The droplet size and the droplet size distribution of placebo and EO-based NEs were analyzed by dynamic light scattering (DLS) technique (Horiba SZ100, Japan). In this analysis, a 532-nm laser and scattering angles of 90 and 173° were used by the HORIBA SZ-100 DLS instrument, depending on sample conditions. Average droplet size was measured, and data were presented as average diameter ± standard deviation (SD). The zeta potential was determined by means of disposable flat-fold capillary zeta cells, and the measurements were performed through the system software. Measurements were repeated three times to obtain the mean millivolt ± SD at 25°C (32).

The microscopic image was used to analyze the shape of the microemulsions using a high-resolution inverted microscope (Leica DM IL LED Fluo, Germany). The results were represented in corresponding figures (33).

The pH value of placebo and EO-based NE formulations was measured at 25 ± 1°C with a digital pH meter (Mettler Toledo FiveGo, Switzerland). The test was achieved in triplicate and displayed as mean ± SD (34).

The electrical conductivity of placebo and EO-based NEs was determined using a conductivity meter (Mettler Toledo FiveEasy Plus). The samples were kept at 25 ± 1°C. The measurements were accomplished in triplicate and pointed out as mean ± SD (35).

A Brookfield cone and plate rheometer were used to measure the shear stress, shear rate, and apparent viscosity of placebo and EO-based NEs in triplicate (Brookfield DV3THACJ0; Brookfield Engineering Labs. Inc., MA, USA) under controlled temperature (25 ± 0.5°C). Rotation speed was adjusted at 10–100 rpm (31).

The cheese sample (10 g) was homogenized with 100 ml distilled water for 1 min using a homogenizer. The pH was carried out using a digital pH meter (Mettler-Toledo, Schwerzenbach, Switzerland), according to the method of Polat Yemiş et al. (39). Color measurements were carried out at room temperature to ensure consistency and minimize temperature-related variations using a Minolta Chroma meter (Chroma Meter CM-5; Minolta Camera Co. Ltd., Osaka, Japan), following the method of Cai et al. (10). The values of L^*, a^*, and b^* were done and the whiteness index (WI) was calculated according to the following equation (40).

Antioxidant activity and total phenol compounds were accomplished by using the method of Shawir et al. (41). For antioxidant activity (ABTS) and total phenolic content, the samples, weighing 5 g, were placed completely in 10 ml of methanol and then centrifuged at 5,000 rpm for 10 min.

The ABTS method was done with slight modifications as described by Re et al. (42). The reaction between the diluted sample and 3.9 ml ABTS radical solution (7 mM; mixed with 2.45 mM potassium persulfate and left for 12–16 h) was carried out kinetically by measuring the change in 734 nm absorbance (Cary 60 UV-Vis Spectrophotometer, Agilent Technologies) for 15 min. Antioxidant activities were expressed as μmol Trolox/L from the absorbances of Trolox standards prepared at different concentrations (6.25–200.0 ppm) determined by the same method.

Total phenolic content (TPC) analysis was done according to the method of Singleton et al. (43) with minor changes. Before analysis, 0.5 ml of Folin–Ciocalteu solution was added to cheese samples prepared according to the procedure of Shawir et al. (41), and the mixture was left for 3 min. After that, 20% sodium carbonate was added to this mixture and allowed to stand for 60 min at room temperature in the dark room. Absorbance values were read at 765 nm with a UV/VIS spectrophotometer (Cary 60, Agilent Technologies). Using the calibration received from the absorbances of standards (gallic acid) prepared at different concentrations (between 31.25 and 500.0 ppm) by the same method, the TPC values were then calculated. Data were presented as mg/g of gallic acid equivalents.

Lactic acid bacteria (LAB), total aerobic mesophilic bacteria (TAMB), and yeast–mold assays were performed on curd cheese samples. The results were calculated as log cfu/g.

One of the most essential properties for stability is droplet size and distribution. The concentration of oil and S_mix has a synergy with the droplet size. Besides the small droplet size, the distribution is also important and is quantified using the polydispersity index (PDI) (26, 62). Wei et al. (63) reported that the droplet size of fenpropathrin NE decreased by increasing the concentrations of surfactant and cosurfactant, and the stability of the emulsion also improved. Similar results were found by Salama et al. (64).

The PDI is a parameter that measures the homogeneity of droplets, ranging between 0.0 and 1.0. A low PDI value indicates closely packed droplet size and homogeneity in the distribution of NE formulations. A PDI value ≤ 0.7 corresponds to a monodisperse formulation, suggesting a uniform NE. A PDI value < 0.25 signifies a narrow size distribution in the system. The other values indicate that the formulations are in acceptable ranges (30, 65). The average droplet size and PDI values of optimum formulations are shown in Table 3.

