**3. Results and Discussion**

#### *3.1. IgG and IgE Binding Abilities*

The IgG and IgE binding abilities of OVM were estimated through indirect competitive ELISA with sera from rabbit and EWA patients, respectively. As shown in Figure 1A, the IgG binding rate of SS-treated OVM samples significantly decreased with the increase of SS temperature and processing time. The IgG binding rate was markedly influenced by high temperatures and long periods of treatments, with a maximum decline to 28% when treated at 200 ◦C for 10 min. The reduction in the IgG-binding ability of OVM may be due to the structural changes derived from amino acid modification and denaturation [15]. The results implied that SS-created extreme high temperatures of up to 200 ◦C might influence the protein structure and destroy IgG allergenicity epitopes.

**Figure 1.** IgG (**A**) and IgE (**B**) binding rates of OVM treated by SS at different temperatures and times.

However, there were no obvious changes in the IgE binding rate of OVM samples treated by SS, except that at 200 ◦C for 8–10 min (Figure 1B). The results were in accordance with the research of Martos, Lopez-Exposito, Bencharitiwong, Berin, and Nowak-Wegrzyn [22], who reported that heat-treated OVM did not induce the symptoms of anaphylaxis in sensitized mice when administered orally. However, being heated by boiling water for 30 min did not completely destroy the IgE binding capacity of OVM. Native and SStreated OVM samples at 120–180 ◦C for 2–10 min and 200 ◦C for 2–6 min showed high IgE

binding rates, suggesting the persistence of linear epitopes recognized by IgE. This could be caused by the fact that OVM possesses high thermal stability and limited denaturation as the structure is made up of nine disulfide bonds and 25% carbohydrate [23]. Interestingly, when the heating temperature reached 200 ◦C and was maintained for 8–10 min, significant reductions of the IgE binding rate of OVM were observed, declining to 60% of that of native OVM when treated for 10 min. Therefore, it was confirmed that high heating temperatures could alter the OVM structure more and reduce the content of the IgE epitope in comparison with lower temperatures. In addition, macrophages are also important effector cells that mediate the immune response. The mediators released from basophils may be responsible for the occurrence of Type I allergic reactions. Therefore, the IgE-mediated allergic response was evaluated using a KU812 cell model in the subsequent experiment.

#### *3.2. Effects of OVM on the Viability and Degranulation of IgE Sensitized KU812 Cells*

To analyze the potential cytotoxic activities of the tested substances, KU812 cells were incubated for 24 h with increasing concentrations of OVM treated by SS at different temperatures and periods. As shown in Figure 2, compared with the control sample, the absorbance (OD450) of cells increased after SS treatment, suggesting that SS heating can promote KU812 cells proliferation. Generally, the absorbance (OD450) of cells increased with the increase of OVM concentration and SS processing period. These suggested that a higher OVM concentration and longer SS processing period promoted the KU812 cells proliferation.

**Figure 2.** Effects of SS-treated OVM at different temperatures and times on the cell viability (**A**–**E**) of the KU812 cells sensitized with sera IgE from patients allergic to egg.

The degranulation rate of basophils and mast cells are key factors that determine the immunoreactivity of allergens. In this work, the effects of SS treatment on the release of β-hex, TNF-γ, histamine and IL-6 in KU812 cells are shown in Figure 3. The varying concentrations of 4 types of I cytokines indicated that SS treatment on OVM affected allergic response at the molecular level [24].

**Figure 3.** Effects of SS-treated OVM at different temperatures and times on the degranulation of KU812 cells sensitized with sera IgE from patients allergic to egg (**A**), the release of β-hex; (**B**), the release of TNF-γ; (**C**), the release of histamine; (**D**), the release of IL-6).

