## 3 Results and Discussion
### Presence of Anisakis allergens  
As shown in Figure 5, the extent of protein recognition by IgE in patient serum varies depending on the fraction analyzed (solid pellet, floating interphase and liquid fraction). However, although the evolution of the three fractions follows a different pattern, the results obtained are related to each other, considering the type of process to which the samples are subjected. Thus, in the solid pellet, no changes were observed during the first days, while in the final days, there was an increase in the binding of IgE to the proteins present in that fraction. This may be because, during the hydrolysis process, the allergenic proteins initially present inside the body of _Anisakis_ become solubilized; consequently, the epitopes (fractions of the protein that recognize the antibodies) change from a state where they are \"hidden\" (days 1-3) to a soluble or superficial state that is more easily recognized by antibodies, gradually increasing IgE binding (days 4-10). These results are also consistent with the degree of liquefaction and the protein content in the liquid fraction (Figures 3 and 4). presented above. On the last day, a decrease in the binding of IgE to _Anisakis_ proteins was observed again due to further hydrolysis of the solubilized proteins. However, IgE binding continued to be stronger than that in the control group. It is necessary to verify that this  
Figure 4: Degree of liquefaction obtained during the 11 days of silage of hake viscera. Figure 5: Evolution of the allergenicity in the different fractions obtained during the ensiling period of the hake viscera: solid fraction (\(\square\)), floating interphase (\(\bullet\)) and liquid fraction (\(\bullet\)). The vertical bars correspond to the SDs (\(n=6\)). downward trend was maintained in the following days and that the IgE epitopes of the _Anisakis_ allergens present were completely hydrolyzed. In the floating interphase, this phenomenon of increased IgE binding due to the initial solubilization of proteins was also observed but, after day 5, there was a rapid decrease in the recognition of the allergens present due to the hydrolysis process that gradually destroyed the epitopes (reduction in the size of the peptides and proteins present). In this case, the reduction in allergenicity can be faster than in the solid pellet since, in this fraction, there are no nematode carcasses (bodies) at the beginning, and the potential allergens present are more accessible to hydrolysis. However, in the last two days, an increase was again observed, possibly related to the increase in allergenicity in the solid and aqueous fractions because the allergens present inside the bodies of _Anisakis_ (solid fraction), initially hidden, are released and consequently migrate to the other fractions. Finally, a significant increase in the IgE binding of the _Anisakis_ proteins present in the aqueous fraction was observed after the first day. This could be due to the advance of hydrolysis, which would cause the proteins to pass to a soluble state while maintaining hydrolyzed protein fragments and peptides still recognizable by the IgEs. Consequently, a high \"allergenic potential\" was observed until the end of the process. These results are also in accordance with previous results, where it has been observed that the passage of proteins from the bodies of _Anisakis_ to a more available and recognizable state is continuous during the period under study. There are currently 14 recognized allergenic proteins of A. simplex [37], although this list may be extended with the identification of new potential allergens. These allergens are classified according to their function and location within the body of _Anisakis_ as somatic antigens (AS) and excretory-secretory antigens (ES), which are produced by the internal glands of the parasite. Some _Anisakis_ allergens have been described as very resistant to heat and pepsin digestion [30; 37; 38]. Most of these allergens are classified in the ES fraction.