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The chicken egg white, making up about 60% of fresh egg weight, is primarily composed of water (88%, w/w) and protein (10%, w/w). Along with eggshell and shell membrane, egg white forms the first line of protection to embryo (in egg yolk) from invading microorganisms, due to its gel-like property and the presence of various antimicrobial proteins. In addition to the antimicrobial activity, egg white proteins were also reported for their antioxidant, anti-viral, anticancer, immunomodulatory and protease inhibitory activities. The objectives of the present work were to identify low molecular peptides presenting naturally in egg white and to determine their potential antioxidant activity.
Unfertilized brown-shell eggs and white-shell eggs laid within 24 h from Hyline Brown hens and Dawu-Jinfeng hens were collected in the morning and used the same day in this study. Egg white peptides were extracted by adding 200 mL 80% ethanol was slowly into ~100 g homogenized egg white while stirring for 15 min (to avoid foaming) and kept overnight at 4 °C. Peptide concentration was measured, molecular weight was defined, peptide profiles were analyzed for peptide identification. Assays were performed in triplicate to determine the presence of antioxidant activity in the peptides.
Peptide samples were subjected to ESI-LTQ-Orbitrap mass spectrometer coupled with online nano-HPLC analysis. A total of 45 peptide sequences were identified from two egg white peptide fractions by using peptidomic analysis, 16 of which were the same, among which 21 and 8 were specific to white-shell and brown-shell eggs respectively. Peptide sequence length ranged from 10 to 31 amino acids, and the mass ranged from 1145.6 to 2923.4 Da. Peptide YPSKPDSPGEDAPAEDMAR, derived from egg white neuropeptide Y. Three most abundant peptides, peptide 7, peptide 6 and peptide 4, were derived from sulfhydryl oxidase 1, zona pellucida glycoprotein C (ZPC) and ovocleidin, respectively. Sulfhydryl oxidase 1 oxidizes sulfhydryl groups to disulfides with the reduction of oxygen to hydrogen peroxide, which involves in protein disulfide bond formation in protein synthesis. The nine peptides identified above were then synthesized to determine their antioxidant activity using H2O2-induced L6 cells. The ROS-mediated fluorescence was significantly enhanced by H2O2 stimulation, but was significantly reduced ranging from 19.4% to 27.8% by the treatment of peptides 1, 2, 3, 5, 7 and 8 at the concentration of 100 μM. Our results suggested that these peptides could provide protection against oxidative stress by increased levels of antioxidant enzymes. The peptides 2 and 8 contain a high proportion of hydrophobic amino acids (such as Ala, Gly, Ile) in the sequence, which may contribute to their ability. The result suggested that these peptides may have the potential as nutraceuticals or pharmaceuticals by abrogation of the superoxide-induced oxidative stress.
There is ongoing interest in identifying novel egg white proteins using proteomics; in comparison, there is a dearth of information in literature about the presence of bioactive peptides in egg white. The presence of Neuropeptide Y was identified for the first time in egg whites, and six of these peptides were found to have antioxidant properties through cellular antioxidant evaluation, providing evidence for the presence of naturally occurring antioxidant peptides in egg white; however, further study is also needed to understand the mechanisms of these antioxidant peptides. Our result suggested that peptides found in egg white may have the potential as nutraceuticals or pharmaceuticals by abrogation of the superoxide-induced oxidative stress.