Bioactive peptides are short amino acid chains liberated from parent proteins that can exert a wide range of activities, including antioxidant, anti-hypertensive, anti-diabetic and anti-inflammatory properties, which are beyond the nutritive value of the protein. Compared with peptides derived from animal proteins, such as egg, milk and fish, the peptides from plant proteins are much less explored. Therefore, the aim of this project was to investigate the biological properties of plant protein peptides using a range of methodologies, including in silico prediction of bioactive properties, in vitro testing of in silico predictions and the application of peptides to cell cultures.
Five oilseed proteins, flaxseed, rapeseed, sunflower, sesame and soybean were selected as peptide sources as well as two well-documented dairy proteins, whey and casein. Three hydrolysis conditions were applied to liberate peptides from these proteins, which were alcalase (pH 8.0) and pepsin (pH 1.3 and pH 2.1), respectively. Based on the frequency indices and potency indices of angiotensin converting enzyme (ACE) and dipeptidyl peptidase-IV (DPP-IV) inhibitory peptides, the in silico analysis predicted oilseed proteins as good sources of ACE inhibitory peptides, but weaker sources of DPP-IV inhibitory peptides. The average scores of the oilseed proteins aligned via the PeptideRanker indicated that oilseed proteins were potentially significant sources of bioactive peptides. When analyzed via Pepsite2, 65 novel peptides exerting ACE and/or DPP-IV inhibitory properties were predicted, based on the binding sites of peptides in ACE and DPP-IV.
The protein hydrolysates, together with their low molecular weight fractions (Mw < 3kDa), separated via ultrafiltration, were tested in several in vitro bioassays. These tests demonstrated that, apart from DPP-IV inhibitory activity, the Mw < 3 kDa fraction showed stronger bioactivity. Limited correlation was found between the in silico predictions and the in vitro findings. This discrepancy was thought to be largely caused by incomplete proteolysis and the lacking cover of protein sequence information in the available protein databases. Nevertheless, oilseed proteins, especially soybean, were confirmed as adequate sources in comparison with whey and casein for liberating ACE and α-glucosidase inhibitory peptides.
Further, anti-inflammatory properties of oilseed peptides were explored using cell culture models. The anti-inflammatory activities of protein hydrolysates and the three different Mw fractions (Mw > 10 kDa, 3-10 kDa and < 3 kDa) were initially screened via RAW BlueTM cells. The fractions with Mw < 3kDa of pepsin (pH 1.3)-treated protein hydrolysates were then selected to confirm their anti-inflammatory activities in RAW 264.7 macrophages. Results showed that rapeseed had the greatest potency to attenuate LPS-induced inflammation via downregulation of pro-inflammatory markers such as IL-6, IL-1β, iNOS and COX-2 and upregulation of anti-inflammatory IL-10, effects which were linked to the modulation of transcription factor NF-κB and supported by evidence for pre-receptor binding to LPS albeit these effects were much weaker as compared to whey and casein.
Taken together, the data indicate that peptides from oilseed proteins, especially rapeseed and soybean, demonstrate biological properties with a potential significance to beneficially affect chronic disease. The current findings should therefore encourage further research into the development of functional foods as well as nutraceutical and pharmaceutical applications.
