## 3 Results and Discussion
### Allergen Characterization and Amino Acid Profile  
Electrophoresis and immunoblotting of the different fractions revealed changes in the protein fraction during hydrolysis. As shown in Figure 6, variations in IgE binding in the different fractions were not associated with any specific protein since, in general, no clear bands were observed in the immunoblotting images; this is probably due to the low sensitivity of this method. Since the _Anisakis_ extract lane (Ani) showed clear reactive bands, the _Anisakis_ protein concentration present in other fractions (where _Anisakis_ proteins make up only a part of the total proteins) was insufficient for detection via this method. However, the electrophoresis images and faint bands in the immunoblot showed differences among the fractions and the different days of evolution, which are in accordance with the ELISA results. The solid pellet (S) fraction analysis revealed a gradual increase in soluble protein concentration (as indicated by more intense bands), and immunoblotting also revealed an increase in IgE binding, albeit somewhat diffuse, until day 10 (Figure 6), in accordance with the results observed in the ELISA analysis (Figure 5). This confirms that proteins are gradually digested and become more soluble and accessible to IgE during silage hydrolysis. In the floating (F) fraction, on the other hand, an increase in protein concentration was observed, except on the last day (Figure 6); however, the allergenicity gradually decreased until day 9 (Figure 5). This is in accordance with the decrease in allergenicity observed in the ELISA. In contrast, in the last two days, although the protein bands detected via  
Figure 6: Images of SDS-PAGE (**left**) and immunoblotting (**right**) of the solid pellet (S), floating interphase (F) and liquid (L) fractions. The first three columns from left to right in each picture correspond to the pattern of molecular weights (St.), an _Anisakis_ extract (Ani) and untreated viscera (Day 0-Control). The other lanes, from left to right, correspond to days 1 to 11 of the silage experiment for the corresponding fraction of the hydrolysate under study. electrophoresis were equal to or less intense than those detected on day 9, an increase in allergenicity was observed. In this sense, in immunoblotting analysis (Figure 6), a slight increase in the reactivity toward proteins with a molecular weight of approximately 50 kDa was observed during the last 2 days; this could explain the increase in IgE reactivity. This reactivity could be related to allergens Ani s 3 (41 kDa), a somatic antigen; or Ani s 11 (55 kDa), but also to fractions of the ES antigen Ani s 7 (139 kDa) [39]. This again confirms that due to hydrolysis, proteins would pass from the body of _Anisakis_ and the soluble (liquid) fraction to other fractions, increasing immunoreactivity. In other studies, the most relevant IgE-binding heat- and pepsin-resistant _Anisakis_ proteins were identified to be Ani s 4 (9 kDa) and Ani s 5 (15 kDa) [30], which seems to coincide with our electrophoresis results. Although these proteins were not reactive to IgE, according to the immunoblotting results, the higher concentrations of these proteins (or protein fragments), especially on the last day of study, could explain the higher IgE binding detected in the ELISA (Figure 5), the most sensitive method. Finally, in the case of the liquid fraction, the increase in reactivity observed from the first day compared to untreated viscera (not yet \"solubilized\") is also related to the fact that the proteins are already in a soluble state from the beginning of the process. In this case, a reactive band of approximately 50 kDa (possibly Ani s 3, a somatic antigen) was also observed in immunoblotting during the experiment, although other proteins could also be involved in IgE immunoreactivity.