## 3 Results
### Lipid Classes
Different letters or an asterisk (*) within each lipid class indicate a statistically significant difference (\(p<0.05\)). Peak areas in an equal content of lipids were determined using liquid chromatography mass spectrometry. protein extraction is often desired. The accumulation of PLs in the pH-shift processed isolates may have contributed to the higher degree of oxidation, as further discussed in Section 3.4. Membrane lipids, consisting mostly of PLs [23], are suggested to be at least partly removed during the first centrifugation step of the pH-shift process, especially when high-speed centrifugation is used. In a study by [PERSON] et al. [15], a high-speed centrifugation step (18,000\(\times\)_g_, 20 min) was seen to reduce the total lipid and PL contents in acidified herring (after solubilization of proteins in acidic pH shift) by 70% and 45%, respectively, by comparing the content of lipids/g of protein. Interestingly, in the same study it was seen that while a longer holding time (65-125 min) between acidification and the first centrifugation step drastically increased overall lipid removal, a holding time of 125 min, compared with 5 min, decreased PL removal from 30% to 0%. It should be noted, however, that when 45% of PLs were removed, the reduction in total lipids was 70%, meaning that after the process the protein isolate contained a higher proportion of PLs of total lipids than the fish raw material. In the present study, the total lipid content of alkaline protein isolate from roach was even higher on a dry weight basis compared with the raw material (19.4 vs. 17.0%), and since the peak area of PLs in lipids of the alkaline isolate was 85% higher than in the roach raw material, the total content of PLs (on d.w. basis) was also higher. In this study, after reaching the target pH values in acid and alkaline solubilization, mixing of the homogenate was continued for a further 15 min to allow more proteins to solubilize. The 15 min delay before centrifugation and centrifugation at low speed (4000\(\times\)_g_, 15 min) could have contributed to the accumulation of PLs in the pH-shifted protein isolates from roach. On the other hand, the enzymatic fractionation of roach and B. herring significantly reduced the proportion of PLs of total lipids (Figure 1), despite the same centrifugation force and time applied as in the case of the pH-shift process. The reduction was especially prominent in the case of roach; lipids in enzymatically produced hydrolyses contained 56-73% less PLs compared with the lipids in the raw material. The hydrolyses produced using Protamex, Neutrase, and Corolase from roach also contained less DAGs, MAGs, and FFAs of total lipids compared with the raw material, whereas the amount of TAGs was higher compared with the raw material. B. herring hydrolyses, compared with roach hydrolyses, showed smaller differences to the B. herring raw material in terms of lipid composition. On the other hand, there were, interestingly, some differences between B. herring hydrolyses produced with different enzymes. Lipids in hydrolysate produced using Neutrase contained statistically significantly less TAGs compared with hydrolyses produced by Protamex and Corolase. The pH-shift-processed isolates from both roach and B. herring contained significantly less TAGs of total lipids compared with the hydrolyses. The lower content of TAGs may be related, in addition to the difference in extracted lipids, to hydrolysis by lipases or changes due to oxidation, and was also accompanied by a higher proportion of FFAs in the pH-shift protein isolates. The alkali-extracted protein isolate of B. herring in particular also contained significantly more FFAs and was found to have the highest PV and peak areas of volatile lipid oxidation indicators (Section 3.4) compared with acid-extracted protein isolate and hydrolyses of B. herring. Extremely alkaline conditions, in addition to lipases, induce the hydrolysis of TAGs [48]. Hydrolysis and oxidation are connected.