Identification of peptides, metal binding and lipid peroxidation activities of HPLC fractions of hydrolyzed oat bran proteins

Abstract

The aim of this study was to evaluate metal binding and antioxidant activities of hydrolyzed oat bran proteins followed by the determination of peptide sequences. Protamex oat bran protein hydrolysates (OBPH) were separated by reverse-phase HPLC into eight peptide fractions (F1-F8) and their abilities to either chelate metals (Fe²⁺, Ca²⁺) or prevent the oxidation of lipids were investigated. In the Fe²⁺ chelation assay, OBPH had significantly (p < 0.05) higher activity (39.7 %) than the best performed fraction F7 (22.8 %). The second most active was F5 with 12.1 % chelating activity and this was higher than the activity of the tripeptide glutathione (5.8 %) used as control. The two most Fe²⁺ chelating fractions (F5, F7) however had weak calcium binding (0.6-1.0 %) properties at peptide concentration ranging from 0.2 to 1.0 mg/mL. In the lipid peroxidation assay, OBPH and all HPLC fractions prevented the oxidation of linoleic acid. More than 60 peptides mainly derived from globulin and avenin proteins were identified using tandem mass spectrometry.

Keywords: Calcium binding; Mass spectrometry; Metal chelation; Oat bran; Protein hydrolysate.

Fig. 1

HPLC chromatogram of hydrolyzed oat bran proteins on C18 column using a linear gradient of 95–5 % water containing 0.1 % acetic acid and methanol. Eight fractions (F1–F8) were collected as indicated

Fig. 2

Metal chelating of oat protein hydrolysate (DP) and its HPLC fractions (F2–F8) obtained using a linear gradient of water and methanol containing 1 % acetic acid. GSH: glutathione. Values are means of triplicates ± SD. Bars with same letters are not significantly different and bars with different letters are significantly different (p < 0.05)

Fig. 3

Inhibition of linoleic acid oxidation by oat protein hydrolysate and peptide fractions. F2–F8 were obtained from after HPLC separation on C18 using a linear gradient of water and methanol containing 1 % acetic acid. DP digested proteins (no separation), GSH glutathione, CTL control (linoleic acid without addition of peptides). Values are means of triplicates ± SD

Fig. 4

Total ion count chromatogram of fraction 5 from LC-qTOF mass spectrometry. Data were collected for 65 min. Signals are concentrated between 25 and 45 min

Fig. 5

TOF MS at 38.15 min. Full scan was performed from 400 to 1250 Da. Subsequence MS/MS analysis was used to determine the sequence of peaks at m/z 556.30, 592.82, 677.01 and 1015.51 as indicated on the figure. Other peaks were not assigned to peptides