All reported values correspond to the mean ± SD of three measurements. The average droplet size for 1%, 3%, and 5% NEs was 154.9 ± 9.6, 165.0 ± 3.6, and 151.3 ± 15.6 nm, respectively. The droplet size distribution and zeta potential graphs for each formulation are presented in Figure 2. The results of all formulations were within the nanometre range and exhibited the desired characteristics. In a previous study, various formulation combinations were designed using ethyl oleate, Cremophor EL, and Tween 80 to develop an NE containing corosolic acid. The distribution of droplet size and PDI of the prepared NE were characterized using DLS (Malvern Instruments, United Kingdom). The smallest droplet size of 181.3 nm and a PDI of 0.424 were obtained in formulations containing oleic acid (60).

The stability of NEs under storage is assessed by the zeta potential, which is an important parameter indicating the electrostatic surface charge of globules or particles. A higher absolute zeta potential value (both positive and negative) represents a higher stability. Stable NEs can also be obtained using non-ionic or polymeric surfactants, with zeta potentials as close to zero as possible (66, 67). The zeta potential measurement results showed that the formulations prepared with Cremophor EL, a non-ionic surfactant, did not exhibit any electrical charge (Table 3).

The results demonstrate that varying the concentration of the active ingredient within the NE 2:1 formulation has a limited impact on most physicochemical characteristics. Zeta potential values were close to zero for all samples, which may indicate steric stabilization provided by Cremophor^® EL rather than electrostatic repulsion. The stable pH and conductivity across all groups suggest that the internal structure of the nanoemulsions was not significantly altered by increasing active ingredient concentrations. Furthermore, viscosity remained within an acceptable range, indicating good flow properties suitable for topical or transdermal application. Overall, the data suggest that the NE 2:1 system is robust and tolerant to variations in active content within the tested range.

The surface morphology of the microemulsion formulations was investigated using a Leica light microscope (DM750). The sample was analyzed to determine the size of the NEs. The microscope images of the NEs revealed globular-shaped droplets with well-defined outlines. There was no aggregation. The sizes of the nanodroplets were consistent with the measurements obtained from the droplet size assessment, further supporting the accuracy of the size analysis (16).

The pH values ranged from 5.0 to 6.0, as shown in Table 3. All formulations had a neutral pH value and did not exhibit highly acidic or highly basic characteristics.

Conductivity is an important parameter to characterize emulsion type. The outcomes of the optimized formulations are presented in Table 3. The conductivity of w/o emulsions was usually low. This proved that the formulation was o/w type, confirming that the oil phase was indeed the inner phase and dispersed in the aqueous phase as desired (37).

The main factors influencing emulsion viscosity are the colloidal interactions, droplet charge, droplet size, and volume fraction of the dispersed phase (26). The viscosity of the NEs incorporating EO at 1%, 3%, and 5% concentrations was 150, 250, and 350 mPa·s (millipascal-second), respectively, whereas the viscosity of the placebo formulation was 100 mPa·s. An ideal viscous emulsion was obtained compared with that in other studies on NEs (61, 62, 67).

The rheological behavior of the rheograms is shown in Figure 3. The shear rate increased proportionally with shear stress, but viscosity remained constant. This invariability confirmed that the NE behaved as a Newtonian fluid, suggesting a well-organized internal structure in which the components maintained their arrangement and resisted significant changes when subjected to applied stress. The linear increase in shear stress with shear rate, which is a characteristic of Newtonian fluids, confirmed the mechanical stability of the NEs. The steady rise in shear stress without deviation highlighted the robustness and consistency of the formulation under mechanical stress. The formulations were developed in accordance with the fluid nature of NEs, exhibiting low viscosity and aligning with the Newtonian fluid model (67–69).

