*3.5. Antigenicity of Jug r 1*

Jug r 1, which is 16.4 kD long and contains 139 amino acids, is the major allergen protein in walnuts, and could reflect the allergenic properties of WPI. As hinted at by Figure 9, the effect of oxidation on the antigenicity of Jug r 1 was assessed. It can be observed that there was a gradual decrease of antigenicity with the AAPH concentration, and the lowest value (13.91 ng/mL) was obtained at 30 mmol/L, indicating a significant effect (*p* < 0.05) on antigenicity by radical oxidation. Combined with the mass spectrometry results, changes in oxidative indicators and secondary structure (Table 1 and Figure 4), the oxidative modification of WPI might be covalent, and the changes of protein side chains led to the destruction of allergen epitopes, which consequently decreased the antigenicity of Jug r 1 [43]. In addition, the unfolding of WPI blocked the epitopes and could not be identified by an antibody. Similar results were exhibited in acrolein-oxidized shrimp tropomyosin [44] and hydroxyl radical-treated β-conglycinin [45], whereas the specific reason for the reduction of antigenicity should be further researched.

**Figure 9.** Effect of oxidation on antigenicity of Jug r 1. AAPH = 2, 2- -azobis (2-amidinopropane) dihydrochloride. Different letters within a column indicate significant differences (*p* < 0.05).

#### **4. Conclusions**

This paper explored the influence of peroxy radicals on structural and functional characters of WPI. The results showed that the carbonyl group of WPI was significantly increased with the concentration of AAPH, whereas the free amino and sulfhydryl groups decreased significantly (*p* < 0.05), which could be due to formation of disulfide bonds and irreversible sulfonic, sulfonic acid groups by radical oxidization. The reduction of fluorescence intensity and increasing of surface hydrophobicity illustrated the unfolding and rearranging of protein tertiary structure; meanwhile, FT-IR analysis also demonstrated alteration of the secondary structure. Besides, large aggregates were observed by particle size distributions and SDS-PAGE analysis, and thus unfolding and cross-linking of WPI were further confirmed. The aforementioned changes of conformation in turn affected the functional properties of WPI, and thus we saw a significant reduction in solubility, whereas different degrees of increase in foaming property and WHC/OHC were observed by mild oxidation. Nevertheless, all of the measured property indicators in this study were found to be significantly decreased after overoxidation. Mass spectrometry analysis revealed numerous modifications of the amino acid side chains, and these modifications are involved in many protein groups. Interestingly, these protein groups were found to be associated with allergies, which implied that oxidative modification could have an effect on the allergenic properties of WPI. The double antibody sandwich ELISA result indicated the gradual reduction of Jug r 1, the major allergen of WPI, and this further confirmed the mass spectrometry results. In conclusion, peroxy radical oxidation altered the conformation of WPI, which in turn caused modification in functional properties correspondingly, including allergenicity.

Proteins are one of the most important components of foods and are easily oxidized during their harvesting, storage, and processing. Although it is well known that protein structure strongly influences its functions, little is known about oxidative modifications and how it affects food protein structure and function. Generally, the preliminary properties, and secondary and tertiary structure of proteins can be investigated via multiple physical, chemical, and biological means, including a chromogenic assay, protein electrophoresis, spectroscopic studies, mass spectrometry, etc. However, they are often studied in isolation, and the results are incoherent. In this study, an integrated approach was used to produce results at multiple levels, seeking to provide a comprehensive understanding of the effect of oxidation induced by peroxy radical on the functional properties of WPI. Our results

suggest that oxidation can lead to the modification of amino acids in proteins, subsequently altering their structure, which in turn changes their functional properties. Although the extent of protein modification is strongly increased with the increase of AAPH concentration, and it appears to perform adversely for the functional properties of the protein, appropriate oxidation is favorable for some properties, such as foaming property, WHC/OHC, and allergenicity. In conclusion, a potential methodology to limit oxidation, including antioxidants, should be taken into account. On the other hand, the above results also revealed the possibility of oxidation as a means to regulate protein functional properties in the future.

**Supplementary Materials:** The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/foods11030385/s1, Table S1: peptide information; Table S2: protein information; Table S3: gene ontology (GO) annotation.

**Author Contributions:** Methodology, X.Z. and Z.W.; validation, X.Y. and Y.L.; formal analysis, X.H.; data curation, X.Y. and Y.L.; writing—original draft preparation, X.Z.; writing—review and editing, Z.W. and J.S.; supervision, X.H.; project administration, J.S.; funding acquisition, J.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Postdoctoral Starting Foundation of Guangxi Academy of Agricultural Sciences (grant number 2018033), National Natural Science Foundation of China (grant number 31760440), and Yunnan Innovative Research Team (grant number 202005AE160017).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

**Acknowledgments:** The authors thank the support of the Applied Protein Technology Co., Ltd. for mass spectrometry support.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