Figure 3A–C showed that SS treatment inhibited the release of β-hex and TNF-γ while promoting the release of histamine from KU812 cells generally. β-hex is an indicator of mast cell degranulation in regard to OVM allergenicity. The release of β-hex was decreased to a minimum of 36% for SS-treated OVM at 180–200 ◦C for 2–10 min, suggesting that SS treatment at high temperatures could lower the basophil degranulation. This might be due to the cross-linking and aggregation of OVM blocking the interaction between IgE and allergens by covering parts of the epitopes, leading to less degranulation [7]. TNF-γ is an important activity mediator of the immune response or allergenicity response produced or increased in inflammatory disease states [25]. The release of TNF-γ decreased by half when OVM was treated at 200 ◦C for 10 min, although there exists the experiment error.

Histamine is another key mediator found in KU812 cell granules which is released during antigen-specific IgE binding and plays an important role in the induction of adverse physiological symptoms of an allergic reaction [25]. Interestingly, compared with β-hex, a higher concentration of histamine was observed with OVM treated by SS at 120–200 ◦C for 2–10 min, except for several samples that showed negligible changes. Previous studies have reported similar findings on changes in the β-hex and histamine release, which suggested that histamine may not be a good indicator of mast cell degranulation in regard to OVM [24].

IL-6 is an important mediator produced in the process of mast cell degranulation. As shown in Figure 3D, no significant increase in the content of IL-6 was detected in KU812 cells, indicating that IL-6 was not stimulated by any antigen from OVM samples.

The above results indicated that the degranulation ability of the basophils was decreased, although there was little change in the IgE binding ability. In conclusion, SS treatment on OVM inhibited the anaphylactic reaction and reduced the release of β-hex

and TNF-γ in the process of basophilic granulocyte degranulation. The allergenicity of SS-treated OVM decreased in the KU812 cell.

### *3.3. Analysis of Oxidation Degree*

Generally, the content of carbonyl is an oxidative indicator of the protein [18]. In this study, the oxidation degree of OVM was analyzed through the determination of the contents of carbonyl and free SH. As shown in Figure 4A, a significant increase of the carbonyl content was observed with the rise of temperature and extension of processing time at 120–160 ◦C for 2–6 min and 200 ◦C for 2–4 min. No significant change was observed in OVM treated by SS at 200 ◦C for 6–10 min. In an environment of high temperature and high humidity, vaporized water molecules could react with oxygen ions, which will induce the protein backbone of amino acid residue to form peroxyl radicals. Subsequently, carbonyl derivatives were produced via an amidation reaction [26]. However, when the temperature was increased up to 200 ◦C and the processing time reached 6–10 min, large amounts of protein aggregation were generated, which influenced the monitoring of the carbony content.

**Figure 4.** Effects of SS on the carbonyl content (**A**), surface hydrophobicity (**B**) and free SH content (**C**) of OVM.

#### *3.4. Analysis of Surface Hydrophobicity*

Surface hydrophobicity is an important index that indicates the binding ability between antibody and antigen. As one type of molecular force involved in immune reactions, surface hydrophobicity plays an important role in assisting in epitope recognition [27]. As shown in Figure 4B, the surface hydrophobicity significantly increased with the increase of temperature and time. The results indicated that SS treatment could induce the unfolding of protein structure, and the internal hydrophobic amino acids were exposed to a nonpolar microenvironment that enhanced the surface hydrophobicity [7]. High temperatures may cause a reduction in steric hindrance against some hydrophobic groups, and ANS is more likely to bind to amino acid residues with cationic groups, such as lysine and arginine. Therefore, it can be concluded that SS treatment could promote the combination of ANS and OVM. It was also concluded that the decrease in IgG binding ability may be related to the increased hydrophobic interactions, while this was not the cause for IgE binding ability.

