Production of egg yolk phosphopeptides and the cellular mechanism of intestinal oxidative stress reduction
Oxidative stress in the gastrointestinal tract has been implicated in the initiation and propagation of inflammatory bowel diseases and other chronic intestinal pathologies. Antioxidants have limited efficacy and are sometimes pro-oxidant. An alternative, novel approach lies in the upregulation of endogenous antioxidative stress mechanisms which can combat oxidative stress and re-establish a stable cellular redox balance. Hen egg yolk-derived phosvitin phosphopeptides have been shown to exert this biological activity; however the preparation of these peptides was not suited to large-scale production. The goal of this work was to prepare bioactive peptides from delipidated egg yolk proteins with industry scale-up potential. In addition, the antioxidative stress activity of the peptides was evaluated, the bioactive fraction was identified and characterized, and the cell signaling pathways involved in influencing bioactivity was elucidated. Egg yolk peptides (EYP) were produced by Alcalase and Protease N digestion of defatted egg yolk proteins. This hydrolysate contained 1.00 ± 0.02% phosphate and significantly reduced (P<0.001) the oxidative stress biomarker, interleukin-8 (IL-8), in hydrogen peroxide-stimulated Caco-2 intestinal cells. EYP significantly (P<0.05) increased glutathione (GSH) and [gamma]-glutamylcysteine synthetase activity and mRNA expression, elevated the activities of antioxidant enzymes (superoxide dismutase, catalase, glutathione reductase, glutathione S-transferase), and lowered malondialdehyde and protein carbonyl concentrations in the duodenum, jejunum, ileum, and colon of piglets subjected to oxidative stress. GSH concentrations in red blood cells were also significantly (P<0.05) elevated in EYP-supplemented animals. Anion separation of EYP resulted in 4 fractions with the last fraction, EYP-F4, having 0.72 ± 0.06% phosphate. EYP-F4 significantly decreased (P<0.001) IL-8 in H202-stimulated cells and this bioactivity was maintained after pepsin and pancreatin digestion of EYP-F4 but not after dephosphorylation. Serine and phosphoserine did not reduce oxidative stress, but [Ser(PO3)]2 and [Ser(PO3)]3 significantly reduced (P<0.001) IL-8. Using mass spectrometry analysis, one of the peptides in the EYP bioactive fraction was identified as SKDSSSSSSKSNSK. This identified peptide is found in the phosvitin portion of vitellogenin-1, a yolk precursor protein. These results suggest a minimum of two contiguous phosphoserines in EYP-F4 peptides influence bioactivity. EYP-F4 and [Ser(PO3)]2 cell treatments increased the mRNA expression of antioxidant genes including glutathione reductase and glutathione peroxidase, and lowered the expression of pro-inflammatory genes such as myeloperoxidase and lactoperoxidase in an oxidative stress and antioxidant defense microarray. It is likely that EYP-F4 and [Ser(PO3)]2 increased antioxidant gene expression by activating the nuclear factor-E2-related factor 2-antioxidant response element pathway and decreased inflammatory gene expression by inhibiting the nuclear factor kappa B pathway. Egg yolk phosphopeptides influence the activation of protective antioxidative stress mechanisms which contribute to the prevention of intestinal oxidative stress.