Abstract/Summary

Research in the production of anti-hypertensive activity demonstrates that several milk/whey derived peptides possess high in vitro angiotensin I-converting enzyme (ACE) inhibitory activity. However, in some cases, poor correlation between the in vitro ACE inhibitory activity and the in vivo anti-hypertensive activity has been observed. The main aim of this study is to investigate whether molecular docking can be used as an effective prediction tool to identify milk protein derived angiotensin I-converting enzyme (ACE) inhibitors, prior to testing the candidate peptides in vivo. Firstly, a molecular docking method was developed to elucidate the structure-activity relationship of peptide sequences present in whey/milk protein hydrolysates with previously reported high in vitro ACE inhibitory activities (Chapter Two). Main amino acid residues at the binding site of the human ACE formed strong hydrogen bonds with whey-derived peptide sequences IPP, LIVTQ, IIAE, LVYPFP, in common with anti-hypertensive drugs such as Sampatrilat, Captopril, Lisinopril and Elanapril. The molecular docking results indicate that these natural peptides may be potent ACE inhibitors. The developed molecular docking method was then employed to investigate the molecular interactions between specific amino acid residues at the binding site of human angiotensin II-converting enzyme (ACE2), and the whey-protein derived peptide sequences (Chapter Three). IPP, LIVTQ, IIAE, and LVYPFP all formed strong hydrogen bonds and salt bridge interactions with key residues in the active site of human ACE2, in common with known potent pharmaceutical ACE2 inhibitors. Therefore, IPP, IIAE, LIVTQ, and LVYPFP are suggested as potential candidates to be used in the treatment of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) via inhibition of the host cell receptor ACE2. Also, in contrast with well-known anti-hypertensive drugs, these peptides have a dual inhibitory action against both ACE and ACE2, and are natural and dietary derived, which may represent advantages in the treatment of coronavirus disease 2019 (COVID-19). To further investigate whether molecular docking can be used as a reliable pre-screening tool to identify milk protein-derived bioactive peptides and predict their ACE inhibitory activities, whey protein isolate was subjected to a simulated gastric digestion procedure that mimics the chemical and physiological conditions of the gastro-intestinal tract to process whey proteins. Whey proteinderived hydrolysates were pre-purified by solid phase extraction, and the various hydrolysate fractions were then characterised in terms of their in vitro ACE inhibitory activities. The chemical characterisation of each fraction was carried out using mass spectrometry (MS) to identify the main peptides contributing to the ACE inhibitory activity measured in each fraction. Some novel sequences were identified, such as AIPPK, TPEVDDE, PFPGPI, and VELLKHKP. To gain insight into which peptide sequences are most likely contributing to the ACE inhibitory activities measured in the various hydrolysate fractions, the whey protein-derived peptide sequences were then docked into the binding site of human ACE. The docking results, together with comparisons with ACE inhibitory drugs, provided strong evidence for the ACE inhibitory activities of whey protein-derived peptides AIPPK, PFPGPI, TPEVDDE and VELLKHKP. Finally, the results obtained indicate that experimental measurement of ACE inhibitory activities and docking results of AIPPK, PFPGPI, TPEVDDE and VELLKHKP are in agreement. This shows the potential effectiveness of molecular docking as a prescreening tool to identify milk protein-derived bioactive peptides. Further research is needed to assess the in vivo anti-hypertensive effects of whey protein-derived bioactive peptides.