2.6.1. Hydroperoxides

Hydroperoxides were analyzed according to Celus et al. [10]. In brief, 0.3 mL of emulsion and 1.5 mL of iso-octane/2-propanol (3:1 v/v) were added to a centrifuge tube and centrifuged (Sigma 3-18KS centrifuge, Germany) at 1000 g and 20°C for 2 min after vortexing three times for 10 s. Then, 0.2 mL of the supernatant was transferred to the test tube, and 2.8 mL of methanol/2-propanol (2:1 v/v) solution, 15 μL of 3.94 M ammonium thiocyanate solution, and 15 μL of 0.0072 M Fe²⁺ solution were successively added and mixed. The absorbance of the mixture was measured at 510 nm after 20 min of storage without light. The peroxide concentration in the emulsion was calculated by dividing the Fe³⁺ concentration by 55.845 (the atomic weight of Fe³⁺), and the Fe³⁺ concentration was determined by reference to the standard curve of FeCl₃ solution.

2.6.2. TBARS

The formation of TBARS was measured according to Xu et al. [11] with slight modifications. In brief, 1 mL of emulsion and 2 mL of thiobarbituric acid (TBA) solution were added to a capped test tube. After boiling for 15 min, cool quickly to room temperature in 10 min. Centrifuge 1000 g for 15 min. The absorbance of the supernatant was measured at 532 nm. The concentration of secondary oxidation products was obtained by a 1,1,3,3-tetraethoxypropane (TEP) standard curve.

3.1.1. Similar Charges on Droplets and Polysaccharides (pH 7.0)

Both RBPH-coated oil droplets and polysaccharides carry negative charges at pH 7.0 [9], which should cause electrostatic repulsion between them.

The emulsion containing XG remained relatively stable without significant phase separation (Figure 1). The control emulsion also showed this stable state when the storage time was less than 21 days (Figure 1). In addition, no large aggregation was observed in the 29-day microscopic images (Figure 2(a)). The mean particle diameter (d_3,2) remained at approximately 0.96 ± 0.28 μm (control emulsion) and 1.55 ± 0.36 μm (containing XG) (Figure 3(a)). This system was stable because it was maintained by electrostatic repulsion between RBPH-coated droplets or RBPH-coated droplets and polysaccharide molecules. In particular, the emulsion with XG always had a high charge, from approximately −30 mV to −40 mV (Figure 4(a)). This result may be related to changes in interface composition during storage. One possible reason for this observation is that the anionic reaction products formed during lipid oxidation accumulated on the surface of the oil droplets, thereby increasing their negative charge. An increase in electrostatic repulsion also reduced the accumulation of oil droplets [12].

Pectin-containing emulsions also showed considerable aggregation and creaming stability at the beginning of the storage period. The d_3,2 value remained at 1.03 ± 0.2 μm (Figure 3(a)), and rapid phase separation was not observed (Figure 1). No substantial amount of aggregation was observed in microscopic images (Figure 2(a)). The initial particle size (1.03 ± 0.21 μm) of pectin-coated emulsions was more extensive than that of the control emulsions (0.96 ± 0.28 μm), probably due to the presence of a layer of pectin molecules on the surface of the oil droplets. The pectin layer can be adsorbed because the anion group carries binds to the cationic region on the surface of the RBPH-coated droplet. This is similar to the reported protein–polysaccharide system above the isoelectric point of proteins [8]. The excellent aggregation stability of pectin-stabilized emulsions may also be due to the synergistic reaction of electrostatic and steric repulsion between the RBPH-coated droplets and the pectin-coated droplet [13].

3.1.2. Opposite Charges on Droplets and Polysaccharides (pH 3.5)

At pH 3.5, the control emulsion was very unstable due to droplet aggregation and creaming: After 29 days of storage, the d_3,2 value increased from 2.35 μm to 11.91 μm (Figure 3(b)). Rapid phase separation occurred after 24 h (Figure 1). Large droplets and flocs were clearly observed under a microscope (Figure 2). The reason for these droplets and flocs was that the pH of the solution was close to the isoelectric point of the adsorbed protein. At the same time, the electric repulsion between oil droplets was not sufficient to overcome van der Waals forces and hydrophobic interactions and thus led to droplet aggregation. The particle size of the control emulsion remained stable for the first 6 days and then increased rapidly (Figure 3(b)).

At pH 3.5, the RBPH-coated oil droplets had a positive charge, while the polysaccharide molecules had a negative charge. The addition of polysaccharides has been shown to promote a strong electrostatic attraction between these molecules [9]. Compared with the control emulsion, the emulsion containing anionic polysaccharide had a smaller particle size (Figure 3(b)), better emulsion stability (Figure 1), and less aggregation (Figures 1 and 2(b)). It can be seen that the aggregation and creaming stability of the emulsion will be increased after the addition of polysaccharide. The results show that the electrostatic repulsion and steric hindrance between the anionic polysaccharides adsorbed on the surface of cationic oil droplets can be increased, and their aggregation stability can be greatly improved [13]. Therefore, this mechanism reasonably explains the phenomenon: the physical stability of the emulsion added with XG and pectin was increased.

