## 3 Results
### Peroxide Values, Protein Carbonyls and Oxidation-Related Volatile Compounds  
Protein carbonyls and peroxide values in the studied protein isolates and hydrolysates are presented in Figures 2 and 3. In general, isolates and hydrolysates from B. herring showed more signs of oxidation compared with roach, in terms of PV and protein carbonyls. B. herring, unlike roach, is a dark-muscled fish and hence contains more heme pigments, known to act as pro-oxidants in fish. The development of lipid oxidation has been observed even at a low lipid concentration of 0.1%, when pro-oxidants in fish blood were present [51]. Protein isolates produced by an alkaline pH shift from both raw materials showed high carbonyl values (14-33 nmol/mg protein), whereas protein hydrolysates showed moderate values (7.7-9.0 nmol/mg protein). Protein isolates produced using pH shift were subjected to a short heat treatment after precipitation (Supplementary Figure S1), which could explain the high carbonyl values. The heat treatment may have contributed to protein and lipid oxidation, indicated by the higher carbonyl values and PVs. In a study by [PERSON] and [PERSON] [52], protein carbonyls in herring protein separated by the alkaline pH-shift process were measured at a level of 2.5 nmol/mg protein and did not show a significant increase compared with the raw material. At the same time, however, lipid oxidation was observed to have significantly increased PV and TBARS. [PERSON] et al. [22] reported the effect of different pretreatments on protein carbonyl values after 1, 2, or 3 h hydrolysis of rainbow trout by-product proteins using Alcalase. The lowest carbonyl value (<3 nmol/mg protein) was detected after 1 h of hydrolysis of the protein isolate without any pretreatments, but all values were below 4 nmol/mg protein, which is slightly less than observed in the present study. Figure 2: Protein carbonyls (nmol/mg protein) in protein isolates produced using pH shift (**A**), quantified using the original carbonyl method [40], and in enzymatically produced hydrolysates (**B**), analyzed using a modified method by [PERSON] et al. [41]. Different letters within the same raw material indicate a statistically significant difference (\(p<0.05\)). In the case of both fishes, the highest PV was found in the lipids of the protein isolate produced using alkaline pH shift. Most previous studies have reported higher rates of oxidation for acidic pH shift compared with the alkaline one [18; 53; 54]. An acidic pH induces the unfolding of hemoglobin [55], making it more pro-oxidative, whereas as alkaline treatment may even have a protective effect compared with native hemoglobin [53]. However, a recent study [21] also reported significantly higher oxidation levels, indicated by higher malondialdehydehehyde levels, for herring processed with alkaline compared with acidic pH shift, as was the case in the present study. The contradiction could be due to species-related differences in hemoglobin autoxidation. Due to the variations in the environmental conditions, such as water temperature, of different fishes, there are electrophoretically distinct forms of hemoglobin in fish that vary in their response to changes in pH [56]. The autoxidation of hemoglobin due to oxygen loss takes place in mildly acidic conditions (Root or Bohr effect), such as the precipitation pH 5.2 in the present study, which plays an important role in its pro-oxidativity [57]. In addition, in the pH-shift process the pH is adjusted in two sequential steps, and even if the precipitation pH is the same for both acidic and alkaline process, the previous solubilization pH is likely to alter the susceptibility of the system to lipid oxidation [58]. Nevertheless, findings from this and previous studies show that oxidation during the pH-shift process is a complex phenomenon and should be studied further. Increased lipid oxidation of alkaline protein isolates of reach and B. herring in the present study likely contributed to the lower levels of PUFAs observed.