Glass vials containing the different SEO nanoemulsions were examined visually against bright light to check their opacity or translucency. The UV-Vis absorption values (A) for all samples were also evaluated spectrophotometrically using Shimadzu (UV-160 1PC spectrophotometer, Japan) at wave length = 600 nm to establish a quantitative measure for the visual examination of transparency of the samples. The absorption values (A) are an average of two measurements from two different formulations.

The particle size of the samples was measured using the dynamic light scattering instrument Zetasizer (Nano-ZS model ZEN3600, Nanoseries, Malvern Instruments, UK). Measurements were taken at 20 ^oC unless otherwise specified, with a fixed angle of 173^o. Before measurement, all samples were filtered through 0.20 µm single-use syringe filter unit (Minisart®, Sartoius Stedium Biotech GmbH Germany) to remove impurities. Each sample was diluted before measurement with distilled water to only 0.05% to prevent multiple scattering. The measurements are based on the Brownian motion of the hydrated particles thus; it provides information on the hydrodynamic diameter (nm) of the SEO particles. The sizes quoted are the z-average mean of the SEO droplet’s hydrodynamic diameter (nm) obtained from 6 measurements for each sample (2 duplicate x 3 measurements each) ±SD, as shown in Table (2). The particle size distribution curves are plotted from the dynamic laser scattering results from duplicate samples.

In a trial to produce ethanol-free nanoemulsion upon dilution into water, a basic tertiary emulsifiable concentrate (Ia) composed of SEO (6.0%), single surfactant (6.0%), water (88.0%) was made (Table 1). No traces of triglyceride oil were used in this formula to see if the desired nanoemulsion could be developed using SEO as the sole oil phase. The results showed that upon dilution of (Ia) emulsifiable concentrate into water, a traditional cloudy emulsion which had high UV-Vis absorbance (A= 3.39, Figure 1A) was obtained. That indicated a failure in the formulation of the desired nanoemulsion. Although the average particle size of that emulsion was 121.3 nm (Table 2), its size distribution analysis showed a second population of particles with an average size of about 5.0 μm (Figure 1A , chart). That makes the emulsion of (Ia) appear cloudier due to enhanced light scattering by the large particle population which also caused a relatively high polydispersibility index (Table 2).

Regarding physical stability, emulsion (Ia) demonstrated poor stability as evident from the appearance of a heavy creamy layer on the walls of the glass vial upon tilting to 45º within only 24h of storage at room temperature (20 °C). These results are likely to be due to the phenomena of Ostwald ripening, (OR).

The incorporation of 1.0 wt% long-chain triglyceride (LCT), such as sunflower oil (set Ib, Table 1) led to the inhibition of OR and positively influenced the appearance, particle size, and shelf-life stability of the obtained SEO nanoemulsion, (Figure 1A and Table 2). This was manifested by a change in UV-Vis absorption of the nanoemulsion of (Ib) to become 0.33, which was in accordance with its less cloudy appearance compared to (Ia), as shown in Figure 1A. In addition, the nanoemulsion of set (Ib) was characterized by a much smaller particle size (38.0±0.5 nm) with better size distribution and lower PDI, as evident from the data in Table 2 and Figure 1A (chart). Unlike the emulsion of set (Ia), which creamed after 24h, the new nanoemulsion of (Ib) which contained LCT overcame OR and showed much higher physical stability. It maintained the same appearance with almost the same UV-Vis absorption even after the 3-month storage period at 20 ºC (Figure 1B). The particle size of nanoemulsion (Ib) after the end of the 3-month storage was 35.0±0.2 nm (Table 2) and its size distribution was monomodal (Figure 1B , chart). The particle size of (Ib) and its distribution pattern were even better after storage than at time zero. The reason behind this result is that storing (Ib) for 3months provided enough time for SEO nanoparticles to equilibrate and attain their final size. That chance was not offered to the same formula at time zero because particle sizing took place shortly after fabrication.

The reader may notice that the particle sizing of emulsions (Ia) and (Ic) are excluded from analysis after the 3-month storage period and seemed absent from Figure 1B (chart). This is because they initially showed early creaming after 24h (Ia), and 1 week (Ic), respectively, as will be shown in the results regarding nanoemulsion of set (Ic).

