*2.1. Stability Tests*

Three level Box–Behnken experiment design with response surface methodology was applied for the experiment setup (Table 1). To evaluate the efficiency of the emulsion preparation, a two-step homogenization process using both a high-speed homogenizer and an ultrasound homogenizer was used. To compare the behavior of the emulsion composition, we determined the emulsification index (EI) [31] which measured the emulsified layer relative to the total volume of the mixture. The EI results were compared with visual observation, which are presented in Figure 1 and show samples 24 h after homogenization. The visual measures confirmed that the two-step preparation process strongly increased the homogeneity of the emulsion in contrast to the one-step process [12,30]. One day after the preparation, no visible differences in samples were produced, and homogeneity was noted within the total volume. Visual observation confirmed a slight color difference in the samples, which were attributed to the different HSO and AHE concentrations, respectively.

**Table 1.** Box–Behnken design samples description; AHE—*Aesculus hippocastanum* L. extract; HSO hempseed oil.


\* Excluded from droplet size distributions tests.

We employed additional centrifugation tests to accelerate evaluation of the sample emulsion. The ratio of the optical density (measured at 600 nm) of the samples before and after centrifugation was used as an indicator for emulsion stability. The results were collected and presented in Table 2. A simple correlation between the concentration of individual emulsion components on emulsion stability after centrifugation could not be established. Only samples with the lowest HSO content (i.e., 1%) were characterized by higher stability. The highest values for this parameter (>50%) were also observed for samples S16 and S17, respectively, with the same concentration of HSO (1%). These results suggest that greater whey concentrations promoted the formation of relatively more stable emulsions.

*Molecules* **2021**, *26*, x FOR PEER REVIEW 4 of 20

**Figure 1.** (**A**) The emulsion samples after 24 h. (**B**) Criteria of selecting the optimal samples composition for further studies. **Figure 1.** (**A**) The emulsion samples after 24 h. (**B**) Criteria of selecting the optimal samples composition for further studies.


We employed additional centrifugation tests to accelerate evaluation of the sample emulsion. The ratio of the optical density (measured at 600 nm) of the samples before and **Table 2.** Preliminary stability tests after centrifugation. ODC/OD0—ratio of the optical density at 600 nm of samples after and before centrifugation.
