Disruption of the intestinal barrier is closely associated with intestinal inflammation and disease development. Ovomucin is a bioactive protein in chicken egg white, and its hydrolysate has been revealed to prevent the adhesion of pathogen to intestinal epithelial cells. However, the effect of ovomucin hydrolysate on intestinal barrier integrity and associated anti- inflammation mechanism has not been demonstrated. The objectives of this study were to investigate the beneficial effects of ovomucin-protex 26L hydrolysate on intestinal barrier integrity and its anti-inflammatory activity in differentiated Caco-2 cells challenged with LPS (Lipopolysaccharide).
Fresh eggs from White Leghorns were collected and fresh egg white was mixed with 3 times volume of Milli-Q water, and stirred constantly for 2 h before the pH was adjusted to 5.0. Then the slurry was placed at 4°C for 24 h, followed by centrifugation at 15,344g for 10 min at 4°C and lyophilization. The ovomucin hydrolysate was prepared by the enzyme Protex 26L (Aspergillus niger origin, Genencor Division of Danisco) for 4 h at 50°C and pH 3.0 in a 1% (ovomucin/Milli-Q water, w/v) slurry. The enzyme was added at the ratio of 2% enzyme/substate, w/w). The process was conducted using a Titrando (842, Metrohm, Switzerland). The suspension was heated in a water bath at 95°C for 15 min, and was centrifuged at 15,344g for 20 min at 4°C after cooled down to room temperature on ice. Finally, the supernatant was obtained, lyophilized, and stored at −20°C for future use. Caco-2 cells were cultured in DMEM with 10% FBS, 1% NEAA, and 1% penicillin − streptomycin solution in a humidified incubator under 5% CO2 atmosphere at 37°C. Ovomucin hydrolysate was diluted in medium to the final concentration of 0.1, 0.5, and 1.0 mg/mL and incubated with differentiated Caco-2 cells for 24 h. Then samples were collected for different analysis.
Ovomucin-protex 26L hydrolysate inhibited endotoxic activity of LPS in a concentration-dependent manner. Ovomucin-protex 26L hydrolysate significantly increased transepithelial electronic resistance (TEER) values, decreased the paracellular FITC-dextran flux permeability, and recovered the expression of occludin and ZO-1 in LPS-stimulated Caco-2 cells. Meanwhile, ovomucin-protex 26L hydrolysate significantly inhibited the LPS-induced activation of NFkB
and MAPK pathways.
In conclusion, this study revealed that ovomucin-protex 26L hydrolysate showed LPS-neutralizing activity and reduced the TJ (tight junction) permeability, and that it mitigated inflammatory response induced by LPS likely via inhibiting the activation of the NF-kB and MAPK signaling pathways in Caco-2 cells. These data indicate the possible application of ovomucin hydrolysate as an intervention strategy to prevent inflammation in pathogenic infection. Further experiments
in animal models of pathogenic infection are necessary to advance our understanding of the activities of ovomucin hydrolysates and support the potential application.
Disruption of the intestinal barrier is closely associated with intestinal inflammation and disease development. Ovomucin is a bioactive protein in chicken egg white, and its hydrolysate has been revealed to prevent the adhesion of pathogen to intestinal epithelial cells. However, the effect of ovomucin hydrolysate on intestinal barrier integrity and associated anti-inflammation mechanism has not been demonstrated. Here, we investigated its activity in lipopolysaccharide (LPS)-induced monolayer dysfunction of differentiated Caco-2 cells to unravel the underlying molecular mechanisms. Ovomucin-protex 26L hydrolysate significantly increased transepithelial electronic resistance (TEER) values, decreased the paracellular FITC-dextran flux permeability, and recovered the expression of occludin and ZO-1 in LPS-stimulated Caco-2 cells. Meanwhile, ovomucin-protex 26L hydrolysate significantly inhibited the LPS-induced activation of NF-κB and MAPK pathways. These results suggested the ability of ovomucin-protex 26L hydrolysate to suppress LPS-induced intestinal barrier function damage via the inhibition of inflammatory responses.