Table 5 demonstrates how the physicochemical characteristics of curd cheese samples changed during 28 days of storage. The pH is one of the most important factors influencing the texture and flavor of cheese, as it affects the solubility of caseins and the activity of enzymes involved in ripening (39). The pH in all cheese samples varied between 6.54 ± 0.04 and 5.42 ± 0.01, indicating that nanoemulsions with T. spicata EO had a significant impact on the pH of curd cheese (p < 0.05). The release of alkaline compounds during proteolysis may explain the slight increase in the pH of the cheese, especially on day 5 (39). Additionally, in a recent study conducted to determine the effect of microencapsulated allspice nanoemulsion on the quality and shelf life of Ras cheese, total acidity increases and pH decreases in cheeses fortified with various concentrations of microcapsules emerging during the ripening period (77). Al-Obaidi (78) showed no significant differences in pH, ash, protein, fat, and moisture between normal cheese and cheese with three different amounts of Curcuma longa (0.1%, 0.2%, and 0.3%) immediately after production. Christaki et al. (79) determined the pH of oregano EO-based nanoemulsion at 4.28 and 6.34 in whey cheese. There were no significant differences in pH and other contents between the control and the trial, respectively, and curcumin nanoemulsion and emulsion-based cheese were also reported by Bagale et al. (80). Cai et al. (10) examined the pH of cheese increased in edible films reinforced with orange EO-based nanoemulsion during storage. The pH of cheese samples packaged with edible films increased slightly from 6.27 ± 0.014 on the first day to 6.465 ± 0.01 on the 12th day, indicating that no spoilage occurred in the cheese after 12 days of storage. The results showed that edible film packaging slowed down the pH changes of cheese, thereby preventing the cheese samples from spoiling. The particle size of nanoemulsions is closely related to WI. Hence, parameters such as droplet size, concentration, and refractive index directly affect the overall optical properties of the emulsion (81). In the study, all nanoemulsions visually exhibited a transparent appearance in cheese. However, WI measurements showed minor differences depending on the concentration of EO used in nanoemulsion (Table 5). In this study, the WI value decreased as the droplet size decreased. This situation reveals that the optical properties of nanoemulsions change depending on the particle size. The optical characteristics of nanoemulsions are an important factor when it comes to food applications (39). In most cases, transparent film-forming solutions are preferred to produce films that almost do not change the color of the food (82). Christaki et al. (79) incorporated nanoemulsions of oregano EO in whey cheese, and they observed that C. citratus or C. reticulata compared to Origanum or Thymus nanoemulsions (44.50 ± 0.17 and 32.94 ± 0.03), which had a significantly lower WI (27.89 ± 0.08 and 26.77 ± 0.49) (79).

TPC and the antioxidant activity of curd cheese fortified with T. spicata EO nanoemulsion during the stored period were given in Table 5. In this study, the antioxidant capacity (15.47 ± 1.05), which was very low on the first day in the control sample, increased on the third day and started to decrease on the following storage days (p < 0.005). T. spicata pure oil has the highest antioxidant activity starting from the third day (Table 5). However, an increasing trend was obtained in antioxidant activity of 1%−5% EO-added nanoemulsion (p < 0.005). The results obtained from this study are in agreement with the literature. The study conducted on the antioxidant activities of Kareish cheese that was supplemented with curcumin nanoemulsions at 1.0, 1.5, and 2.0 mg/g showed the highest antioxidant activity in the nanoemulsion at 2.0 mg/g. Shawir et al. reported that the antioxidant activity of kareish cheese was between 19.23 and 42.31% and the antioxidant effects of kareish cheese applied with curcumin nanoemulsions at 1.0, 1.5, and 2.0 mg/g were 33.40%, 37.80%, and 42.31%, respectively (41).

Although all antioxidant activities decreased over time during storage, significant activity was detected in T. spicata oil and 1%, 3%, and 5% nanoemulsions on the 28th day of storage than the control cheese sample. In addition, the change in antioxidant capacity results was correlated with the total phenolic content of nanoemulsions (Table 5). Moreover, in a study, El-Sayed et al. (83) aimed to increase the phenolic compound and antioxidant content in Ras cheese by the addition of microencapsules containing nanoemulsions of allspice fruit extracts. The results revealed that the control cheese had lower TPC content (6.26 mg/100 g) and antioxidant activity (9.89%) than the supplemented cheese samples. TPC and antioxidant activity increased significantly (p < 0.05) with increasing microcapsule concentrations in Ras cheese. The total antioxidant activity, total phenolic content, and radical scavenging activity of T. spicata were analyzed by Bener (84). All models calculated for the three responses that are total phenolic, antioxidant, and radical scavenging activities were noteworthy (p < 0.0001) and showed that there was a significant relationship between the response and the independent parameters (84). The antioxidant capacity of T. spicata EO-based nanoemulsions on cheese has not been studied, but it has been studied on cheese with nanoemulsions containing EO from different plant species. Pérez-Soto et al. incorporated phenolic compounds and the antioxidant capacity of microemulsions from Opuntia oligacantha to fresh cheese, and they observed that phenolic compounds decreased during storage. As observed with ABTS, the microemulsion maintained a higher antioxidant acitivity with DPPH from day 0 (19.50 ± 0.43mg AAE/g). This behavior was observed up to 45 days (85). Shabani et al. (86) investigated the antioxidant activities of Urtica dioica EO-based nanoemulsions on L. monocytogenes and E. coli and reported that the values of DPPH were identified in EO-based nanoemulsions as 33.7 ± 12.2. Faramarzi et al. investigated a similar study with Shabani et al. (86) and applied it to the same bacteria, and the effect of Urtica dioica EO and nanoemulsions was identified in the nanoemulsions as 31.25 ± 1.50 % (11).

