Maintaining the physicochemical stability of oil emulsions is crucial in the food industry, especially for valuable omega-3 fatty acids found in fish oil, which are prone to oxidation and degradation. Emulsions enhance lipid bioavailability, making their stability critical. While the effects of hydrolases (like lipases and proteases) on emulsion stability are well-studied, the role of oxidoreductases remains less understood. Previous research showed that catalase caused demulsification in fish oil emulsions. This study focuses on HRP and SOD, two metal ion-containing oxidoreductases. HRP, a heme protein, was chosen for its similarity to catalase, while SOD was selected for its potential antioxidant and stabilizing effects. Both enzymes are common in foods and interact with lipids and emulsifiers. The study uses a submicron fish oil-in-water emulsion stabilized with polysorbate 80 to investigate the impact of HRP and SOD on its physicochemical stability.

Existing literature highlights the impact of enzymes on emulsion stability. Hydrolases such as lipase, chymotrypsin, and trypsin are extensively studied, with some enhancing stability while others promoting destabilization. Oxidoreductases have also been investigated; for example, SOD showed antioxidant effects in milk fat systems, while catalase alone caused rapid demulsification in fish oil emulsions in previous studies. The influence of HRP and SOD on lipid-rich emulsions, particularly considering their prevalence in food products, is less understood and requires investigation.

Submicron fish oil-in-water emulsions (FOE) were prepared using polysorbate 80 as an emulsifier and high-speed homogenization. Different concentrations of HRP and SOD (0 µM, 0.16 µM, 0.32 µM, 0.48 µM, 0.64 µM, 0.8 µM, 1.6 µM, 2.4 µM) were added to the emulsions. The emulsions were incubated at 37°C for 7 or 14 days. Physicochemical stability was assessed through visual observation, microscopy (TEM), turbidity measurements, centrifugation tests to determine stability, particle size analysis (dynamic light scattering), zeta potential measurements, and analysis of lipid oxidation (hydroperoxide content and TBARS levels). Enzyme activity assays were performed for HRP and SOD in both pure solutions and within the emulsions. Statistical analysis (one-way ANOVA or t-test) was used to determine significant differences.

Visual observations and turbidity measurements revealed that HRP caused concentration-dependent demulsification and precipitation after 3-7 days of incubation. In contrast, SOD did not induce demulsification even after 14 days. Particle size analysis showed that HRP led to aggregation and enlargement of oil droplets, while SOD maintained the nano- and submicron size distribution. Zeta potential measurements showed a decrease in the presence of HRP, consistent with destabilization, and an increase in the presence of SOD. TEM images confirmed the aggregation of oil droplets and the presence of HRP precipitates in the HRP-treated emulsions. HRP initially increased hydroperoxide levels, followed by a decrease, while TBARS levels increased. SOD significantly reduced hydroperoxide and TBARS levels, indicating its antioxidant activity. The activity of HRP decreased significantly in the emulsion compared to its pure solution, while SOD activity also decreased, albeit less dramatically.

The results demonstrate that HRP's demulsifying effect is likely due to its catalytic oxidation activity and interactions between the enzyme and emulsion components. The heme iron in HRP likely catalyzes the generation of free radicals, leading to increased lipid oxidation and the formation of hydroperoxides. These changes may alter the interfacial properties of the emulsion, contributing to demulsification and protein precipitation. In contrast, SOD's antioxidant activity prevents lipid oxidation and maintains emulsion stability. The different effects of HRP and SOD highlight the crucial role of the enzyme's redox properties in influencing emulsion stability. The study supports the hypothesis that lipid peroxidation alone is not sufficient to cause demulsification, suggesting a role for protein-lipid interactions at the interface.

This study clearly demonstrates that HRP destabilizes fish oil emulsions, while SOD stabilizes them. HRP's action involves oxidation catalysis, and interactions between the enzyme's heme group and lipids. SOD's stabilizing effect stems from its antioxidant activity. These findings contribute significantly to the understanding of oxidoreductase effects on food emulsions and open avenues for exploring the detailed mechanisms and developing strategies for enhancing the stability of lipid-rich food products.

The study used a specific model emulsion system. The results may not be directly generalizable to all types of fish oil emulsions or other lipid-based emulsions. Further studies are needed to investigate the influence of other factors (surfactants, fatty acid composition, pH) on the effects of HRP and SOD on emulsion stability. Also, the exact mechanisms involved in protein-lipid interactions warrant further investigation.