, 4 mL of UCON solution (40%, w/w), 4 mL of KH2PO4 solution (15.5%, w/w), 2 mL of WPI hydrolysate and 0.40 g/10 mL of NaCl. Reversed-phase high-performance liquid chromatography (RP-HPLC), amino acid analyzer and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) were used to characterize the purified peptides separated by the ATPS. The peptides in top phase were less polar than those in bottom phase. More antioxidative and hydrophobic amino acids were extracted to the top phase of ATPS, and the peptides with the amino acid sequences with antioxidant activities moved to the top phase as well. In conclusion, antioxidant peptides were successfully separated from the WPI hydrolysate by UCON/phosphate ATPS.

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

“…Xie et al discovered that the highly hydrophobic peptides from casein had excellent antioxidant activity [22]. Jiang et al reported that the highly hydrophobic and less polar peptides had excellent antioxidant activity [9]. Therefore this indicates that peptides from the bottom phase of ATPE had little antioxidant activity and peptides from the top phase of ATPE had excellent antioxidant activity.…”

“…Table 3 showed that the special VGINYWLAHKALCSEK, KILLDKVGINYWLAHK, ILLDKVGINYWLAHK, IIAEKTKIPAVFK, KIIAEKTKIPAVFK, and VYVEELKPTPEGDLEILLQK sequences found in the peptides of the top phase of ATPF contained the AHK, IPAVF [27], YVEEL [9] peptides, all of which have antioxidant activity.…”

“…After hydrolysis, the trypsin was deactivated in a boiling water bath for 10 min. The system was cooled to room temperature and centrifuged (4500x g, 30 min) with an ultrafiltration centrifuge tube (10 kDa MWCO) to obtain WPI tryptic peptides [9], which were stored at −20 °C for further analysis.…”

“…Advantages of ATPE include enhanced selectivity, scale-up, process integration, continuous operation, low toxicity and biocompatibility [8]. Jiang et al reported that the antioxidant peptides from the hydrolysate of WPI were separated using ATPE formed by a propylene oxide polymer (EOPO) co-polymer and phosphate [9]. Hence, purifying bioactive peptides from the mixed peptides by ATPE is achievable.…”

A Review on Health-Promoting, Biological, and Functional Aspects of Bioactive Peptides in Food Applications

“…The antioxidant activity of peptides can be influenced by amnio acid content and position within the chain. The amino acids histidine, tyrosine, lysine, phenylalanine, valine, and cysteine were significantly higher in whey protein-derived peptides than in WPI hydrolysate, suggesting that these amino acids are essential for antioxidant activity [ 58 ]. The hexapeptide YFYPEL is generated by pepsin hydrolysis of bovine casein and scavenges superoxide radicals [ 51 ].…”