Advances in Understanding the Antioxidant and Antigenic Properties of Egg-Derived Peptides

Abstract

Pepsin, trypsin and proteinase K were used in the present study to hydrolyse the proteins from whole eggs, yolks or whites, and the resulting hydrolysates were characterised in terms of antioxidant and IgE-binding properties, using a combination of in vitro and in silico methods. Based on the degree of hydrolysis (DH) results, the egg yolk proteins are better substrates for all the tested enzymes (DH of 6.2-20.1%) compared to those from egg whites (DH of 2.0-4.4%). The SDS-PAGE analysis indicated that pepsin and proteinase K were more efficient compared to trypsin in breaking the intramolecular peptide bonds of the high molecular weight egg proteins. For all the tested substrates, enzyme-assisted hydrolysis resulted in a significant increase in antioxidant activity, suggesting that many bioactive peptides are encrypted in inactive forms in the parent proteins. The hydrolysates obtained with proteinase K exhibited the highest DPPH radical scavenging activity (124-311 µM Trolox/g protein) and the lowest residual IgE-binding capacity. The bioinformatics tools revealed that proteinase K is able to break the integrity of the main linear IgE-binding epitopes from ovalbumin and ovomucoid. It can be concluded that proteinase K is a promising tool for modulating the intrinsic properties of egg proteins.

Keywords: antigenic properties; antioxidant activity; egg proteins; hydrolysis degree; proteolytic enzymes.

Figure 2

The SDS-PAGE profile of a whole egg, egg yolk and egg white after 24 h of assisted hydrolysis with pepsin, trypsin and proteinase K. Lanes 1, 2 and 3—the patterns of undigested whole egg (WE), egg yolk (EY) and egg white (EW), respectively; λ—Dual Xtra Standard (Bio-Rad, Hercules, CA, USA); lanes 4, 5 and 6—WE, EY and EW digested with pepsin; lanes 7, 8 and 9—WE, EY and EW digested with trypsin; lanes 10, 11 and 12—WE, EY and EW digested with proteinase K; OVM—the profile of the commercial ovomucoid; OVA—the profile of the commercial ovalbumin. The arrows on lane 3 are used to mark the main egg white proteins: (a) ovotransferrin (MW of ~78 kDa; [24]), (b) ovalbumin (MW of ~43 kDa [24], comparable with the pattern developed by the commercial OVA), (c) ovomucoid (MW of 30–40 kDa [25] matching the pattern of the commercial OVM) and (d) lysozyme (MW of 14.3 kDa, [24]).

Figure 6

The IgE-binding epitopes of ovomucoid (represented in grey in New Cartoon style) reported by (a) Cooke and Sampson [42] (the epitopes (25–44), (73–74), (109–120), (139–146) and (175–186) are represented in orange, blue, magenta, cyan and red, respectively), (b) Besler et al. [43] (the epitopes (114–145) and (158–210) are represented in magenta and blue, respectively); (c) Holen et al. [44] (the epitopes (25–38), (35–48), (55–68), (75–88), (85–98), (125–138) and (145–158) are represented in orange, ice blue, cyan, magenta, blue, violet and red, respectively); (d) Mine and Zhang [45] (the epitopes (55–66), (64–74), (80–90), (95–99), (104–114), (125–129), (145–154) and (183–198) are represented in red, blue orange, magenta, ice blue, cyan, pink and green, respectively); (e) Jarvinen [46] (the epitopes (25–34), (35–44), (71–80) and (137–146) are represented in blue, magenta, red and orange, respectively); (f) Martínez-Botas et al. [41] (the epitopes (28–44), (70–83) and (115–128) are represented in blue, magenta and pink, respectively).

Figure 1

Comparison of the degree of hydrolysis (DH) of the whole egg (WE), egg yolk (EY) and egg white (EW) after 24 h of assisted hydrolysis with pepsin, trypsin and proteinase K. For an egg derivative (WE, EY and EW), different letters (a, b, c) assigned to the mean values indicate significant differences among enzyme treatments, at p < 0.05, based on Tukey post-hoc test and 95% confidence.

Figure 3

Influence of hydrolysis with pepsin, trypsin and proteinase K on the DPPH radical scavenging (DPPH-RSA) activity (a) and Trolox equivalent antioxidant capacity (TEAC) (b) of a whole egg (WE), egg yolk (EY) and egg white (EW). Controls were prepared with no enzyme addition. For an egg derivative (WE, EY and EW), different letters (a, b, c, d) assigned to the mean values indicate significant differences among enzyme treatments, at p < 0.05, based on Tukey post-hoc test, and 95% confidence.

Figure 4

The residual IgE-binding capacity of the protein hydrolysates obtained through the extensive hydrolysis of the whole egg, egg yolk and egg white with pepsin, trypsin and proteinase K. For an egg derivative (WE, EY and EW), different letters (a, b, c) assigned to the mean values indicate significant differences among enzyme treatments, at p < 0.05, based on Tukey post-hoc test, and 95% confidence.

Figure 5

The IgE-binding epitopes of ovalbumin (represented in grey in New Cartoon style) reported in human patients by (a) Mine and Rupa [36] (the epitopes (39–50), (96–103), (192–201), (244–249) and (252–261) are represented in red, orange, cyan, purple and blue, respectively), (b) Elsayed et al. [37] (the (2–11) epitope is represented in magenta) and Elsayed and Stavseng [38] (the (12–20) epitope is represented in blue); (c) Kahlert et al. [39] (the epitopes (42–173) and (302–386) are represented in magenta and blue, respectively); (d) Honma et al. [40] (the (358–367) epitope is represented in blue).