Despite the high physical stability of the nanoemulsion of (Ib) at 20 ºC, it showed temperature sensitivity when stored at 40 ºC for only 24h. That was manifested by the conversion of its appearance into milky white (Figure 1C), indicating an increase in particle size and loss of the nanostructure.

The incorporation of 1.0 wt% of another triglyceride oil with medium-chain fatty acids (MCT) such as C₈ and C₁₀ (set Ic, Table 1) also led to the inhibition of OR. However, the quality of the obtained emulsion of (Ic) was inferior compared to the nanoemulsion of (Ib) which was formulated using LCT. For instance, Figure 1A indicates that the appearance of nanoemulsion (Ic) was cloudy with a much higher UV-Vis absorbance value compared to (Ib). The particle size of (Ic) was 96.3±0.8 nm which represents more than double the size of nanoemulsion (Ib), as shown in Table 2. The particle size distribution of (Ic) also showed a bimodal pattern which justified its relatively high PDI (0.41, Table 2). The shelf-life stability of emulsion (Ic) was also inferior compared to (Ib). After only 1 week of storage at room temperature, the nanoemulsion of (Ic) showed a thin creamy layer on the side of the glass vials upon tilting to 45º. Regarding thermal stability, the emulsion of (Ic) became even cloudier after storage for 24h at 40 ᵒC (Figure 1C).

An additional emulsifiable concentrate (Id) was formulated with the incorporation of 1.0 wt% propylene glycol (PG) as a cosurfactant (Table 1). Results of the effect of PG on the formation and stability of SEO nanoemulsion showed that PG increased the transparency of the nanoemulsion (Id) to become the clearest among those of set (I), (Figure 1A). The average particle size of that nanoemulsion was 28.2±0.1 nm which is considered to be the smallest among all nanoemulsions of set (I) as shown in (Table 2). This result demonstrates the potential of PG at only 1.0 wt% of the formula to act as an efficient cosurfactant that can replace ethanol for enhancing the formulation of SEO nanoemulsion with an ultra-fine particle size.

The helf-life stability study indicated that the nanoemulsion which contained PG was physically stable for 3(+) months as long as the temperature was ≤ 20 ºC. No noticeable change in transparency or in the average particle size after storage (25.9±0.1 nm) was detected as shown in Table 2, and Figure (1B, chart). It is worth noting that these results cannot be attained with PG concentration < 1.0%, as revealed in a preliminary experiment in the current study (data not shown).

Unfortunately, despite the distinctive effect of PG on the formulation and stability of SEO nanoemulsion of (Id), the formula showed temperature sensitivity when stored at 40 ºC for only 24h. That was manifested by a radical change in appearance from the most translucent nanoemulsion into milky white macroemulsion (Figure 1C). That indicates a dramatic increase in particle size and loss of nanostructure.

A fifth emulsifiable concentrate (Ie) was made among the formulas of set (I) using the ingredients of formula (Id) after reducing its S/O ratio from 1.0 to 0.5 (Table 1). The effect of surfactant reduction on the stability of the ethanol-free SEO nanoemulsion was studies. The results in Figure 1A show that the obtained nanoemulsion of (Ie) had an opaque appearance with a monomodal particle size distribution. The average particle size was 62.4±0.2 nm (Table 2), which is about double the size of the nanoemulsion of (Id) in which the S/O ratio was higher (equals 1.0). After a 3-month storage period at 20 ᵒC the particle size of (Ie) slightly changed from 62.4 nm to 71.9±0.7 nm (Table 2), with monomodal size distribution as shown in Figure 1B, (chart).

There was also no detected separation or formation of creamy layer after the 3-month storage period. This result is considered to be satisfactory for a stable nanoemulsion because the particle size remained < 100 nm after storage, with no signs of creaming. The reader can also take into consideration that this result was obtained at 50.0% less surfactant with respect to the most ideal physically stable nanoemulsion of (Id).

Subjecting the nanoemulsion of (Ie) thermal stress by storage at 40 ºC led to the conversion of the nanoemulsion into a milky white macroemulsion after only 24h (Figure 1C).

