*2.4. FTIR*

The FTIR spectra were recorded with air or water as the background. Both series of results are presented in Figure 5.

The HSO spectrum shows clear signals originating from the C–H bonds (around 2950 cm−<sup>1</sup> ) and C = O (at 1700 cm−<sup>1</sup> ), respectively. The absence of signals that are characteristic of hydroxyl group bonds proves that there were no significant amounts of free fatty acids and hydroxylated acids in the oil. A relatively weak but distinctive signal slightly above 3000 cm−<sup>1</sup> confirms the existence of double bonds between carbon atoms, which most likely originated from unsaturated fatty acid residues in HSO, as confirmed by others [49].

Among the signals common to all emulsion samples, those arising from the O–H bonds (at about 3250 cm−<sup>1</sup> ), aliphatic C–H (at 2800–2950 cm−<sup>1</sup> ) and C = O (at 1700 cm−<sup>1</sup> ) should be distinguished. Although the differences in the intensities of the individual bands can be explained by the different composition of the emulsions, it is important to note that these differences were not only in intensity, but also in the position of the signals in the 1050–1150 cm−<sup>1</sup> range. They could originate from vibrations of the C–O bonds (to a lesser

extent, of C–N, which occurs in the protein structure). A similar shift, visible as a change in shape, is present for vibrations from the O–H group. This shift may indicate a variation in the strength and configuration of the hydrogen bonds between polar components of the tested emulsions, suggesting the complex interactions between the emulsion components, which may then have impact on their physicochemical properties [50,51] and plausible biological activity of the samples [52,53]. *Molecules* **2021**, *26*, x FOR PEER REVIEW 11 of 20

**Figure 4.** Rheological test results of the S02, S08, S13, and S14 samples: (**a**) flow curve, (**b**) dynamic viscosity changes, (**c**) power law model. **Figure 4.** Rheological test results of the S02, S08, S13, and S14 samples: (**a**) flow curve, (**b**) dynamic viscosity changes, (**c**) power law model.

The FTIR spectra were recorded with air or water as the background. Both series of

*2.4. FTIR* 

results are presented in Figure 5.

**Figure 5.** FTIR spectra of investigated samples with (**a**) air or (**b**) water as the background. **Figure 5.** FTIR spectra of investigated samples with (**a**) air or (**b**) water as the background.

#### The HSO spectrum shows clear signals originating from the C–H bonds (around 2950 *2.5. Color and Refractive Index Analysis*

cm−1) and C = O (at 1700 cm−1), respectively. The absence of signals that are characteristic of hydroxyl group bonds proves that there were no significant amounts of free fatty acids and hydroxylated acids in the oil. A relatively weak but distinctive signal slightly above 3000 cm−1 confirms the existence of double bonds between carbon atoms, which most likely originated from unsaturated fatty acid residues in HSO, as confirmed by others [49]. Among the signals common to all emulsion samples, those arising from the O–H bonds (at about 3250 cm−1), aliphatic C–H (at 2800–2950 cm−1) and C = O (at 1700 cm−1) should be distinguished. Although the differences in the intensities of the individual bands can be explained by the different composition of the emulsions, it is important to note that these differences were not only in intensity, but also in the position of the signals in the 1050–1150 cm−1 range. They could originate from vibrations of the C–O bonds (to a The results of the *L\*a\*b\** color analysis of samples in Figure 6 showed that the S02 sample represented more lightness, followed by S13, S08 and S14. Considering the ratio of ingredients in samples S02, S13 and S08, it was determined that the *L\** value decreased with higher concentrations of HSO or the AHE, respectively. However, sample S14 having a low content of HSO (1%) and the AHE (1%) featured a low *L\** value. We hypothesize that other factors might affect the lightness of samples in addition to the concentration of ingredients. Based on droplet size distribution studies, the PDI and Z-ave of S14 were lower compared to other samples (Table 1). In addition, according to McClements and Demetriades (1998), interactions between the ingredients within the emulsion and some other factors such as transmission, reflection, scattering and absorption can also affect the color and appearance of the emulsion [54].

lesser extent, of C–N, which occurs in the protein structure). A similar shift, visible as a change in shape, is present for vibrations from the O–H group. This shift may indicate a variation in the strength and configuration of the hydrogen bonds between polar components of the tested emulsions, suggesting the complex interactions between the emulsion components, which may then have impact on their physicochemical properties [50,51] and plausible biological activity of the samples [52,53]. It was also found that the difference between the *a\** values was negligible. In this analysis, sample S14 showed lower *b\** values. This observation may also relate to the higher ratio of whey to HSO in this formulation, which caused a shift in favor of a yellow shade. To monitor the color of the emulsions with a unique index, the WI values of the samples were calculated based on the *L\**, *a\** and *b\** values. The WI values were found to be 42.68, 35.86, 40.00 and 29.87 for S02, S08, S13 and S14, respectively, indicating marked difference between sample appearance.

