**2. Materials and Methods**

### *2.1. Chemicals and Materials*

OVM, D-glucose, goat antirabbit IgG-HRP conjugate, goat antihuman IgE-HRP conjugate and trypsin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dithiothreitol (DTT) was purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA). The KU812 cells were obtained from iCell Bioscience (Shanghai, China). All other reagents used were of analytical reagent grade. Ultrapure water from a water purification system (Millipore; Billerica, MA, USA) was used throughout this study.

The sera of six egg white allergy (EWA) patients were purchased from Plasma Lab International. The specific IgE levels of six groups of sera were 10.8, 26.3, 24.9, 13.5, 16.5 and 12.8 kU/L. The six groups of sera were mixed and served as human anti-OVM IgE serum and stored at −80 ◦C until used.

Preparation of rabbit anti-OVM IgG serum: Male Japanese rabbits (100 days) (permission number, SCXK (Gan) 2014–0005) were purchased from Longping (Nanchang, China). After acclimatizing in a breeding room for 10 days, the rabbits were intravenously injected with 1 mL OVM (0.8 mg/mL) emulsified with the Freund's complete adjuvant (*v/v*, 1:1). The booster immunization was taken after one, two and three weeks at a dose of 0.4 mL, respectively. After the rabbits were anesthetized, the rabbit anti-OVM IgG plasma was

collected. After being centrifuged at 4 ◦C, the rabbit anti-OVM IgG serum was separated and stored at −80 ◦C until used.

#### *2.2. SS Processing*

An SS processing system developed by the Food Engineering Center of Nanchang University was used in this study. The schematic diagram was detailed and shown in the report of Wu et al. [13]. A total of 200 mg OVM powder was spread in a culture dish. When the temperature in the SS chamber was kept stable, the culture dish was inserted into the processing chamber. Processing of OVM samples was conducted at atmospheric pressure. The flow velocity of SS was 1.0 m/s. The treatment temperatures were 120, 140, 160, 180 and 200 ◦C. The processing times were 2, 4, 6, 8 and 10 min. After being treated for the set time, the OVM sample was taken out of the processing chamber and then cooled in an ice bath. Native OVM was served as the control. All the samples were stored at 4 ◦C until used.

#### *2.3. IgG and IgE-Binding Abilities*

The IgG and IgE-binding abilities of the OVM samples were estimated with indirect competitive ELISA according to our previous report [14]. Rabbit anti-OVM IgG serum and human anti-OVM IgE serum were used to determine IgG-binding ability and IgE-binding ability, respectively. A 96-well microplate was coated with 2 μg/mL of native OVM at 37 ◦C for 1 h. Then residual free binding sites were blocked with 1% fish gelatin solution and incubated at 37 ◦C for 1 h. Next, the diluted samples and the serum of rabbit or EWA patients were injected into the microplate and incubated at 37 ◦C for 1 h. After removing the solutions, the microplate was washed with PBST five times. Goat antirabbit IgG-HRP conjugate (100 μL, diluted at 1:2500) or goat antihuman IgE-HRP conjugate (100 μL, diluted at 1:700) was injected and then incubated at 37 ◦C for 1 h. Finally, tetramethylbenzidine (TMB, 100 μL) was immediately added to each well and purged with sulfuric acid. The absorbance was measured at 450 nm and the decline rate was calculated using the equation:

$$\text{Inhibitation (\%)}=(1 - \text{B/B}\_0) \times 100\text{\%} \tag{1}$$

where B0 and B indicate the optical density (OD) value of a well with the control and SS-treated OVM, respectively.