XG molecules adhere more strongly to the surface of the oil droplets and generate greater electrostatic repulsion. In contrast, pectin has weak adhesion to the surface of the droplet. When some pectin molecules are separated from one droplet and adsorb to the surface of another droplet, bridging flocculation occurs [14]. In addition, the electrostatic repulsion between the droplets coated with pectin was weaker than that of the droplets coated with XG, which was also the reason for the greater droplet aggregation. The ζ-potential also reflected that the droplets coated with XG carried a significantly higher charge than those coated with pectin (Figure 4(b)). It is interesting that at pH 3.5, the ζ-potential coated by an anionic polysaccharide did not change over time, suggesting that their interface composition remained relatively constant.

3.2.1. Similar Charges on Droplets and Polysaccharides (pH 7.0)

During preservation at neutral pH (pH 7.0), although the primary oxidation products of the emulsion at the initial stage increased, lipid oxidation occurred, but the addition of polysaccharides had a certain delay effect on the increase in peroxidation value. During the initial 16 days of storage, lipid hydroperoxides and TBARS formed very similarly in all emulsion systems (Figures 5(a) and 5(b)) and increased slowly. However, during longer storage periods, the degree of lipid oxidation in the samples showed significant differences, and the general trend was XG-containing emulsion > control emulsion > pectin-containing emulsion. The rapid formation of TBARS occurs after the formation of lipid hydroperoxides because of the decomposition of primary oxidation products before the formation of secondary oxidation products [15]. The results showed that under these conditions, XG played a small role in promoting oxidation, while pectin played a small role in antioxidation. One possible reason is that the negatively charged polysaccharides in the aqueous phase can bind to positively charged transition metals (e.g., Fe²⁺), preventing them from binding to the ω-3 PUFAs contained in the droplets [10]. However, a similar phenomenon was observed in emulsions stabilized with whey protein: the rate of lipid oxidation in emulsions containing XG was significantly higher than that in control emulsions [10]. One possible reason for this observation is that XG increased the water solubility of metals at this pH. Metal ions are poorly water soluble at a neutral pH of 7.0, which may reduce their oxidation-promoting activity [16]. Therefore, if the highly anionic XG molecule increases the solubility of iron ions, it can increase the oxidation capacity of lipids. The addition of high anionic phospholipids to a neutral aqueous solution containing iron has a similar effect [17].

3.2.2. Opposite Charges on Droplets and Polysaccharides (pH 3.5)

As shown in Figure 6, the curves of the control emulsion oxidation products with RBPH stabilization were all above the curves of emulsions containing a polysaccharide (pectin, XG), which demonstrated the inhibition of polysaccharide addition on oxidation at pH 3.5. The lipid hydroperoxide and TBARS contents increased initially but decreased with prolonged storage time (Figure 6). The possible reasons for this effect were the initial formation of these reaction products and their degradation as lipid oxidation progressed. lipid oxidation increased in the order of XG-containing emulsion < pectin-containing emulsion < control emulsion. This pattern was consistent with previous reports that the addition of XG or pectin under acidic conditions can reduce lipid oxidation in emulsions [18, 19]. At pH 3.5, both XG and pectin can inhibit lipid oxidation to a certain extent, but XG has a stronger inhibitory capacity than pectin.

Previous studies have shown that iron is more soluble under acidic conditions than under neutral conditions [14]. Therefore, under these conditions, anionic polysaccharides have a strong binding ability with cationic transition metal ions, which may reduce their ability to interact with oil droplets rich in omega-3 unsaturated fatty acids. An important reason for the antioxidant activity of anionic polysaccharides is that the negative electric groups carried by them can chelate with cationic metals [20]. Under acidic conditions, because XG has a more negative charge than pectin [9], it has a stronger ability to bind transition metals. XG can inhibit lipid oxidation through physical and chemical effects. XG can chelate metal ions via the negatively charged pyruvate moiety and quench peroxides in the oil by passivation of Fe²⁺ ions. This is why it is a more potent oxidant than pectin under these conditions. Under acidic conditions, the control emulsions were extremely unstable and tended to aggregate compared with those containing polysaccharides (Figure 1). Lipid oxidation may occur more rapidly when the oil droplets are relatively close together because free radicals produced in one oil droplet may be more easily transported to adjacent oil droplets. This explains why emulsions containing XG have greater antioxidant capacity than emulsions containing pectin. Similar conclusions also appeared in the study of the hydrolyzate of rice gluten [11], suggesting that the main role of rice bran protein may be gluten.