Microbiological analyses of curd cheeses were performed on each storage day. Total mesophilic aerobic bacteria (TMAB) counts, lactic acid bacteria (LAB), and yeast counts of curd cheeses during storage were given in Figures 5a–c, respectively. TMAB counts were found between 7.58 and 8.18 log cfu/g in curd cheese with pure oil added, 6.97 and 8.29 log cfu/g in control curd cheese sample, 7.64 and 8.04 log cfu/g in placebo added curd cheese, and 7.52 and 8.17, 7.55 and 8.05, and 7.68 and 8.01 log cfu/g in curd cheese sample containing 1%, 3%, and 5% T. spicata EO nanoemulsion, respectively. The total number of aerobic mesophilic bacteria in the curd cheese to which different EOs (1%, 3%, and 5%) containing nanoemulsions were added before storage decreased slightly. LAB counts were found between 2.80 and 5.41 log cfu/g in curd cheese with pure oil added, 2.21 and 5.41 log cfu/g in control curd cheese sample, 3.02 and 5.42 log cfu/g in placebo added curd cheese, and 2.84 and 4.85, 2.91 and 5.26, and 2.81 and 5.05 log cfu/g with 1%, 3%, and 5% T. spicata EO nanoemulsion, respectively. Yeast counts were found between 4.17 and 7.54 log cfu/g in pure oil-added curd cheese, 3.82 and 7.49 log cfu/g in control curd cheese sample, 4.35 and 7.72 log cfu/g in placebo-added curd cheese, and 4.13 and 7.70, 4.13 and 7.54, and 3.59 and 7.44 log cfu/g in 1%, 3%, and 5% T. spicata EO nanoemulsions, respectively. It was observed that the number of yeasts increased as the storage was prolonged. No mold growth was observed in the curd cheese sample. El-Sayed et al. reported the preservation effect of cumin EO nanoemulsion as a brined solution on the microbiological properties of white soft cheese during 60 days of storage time (77). The total mesophilic bacterial counts and lactic acid bacteria counts of white soft cheese treatments gradually reduced significantly in the presence of cumin EO nanoemulsion. However, it is observed that this decrease was more noticeable after a 30-day storage period. The reason for the small change in this study could be related to the shorter period of storage time for cheese samples. Elsherif and Al Shrief studied the effectivity of Szygium aromaticum and Cuminum cyminum EOs and its nanoemulsions against foodborne pathogens after inoculation in manufactured cheese at different concentrations. Complete reduction of L. monocytogenes was observed after 2 weeks of treatment with carvacrol nanoemulsions at 0.78 and 1.56% (2).

Gadallah et al. showed antimicrobial and antioxidant properties of Rosmarinus officinalis EO-based nanoemulsions as a natural alternative to Karish cheese. The results demonstrated a complete reduction of A. flavus, B. cereus, and L. monocytogenes, on days 5, 7, and 10, respectively, and a 96.93% reduction of P. aeruginosa at the end of the storage time. In the viability study of Karish cheese, C. albicans, A. flavus, and P. aeruginosa were completely reduced on days 10, 10, and 15 of storage, respectively (4).

Abd El Gwad et al. developed Thymus vulgaris and Syzygium aromaticum EOs-based nanoemulsions to improve the quality of soft cheese. The results showed that different concentrations of T. vulgaris nanoemulsion (0.03%, 0.06%, and 0.1%), as well as S. aromaticum nanoemulsions (0.06%, 0.1%, and 0.25%), were used to preserve the cheese. During a cold storage period of 50 days, the microbiological analysis and sensory evaluation of different cheeses were evaluated. The results showed that the droplet size of the prepared NEs was 94.85 and 68.67 nm for T.vulgaris and S. aromaticum nanoemulsions, respectively (3). Kamal et al. investigated the inhibitory effect of thyme oil against C. sakazakii in Tallaga cheese. Furthermore, thyme oil also inhibited the growth of Salmonella and mesophilic aerobic bacteria in other food products, maintained until the end of storage at 4°C (87). Shawir et al. showed that increasing the concentration of Curcumin nanoemulsions (1.0, 1.5, and 2 mg/ml) can be used in the preservation of Kareish cheese to enhance antimicrobial protection (41).