*2.5. Color and Refractive Index Analysis*  The results of the *L\*a\*b\** color analysis of samples in Figure 6 showed that the S02 sample represented more lightness, followed by S13, S08 and S14. Considering the ratio of ingredients in samples S02, S13 and S08, it was determined that the *L\** value decreased The refractive index recorded for the dispersed phase represented the average refractive indices of individual droplets (Table 5). The theory that the thickness of the phase interface is small enough comparative to the wavelength of the related light would indicatethat the interface of droplets plus the oil phase act as an individual dispersed phase [55].

with higher concentrations of HSO or the AHE, respectively. However, sample S14 having a low content of HSO (1%) and the AHE (1%) featured a low *L\** value. We hypothesize that other factors might affect the lightness of samples in addition to the concentration of ingredients. Based on droplet size distribution studies, the PDI and Z-ave of S14 were lower compared to other samples (Table 1). In addition, according to McClements and Demetriades (1998), interactions between the ingredients within the emulsion and some

color and appearance of the emulsion [54].

**Figure 6.** *L\*a\*b\** color analysis results of the samples S02, S08, S13, S14. **Figure 6.** *L\*a\*b\** color analysis results of the samples S02, S08, S13, S14.

It was also found that the difference between the *a\** values was negligible. In this **Table 5.** Refractive index of the investigated samples.


other factors such as transmission, reflection, scattering and absorption can also affect the

Refractive index accuracy was estimated to be 0.0001.

be 42.68, 35.86, 40.00 and 29.87 for S02, S08, S13 and S14, respectively, indicating marked difference between sample appearance. **Table 5.** Refractive index of the investigated samples. **Sample Name S02 S08 S13 S14**  Refractive index 1.3368 1.3400 1.3384 1.3349 Refractive index accuracy was estimated to be 0.0001. The refractive index recorded for the dispersed phase represented the average refractive indices of individual droplets (Table 5). The theory that the thickness of the The total appearance of an emulsion can be determined by both light scattering and absorption. Scattering is mainly accountable by recording the turbidity or lightness of an emulsion, while absorption determines the chromatic properties (redness, blueness, greenness, etc.) [56]. The relationship between the color of an emulsion and the refractive index ratio has an important role in foods containing high proportions of the aqueous phase. The refractive index of the samples (Table 4) was similar to the RI of the dispersed phase (water). This property allowed us to use an optical transmission method to analyze emulsions. The level of lipid oxidation in an emulsion, for example, can be determined by adding some chemicals (such as glycerol) to adjust the RI to 1.0, then measuring the spectra of the absorption [57].

#### phase interface is small enough comparative to the wavelength of the related light would indicate that the interface of droplets plus the oil phase act as an individual dispersed *2.6. Biological Activity*

phase [55]. The total appearance of an emulsion can be determined by both light scattering and absorption. Scattering is mainly accountable by recording the turbidity or lightness of an emulsion, while absorption determines the chromatic properties (redness, blueness, greenness, etc.) [56]. The relationship between the color of an emulsion and the refractive To assess the microbiological safety of optimized emulsions, we determined the effect of emulsions on the *Lactobacillus* sp. 2675 strain, a common probiotic (Figure 7). As a reference sample, cultures without an emulsion were considered (with 0% changes in cell metabolic activity). The highest increase in the bacterial cell metabolic activity (84%) was noticed for sample S08. The observed increase for sample S13 was relatively lower (66%). The increase in metabolic activities were lowest for samples S14 and S02 (37% and 31%, respectively). It can be assumed that higher concentrations of the AHE promoted

higher probiotic bacteria growth; however, the small number of experiments needs further confirmation of results.

Nevertheless, the critical observations were that all samples showed a positive effect on the metabolic activity of *Lactobaccilus* sp. 2675, indication non-toxic properties of the emulsions. Our results correspond directly with results presented by Gharehcheshmeh et al. [58], who reported no effect of a sweet almond and sesame oil nanoemulsions on the growth of *L. delbrueckii* subsp. *bulgaricus*. There are however, few studies showing an effect of AHE on probiotic bacteria. However, former studies showing an impact on probiotic bacteria of polyphenol-rich extracts from blueberry [57,58] or apples [59] agree with our results.

Nevertheless, the crucial observations are that all the samples exhibited a positive effect on the metabolic activity, which proves the nontoxic properties of the emulsions. Our results correspond directly with the results presented by Gharehcheshmeh et al. who studied the impact of sweet almond and sesame oil nanoemulsions on the growth of *L. delbrueckii* subsp. *bulgaricus*. They did not observe any inhibitory effect of nanoemulsions.

It is worth adding that the blueberry (*Vaccinium corymbosum*) extract [59,60] also showed beneficial effects on probiotic microorganisms due to the presence of polyphenols, which is also a distinguishing feature of the AHE we used [31]. It should also be noted that plant-derived substances can become an additional nutrient for probiotic microorganisms while providing a protective substance against the adverse effects of the external environment. Ahmad et al. [61] obtained promising results using polyphenols extracted from apples to protect the *Bifidobacterium lactis* bacteria during freezing. In the light of these studies, we can see the great potential of our obtained emulsions to stimulate growth and protect probiotic bacteria. *Molecules* **2021**, *26*, x FOR PEER REVIEW 15 of 20

**Figure 7.** Changes in the metabolic activity of the *Lactobacillus* sp. 2675 cells exposed for 24 h to emulsion samples. **Figure 7.** Changes in the metabolic activity of the *Lactobacillus* sp. 2675 cells exposed for 24 h to emulsion samples.
