*Article* **Effects of Superheated Steam Treatment on the Allergenicity and Structure of Chicken Egg Ovomucoid**

**Ping-Wei Wen 1,2,3, Zong-Cai Tu 1,2,3,\*, Yue-Ming Hu <sup>3</sup> and Hui Wang <sup>3</sup>**


**Abstract:** The aim of this study was to explore the effects of an emerging and efficient heating technology, superheated steam (SS), on the allergenicity and molecular structure of ovomucoid (OVM). OVM was treated with 120–200 ◦C of SS for 2 to 10 min. The allergenicity (IgG/IgE binding abilities and cell degranulation assay) and molecular structure (main functional groups and amino acids modification) changes were investigated. The IgG-binding ability of OVM decreased and the releases of β-hex and TNF-γ were inhibited after SS treatment, indicating that the protein allergenicity was reduced. Significant increases in oxidation degree, free SH content and surface hydrophobicity were observed in SS-treated OVM. The protein dimer and trimer appeared after SS treatment. Meanwhile, obvious changes occurred in the primary structure. Specifically, serine can be readily modified by obtaining functional groups from other modification sites during SS treatment. Moreover, the natural OVM structure which showed resistance to trypsin digestion was disrupted, leading to increased protein digestibility. In conclusion, SS-induced OVM aggregation, functional groups and amino acids modifications as well as protein structure alteration led to reduced allergenicity and increased digestibility.

**Keywords:** ovomucoid; superheated steam; modification; allergenicity; mass spectrometry

#### **1. Introduction**

Egg is the main daily protein source for human beings, and also one of the major ingredients in the food industry [1]. However, egg allergy is common among infants and young children, and a high proportion of patients suffer the egg allergy throughout their lives [2]. Egg allergens are concentrated in egg white proteins, including ovomucoid (OVM), ovalbumin, ovotransferrin and lysozyme, among which, OVM is considered as the predominant component of egg allergens [3]. It is urgent to explore an effective and safe technology to reduce the allergenicity of eggs.

Previous studies have shown that heating, high pressure, glycation and hydrolysis can decrease the protein allergenicity [1,4,5]. Heat processing includes dry-heating and heatmoisture treatments. There are two drawbacks of traditional heating approaches for protein modification. One is that they often consume long periods of time, and the other is that the protein solubility is always decreased. Moreover, the traditional heating treatments are not drastic enough to alter specific sequences and conformation of allergenic epitopes and could not significantly decrease the protein allergenicity. Among the egg protein components, OVM showed the strongest heat resistance, and even 100 ◦C of heating for 60 min could not lead to the denaturation of OVM [6]. It is necessary to explore new high temperature heating methods to efficiently alter the OVM structure and modify/disrupt/bury the allergen epitopes of protein.

**Citation:** Wen, P.-W.; Tu, Z.-C.; Hu, Y.-M.; Wang, H. Effects of Superheated Steam Treatment on the Allergenicity and Structure of Chicken Egg Ovomucoid. *Foods* **2022**, *11*, 238. https://doi.org/10.3390/ foods11020238

Academic Editor: Jan Mei Soon

Received: 14 December 2021 Accepted: 12 January 2022 Published: 17 January 2022

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Superheated steam (SS) treatment is an emerging heating method and has attracted more and more attention for its advantages, including higher heat transfer efficiency, lowoxygen and pollution-free environment as well as good quality of processed products [7]. During SS processing, a large amount of heat is transferred to food when steam condenses on food surfaces, which rapidly increases the food temperature. The efficiency of SS has recently been demonstrated in several different food processing fields by dramatically reducing the processing time from days and h to min or even s [8]. SS-treated vegetables, grains, meats, etc. showed better functional and textural properties as well as higher nutritional qualities than those treated by conventional heating methods [9,10]. SS was also proven to improve the physicochemical and nutritional properties of biological macromolecules, including starch and protein, etc. [11,12]. Hu, Wang, and Li [7] reported that SS treatment could promote the covalent and non-covalent interactions as well as alter the conformation of wheat gluten. However, to date, the application of SS on the modification of natural structure and nutrition of food components is still lacking. Particularly, the effects of SS processing on allergenicity and the molecular structure of egg proteins have rarely been reported.

The objective of the study is to find a new, easily operated, efficient and safe technology to reduce the allergenicity of proteins. Our preliminary experiments showed that SS is an effective approach to modifying egg proteins and reducing the allergenicity. It is necessary to perform experiments to prove those effects.

Previous research demonstrated that egg proteins allergies are mainly originated from immunoglobulin G (IgG) and immunoglobulin E (IgE)-mediated rapid allergic reaction. In addition, macrophages are also important effector cells that mediate immune response [2]. The mediators released from basophils may be responsible for the occurrence of Type I allergic reactions. Therefore, in this study, the allergenicity changes of SS-treated OVM were revealed by the following experiments: (1) IgG and IgE binding abilities analysis through enzyme-linked immunosorbent assay (ELISA) with rabbit polyclonal antibodies and sera from patients allergic to egg, respectively. (2) Histamine, β-hexosaminidase (β-hex), TNF-γ, and interleukin-6 (IL-6) releases determination using a basophil cell line (KU812) model.

The protein allergenicity changes are reported related to the alteration in molecular structure in terms of primary structure, main functional groups and degree of protein aggregation [1–5]. Therefore, in this study, the changes in the surface hydrophobicity, free sulfhydryl (SH) content, molecular weight and amino acids modification of OVM were investigated to preliminary interpret the mechanism of SS-induced protein allergenicity alteration.
