Ovomucoid hydrolysates produced by pepsin stimulate immune activity of RAW 264.7 macrophages via the MAPK/NF-κB pathway

Ovomucoid (OVM) was hydrolyzed by proteolytic enzymes to increase immune-enhancing effects for health. Bromelain, Neutrase®, papain, and pepsin were used to hydrolysis and immunostimulatory properties of OVM hydrolysates were evaluated in RAW 264.7 macrophages in this study. Among the OVM hydrolysates, pepsin hydrolysate (OMPH) produced peptides with 10–20 kDa molecular weight, as confirmed by SDS-PAGE. In RAW 264.7 macrophages, OVM hydrolysates at 500 μg/mL or less exhibited high cell viability, exceeding 80 %. In the Griess assay, OMPH produced higher levels of nitric oxide (NO) compared to other OVM hydrolysates. Additionally, OMPH upregulated inducible nitric oxide synthase (iNOS) mRNA expression in a concentration-dependent manner. Similarly, the expression of TNF-α and IL-6 was increased by OMPH. Furthermore, OMPH activated mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-κB), with increased phosphorylation of p38, JNK, ERK, p65, and IκB-α, in particular exhibiting high ERK and IκB-α phosphorylation levels. Furthermore, RAW 264.7 macrophages treated with OMPH exhibited an enlarged cell morphology and a dendritic-like shape compared to the control. Finally, liquid chromatography-tandem mass spectrometry (LC-MS/MS) fractionation of OMPH identified nine peptides, including the EGKDVLVCNK, which was determined to be immunostimulatory properties. These results suggest that OMPH can be used as a natural bio-functional ingredient for potential enhancing human immunity in both the functional food and pharmaceutical industries.