## 2 Materials and Methods
### Allergen Characterization  
SDS-Electrophoresis in polyacrylamide gel (SDS-PAGE) was used to investigate the presence of proteins with a molecular size compatible with known _Anisakis_ allergens and to monitor their presence throughout the silage trial. Immunobloting (IB) was used to detect immunoreactive proteins or their fragments within those identified via electrophoresis (EF).  
\begin{table}
\begin{tabular}{l l l} \hline \hline
**Category** & **Value** & **Description** \\ \hline Live & 1 & Vigorous motility without needle stimulation \\ Reduced motility & 0.5 & Motility requiring needle stimulation \\ Dead & 0 & Null motility when stimulated with the needle \\ \hline \hline \end{tabular}
\end{table}
Table 1: Description of qualitative (category) and quantitative (value) criteria for estimating the mortality rate of _Anisakis_ after treatment.  
Electrophoresis provides information on the molecular weight of the proteins in suspension and makes it possible to identify most allergens found in the samples compared with the known allergens of _Anisakis_. It also determines whether they are somatic antigens (SAs) or secretory antigens (SEs).  
The electrophoresis of the proteins present in each fraction throughout the digestion period of the viscera (days 1-9) was performed on gels with a gradient of 4 to 20% polyacrylamide in a Mini-PROTEAN Tetra Cell (Bio-Rad, Hercules, CA, USA) system according to the conditions set for these samples. The different samples were diluted in 10 mM Tris-HCl buffer (pH 8.0) with 2.5% SDS and 50 mM DTT as the reducing agent and heated in a water bath for 6 min at 95 \({}^{\circ}\)C. The proteins were separated at 125 V for 1 h. In each gel, a molecular weight standard (10-250 kDa, Precision Plus Protein(tm) Dual Color Standards, Bio-Rad) was used, and a sample of a complete _Anisakis_ protein extract was also included as a reference. After separation, the proteins were stained by immersing the gels in a solution of Coomassieie blue for 1 h. The excess dye was removed by immersion in distilled water for 2 h until the background was colorless. The gels were photographed and analyzed in an EZ Gel Doc(tm) system with Image Lab(tm) software version 6.0.1 build 34, 2017 (Bio-Rad Laboratories Inc., Hercules, CA, USA).  
Immunoblotting makes it possible to identify the specific proteins or the proteins recognized by antibodies, determining whether there is a modification throughout the digestion process of the viscera and also determining the degree of hydrolysis (measured by molecular weight size) in which the specific IgE antibodies cannot recognize the hydrolyzed proteins.  
First of all, the proteins were separated via electrophoresis under the same conditions as those described above. The separated proteins of each fraction were transferred to a nitrocellulose membrane under the current conditions of Am, V for 7 min. Immunoblotting was performed with a commercial Amplified Opti-4 CN Substrate Kit (Bio-Rad) following the manufacturer's instructions and optimized for the conditions of this assay. The nitrocellulose membranes were allowed to dry completely, and the free sites were blocked with the provided blocker diluted 3% in PBST for 1 h. After blocking, each membrane was washed twice with PBST and incubated with the pooled sera diluted 1/100 in a 1% solution of bovine serum albumin (BSA) in PBST. Subsequently, the membranes were washed again and incubated with the secondary antibody labeled with peroxidase (the same as for the ELISA) diluted 1/4000 in PBST. The following steps were performed according to the instructions of the aforementioned kit for signal amplification and colorimetric detection with 4-chlor-1-naphthol substrate.