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A basic requirement for low oxidative sensitivity is that the materials used for emulsification, in particular the oil phase and the emulsifier, contain the lowest possible levels of hydroperoxides (as they are a source of free radicals upon decomposition) and iron and copper (as they lead to decomposition of hydroperoxides) (McClements and Decker 2000). For example, Mancuso et al. (1999a) found 4–35 µmol peroxide/g surfactant in lecithin, Brijs and Tweens.

The presence of effective chelators in the system makes metal ions unavailable for the lipid oxidation process. The addition of artificial chelators such as EDTA has a marked

antioxidative effect. EDTA can also be added legally as an antioxidant in food O/W emulsions (e.g. in the USA with a permitted dose of 75−100 ppm (FDA 2007)). Chelating properties of proteins have also been reported, as discussed later. Not only the continuous phase but also the oil phase can be a source of metal ions (water, oil, surfactants, buffer substances (Mei et al. 1998a)). Proteins naturally contain minerals and water may also be a source of pro-oxidative metal ions. Food oil can be contaminated with low levels of metals from the production process (Benjelloun et al. 1991). The location of chelators is therefore a relevant factor for an effective chelation process.

The solubility of iron in water increases with decreasing pH (Donnelly et al. 1998; Graf et al. 1984; Mancuso et al. 1999b). Thus, more lipid oxidation occurred in a Tween-20- based emulsion at pH 3 than at pH 7, when oxidation was promoted by Fe3+ and ascorbate, creating Fe2+. Metal repulsion would have been similar at both pH conditions because of a non-ionic droplet surface (Donnelly et al. 1998). In another study, in which Tween 20 was also used as the emulsifier and no iron was added, lipid oxidation was greater at pH 7 than at pH 3. An increased iron concentration on the droplet surface was found at pH 7, which indicated that more iron had precipitated on the droplet surface at the higher pH (Mancuso et al. 1999b). An explanation for these seemingly contradictory results is offered by Mei et al. (1998a); the solubility of ferric iron increases from pH 7 to pH 3, whereas the solubility of ferrous iron increases from pH 3 to pH 7. In contrast, ferrous iron is apparently still 1017 (pH 7) and 1013 (pH 3) times more water soluble than ferric iron (Decker et al. 2002). The concentration of the respective redox form therefore appears to be a relevant factor for how much iron can precipitate on the droplets at a certain pH, affecting the oxidative stability of the emulsion.

Iron on the droplet interface can also be removed by continuous phase chelators. This was shown in a study of Mei et al. (1998a), as the addition of EDTA or phytate to an SDS-stabilised hexadecane O/W emulsion with ferrous iron lowered the zeta-potential on the droplets. In the same study, the lipid oxidation rate in SDS-stabilised salmon O/W emulsions with ferrous iron decreased when EDTA or phytate was added. An example of a real food product is mayonnaise, stabilised with the protein phosvitin from egg yolk. Phosvitin has a high iron content and lipid oxidation may be accelerated by

the release of iron at the droplet interface under acidic conditions. EDTA can remove iron from phosvitin at the interface and thus inhibit lipid oxidation (Jacobsen et al. 2001; Thomsen et al. 2000). In a study of Tong et al. (2000b), the addition of ferrous iron to a hexadecane O/W emulsion (pH 7) stabilised with bovine serum albumin (0.1 %) led to an increase in the zeta-potential at the droplet surface, indicating iron association. When increasing levels of a high molecular weight fraction of whey (molecular weight ≥ 3500) were incorporated, the zeta-potential decreased. At the highest whey protein level (14.0 µg/ml), the zeta-potential had decreased more than when 20 µmol EDTA/l was added and the level was below the initial level before iron addition. It was concluded that both components were able to remove iron from the interface by chelation. In the same study, the lipid oxidation rate in a Tween-20- stabilised salmon O/W emulsion (pH 7) to which the same whey fraction had been added also decreased. This pointed to the possibility that the removal of iron from the interface into the continuous phase may have contributed to the improved oxidative stability. Other milk proteins also exhibit metal-binding abilities and can thus enhance the oxidative stability of emulsions. This is discussed in more detail in a later section.

The surface charge of the droplets also plays an important role in the lipid oxidation process. Pro-oxidative cations can be electrostatically repelled into the continuous phase by a positive surface charge, away from the interface where lipid hydroperoxides are prone to metal-induced decomposition. In contrast, a negatively charged droplet surface can promote lipid oxidation by attracting pro-oxidative cations (Genot et al. 2003; McClements and Decker 2000). These effects were shown when emulsions stabilised with an anionic surfactant (SDS) oxidised more than emulsions stabilised with non- ionic (Tween 20 or Brij 35) or cationic (dodecyltrimethylammonium bromide) surfactants, when iron (Mei et al. 1998b) or no iron (Mancuso et al. 1999b) was added. In a study of Mei et al. (1998b), sodium chloride (1.0 %) reduced iron-promoted lipid oxidation in an SDS-stabilised emulsion with a net negative surface charge by 20 %, but had no effect in emulsions with non-ionic or cationic surfactants. The authors pointed to the possibility that other cations, which are not pro-oxidative, such as sodium ions in this particular case, may compete with iron ions for negatively charged binding sites on the droplet surface. This can lead to lower iron adsorption on the droplets and thus decreased lipid oxidation.

It was also found that the access of antioxidants from the continuous phase to the interface can be limited, and their effectiveness reduced, if their electric charge has the same sign as the droplet surface charge. For example, partially negatively charged galloyl derivatives did not associate with SDS-stabilised droplets with a negative surface charge at pH 7.0 (Mei et al. 1999).

In general, the repelling or attracting force on the droplet surface depends on the surface charge density (McClements and Decker 2000). The pH determines the surface charge of protein molecules. In protein-based emulsions, the pH is therefore an influential factor on the oxidative stability. Proteins are more likely to repel iron and copper ions when they are positively charged at pH conditions below their isoelectric point. At the interface, metal ions from the continuous phase would be repelled, but continuous phase proteins are also less capable of binding metal ions. When a fraction of acid whey (molecular weight ≤ 5000) was added to a phosphatidylcholine liposome system, the inhibition of TBARS formation increased with increasing pH from pH 5 to pH 7. This might have been due to greater metal-binding capacity, keeping metal ions away from the liposomes (Colbert and Decker 1991). In a study of Donnelly et al. (1998), WPI- stabilised menhaden O/W emulsions with iron had more oxidative stability at pH 3.0 than at pH 7.0. This observation was attributed to the greater repulsion of iron from the net positively charged protein interface at low pH. Similar results were obtained by Faraji et al. (2004); less lipid hydroperoxides and propanal were produced in menhaden O/W emulsions stabilised with 0.5 % WPI at pH 3 than at pH 7. In a study by Hu et al. (2003a), various whey proteins (WPI, sweet whey, α-lactalbumin, β-lactoglobulin) were used to produce salmon O/W emulsions. Increasing pH led to decreasing droplet surface charge (indicated by a decrease in the zeta-potential). At the same time, the lipid oxidation rates increased. However, amongst the emulsions prepared with different proteins (pH 3), the order of oxidative stability and zeta-potential did not totally correspond, showing that the droplet surface charge was not the only factor that influenced the oxidative stability.