Effects of peroxidase and superoxide dismutase on physicochemical stability of fish oil-in-water emulsion

The study addresses how oxidoreductases affect the physicochemical stability of fish oil-in-water emulsions, which are used to improve the bioavailability of ω-3 polyunsaturated fatty acids but are prone to oxidation and destabilization. Prior work showed catalase rapidly demulsifies fish oil emulsions. Here, the authors hypothesize that horseradish peroxidase (HRP), a heme peroxidase, may similarly destabilize emulsions by promoting lipid oxidation and interacting with droplets, whereas copper/zinc superoxide dismutase (SOD) may act antioxidatively to enhance stability. The purpose is to compare HRP and SOD effects on emulsion physical stability (turbidity, size, ζ-potential, morphology) and chemical stability (hydroperoxides, TBARS), thereby clarifying enzyme–emulsion interfacial interactions relevant to food systems rich in unsaturated fats.

- Emulsions can enhance lipid bioavailability but stability is challenged by oxidation and environmental factors. - Hydrolases (lipase, chymotrypsin, trypsin) can alter emulsion stability; e.g., trypsin hydrolysates can delay lipid oxidation and extend shelf-life. Phospholipase and protease can demulsify certain food emulsions. - Oxidoreductases affect emulsions: prior work found SOD shows antioxidant effects in milk fat systems and with catalase can improve stability, though SOD could not inhibit hemin-catalyzed oxidation in high-oil emulsions. Catalase alone caused rapid demulsification of fish oil–polysorbate 80 emulsions. - Heme proteins (hemoglobin, myoglobin, peroxidases) can initiate peroxidation of PUFAs via radical chain reactions. HRP is robust and present in foods; SOD dismutates superoxide to O2 and H2O2 and is used as a food antioxidant. - Unclear how HRP and Cu/Zn-SOD, common in foods, modulate emulsions containing unsaturated lipids through noncovalent/covalent interactions, motivating this comparative study.

- Materials: Fish oil (EPA 18.3%, DHA 12.3%), polysorbate 80, HRP, Cu/Zn-SOD; deionized water. - Emulsion preparation: 1% v/v polysorbate 80 in water; 5% v/v fish oil added dropwise; homogenized at 18,000 rpm for 5 min at 40°C; filtered (0.22 μm PES) to yield submicron emulsion (FOE), pH 6.3. Initial droplet D_h ~130.6 nm; ζ-potential ~ −20 mV. - Enzyme treatments: HRP or SOD at 0, 0.16, 0.32, 0.48, 0.64, 0.8, 1.6, 2.4 μM. Incubation at 37°C for up to 7 or 14 days. - Physical stability assessments: - Visual inspection and microscopy over time. - Turbidity: Abs600 after 20× dilution at 25°C; mean of triplicates. - Centrifugation-based stability: 3000 rpm, 10 min; stability (%) = A_b/A_a ×100. - Dynamic light scattering for droplet size distribution and ζ-potential (Zetasizer Nano-ZS) at 25°C; triplicate after 60 s equilibration; dilute if necessary. - TEM: negative staining with 1% phosphotungstic acid; observed at 80 kV to visualize droplet morphology and protein precipitates. - Enzyme activity assays: - HRP activity: ABTS/H2O2 assay, Abs414 over 1 min; 1 U = ΔAbs414 of 0.01/min. - SOD activity: inhibition of hydroxylamine oxidation in xanthine/xanthine oxidase system; Abs535; activity computed as (A_blank − A_sample)/A_blank ÷ 50% × B × C. - Chemical stability (oxidation) measurements: - Hydroperoxides: FOX assay (Fe2+ to Fe3+ with xylenol orange), Abs560. - Lipid peroxidation (TBARS): TCA/TBA reaction, boil 15 min, centrifuge, Abs532; quantified vs 1,1,3,3-tetraethoxypropane standard. - Statistics: triplicate measurements; one-way ANOVA or t-test; p<0.05 significant, p<0.01 highly significant.