#### *3.5. Analysis of Free SH Content*

OVM consists of three structurally independent tandem homologous domains and possesses nine intramolecular disulfide bridges but lacks interdomain disulfide bonds. This is the reason that OVM is resistant to heat denaturation and structural change. The surface hydrophobicity analysis suggested that an unfolding of OVM conformation could occur after SS treatment, which was related to the reduction of disulfide bridges. As shown in Figure 4C, the free SH content significantly increased with the increasing in SS temperature and processing time, and a maximum value was observed when the temperature and processing time reached 200 ◦C and 10 min. The native OVM has a free SH content of 1.1 nmol/mg. After SS treatment at 120–180 ◦C for 2–10 min, the free SH content increased to about 1.5 nmol/mg. It is worth noting that at a temperature of 200 ◦C, the free SH content drastically increased with the increase in processing time. Generally, high temperature treatment did not cleave the disulfide bridges. However, 200 ◦C SS treatment could catalyze the oxidation of the disulfide bridges to free SH, leading to the unfolding of the protein. Previous studies have reported that reducing the disulfide bonds in OVM can lower allergenicity in vitro [28], which was in accordance with the IgG-binding ability change in this study.

#### *3.6. Molecular Weight Analysis*

OVM has a molecular mass of 28.0 kDa, comprising 186 amino acids and 20–25% of carbohydrate [29]. Heat treatment could induce the oxidation or aggregation of OVM, increasing the molecular weight as well as forming dimer, trimer or polymer [21,30]. MALDI TOF MS was used to measure the molecular weights of SS-treated OVM samples. The dimer, trimer and tetramer molecule of OVM were observed after SS treatment at 120, 140, 160 and 180 ◦C for 10 min (Figure 5), indicating that covalent-binding reactions had occurred. However, there were no obvious changes in dimer among the native and SS-treated OVM samples at 120, 140, 160, 180 and 200 ◦C for 10 min. Interestingly, the single protein molecule and trimer disappeared in the MALDI TOF MS spectrometry of OVM treated at 200 ◦C for 10 min. It was presumed that SS treatment at higher temperatures for longer periods might markedly promote the aggregation of proteins, which was not detected by MALDI TOF MS.

#### *3.7. Modification Sites of OVM*

In order to identify the specific protein sites susceptible to SS treatment and explore the mechanism of the decreasing in OVM allergenicity, it is necessary to find out the precise modification sites of OVM. Previous studies have suggested that the antigenic epitopes in protein sequence mainly originated from hydrophobic amino acids and amino acids containing sulfhydryl groups [21,31]. Therefore, trypsin was used to digest native and SStreated OVM at 120, 140 and 200 ◦C for 10 min. Table 1 showed the main modified peptides and sites of OVM. It is worth noting that the coverage of the control was 86.67%, much lower

than SS-treated OVM, reaching 99%, 99% and 100%, respectively. This result indicated that although OVM was a trypsin inhibitor, it could also be hydrolyzed. Only a few structural domains were resistant to trypsin digestion. It also indicated that SS treatment could destruct the native structure of OVM and weaken the anti-digestion property.

**Figure 5.** MALDI TOF MS of OVM treated by SS at different temperatures (**Control**, native OVM; (**A**–**E**), OVM treated with SS at 120, 140, 160, 180 and 200 ◦C, respectively, for 10 min).

OVM has a signal sequence of 24 amino acids and a protein sequence of 186 amino acids. Therefore, in this work, the SS-induced changes in 210 amino acids of OVM were explored and mainly concentrated in oxidation, nitro, phospho, carboxymethyl, glygly and sulfo modifications. Oxidative modification is typically associated with lower protein solubility, which can significantly hinder and confound the identification [21,32]. Therefore, only signal sequence M3 produced after treatment at 140 and 200 ◦C for 10 min and protein sequence M84 after treatment at 120, 140 and 200 ◦C for 10 min were analyzed. From the results, it can be concluded that SS could catalyze the oxidation of OVM (Table 1). It was also found that nitro modification occurred on some specific amino acids, such as Y73 and Y161. However, the mechanism of the reaction under SS treatment is not yet clear.

Interestingly, it was observed that the phospho modification of OVM occurred after treatment at 120, 140 and 200 ◦C for 10 min. There were two phospho sites S6 and T38 in native OVM, and three new phospho sites were observed in OVM treated by SS at 120, 140 and 200 ◦C for 10 min. It is worth noting that no phosphate groups participated in this reaction. Therefore, this result indicated that the phospho groups have shifted from phospho sites to non-phospho sites, which was similar to the observation in our previous research [33]. This finding provides a new method to promote the phospho modification of proteins using SS technology.


