*2.2. Droplet Size Studies*

Droplet size and PDI (polydisperisty index) are key factors affecting emulsion stability. Based on the experimental design used for preliminary studies (Table 3), different concentrations of HSO, AHE and whey protein were used to find the optimal droplet size distribution of nanoemulsion formulations. According to our previous studies [9,32], we decided to eliminate runs S06, S12 and S15 from the experimental design model (Table 1) for the evaluation of the droplet size. Analysis of the results presented in Table 3 for the center points of the Box–Behnken design were performed on samples S01, S05, S06, S12, S15, and similar results for Z-ave, PDI, average peak maximum by intensity were recorded for the S01 and S05 samples (see the standard deviation range of three replications).

In contrast to previous studies [33,34], we determined the droplet size of the emulsion systems as delivered after preparing the emulsion without additional dilution since dilution may affect the emulsion stability. Furthermore, based on the principles of the dynamic light scattering (DLS) method used herein and apparatus limitations, we chose to round droplet size numbers up to a full number. Moreover, based on previous experience from studies with different high-PDI systems [35], we considered droplet/particle size by intensity and number. Detailed studies of droplet size distributions clearly showed that even a small fraction of larger particles/droplets or accidental contaminations impacted the Z-ave and size distribution. Results presented in Table 2 exhibit that the major percentage of droplets were much smaller than average size distribution by intensity peak maximum. For example, the average maximum of the peak by number for sample S08 was 23 nm; however, its light scattering intensity was 318 nm.


**Table 3.** Preliminary tests, droplet size distribution results: Z-ave (nm); PDI—polydisperisty index; average size distribution by intensity peak maximum (nm) and average size distribution by number peak maximum (nm).

N/A: not applicable; ±SD (standard deviation of three replicates).

According to the regression analysis and the ANOVA, we were able to predict Z-Ave for samples using a quadratic model (*p* < 0.05). The relative significance in order of individual factors according to the greatest effect was determined to be as follows: HSO (%) > whey (%) > AHE. The interaction of HSO and whey was a significant factor affecting Z-ave of samples (*p* < 0.05). The significant interaction effect of HSO and a poloxamer used as a surfactant to produce an HSO-based nanoemulsion was previously reported [36]. The ANOVA and the regression analysis also showed that the PDI of nanoemulsion samples could be fit to a linear model with HSO (%) as the only significant factor (*p* < 0.05). To find an optimal and stable formulation and further possible applications of the emulsion

system, other characterizations such as droplet size distribution, visual, centrifugation and microscopic properties of the emulsion need to be considered in addition to Z-ave and the PDI. Additional stability studies, such as identification of destabilization mechanisms including creaming, sedimentation, flocculation, coalescence can also confirm the stability potential of an emulsion system. Based on the preliminary stability tests as well as droplet size distribution, we selected four samples for further analysis (Figure 1B).

Figure 2 presents detailed analysis of droplet size distribution from four selected samples. All the selected samples contained a whey concentration of 4, except for S14, which had a whey concentration of 2.5%. According to the DLS results of S14 (Figure 2D), the scattered light intensity and droplet size by number were not different. This result was confirmed by a low PDI value (0.190). Microscopic observation of sample S14 (Figure 2D) also confirmed homogeneity of the samples. In addition, in comparison with other samples, single droplets were easy to distinguish at low magnification (e.g., ×40).

Based on our previous observations for the HSO emulsion system stabilized by the AHE [12], we decided to evaluate emulsion droplet distribution and sample homogeneity using light microscopy with an inverted microscope. To prevent fast evaporation of samples, the emulsions were inserted in a corvette equipped with slide channels. The images presented in Figure 3 were taken a few minutes after the emulsion injection. This method prevents the flow of injected liquid artefacts. For unstable systems, a short break in time enabled the observed coalescence of the droplets. With lower magnification, high homogeneity of samples S02, S08 and S13 was observed (Figure 3A–C). Only a small fraction of larger droplets could be distinguished. This observation corresponded with the DLS results in case of droplet size distribution by intensity, where a more intensive signal was detected for the samples with larger hydrodynamic diameters (Figure 2A–C). The presence of the smaller droplets fraction determined by DLS was probably caused by two-step processing using ultrasound treatment for preparing this emulsion. Some of the studies suggested that ultrasound treatment as well as ultrahigh-pressure homogenization caused an increase in the α-helix structure content of whey protein and a decrease in the β-sheet component of whey protein [37,38].

Microscopic imaging for nanoemulsion systems with proteins presented by Zhu et al. [39] corresponded to our findings. In their studies, optical microscopic imaging was compared with confocal laser scanning microscopy (CLSM). It was suggested that using CLSM allowed for the determination of a core–shell structure in the emulsion systems where proteins were located at the surface of the emulsion droplets. More detailed studies are needed for our nanoemulsion systems. First of all, signals from all the fluorescent structures should be identified and separated (i.e., from HSO, AHE) [12,31]. Then, additional studies on the impact of whey protein on the fluorescent behavior of the components should be investigated. Ren and Giusti [40] showed that anthocyanin-rich extracts decreased the fluorescence intensity of whey protein while increasing λmax. The study concluded that thermally induced whey protein was effective in protecting anthocyanin from color degradation. Using an optical microscope, we focused only on the verification of the homogeneity of the samples as well as possible coalescence (which was not observed in the optimal nanoemulsions). Nevertheless, we strongly recommend using more than one technique for the analysis of the emulsion systems droplets size.

a decrease in the β-sheet component of whey protein [37,38].

homogeneity of samples S02, S08 and S13 was observed (Figure 3A–C). Only a small fraction of larger droplets could be distinguished. This observation corresponded with the DLS results in case of droplet size distribution by intensity, where a more intensive signal was detected for the samples with larger hydrodynamic diameters (Figure 2A–C). The presence of the smaller droplets fraction determined by DLS was probably caused by twostep processing using ultrasound treatment for preparing this emulsion. Some of the studies suggested that ultrasound treatment as well as ultrahigh-pressure homogenization caused an increase in the α-helix structure content of whey protein and

**Figure 2.** Droplet size distribution by intensity and by number: (**A**) S02, (**B**) S08, (**C**) S13, (**D**) S14. **Figure 2.** Droplet size distribution by intensity and by number: (**A**) S02, (**B**) S08, (**C**) S13, (**D**) S14.
