2.6.2. Freeze-Thaw Stability of Pickering Emulsions

The freeze-thaw stability of Pickering emulsions was measured by the previous method of Zhu [33]. A total of 15 mL of the emulsion was shifted into freeze-resistant glass bottles and then placed in a freezer at −18 ◦C for 22 h. Then the emulsions were naturally defrosted at room temperature for 2 h. The process was repeated thrice, the creaming index and oiling off index of the emulsions were recorded after each cycle.

#### *2.7. Droplets Size of Pickering Emulsions*

Droplet size distributions of emulsions were measured by a Mastersizer 3000 (Malvern Instruments Co., Ltd., Worcestershire, UK). The Mie theory was applied by considering the following optical properties for corn oil droplets: a refractive index of 1.46 and absorption of 0.001, and for the deionized water, a refractive index of 1.33. Add the sample drop by drop to the deionized water with automatic mixing until an obscuration rate of between 10% and 20% [34]. Prior to the measurements, the samples were thermostated at 25 ◦C for 5 min and all measurements were carried out over at least 3 times. All emulsions were analyzed 24 h after the emulsification process. The volume-weighted mean diameter (D4,3) was reported.

#### *2.8. Microstructure Analysis*

The size, shape, and aggregate state of emulsion droplets were quantified by the optical microscopic method described by Yang et al. [27] with slight modifications. Approximately 20 μL of emulsions were deposited on the microscope slide and covered with a coverslip. The morphology of the Pickering emulsion droplet was observed using an inverted optical microscope (CKX41, Olympus, Tokyo, Japan) at 200× magnification.

Confocal Laser Scanning Microscopy (CLSM) were determined by using a Nikon Ti-E-A1R confocal microscope (Nikon Instruments Inc, Tokyo, Japan) to observe the microstructure of the emulsions. The CLSM was operated in the fluorescence mode. Nile red was used to stain the oil phase. All of the stain solutions were prepared at room temperature and stored in a dark place. Approximately 1.0 mL of the samples were completely mixed with a 10 μL aliquot of the Nile red (0.1%, *w*/*v*). The mixtures were equilibrated for 30 min before observation. Approximately 20 μL of the stained samples was placed on slide and carefully covered with a coverslip, ensuring that there was no trapped air gap or bubbles between the mixture and the cover slip. The images were collected using 488 nm excitation wavelengths for Nile red. Each image contained 1024 × 1024 pixels.

#### *2.9. Statistical Analysis*

All experiments were performed in triplicate, and the value was expressed as mean value ± standard deviation. The statistical differences among means were evaluated using a one-way analysis of variance and Duncan's test (*p* < 0.05).

#### **3. Results and Discussion**
