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Ovomucoid hydrolysates produced by pepsin stimulate immune activity of RAW 264.7 macrophages via the MAPK/NF-κB
Seung-Woo Yu1, Hyeong-Jin Kim1, Jin-Hong Jang1
1Dept. Food Science and Biotechnology of Animal Resources, Konkuk University, Neungdongro 120, Seoul 05029, Republic of Korea.
Abstract:
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.
Insights
Pepsin-hydrolyzed ovomucoid (OVM) peptides show significant immune-enhancing effects. This study found pepsin hydrolysate (OMPH) boosts nitric oxide and cytokine production in macrophages, suggesting its use as a functional ingredient.
Area of Science:
- Food Science
- Immunology
- Biochemistry
Background:
- Ovomucoid (OVM) is a glycoprotein with potential health benefits.
- Hydrolysis of OVM can yield bioactive peptides with enhanced properties.
- Understanding the immunomodulatory effects of OVM hydrolysates is crucial for developing functional foods and pharmaceuticals.
Purpose of the Study:
- To investigate the immune-enhancing effects of OVM hydrolysates produced by different proteolytic enzymes.
- To evaluate the specific immunomodulatory activities of pepsin hydrolysate (OMPH) in RAW 264.7 macrophages.
- To identify bioactive peptides within OMPH responsible for its immunostimulatory properties.
Main Methods:
- OVM was hydrolyzed using bromelain, Neutrase®, papain, and pepsin.
- Hydrolysates were analyzed for peptide molecular weight using SDS-PAGE.
- Immunostimulatory properties were assessed in RAW 264.7 macrophages, including cell viability, nitric oxide (NO) production (Griess assay), iNOS, TNF-α, and IL-6 mRNA expression.
- MAPK and NF-κB signaling pathways were investigated via Western blotting.
- Peptide identification was performed using LC-MS/MS.
Main Results:
- Pepsin hydrolysate (OMPH) yielded peptides in the 10-20 kDa range.
- OMPH demonstrated high cell viability (>80%) at concentrations ≤ 500 μg/mL.
- OMPH significantly increased NO production, upregulated iNOS, TNF-α, and IL-6 mRNA expression.
- OMPH activated MAPK and NF-κB pathways, evidenced by increased phosphorylation of p38, JNK, ERK, p65, and IκB-α.
- Morphological changes in macrophages (enlargement, dendritic shape) were observed after OMPH treatment.
- LC-MS/MS identified nine peptides in OMPH, including EGKDVLVCNK, with immunostimulatory properties.
Conclusions:
- Pepsin hydrolysis effectively generates OVM peptides with potent immunostimulatory activities.
- OMPH exhibits significant immunomodulatory effects by enhancing NO and cytokine production and activating key signaling pathways.
- OMPH contains bioactive peptides, such as EGKDVLVCNK, contributing to its immune-enhancing capabilities.
- OMPH is a promising natural bio-functional ingredient for the food and pharmaceutical industries to boost human immunity.
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