Generation and characterization of bioactive peptides from flaxseed (Linum usitatissimum L.) proteins

The potential of flaxseed (Linum usitatissimum L.) protein to release bioactive peptides upon enzymatic hydrolysis was evaluated. Flaxseed protein released angiotensin I-converting enzyme inhibitory (ACEI) peptides during in vitro simulated gastrointestinal (GI) digestion in a static (no removal of digested products) and a dynamic model (removal of <1 kDa molecules in the intestinal phase). The ACEI activity of the gastric plus intestinal digest (absorbed fraction-IC50: 0.04 mg N/mL; retained fraction-IC50: 0.05 mg N/mL; degree of hydrolysis, DH: 46.78 %) of the dynamic model was significantly higher (P<0.05) than that of the static model (IC50: 0.39 mg N/mL; DH: 43.95 %). Polypeptides of 48, 41, 29 and 20 kDa could be releasing these ACEI peptides. Six peptides in the highest ACEI fraction (0.5-1 kDa) of the absorbable gastric plus intestinal digest were identified via de novo sequencing. Only digests of the static model exhibited hydroxyl radical (OH ·) scavenging activity (IC50: 0.40 mg N/mL), suggesting the inappropriateness of such models in this type of research. Presence of mucilage and oil interfered with the in vitro digestibility of flaxseed protein, which could limit the release of ACEI peptides during GI digestion. The protein digestibility of milled whole flaxseed (12.61 %) was significantly improved (P<0.05) with the removal of mucilage (51.00 %) and oil together with mucilage (66.79 %). The digestibility of isolated flaxseed protein was 68.00 %.;Flaxseed protein, hydrolyzed (DH: 11.94-70.62 %) with Flavourzyme RTM in a central composite rotatable design, possessed bioactivities with identified optimum enzyme/substrate and time of hydrolysis combinations, including ACEI activity (71.59-88.29 %, 83.7 LAPU/g protein, 19.9 h), scavenging of OH· (12.48-22.08 %, 30.2 LAPU/ g protein, 1.5 h) and superoxide radical (O2·-) (26.33-39.41 %, 4.9 LAPU/ g protein, 16.3 h) and inhibiting linoleic acid oxidation (0.71-94.33 %, 1.6 LAPU/ g protein, 12.6 h). The degradation pattern of polypeptides during enzymatic hydrolysis indicated that 48 and 13 kDa molecules could be releasing these bioactive peptides. De novo sequencing identified two ACEI and five OH· scavenging peptides in the hydrolysate fractions (0.5-1.05 kDa) with the highest bioactivities. The findings suggest the importance of flaxseed protein as a source of cardioprotective bioactive peptides.