In Vitro Acetylcholinesterase-Inhibitory Properties of Enzymatic Hemp Seed Protein Hydrolysates

The aim of this work was to characterize the structural and functional properties of hemp seed protein-derived acetylcholinesterase (AChE)-inhibitory enzymatic hydrolysates. Hemp seed protein isolate hydrolysis was performed using six different proteases (pepsin, papain, thermoase, flavourzyme, alcalase and pepsin + pancreatin) at different concentrations (1-4 %). The degree of hydrolysis was directly related to the amount of protease used but had no relationship with AChE-inhibitory activity. Amino acid composition results showed that the hemp seed protein hydrolysates (HPHs) had high levels of negatively charged amino acids (39.62-40.18 %) as well as arginine. The 1 % pepsin HPH was the most active AChE inhibitor with similar to 6 A mu g/mL IC50 value when compared to 8-11.6 A mu g/mL for the other HPHs. Mass spectrometry analysis showed that most of the peptides in all the hydrolysates were less than 1000 Da in size. However, the pepsin HPHs contained larger-sized peptides (244-1009 Da) than the papain HPHs (246-758 Da), which in turn was larger than the alcalase HPH (246-607 Da). The higher AChE-inhibitory effects of the pepsin HPHs may be due to increased synergistic effects from a wider peptide size range when compared to the papain and alcalase HPHs that had narrower ranges. The narrow peptide size range in the alcalase HPH confirms the higher efficiency of this protease in releasing small-sized peptides from food proteins.