Prevention of hemolysis as a novel strategy to limit hemoglobin-mediated lipid oxidation in fish - towards a more sustainable use of fish raw materials

Hemoglobin (Hb) has been recognized as a main pro-oxidant in fish, causing fast and intense lipid oxidation. This thesis explores the hypothesis that maintaining red blood cells (RBCs) intact for an extended time during fish processing could delay Hb-mediated lipid oxidation. The aims were to (i) unravel how selected endogenous and exogenous parameters affect fish RBC-stability, (ii) confirm the relation between hemolysis and lipid oxidation and (iii), evaluate antioxidative strategies to prevent oxidation caused by released Hb.In Study I, washed and resuspended trout RBCs (wr-RBC) and whole trout blood (WB) were used to study how temperature, salinity and mechanic pressure affect hemolysis. Cold temperatures, 0-6 \ub0C, and physiological saline were advantageous for the RBC stability while hyper- (3% NaCl) or hypotonic (tap water) conditions as well as simulated mechanic pressure hemolysis. Study II revealed that typical post-mortem pH’s, 6.4 and 6.8, yielded a higher hemolysis rate of wr-RBCs compared to pH 7.2-8.0, and stimulated lipid oxidation of the RBC membrane. Similarly, in RBC-spiked washed cod mince (WCM), pH 6.4/6.8 stimulated both lipid oxidation and hemolysis compared to pH 7.2/7.6 (Study III). Hb-analyses of the soluble phase revealed that only low levels of released Hb (7.1 \ub5M) were needed to initiate lipid oxidation at pH 6.4-6.8, which was not the case at high pH, even when >50% of the Hb was in the metHb form. In experiments with wr-RBCs, re-addition of blood plasma (12-75% v/w) largely increased the RBC stability (Study I-II). The stabilizing effect was partly ascribed to glucose, albumin, and ascorbic acid (AA). Adding blood plasma to the RBC-spiked WCM system 3% (v/w), delayed the onset of lipid oxidation at pH 6.8 with 3-4 days without delaying hemolysis; the latter most likely due to the high plasma dilution. To clarify the effect of hemolysis on WCM lipid oxidation, different ratios of intact and lysed RBCs were added to a WCM system at pH 6.8 (Study III). Samples with 25-100% lysed RBCs oxidized rapidly within the first day of storage, whereas the sample with 100% intact RBCs provided a 1-day delay in the onset of lipid oxidation. Further, the sample with initially fully intact RBCs resulted in 59.5\ub1 2.9% and 48.1\ub1 2.9% reduced maximum peroxide value (PV) and thiobarbituric acid reactive substances (TBARS), respectively. Short pre-incubation of herring co-products in 0.9% NaCl removed 6.6-18.0% of their Hb and slightly reduced the intensity of TBARS development during ice storage of the minced product, compared to pre-incubation in tap water or no pre-incubation (Study IV). Refrigerated storage of herring co-products while submerged in tap water, 0.9% NaCl or phosphate buffered saline (PBS) (pH 6.5 or 7.5) also reduced the lipid oxidation intensity compared to air-storage; however, without any effect from pH or salinity. When adding the commercial antioxidant mixture Duralox-MANC to the pre-incubation or submerging solutions (2% vs 0.5%) the lipid oxidation lag phase was largely prolonged; e.g., from < 1 day in controls to >6 days. Direct fortification of minced herring co-products with 0.5 % Duralox-MANC confirmed its antioxidative efficiency as lipid stability was prolonged from <1 day to >8 days.Altogether, this thesis confirmed that delaying hemolysis can be a route to delay lipid oxidation. It also revealed that current conditions used in the early handling/processing of fish, such as subjection to crowding, pumping, refrigerated sea water (RSW)-storage or tap water rinsing do not favor RBC stability and call for adjustments to limit hemolysis; and thereby Hb-mediated lipid oxidation. Intact RBCs, presence of blood plasma and elevated pH’s delayed lipid oxidation in a WCM system, providing an important window of time which could allow for better utilization of blood-rich fish raw materials currently leaving the food chain. To further enhance the lipid stability of such materials, subjecting them to rosemary-derived antioxidants via pre-incubation, submerging or direct addition is recommended. Results presented can contribute to an increased robustness of our global food systems towards unprecedented challenges such as pandemics, war, and climate threats. They also contribute to the ongoing dietary protein shift in which aquatic foods as small pelagic fish and fish rest raw materials have an immense potential if correctly preserved by updated procedures