Formula (Ia), which contained no traces of triglycerides (Table 1), failed to produce SEO nanoemulsion and showed poor physical stability in less than 24h. One should bear in mind that the S/O ratio of emulsion (Ia) equals 1.0. That means the presence of a high amount of surfactant, enough to support the formation of a stable emulsion even without using high energy shear equipment in the emulsification process. Therefore, there should be a factor which caused such rapid creaming in this formula which is likely to be the phenomena of Ostwald ripening (OR), (McClements et al., 2012). OR occurs in nanoemulsions (or emulsions) when the oil phase is slightly soluble in water, such as the case with essential oils. OR takes place as a result of the growth of large oil droplets at the expense of tiny ones. That happens due to the continuous migration (through water) of oil from the tiny droplet to the larger droplets under the effect of the high curvature pressure “Laplace pressure” which is exerted on the tiny droplets. Therefore, large droplets become larger and finally float to the surface causing a creamy layer in a short period of time, just a few hours after formulation (McClements et al., 2012).

EO emulsions and nanoemulsions are a typical example of the manifestation of OR due to the slight water solubility of their ingredients (McClements et al., 2012). This was clearly proven in the current study in the case of formula (Ia) due to the slight water solubility of the major volatile constituents of SEO. For instance, the water solubility of l-carvone, d-limonene and 1,8-cineol, which are the three major constituents of SEO, was 27.0 mg·l^-1, 13.8 mg·l^-1 and 552.0 mg·l^-1, respectively, as indicated by the official database (PPDB & BPDB, web sites). These three constituents together represented about 81.2% of the total composition of SEO, as was revealed in the current study by GC analysis. Therefore, the author concludes that OR contributes significantly to the observed early creaming of formula (Ia).

In a trial to inhibit OR and to develop more stable SEO nanoemulsion, we incorporated equal amounts (1.0wt %) of two different triglyceride oils into formula (Ib) and (Ic). The first was sunflower oil, which is considered to have long-chain triglycerides (LCT, set Ib), while the second was synthetic medium-chain triglycerides (MCT) made of C₈-C₁₀ (set Ic), as shown in Table 1.

The entropy of mixing of SEO with these triglyceride oils provided chemical potential against OR and counterbalanced the driving force for EO migration from tiny particles to large ones (McClements et al., 2012).

This treatment showed a noticeable improvement in the appearance, particle size and storage stability of the SEO nanoemulsion, as shown in the results section (Table 2) and (Figure 1A, B). However, it was clear from the results that LCT was a much better excipient than MCT in the formulation of stable SEO nanoemulsion with respect to two aspects: first, better inhibition of OR; and second, greater influence on the reduction of SEO particle size. Detailed justifications of these two findings are discussed in the next passage.

First, the ability of LCT to inhibit OR to a better degree compared to MCT is due to its strong hydrophobic nature (HLB 4.0) compared to that of MCT (HLB 8.0), (Wooster et al., 2008). The strong hydrophobicity of LCT originates from its long chain fatty acids which include linoleic (C18:2), oleic (C18:1) and stearic (C18:0) acids. These fatty acids constitute the composition of the sunflower oil which was used as LCT. Therefore, the spiking of SEO with such a small amount (1.0 wt%) of LCT, (as in nanoemulsion of set Ib) can reduce the water solubility of its volatile ingredients to a great extent compared to MCT, leading to better inhibition of OR. That conclusion came in accordance with another study (McClements et al., 2012) which further concluded that the hydrocarbon chain length of the triglyceride can affect the interfacial tension between EO droplets and water, and consequently, affect the overall nanoemulsion stability. It is worth indicating that MCT is composed of medium-chain caprylic (C₈) and capric (C₁₀) acids which are less hydrophobic than the other long-chain fatty acids.

On the other hand, our second finding concerning the greater influence of LCT on the reduction of the particle size of SEO nanoemulsion of set (Ib) compared to (Ic) can be justified based on the physical affinity between the oil phase mixture (SEO/triglyceride) and the utilized surfactant. The high affinity between these ingredients leads to a high tendency toward the production of nanoemulsion with ultra-fine particle size (Anton and Vandamme, 2009). Our results indicated that the affinity between (SEO/LCT) and Tween 80, which is used as a surfactant, is higher than that of (SEO/MCT) and the same surfactant. This conclusion is based on the ability of (SEO/LCT) to produce nanoemulsions (Ib) with smaller particle size (38.0 nm) compared to (Ic, 96.0 nm) which was made of (SEO/MCT), as shown in Table 2.

