## 3 Results
### Peroxide Values, Protein Carbonyls and Oxidation-Related Volatile Compounds
Interestingly, the protein hydrolyses produced from roach using the three enzymes, Protamex, Neutrase, and Corolase, had similar PVs, but in the case of B. herring, treatment with Neutrase resulted in a higher PV in the hydrolysate. B. herring hydrolysate produced using Neutrase also showed a lower proportion of TAGs (Figure 1) and PUFAs (Table 3) compared with Protamex and Corolase, which was likely linked to oxidation. When Nile tilapia stored on ice for 0 or 18 days was hydrolyzed using Alcalase without antioxidants, the PV of the hydrolysate was 6-fold and 3-fold, respectively, higher compared with when hydrolysis was performed with the addition of antioxidants (Trolox and EDTA) [24]. When the muscle was stored on ice for 18 days prior to hydrolysis, the hydrolysate produced with antioxidants showed a similar PV to the hydrolysate produced from fresh muscle without antioxidants, highlighting the significance of both antioxidants and the use of fresh raw material for limiting lipid oxidation. Figure 3: Peroxide values (meq/kg protein isolate) in protein isolates produced using acidic or alkaline pH shift and hydrolyses produced using Protamex, Neutrase, or Corolase. Different letters within the same raw material (roach or B. herring) indicate a statistically significant difference (\(p<0.05\)). The volatiles of the raw materials and the protein isolates and hydrolyses were measured as another group of markers for oxidation. The most abundant volatiles found from roach raw material were propanal, 1-penten-3-ol, hexanal, heptanal, octanal, 3,5(_E_,_E_)-octalien-2-one and nonanal. All of these volatiles are formed from the oxidation of PUFAs. In the B. herring raw material, the most abundant volatiles were 2-methylbutanal, 3-methylbutanal, 1-penten-3-ol, 2,3-pentanedione, hexanal and heptanal. These Strecker aldehydes and volatile secondary lipid oxidation products were previously observed from fresh B. herring [37]. For the assessment of lipid oxidation in protein isolates and hydrolyses, seven volatile secondary lipid oxidation products were selected and semi-quantified as oxidation indicators. Propanal, 2-ethylfuran, 2-pentylfuran, 1-penten-3-ol, hexanal, 2(_E_)-hexenal, heptanal and 2,6(_E_,_E_)-nonadien were chosen, since they were among the most abundant volatiles in the isolates and hydrolyses, and have been shown to be common in oxidized fish oil or other oils rich in n-3 or n-6 PUFAs [38; 59]. Some of these volatiles have been previously proposed as oxidation indicators, e.g., Saiib and Undeland [60] recently suggested 2-pentylfuran as the best volatile marker for lipid oxidation in the production of silage from herring by-products, whereas 1-penten-3-ol and 2(_E_)-hexenal showed good potential to be used as indicator compounds for lipid oxidation in EPA- and DHA-rich products [38]. Most of the selected volatiles have also been associated with fishy and rancid odors [61]. The peak areas of the indicator volatiles showed a higher volatile formation in protein isolates and hydrolyses from B. herring compared with ones from roach (Figure 4). The higher content of DHA in isolates and hydrolyses from B. herring compared with the level in the isolates and hydrolyses from roach (Table 3) and an extra processing step (additional centrifugation; Supplementary Figure S1), as well as possible differences in levels of pro-oxidants, may have contributed to this difference in the development of oxidation status. The highest formation of lipid-oxidation-derived volatiles was seen in alkaline pH-shift-processed isolate, in the case of both fishes, followed by acid-treated isolate. Hexanal was the most abundant volatile followed by heptanal and 2-ethylfuran in the pH-shift isolates of both fishes.