Value added fish by-products - Nordic Innovation

2.5.4 Antioxidative activity of fish proteins in liposome system
The antioxidant activity of proteins was determined using cod roe phospholipids liposomes
that were made as described by Slizyte et al. (2009). Lipid oxidation was performed in a
liposome assay containing 6 mg/ml phospholipids. Fe 3+ and bovine haemoglobin
(hemoglobin from bovine blood (lyophilized powder), Sigma-Aldrich) (Hb), was used to
catalyse lipid oxidation. The consumption of dissolved oxygen by liposomes in a closed,
stirred, water jacketed cell was used as a measure of lipid oxidation. The concentration of
dissolved oxygen was measured continuously by a polarographic oxygen electrode
(Hansatech Instrument Ltd., Norfolk, UK).
The antioxidative activity of proteins against Fe 3+ (15µM) induced oxidation was studied as
described by Slizyte et al. (2009). The antioxidant behaviour of proteins was studied by
analysing the effectiveness in reducing oxygen uptake induced by iron.
Hb induced oxidation led to non-linear oxygen uptake by liposomes (Figure 2.1, A). Due to
this, protein solution was injected to liposome solution before addition of Hb solution as
shown in Figure 2.1, B. The antioxidative activity of proteins (4mg/ml) against Hb (0.05
mg/ml) induced oxidation was calculated following: I(%) = 100 – (rprotein/rHb)×100, where:
rprotein - oxygen uptake by liposomes after injection of protein and Hb solutions; rHb - oxygen
uptake by liposomes after injection only Hb solutions (control for protein effect).
The influence of pH on protein antioxidative activity both on iron and Hb induced oxidation
was performed by making protein solution with different pH. The pH of the liposome
solution was adjusted by replacing some of the MES buffer that was used to dilute the 30
mg/ml liposome solution with different concentration of NaOH and HCl solution. Fe 3+
solution used to catalyse oxidation was with pH 2 for all experiment. Hb solution used to
initiate oxidation was made with the same pH as liposome solution. The pH of experiment
was verified after the oxidation experiment. Oxidation experiments were performed in
triplicates.
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