**Table 1.** Modified peptides and sites of the OVM treated by SS.

There was also an important finding in regard to glygly modification, which plays an important role in life activities. Similarly, three new glygly sites S71, S72 and S78 were observed in OVM treated at 120, 140 and 200 ◦C for 10 min, without the participation of the glygly group. The internal transfer might be the reason for the generation of new glygly modification sites in the OVM molecule. Finally, the sulfo modification sites among native and SS-treated OVM at 120, 140 and 200 ◦C for 10 min were also compared. There were four new sulfo sites (S6, S47, S156 and S174) observed in OVM treated at 200 ◦C for 10 min. The sulfuric groups may be originated from cysteine or other sulfo sites. Interestingly, serine could be readily modified with the acquisition of functional groups from other modification sites during SS treatment.

OVM molecule is organized into three well-separated domains which play an important role in inducing allergic reactions. It is important to explore the relationship between the sensitive modification sites and the three-dimensional (3D) structure of SS-treated OVM. The modification sites of OVM treated at 120 ◦C, 140 ◦C and 200 ◦C for 10 min are shown in Figure 6. OVM treated at 120 ◦C for 10 min exhibited similar modification sites with that treated at 140 ◦C for 10 min, and the 3D diagrams of three domains are presented in Figure 6A. The content of modification sites was higher in OVM treated at 200 ◦C for 10 min (Figure 6B). Interestingly, the modification sites including T52, T12, S71, S72, S78, M84 and T160 were all found in the β-sheet structure of SS-treated OVM at 120 ◦C and 140 ◦C for 10 min. However, for the SS-treated OVM at 200 ◦C for 10 min, S47 was found in the α-helix structure, and S156 was found in the structure. From these results, it suggested that the low temperature of SS always modifies the amino acids located on β-sheet which were relatively fragile. Meanwhile, the higher temperature treatment of 200 ◦C could also attack the amino acids located on α-helices and β-turn of OVM and promote the modification reaction.

**Figure 6.** Modification sites in 3D diagrams of the three domains of SS-treated OVM. (**A**), 120 ◦C for 10 min and 140 ◦C for 10 min; (**B**), 200 ◦C for 10 min; purple, oxidation modification; blue, nitro modification; red, phosphorylation; orange, glygly modification; green, sulfo modification.

SS treatment, particularly at high temperatures and long processing times, could induce the OVM aggregation, increase surface hydrophobicity, modify the functional groups and amino acids in OVM. These phenomena disrupted and buried some allergen epitopes and made it difficult for the combination of antibodies and antigens [14,15]. Therefore, the allergenicity of OVM was decreased and the anaphylactic reaction in the KU812 cell was inhibited.

#### **4. Conclusions**

To our knowledge, this is the first study investigating the allergenicity and structure changes of OVM under SS treatment. A decrease in the IgG-binding ability was observed for SS-treated OVM with no significant changes in the IgE binding ability. SS treatment reduced the release of β-hex and TNF-γ, promoted the release of histamine, while having no significant effect on IL-6 release in KU812 cells. An obvious increase in the oxidation degree, free SH content and surface hydrophobicity were observed. Trimer and tetramer aggregations generated after SS treatment. In addition, there were obvious changes in the primary structure. Furthermore, some amino acids could be readily modified by obtaining functional groups from other modification sites during SS treatment. It can be inferred that SS-induced OVM aggregation, functional groups and amino acids modifications as well as protein structure alteration led to the reduction in allergenicity and increase in digestibility.

**Author Contributions:** P.-W.W.: Writing—original draft, Methodology, Validation, Formal analysis. Z.-C.T.: Funding acquisition, Validation, Resources. Y.-M.H.: Formal analysis, Supervision. H.W.: Supervision, Software, Visualization. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by National Natural Science Foundation of China (No. 32101946) and Jiangxi Provincial Natural Science Foundation (20212BAB215037).

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.

**Conflicts of Interest:** The authors declare no competing financial interest.
