## 3 Results and Discussion
### Allergen Characterization and Amino Acid Profile
On the other hand, DNA analyses detected the presence of _Anisakis_ only in solid pellet samples at any silage time (from 24 h to 264 h), while other fractions (liquid and interphase) did not present detectable traces of parasite DNA. This finding confirmed that parasite bodies remained mainly in the solid pellet, while during the hydrolysis process, only proteins and their fragments were solubilized. Additionally, long-term resistance of _Anisakis_ allergenic proteins to the action of digestive enzymes present in the silage process was observed in three of the experimental silage samples that were kept for up to 12 months (V3) or 15 months (F2, F5). The differences between the samples were also in the grinding step (F2 and F5 were intensively ground, while V3 was slightly crushed) and in the pH (3.6 in V3 and 2.2 and 2.5 in F2 and F5, respectively). After this period, up to 82% of the total protein became part of the liquid fraction, 6% remained in the undigested pellet, and 12-13% remained in the floating interphase fraction. According to the SDS-PAGE profile, the solid fraction (S) and floating interphase (F) were fully hydrolyzed, and protein bands above 10 kDa were not observed (Figure 7). However, in the liquid fraction (L), there were still faint protein bands (>10 kDa) in all three samples, which also showed slight immunoreactivity in immunoblotting, especially in silages F5 and V3, confirming the presence of highly resistant allergens. Figure 7: Images of electrophoresis (**A**) and immunoblotting (**B**) of the three fractions: S (solid pellet), L (liquid fraction) and F (floating interphase) of silages F2, F5 and V3. In B (immunoblotting), the first three columns from left to right correspond to the molecular weight pattern (St), an _Anisakis_ extract (Ani) and untreated viscera (0). The results of the SE-HPLC were in accordance with the high degree of hydrolysis observed in V3, F2 and F5 (Figure 8). The average molecular size was smaller in V3 (Table 3). However, in V3, no peptides were observed in the range that could correspond to the bands of higher molecular size and the 20 and 15 kDa bands captured in the EF. This could be due to differences in the pretreatment of the sample and/or to the very low concentration of these fragments in the hydrolysate. As discussed later, in V3, most of the protein is hydrolyzed into small peptides and free amino acids. Regarding the nutritional quality of silage for further use, prolonged silage could lead to partial deamination and loss of protein and some essential amino acids (EAA); however, other authors argue that maintaining silage at lower pH can minimize these losses even over very long storage periods [40]. Previous studies reported protein losses of 17.91% after 91 days of storage at pH 4-4.5 and a reduction in EAA mainly during the first month of silage [41]. In this study, the average protein content (total N2 \(\times\) 6.25) in the liquid fraction of the hydrolyses on day 9 of silage was 45.73 \(\pm\) 2.48% (dry matter basis, n = 4), 73.38 \(\pm\) 0.38% after 12 months and 54.83 \(\pm\) 1.23% after 15 months of storage. The amino acid profile was not determined at the beginning of the experiment, but the three silage samples after 12 and 15 months contained a similar pattern of amino acids (Table 3). However, there was a big difference in total amino acid (AA) and free amino acid (FAA) contents between the two groups. Similarly, the ratios of essential (EAA) to nonessential (NEAA) amino acids were 0.9 and 1.0; this is similar to what was observed in the aforementioned studies by [PERSON] et al. [36] and [PERSON] et al. [42], respectively, for the autolysis of salmon viscera at 52 \({}^{\circ}\)C and 120 min, which may indicate that no significant losses of amino acids were produced despite the long duration of silage. The FAA/AA (total amino acid) ratio in the V3 hydrolysate was quite high (0.85) (Table 4). This ratio may indicate the degree of hydrolysis achieved in the V3 sample. The ratios in F2 and F5 were only 0.25 and 0.28, respectively.