The test was performed by stain diffusion at 25°C with the addition of 1 mg/ml of water-soluble methylene blue and oil-soluble Sudan IV solutions to G-NE. If the diffusion rate of methylene blue base was larger than that of Sudan red, it was an O/W type; otherwise, it was a W/O type (He et al., 2017).

The mean particle size, polydispersity index (PDI), particle size distribution and zeta potential of the samples were analyzed at 25°C with a laser particle size/potential analyzer (Delsa Nano C, Beckman Coulter, USA). The results of the analysis for each recorded measurement are the average of 3 scans.

The viscosity of G-NE was measured at 25°C using a digital viscometer (SNB-2, Shanghai Jingtian Electronic Instruments Co., Ltd., China).

The pH of G-NE was measured at 25°C using a handheld pH meter (OHAUS Corporation, USA).

Measure the color of G-NE at 25°C using a colorimeter (CR-10 PLUS, Konica Minolta Sensing Co., Ltd., Osaka, Japan), determine L*, a* and b* values, and calculate the whiteness index (WI).

The surface tension of G-NE was measured at 25°C using a contact angle meter (SL200L2, Shanghai Soren Information Technology Co., Ltd., China).

The contact angle of G-NE was measured on a smooth glass surface using a contact angle measuring instrument (SL200L2, Shanghai Soren Information Technology Co., Ltd., China).

The copper mesh containing the support film was placed on a wax plate, 5 μl of G-NE was added dropwise on the film and dried naturally, then 5 μl of 2% phosphotungstic acid was added dropwise and the excess liquid was blotted up with filter paper, and the morphology of the microemulsion was observed under a transmission electron microscope (JEM2100, Nippon Electron Co., Ltd., Japan) after drying naturally.

Take 500 μl of G-NE into a 10 ml volumetric flask, add appropriate amount of anhydrous ethanol to dissolve, then ultrasonic for 15 min, and continue to fix the volume with anhydrous ethanol to the scale line. Take 0.1 ml of the ultrasonicated solution into a 10 ml volumetric flask and dilute to the scale with anhydrous ethanol. The absorbance was measured by UV spectrophotometer (UV-2600i, Shimadzu, Japan), and the content of total active substance (W_t) was calculated according to the standard curve and the relationship between dilution times.

Take 500 μl G-NE and centrifuge it in an ultrafiltration tube at 10,000 r/min for 15 min to obtain the clarified centrifugal solution. The filtrate was aspirated, dissolved with anhydrous ethanol, and fixed into a 10 ml volumetric flask. The concentration of free active ingredient (W_f) was determined using the standard curve and dilution multiple relationship after the absorbance was measured using a UV spectrophotometer. The encapsulation efficiency (EE) was calculated according to the formula.

The inhibition effect of G-NE on four foodborne pathogenic microorganisms, Staphylococcus aureus (S. aureus), Escherichia coli (E. coli), Salmonella typhimurium (S. typhimurium) and Listeria monocytogenes (L. monocytogenes), was determined by the punching method. 100 μl of bacterial broth with a concentration of 1 × 10⁶ CFU/ml was spread on NA medium, 4 wells were punched on the medium, and then 90 μl of geraniol (G), G-NE, emulsion without geraniol (C-NE), and blank control (C) were added to the wells, respectively. The treated petri dishes were sealed and incubated at 37°C for 24 h. The size of the BIC is measured and photographed (Kotan et al., 2007). Every experiment’s material and tool are autoclaved, and the ultra-clean bench is used for all operations.

The minimum inhibitory concentration (MIC) of G-NE was measured by micro-broth dilution method by referring to the experimental method of Miladinović et al. with slight modifications (Miladinovic et al., 2014). 100 μl of LB medium mixed with sterile TTC solution (0.5%) was added to a sterile 96-well plate, 100 μl of G-NE was added to the first well, and then the mixture was diluted sequentially to the twelfth well by twofold dilution method, and finally 100 μl of bacterial solution (1 × 10⁶ CFU/ml) was added to each well. Each row of 12 wells is a group of experimental groups, the blank group is added to the medium, and the control group is added to C-NE, and the procedure is repeated three times. The MIC of the experimental bacteria treated with geraniol was the same as above.

Referring to the experimental method of Amrutha et al. with slight modifications (Amrutha et al., 2017), 200 μl of LB medium was added to a 96-well plate, followed by additions of 0.5 MIC, 1 MIC, and 2 MIC concentrations of G-NE and a thorough shaking. 20 μl of bacterial solution (1 × 10⁶ CFU/ml) was added to each well and the culture was incubated at 37°C, the OD value at 600 nm was measured and recorded in every 2 h. Three biological replicates were performed for each group.

The prepared G-NE is slightly turbid with blue opalescence as shown in Figure 1B. After adding methylene blue dye and Sudan IV dye dropwise to the emulsion, the diffusion rate of methylene blue dye in the emulsion was significantly faster than that of Sudan IV dye, so the emulsion prepared in this test can be judged as O/W type nanoemulsion.

