Use of Different Proteases to Obtain Flaxseed Protein Hydrolysates with Antioxidant Activity

Abstract

The antioxidant activity of flaxseed protein hydrolysates obtained using five different enzymes was evaluated. Proteins were isolated from flaxseed cake and were separately treated with papain, trypsin, pancreatin, Alcalase and Flavourzyme. The degree of hydrolysis (DH) was determined as the percentage of cleaved peptide bonds using a spectrophotometric method with o-phthaldialdehyde. The distribution of the molecular weights (MW) of the hydrolysis products was profiled using Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Tricine-SDS-PAGE) and size exclusion-high performance liquid chromatography (SE-HPLC) separations. The antioxidant activities of the protein isolate and hydrolysates were probed for their radical scavenging activity using 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonate) radical cation (ABTS(•+)) and photochemiluminescence (PCL-ACL) assays, and for their ferric reducing antioxidant power (FRAP) and ability to bind Fe(2+). The hydrolysates were more effective as antioxidants than the protein isolate in all systems. The PCL-ACL values of the hydrolysates ranged from 7.2 to 35.7 μmol Trolox/g. Both the FRAP and ABTS(•+) scavenging activity differed among the hydrolysates to a lower extent, with the ranges of 0.20-0.24 mmol Fe(2+)/g and 0.17-0.22 mmol Trolox/g, respectively. The highest chelating activity (71.5%) was noted for the pancreatin hydrolysate. In general, the hydrolysates obtained using Alcalase and pancreatin had the highest antioxidant activity, even though their DH (15.4% and 29.3%, respectively) and the MW profiles of the peptides varied substantially. The O₂(•-) scavenging activity and the ability to chelate Fe(2+) of the Flavourzyme hydrolysate were lower than those of the Alcalase and pancreatin hydrolysates. Papain was the least effective in releasing the peptides with antioxidant activity. The study showed that the type of enzyme used for flaxseed protein hydrolysis determines the antioxidant activity of the hydrolysates.

Keywords: antioxidant activity; enzymatic hydrolysis; flaxseed cake; proteases; protein hydrolysates.

Figure 1

Degree of hydrolysis (DH) of flaxseed protein hydrolysates.

Figure 2

Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Tricine-SDS-PAGE) separation of flaxseed protein isolate and its hydrolysates. MW standard: molecular weight standard.

Figure 3

Size exclusion-high performance liquid chromatography (SE-HPLC) chromatograms of flaxseed protein isolate and its hydrolysates.

Figure 4

Antiradical activity of flaxseed protein isolate and its hydrolysates towards (a) 2,2′- azino-bis-(3-ethylbenzothiazoline-6-sulfonate) radical cations (ABTS^•+) and (b) superoxide radical anions (O₂^•−) determined by a photochemiluminescence assay. The bars with different letters (a–e) are significantly different at p < 0.05.

Figure 5

(a) Ferric reducing antioxidant activity (FRAP) and (b) ability to chelate Fe²⁺ of flaxseed protein isolate and its hydrolysates. The bars with different letters (a–f) are significantly different at p < 0.05.