Yu Seung-Woo, Kim Hyeong-Jin, Jang Jin-Hong, Kim Kee-Tae, Ahn Dong Uk, Paik Hyun-Dong
Dept. Food Science and Biotechnology of Animal Resources, Konkuk University, Neungdongro 120, Seoul 05029, Republic of Korea.
Dept. Food Science and Biotechnology of Animal Resources, Konkuk University, Neungdongro 120, Seoul 05029, Republic of Korea; WithBio, Inc., Neungdongro 120, Seoul 05029, Republic of Korea.
Enzyme Microb Technol. 2025 Oct;190:110710. doi: 10.1016/j.enzmictec.2025.110710. Epub 2025 Jul 9.
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.
卵类黏蛋白（OVM）通过蛋白水解酶进行水解，以增强对健康的免疫增强作用。本研究中使用菠萝蛋白酶、中性蛋白酶®、木瓜蛋白酶和胃蛋白酶对OVM进行水解，并在RAW 264.7巨噬细胞中评估了OVM水解产物的免疫刺激特性。在OVM水解产物中，经SDS-PAGE证实，胃蛋白酶水解产物（OMPH）产生了分子量为10 - 20 kDa的肽。在RAW 264.7巨噬细胞中，500μg/mL及以下的OVM水解产物表现出较高的细胞活力，超过80%。在Griess试验中，与其他OVM水解产物相比，OMPH产生了更高水平的一氧化氮（NO）。此外，OMPH以浓度依赖的方式上调诱导型一氧化氮合酶（iNOS）mRNA的表达。同样，OMPH使TNF-α和IL-6的表达增加。此外，OMPH激活了丝裂原活化蛋白激酶（MAPK）和核因子κB（NF-κB），p38、JNK、ERK、p65和IκB-α的磷酸化增加，特别是ERK和IκB-α的磷酸化水平较高。此外，与对照组相比，用OMPH处理的RAW 264.7巨噬细胞呈现出细胞形态增大和树突状形状。最后，通过液相色谱-串联质谱（LC-MS/MS）对OMPH进行分级分离，鉴定出9种肽，其中包括EGKDVLVCNK，该肽被确定具有免疫刺激特性。这些结果表明，OMPH可作为一种天然的生物功能成分，在功能性食品和制药行业中潜在地增强人体免疫力。
