3.2.1. Effect of HLB Value on the Mean Droplet Size of LEO-NEs

The HLB value of the surfactant can assist in identifying the best-suited stabilizer. When the HLB value of the STmix couple matches the HLB value required for the EO to form nanoemulsions, NEs with a small droplet size can be produced [22]. It was a crucial step to select an appropriate HLB value to obtain LEO-NEs with the smallest particle size. In the present work, the impact of the HLB value on the mean droplet size of LEO-NEs was studied first. As indicated in Figure 1a, when the HLB value changed from 8–12, the mean droplet size progressively declined, while the mean droplet size exhibited an upward trend when the HLB value was above 12. Furthermore, the particle size of LEO-NEs grew considerably when the HLB value increased from 14 to 15. Therefore, the optimum HLB value for the smallest droplet of LEO-NEs was 12.

**Figure 1.** Effects of HLB value (**a**), essential oil concentration (**b**), Surfactant concentration (**c**), ultrasound time (**d**) and ultrasonic power (**e**) on the mean droplet size of NEO-NEs.

## 3.2.2. Effect of Essential Oil Concentration on the Mean Droplet Size of LEO-NEs

To explore the effect of essential oil concentration on the mean droplet size of LEO-NEs, the formulation was performed with different LEO concentrations (ranging from 0.05 to 0.1 g/mL). As shown in Figure 1b, a significant increase in the mean droplet size was observed when the LEO content was changed from 0.05 g/mL to 0.1 g/mL. The nanoemulsion with a low concentration of LEO was more suitable for production applications. According to our results, the essential oil concentration of 0.05 g/mL in LEO-NEs was finally chosen for the subsequent experiments.

#### 3.2.3. Effect of Surfactant Concentration on the Mean Droplet Size of LEO-NEs

STmix with different concentrations was used in the NEs system. As shown in Figure 1c, a sharp decrease in the mean droplet size from 133.71 to 75.66 nm was observed when STmix concentration increased from 0.0125 to 0.1 g/mL. On the other hand, it remained almost constant when increasing the surfactant concentration from 0.1 to 0.2 g/mL. Therefore, the 0.1 g/mL surfactant concentration was selected for subsequent experiments.

#### 3.2.4. Effect of Ultrasonic Time on the Mean Droplet Size of LEO-NEs

Various ultrasonic time was used to prepare the NEs, with the aim of investigating the effects on the mean droplet size of LEO-NEs. As shown in Figure 1d, when the ultrasonic time was 0, which means that the emulsion was not treated by ultrasound, the mean droplet size fluctuated over a wide range, and the repeatability of the experiment was poor. Meanwhile, a layering phenomenon was observed after staying still at room temperature overnight. The smallest particle size was observed at the ultrasonic time of 20 min. The increase in ultrasonic time can promote the integration of water and oil. Longer ultrasonic times, on the other hand, may result in higher degradation or disintegration of bioactive chemicals in LEO, as well as energy waste [23]. Therefore, ultrasonic time of 20 min was selected for the subsequent studies considering both saving energy and achieving the best results.
