## 2 Materials and Methods  
### Materials  
#### 2.1.1 Raw Materials and Processing  
Freshly caught B. herring (caught in September from the Archipelago Sea) with no preprocessing and descaled roach (caught in June from the Bothnian Sea) were bought from AK Foods Arvo Kokkonen Oy (Finland). The B. herring used as raw material were of smaller size (\(<\)17 cm) than the portion of the catch directed for food use. Roach were on average 20 cm long. Both fish batches were ground as whole within 24 h after catching and stored frozen at \(-\)22 \({}^{\circ}\)C until being processed (3 and 1 months, in the case of roach and B. herring, respectively). Protein isolates and hydrolysates were produced at the VTT Technical Research Centre of Finland (Espoo, Finland), and the processes have previously been described in detail by [PERSON] et al. [29]. Briefly, pH-shift fractionation was performed using both acidic (A, pH 2.5) and alkaline (B, pH 11.5) solubilization, followed by precipitation at pH 5.2. Homogenized fish was mixed with tap water in a 1:4 ratio (1 part fish + 3 parts water), and 6 M or 1 M (for fine adjustment) hydrochloric acid or sodium hydroxide was added under continuous mixing (75 rpm) until target pH was reached. After reaching the solubilization pH, mixing was continued for 15 min to allow enough time for solubilization. Phase separation after solubilization and precipitation was achieved by centrifugation at 4000\(\times\)_g_ for 15 min. In the case of B. herring, which contained more lipids, the centrifugation of the supernatant after protein solubilization was repeated to enhance lipid removal. The mixture was kept at 4 \({}^{\circ}\)C during solubilization and precipitation, but as a precaution to prevent microbial spoilage, after pH adjustment to 5.2 and prior to separation of precipitated proteins, the suspension was heated until 75 \({}^{\circ}\)C was reached and was held for 1 min, after which the suspension was again cooled down to 4 \({}^{\circ}\)C (Supplementary Figure S1). The pH shift itself is a mild non-thermal \"pasteurization\" due to a high or low pH used [30; 31], and hence pasteurization by heat is often not necessary. In the present study, the heating step was, however, included, since whole unguttted fish were used, and the presence of intestinal microbes and enzymes of the fish could have been detrimental to the stability of the protein isolates.  
Enzymatic hydrolysis was conducted using three different endoproteases, Protamex (Novozymes, Bagsvaerd, Denmark), Neutrase (Novozymes), and Corolase 7089 (AB Enzymes, Darmstadt, Germany). Homogenized fish was diluted in tap water in 1:2 ratio (1 part fish + 1 part water). Enzymes were added at a dose of 11.4 hkat/g. Hydrolysis was conducted at 50 \({}^{\circ}\)C for 30 min under continuous mixing, after which enzymes were inactivated at 75 \({}^{\circ}\)C for 15 min. After cooling the mixture to 4 \({}^{\circ}\)C, centrifugation at 4000\(\times\)_g_ for 15 min was applied to separate insoluble matter and lipids, and this step was repeated for the supernatant in the case of B. herring. All protein fractions (from pH shift and enzymatic fractionation) were freeze-dried. All treatments were performed in triplicate. A detailed graphical description of both processes can be found in Supplementary Figure S1. The protein isolates and hydrolysates and raw materials analyzed in this study have been previously characterized for their protein and lipid content [29] and the results are cited in Table 1. Freeze-dried isolates and hydrolysates were stored at \(-\)80 \({}^{\circ}\)C until analysis.