Pulsed Electric Field-Assisted Alcalase Treatment Reduces the Allergenicity and Eliminates the Antigenic Epitopes of Ovomucoid

Abstract

Physically assisted chemical modifications can effectively reduce the allergenicity of ovomucoid (OVM). However, only a few studies have used pulsed electric field (PEF)-assisted alcalase hydrolysis to reduce the allergenicity of OVM. Herein, we investigated the effect of PEF-assisted alcalase treatment on the spatial conformation, allergenicity, and antigenic epitopes of OVM based on multispectroscopic analyses, bioinformatics, and mass spectrometry. The results showed that PEF-assisted alcalase treatment promoted the hydrolysis of OVM; moreover, the α-helix content and surface hydrophobicity of OVM significantly decreased, which disordered its spatial conformation and weakened its intermolecular interactions. Additionally, enzyme-linked immunosorbent assay (ELISA) results showed that the PEF-assisted alcalase treatment significantly reduced the binding levels of IgE and IgG1, which were 47.66 and 36.41%, respectively. Finally, eight epitopes of OVM were obtained by immunoinformatic tools. Nano-high performance liquid chromatography coupled to tandem mass spectrometry (nano-HPLC MS/MS) results showed that the hydrolysate of OVM and alcalase (HOVM) had nine more peptide-containing epitopes than the hydrolysate of PEF-treated OVM and PEF-treated alcalase (HOVM-PP′), indicating that PEF could promote the elimination of linear epitopes in OVM, thereby reducing OVM allergenicity.

Mechanism of Targeted Regulation of Ovalbumin Epitopes by Pulsed Electric Field-Assisted Alcalase Treatment

“…35 H 0 , which was a measure of the hydrophobic amino acid residues on the surface of a protein connected to the polar aqueous area, was another important parameter for characterizing the tertiary structure. 18 The influence of PEF-assisted Alcalase hydrolysis on the H 0 value of OVA samples was depicted in Figure 3D. The H 0 value of HOVA was 6.62 times that of OVA, indicating that Alcalase hydrolysis broke the peptide bonds in OVA and exposed numerous hydrophobic groups.…”

“…H 0 , which was a measure of the hydrophobic amino acid residues on the surface of a protein connected to the polar aqueous area, was another important parameter for characterizing the tertiary structure . The influence of PEF-assisted Alcalase hydrolysis on the H 0 value of OVA samples was depicted in Figure D.…”

“…Xue et al found that hydrolysis of Alcalase and papain significantly reduced the binding level of specific IgE and histamine concentration by regulating the secondary and tertiary structure of glutenin, improved the Treg/Th17 imbalance caused by allergy, and significantly eliminated glutenin-induced allergic reactions . Our previous study have shown that PEF-assisted Alcalase treatment significantly eliminated OVM-induced allergic reactions by disrupting conformational and linear epitopes of allergens . However, there are significant differences in the amino acid composition and sequence of the four major allergens in egg white, resulting in spatial conformation and the degree of allergic reaction.…”

“…25 Nano-HPLC MS/MS Analysis. Based on the method of Li et al, 18 the influence of PEF-assisted Alcalase treatment on the linear epitopes of allergen OVA was determined, and the mechanism of the regulation of potential allergenicity by PEF-assisted Alcalase treatment was revealed.…”

“…17 Our previous study have shown that PEFassisted Alcalase treatment significantly eliminated OVMinduced allergic reactions by disrupting conformational and linear epitopes of allergens. 18 However, there are significant differences in the amino acid composition and sequence of the four major allergens in egg white, resulting in spatial conformation and the degree of allergic reaction. Therefore, in this study, OVA, which is the most abundant in egg white, was used as the research object to reveal the mechanism of PEF-assisted Alcalase treatment to inhibit OVA-induced allergic reactions through serology, spectroscopy, bioinformatics, and mass spectrometry.…”