Ethanol is usually used as cosurfactant in a relatively high amount to facilitate the formulation of EO nanoemulsions. It can be left in the nanomemulsion after fabrication (Zhang et al., 2017), or removed by evaporation in a process known as solvent displacement (Katata et al., 2017). However, the presence of ethanol in food and beverages is met with objection from some consumers who seek solvent-free products.

Therefore, out of the desire to eliminate ethanol from food or beverages, we replaced ethanol with only 1.0 wt% PG as cosurfactant. PG is a food-permitted ingredient which has a wide safety margin that can reach up to 25mg/Kg body wt./day (Joint FAO/WHO, 2002). Therefore, another emulsifiable concentrate (set Id, Table 1) was developed based on the ingredients of the promising formula (Ib) with the incorporation of 1.0 wt% PG as cosurfactant (Table 1). As a result, a new SEO nanoemulsion (Id) with better transparent appearance (Figure 1A), smallest particle size (28.2 nm, Table 2) and high stability was obtained.

The mechanism of the action of PG as cosurfactant which is able to facilitate the formation of nanoparticles and to enhance its physical stability is similar to ethanol and other short chain alcohols (Garti et al., 2001). PG can arrange itself along with the main surfactant at the interfacial layer between SEO and water, leading to the formation of a mixed surfactant film. This behavior lends flexibility to the film around SEO droplets, leading to an ease in attaining the maximum curvature required for ultra-fine oil particles to be developed. In addition, PG can modify the hydrophobicity of the main surfactant, making it more compatible with the SEO phase.

It is important to note that the role of PG in the formation of ethanol-free nanoemulsion cannot be generalized to include all other types of essential oils. This statement was concluded by the author himself who found that PG does not make any improvement in the formulation of clove or cinnamon EO nanoemulsions (data not published). The particle size of these nanoemulsions increased in the presence of PG rather than decreased as expected. At this point, the real reasons behind that peculiar behavior are not known. However, it can be speculated that the chemical composition of the EO as well as its interaction with the chosen surfactant must have an effect. Further investigations are required in this respect to unravel the scientific basis behind the effect of PG on the formation of various types of EO nanoemulsions.

The S/O ratio is considered to be a key factor in the fabrication of EO nanoemulsions using the low energy spontaneous emulsification method. This ratio represents the product of dividing the mass of surfactant by that of the oil phase. The common ratio which is usually used in the art to induce the formation of nanoemulsion is 1.0 (i.e. equal masses of surfactant and oil). However, this ratio can be increased in the case of other types of formulations to become 2.0 (Komaiko and McClements, 2015). This means doubling the amount of surfactant relative to EO.

In the current study the S/O ratio equal to 1.0 was used in the four emulsifiable concentrates of set (I) from (Ia-Id). However, a reduction in the amount of surfactant is generally appreciated in order to fulfill the requirements of legislation in food and beverages and also to proceed economically. However, that trend is considered to be a challenge for formulators due to the crucial role of the amount of surfactant in the stability of nanoemulsions formulated according to the low energy method.

This issue was addressed in the current study by the fabrication of a fifth emulsifiable concentrate (Ie) which was made using ingredients from formula (Id) after the reduction in its S/O ratio from 1.0 to 0.5 (Table 1). That means a 50.0% reduction in the amount of surfactant relative to the four other emulsifiable concentrates of set (I). The results showed that this nanoemulsion was physically stable and had particle size < 100 nm even after a 3-month storage period at 20 °C. It is important to note that this result is unattainable in the absence of PG as cosurfactant. This affirms the importance of this compound as a cosurfactant in the formation of physically stable and ethanol-free SEO nanoemulsion, especially at a reduced S/O ratio. On the other hand, at 40 °C, the appearance of this nanoemulsion changed to milky white within 24h, indicating a loss in the nanostructure (Figure 1C). This elicits the temperature sensitivity of (Ie) nanoemulsion as is the case with all nanoemulsions of set (I).