2.4. Emulsion Preparation

Native WPI powder was gently dispersed in phosphate buffer (10 mM, pH 7.0) and stirred magnetically at 500 rpm for 2 h and then stored overnight at 4 °C to produce a fully hydrated stock WPI solution (2.0%, w/v). In order to avoid droplet flocculation of WPI-stabilized O/W emulsions under acidic conditions since the isoelectric point of WPI is approximately 4.6 [23], the neutral pH of the WPI solution was selected in our experiment. Moreover, as indicated by the results of Chen et al. [20], the O/W emulsion prepared with 2.0% (w/v) WPI solution showed the highest physical stability during the storage.

Before emulsion preparation, the fully hydrogenated coconut oil and the WPI stock solution were kept at 40 °C by a water-bath heating method to prevent coconut oil solidification. To prepare the O/W emulsions, 5.0 mL each of coconut oil was expeditiously mixed with different concentrations of 13-HPODE solution or MDA solution (0, 1, 10, 100, and 1000 mmol/L emulsion) by using a vortex oscillator (Vortex-Genie2, Scientific Industries, Bohemia, NY, USA) at a speed of “5” for 10 s. Then, 45.0 mL of the WPI solution was mixed with the above coconut oils by using a high-shear homogenizer (IKA T18 basic, IKA-Werke GmbH & Co., Staufen, Germany) at 13,500 rpm for 2 min. After that, the coarse emulsions were further homogenized by an ultrasonic probe (FS-100T, Shanghai, China) according to the method of Chen et al. [20]. Sodium azide (0.02%, w/v) was added to the emulsions to prevent microbial growth. The emulsions were quickly transferred to 50 mL sealed plastic tubes and stored in the dark at 37 °C for pending analysis. The indicators of protein oxidation in each of the O/W emulsions were measured at 24 and 48 h.