- HRP caused concentration- and time-dependent demulsification: - Visual phase separation and precipitation appeared by Day 7 at 37°C; turbidity decreased with higher HRP; demulsification onset preceded by rapid HRP activity loss in FOE (activity diminished by Day 3), whereas HRP in buffer remained largely active. - Stability (centrifugation) decreased dose-dependently up to ~0.64 μM HRP by Day 7; at ≥0.8 μM measurements were unreliable due to severe demulsification/precipitation. - DLS/TEM: At low HRP (≤0.32 μM), size distribution ~100–300 nm; at higher HRP (≥1.6 μM) by Day 7, major size shifted to micrometers; TEM showed droplet aggregation to submicron on Day 3 and micron-sized droplets by Day 7 surrounded by HRP layers; chain-like HRP precipitates observed. - ζ-potential: Mild decrease at high HRP on Day 0 (consistent with HRP's positive charge at pH 6), increased on Day 3 (attributed to lipid peroxidation and carboxyl formation), then decreased by Day 7 at high HRP (consistent with demulsification and presence of polysorbate colloids). - Hydroperoxides: Control FOE Day 0 <200 μM; fell <30 μM by Day 2; spiked on Day 3 and plateaued Day 7. HRP increased hydroperoxide nearly 3× immediately (Day 0); even 0.32 μM HRP raised >500 μM; by Day 7, hydroperoxides significantly dropped at 0.64 and 1.6 μM HRP (consistent with demulsification removing dispersed lipids). - TBARS: HRP elevated TBARS on Day 0 and Day 3; peak at 0.32 μM on Day 3 with declines at ≥0.64 μM; by Day 7 TBARS decreased at >0.32 μM despite ongoing demulsification. - SOD enhanced physical and chemical stability: - No demulsification or precipitation up to 14 days; turbidity and stability unchanged across 0–2.4 μM. - SOD activity in buffer declined slightly by Day 3 then stable; in FOE, slight decline through Day 5 then >90% loss by Day 7, likely due to inactivation by lipid hydroperoxides; despite inactivation, emulsion remained physically stable. - DLS/TEM: Droplets remained nano/submicron-sized; no protein precipitates. ζ-potential increased on Day 3 and Day 7 at all SOD concentrations without changes on Day 0, suggesting stabilization mechanism not due to direct binding nor increased carboxyls. - Hydroperoxides: 1.6 μM SOD immediately reduced Day 0 hydroperoxides and inhibited formation by up to ~80% during first 3 days; by Day 7, hydroperoxides were ~50% of control, with weakened inhibition consistent with SOD inactivation. - TBARS: SOD inhibited lipid peroxidation at all concentrations; higher SOD (≥1.6–2.4 μM) showed slightly higher TBARS than lower SOD (0.16–0.8 μM), possibly due to more H2O2 generation. - Mechanistic insights: HRP heme iron catalyzes radical formation and peroxidation at the oil/water interface, promotes droplet aggregation, protein precipitation, and demulsification; SOD counters oxidative stress (superoxide), thereby maintaining droplet charge and size and stabilizing the emulsion.

Findings show contrasting roles of two metalloenzymes at the oil/water interface of fish oil–polysorbate emulsions. HRP rapidly increased hydroperoxides (likely via heme iron-mediated radical chemistry), enhanced peroxidation (TBARS) early, and subsequently led to droplet growth, protein precipitation, and macroscopic demulsification after its apparent deactivation. Demulsification correlated with increased droplet size, reduced stability, and altered ζ-potential, and is attributed to HRP-lipid electrostatic/hydrophobic interactions and redox-driven disruption of the lipid/polysorbate interfacial assembly. The delayed yet pronounced demulsification versus catalase likely reflects fewer heme centers per protein and lower hydrophobicity of HRP. In contrast, SOD suppressed primary and secondary lipid oxidation (up to ~80% reduction in hydroperoxides during early incubation and reduced TBARS), increased ζ-potential over time without direct binding evidence, and preserved droplet size and macroscopic stability even after substantial SOD inactivation. This suggests superoxide control is sufficient to mitigate oxidative interfacial damage and maintain emulsion integrity. Overall, the results support a model where heme peroxidase activity at the interface promotes radical propagation and structural weakening of PUFA-rich droplet shells, whereas SOD attenuates oxidative stress and stabilizes interfacial properties.

Horseradish peroxidase, but not superoxide dismutase, initiates and accelerates demulsification of polysorbate 80-stabilized fish oil submicron emulsions. HRP elevates hydroperoxides and TBARS early, then, via redox and interfacial interactions, causes droplet growth to micrometer scale, protein precipitation, and phase separation over 3–7 days. SOD reduces hydroperoxides and lipid peroxidation and maintains physical stability over 14 days. The heme/iron center of HRP and electrostatic/hydrophobic protein–lipid interactions likely underlie demulsification, while SOD’s superoxide dismutation supports stabilization despite eventual inactivation. Findings highlight enzyme-specific impacts on PUFA-rich emulsions and motivate mechanistic studies of radical species and interfacial electron transfer, as well as the influence of surfactant type, fatty acid composition, ions, and pH on enzyme-mediated emulsion stability.

- The exact mechanism of HRP-induced demulsification remains unresolved; radical species and interfacial electron-transfer pathways were inferred but not directly quantified. - Causes of SOD-induced ζ-potential increase without evident binding are unclear and require further study. - Experiments used a single emulsifier (polysorbate 80), oil (fish oil), pH (~6.0–6.3), and temperature (37°C); generalizability to other surfactants, lipid compositions, ionic strengths, and pH conditions was not tested. - High SOD concentrations may generate more H2O2, potentially influencing peroxidation; this was hypothesized but not directly measured. - Long-term stability beyond 14 days and in real food matrices was not evaluated.