Characterisation of storage proteins and their digesta from oat bran, wheat bran and barley, and identification of bioactive peptides derived from oat bran : A thesis submitted in partial fulfilment of the requirements for the Degree of Doctor of Philosophy at Lincoln University

Aims: The aims of this thesis were to characterise storage proteins extracted from oat bran, wheat bran and barley and the digesta produced from them in an in vitro digestion model, followed by investigating the antioxidant and angiotensin-converting enzyme (ACE) inhibitory activity of the digesta, and finally to identify novel peptides with antioxidant and ACE inhibitory activity in oat bran intestinal digesta. Method: Storage protein concentrates of oat bran, wheat bran and barley were obtained by alkaline extraction and isoelectric precipitation. In vitro digestion model was adapted to generate their digesta protein where their protein/peptide profiles were analysed using sodium dodecyl-sulfate- polyacrylamide gel electrophoresis (SDS-PAGE). The digestibility, degree of hydrolysis and the release of the free amino acids (FAAs) were determined on these digesta. Antioxidant assays including 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid (ABTS), total phenolic content (TPC), ferric ion reducing antioxidant power (FRAP) assay and oxygen radical absorbance capacity (ORAC) assay were conducted to evaluate the overall antioxidant activity of the crude protein and digesta. The samples were also assessed for ACE inhibitory activity.The storage proteins in oat bran were selected for further analysis by two-dimensional electrophoresis (2-D electrophoresis). The small-intestinal digesta of oat bran protein with the molecular weight (MW) of < 3 kDa was purified by anion-exchange chromatography and the selected fractions were subjected to mass spectrometry (MS) to determine the peptide sequences. Findings: The major storage protein in oat bran protein concentrate were globulin, albumin and avenins, while prolamin, gliadin and albumin predominated in wheat bran protein concentrate, and hordein, albumin and globulin in the barley protein concentrate. These proteins were mostly degraded into polypeptides and smaller protein fractions by the end of intestinal digestion. This process resulted in significantly higher overall antioxidant activity and ACE inhibitory power in digesta. Oat gastric and intestinal digesta showed the highest ABTS scavenging activity of 2 Trolox equivalent mM/g sample. Oat digesta also showed a higher TPC value (200 gallic acid equivalent mg/g sample) compared to wheat bran and barley digesta (150 gallic acid equivalent mg/g sample and 40 gallic acid equivalent mg/g sample). The ACE inhibitory power of all three intestinal digesta was greater than 80%. Because oat digesta showed the highest overall antioxidat and ACE inhibitory activity, oat intestinal digesta was selected for separation by anion exchange chromatography. In all elutes, the highest ABTS scavenging activity reached to 275 Trolox equivalent µM/g sample and the highest ORAC scavenging activity was 170 Trolox equivalent µM/g sample. Four antioxidant anionic fractions and two ACE inhibitory fractions along with the digesta < 3 kDa were subjected to reverse-phase high performance liquid chromatography-tandem mass spectrometry (R-HPLC-MS/MS). Peptides with antioxidant and ACE inhibitory potentials were identified. Conclusion: These investigations have shown that storage proteins derived from oat bran, wheat bran and wholegrain barley are ideal sources to produce bioactive digesta after digestion. The peptides identified in oat bran intestinal digesta displays bioactive nature as an intermediate ACE inhibitor, with the potential of future applications in improving human health.