## 3 Results and Discussion
### Allergen Characterization and Amino Acid Profile
In previous studies, other authors reported a ratio of 0.49 for the autolysis of salmon viscera [42] and 52-62% after 91 days of silage, as measured by the OPA method [41]. The latter gives slightly higher results, as it measures the total number of peptide bonds cleaved (FAA plus peptides). The difference between the V3 and F batches cannot be explained by the difference in pH conditions, as acid proteases in fish viscera are reported to work optimally at pH 2.0-4.0 [34]. However, when formic acid is used, it is not necessary to work at such an acidic pH to preserve silage from microbial growth as when mineral acids are used. As no differences in the amino acid profiles between the V3 and F samples were observed, the difference in the degree of hydrolysis could be due to differences in the composition of the initial sample or differences in the feeding activity of the fishes, which could also result in differences in enzymatic activity. \begin{table}
\begin{tabular}{c c c c} \hline  & **F2** & **F5** & **V3** \\ \hline \(>\)10 kDa & 0.27 & 0.74 & 0.00 \\
10 \(>\) 6 kDa & 2.10 & 2.13 & 0.00 \\
6 \(>\) 3 kDa & 13.44 & 14.18 & 0.00 \\
3 \(>\) 1 kDa & 52.19 & 52.88 & 35.76 \\
1 \(>\) 0.3 kDa & 25.34 & 23.45 & 38.07 \\ \(<\)0.3 kDa & 6.66 & 6.72 & 26.17 \\ Average mw (kDa) & 1.98 & 2.02 & 0.96 \\ \hline \end{tabular}
\end{table} 
Table 3: Distribution of the molecular size profiles as a percentage of the defined range for each hydrolysate obtained in the long-term silage experiments. Figure 8: SE-HPLC profile of the liquid fraction of silage V3 (12 months of storage), F2 and F5 (15 months of storage). The standard molecular sizes corresponding to the retention times were as follows: 45 kDa, 6.297 min; 17 kDa, 7.103 min; 6.7 kDa, 8.223 min; 1.0 kDa, 10.860 min; 0.379 kDa, 12.880 min. The points used to define the selected intervals were 10 kDa, 7.968 min; 6 kDa, 8.683 min; 3 kDa, 9.652 min; 1 kDa, 11.189 min; and 300 Da, 12.873 min. Compared with the amino acid requirements of juvenile common carp listed by the NRC [43], phenylalanine and tryptophan were present in less than the required proportions, while the other amino acids were present and even exceeded the necessary relative contents. Tryptophan is usually known as the first limiting amino acid in fish silage because it is acid sensitive [44]. Similar results were observed by [PERSON] et al. [36] in an experiment in which fish viscera were hydrolyzed with Alcalase(r), except that they reported methionine as well as a limiting amino acid. There is only one published feeding trial investigating whether the protein fragments of Anisakis ingested by zebra fish can be found in their tissues. After a two-week feeding experiment, Anisakidae peptides were detected in zebrafish tissue [25]. However, this approach was based on the percentage of intact Anisakis larvae included, which was much greater than what could be expected from fishmeal from naturally infested fish. In a survey of farmed salmon and several processed seafood products in the Norwegian market, only trace amounts (<10 mg/kg) were detected, and the authors considered that this contamination does not represent a health risk [24]. Whether or not the resulting resistant fragments detected from silage of infested viscera may be a risk for consumers through their inclusion in fish feed must be investigated by means of an adequate exposure assessment [45].