Identification of short peptide sequences in the nanofiltration permeate of a bioactive whey protein hydrolysate

Short peptides in food protein hydrolysates are of significant interest as they may be highly bioactive whilst also being bioavailable. A dipeptidyl peptidase IV (DPP-IV) inhibitory whey protein hydrolysate (WPH) was fractionated using nanofiltration (NF) with a 200Da MWCO membrane. The DPP-IV half maximal inhibitory concentration of the NF permeate (IC₅₀=0.66±0.08mgprotein equivalentmL^-1) was significantly more potent (P>0.05) than that of the starting WPH (IC₅₀=0.94±0.24mgprotein equivalentmL^-1) and associated retentate (IC₅₀=0.82±0.13mgprotein equivalentmL^-1). This confirmed the contribution of short peptides within the NF permeate to the overall DPP-IV inhibitory activity. An hydrophilic interaction liquid chromatography (HILIC-) and reverse-phase (RP-) liquid chromatography tandem mass spectrometry (LC-MS/MS) strategy, based on two retention time models, allowed detection of eight free amino acids and eight di- to tetrapeptides in the NF permeate. The potential sequences of the peptides within the NF permeate were then ranked on the basis of their highest probability of occurrence. A confirmatory study with synthetic peptides showed that valine-alanine (VA), valine-leucine (VL), tryptophan-leucine (WL) and tryptophan-isoleucine (WI) displayed DPP-IV IC₅₀ values <170μM. The NF and LC-MS strategies employed herein represent a new approach for the targeted identification of short peptides within bioactive food protein hydrolysates.