The mean particle size, PDI, zeta potential, and particle size distribution are very important indicators of nanoemulsions, which can describe the size, stability, uniformity, and dispersion of nanoemulsions. The greater the zeta potential, the greater the mutual repulsion between particles, and the better the stability of the dispersion system. The experimental results obtained by a laser particle size/potential analyzer (Delsa Nano C) are shown in Table 1, where the mean particle size of G-NE is 90.33 ± 5.23 nm, the PDI is 0.058 ± 0.0007, and the zeta potential is − 17.95 ± 5.85 mV. The particle size distribution as shown in Figure 2, the peak width is very narrow, indicating that it has uniform droplet size. The results show that the system is stable, the G-NE mean particle size is small, the droplet dispersion is good, the size is uniform, and there is essentially no aggregation.

The results are shown in Table 2, where obtained G-NE viscosity was 5.14 ± 0.35 mPa⋅s, pH was 5.8 ± 0.2, whiteness index was 31.78 ± 0.26, contact angle was 8.99 ± 0.03°, and surface tension was 21.43 ± 0.11 mN/m. The results are consistent with the mean particle size since the lower viscosity suggests that the surfactant molecules move more quickly and easily, resulting in smaller particles. According to Jimenez et al. (2018), WI values close to 100 correspond to opaque/white systems. Nanoemulsions are slightly turbid systems and the appearance of nanoemulsions is largely influenced by the concentration and diameter of the oil droplets, therefore, as the diameter of the oil droplets increases, light scattering is stronger and the nanoemulsion tends to be opaque. In general, lower surface tension formulations typically give good spreading capabilities (Teixeira et al., 2017). The interfacial tension is one of the elements influencing how quickly Oswald ripens, and the lower it is, the more it delays the start of ripening. However, for an actual influence, the interfacial tension needs to be decreased by several orders of magnitude (Tadros et al., 2004).

According to Figure 3, prepared G-NE droplets are well dispersed as they are evenly distributed, spherical, and of a uniform size when viewed by TEM.

As Figure 4A shows the UV absorption spectrum of geraniol, it has good UV absorption, with the maximum absorption at 208.9 nm. As Figure 4B shows the standard curve of geraniol, the regression equation was obtained: y = 0.0943x − 0.0719, and the correlation coefficient R² = 0.9994, the regression straight line is a good fit to the observed values. The geraniol content and encapsulation efficiency (EE) of G-NE were 2.32 ± 0.13 and 93.22 ± 1.53%, respectively, which are substantial compared to most reported values (Zhang and Zhong, 2020; Zhuang et al., 2022).

The stability of nanoemulsions can be determined in large part by the size and composition of their particles. The storage stability experiment provides a reference for the storage conditions and expiration date of microemulsions. G-NE was stored at 4 and 25°C for 28 days, and the changes of mean particle size and geraniol content were tested to evaluate its physicochemical stability. As shown in Figures 5A–D, with the increase of storage time, there was no significant difference in the mean particle size and geraniol content of G-NE after 28 days of storage at 4°C, with uniform particle size distribution and good storage stability. The mean particle size of G-NE rose from 89 to 197 nm after 28 days of storage at 25°C, its geraniol concentration declined from 2.30 to 1.85%, and the particle size distribution was not concentrated and the emulsion was stratified, which indicated its poor stability in storage. The analysis may be due to the slow droplet motion at 4°C and the nanoscale particle size which makes the effect of gravity on it greatly weakened (Li et al., 2013), the interaction forces between the components of the system reach equilibrium and form a stable dispersion system, so that instability phenomena such as creaming, precipitation, flocculation, agglomeration, and Ostwald maturation leading to phase separation are less likely to occur during the storage process (McClements, 2012). In conclusion, the nanoemulsions stored at a constant temperature of 4°C were able to maintain better stability and were more conducive to the storage of G-NE.

The effects of rotational speed on the mean particle size, geraniol content, and particle size distribution of G-NE are shown in Table 3. All samples showed no delamination, no significant difference in content and particle size, and concentrated particle size distribution after centrifugation at different speeds for 20 min, which suggested that the G-NE gained had relatively favorable centrifugal stability. It might be related to the nanoparticle size and the corresponding viscosity of the microemulsion system (Grit and Crommelin, 1993).

G-NE was heated at different temperatures for 30 min to test the stability of microemulsions at different temperatures and to provide a basis for its practical application. Figure 6 shows that after 30 min of heating G-NE at 60, 70, and 80°C, the emulsion turned milky white and oil-water separation took place. The geraniol content of G-NE dropped as the mean particle size increased and the particle size distribution became more uneven. At this point, the nanoemulsion’s structure was irreparably damaged, and even after cooling to ambient temperature, it was impossible to return it to its previous state. G-NE stratified and the top layer turned milky white and solidified after 30 min of 90°C heating. This indicates that G-NE might be poorly stabilized at high temperatures and the low temperature environment is more suitable for storing it.

The significant increase in average droplet size can be attributed to agglomeration and/or Ostwald maturation phenomenon (Chang and McClements, 2014). Initially, nanoemulsions consist of small droplets (nanoscale). However, these nanoemulsions are quite unstable and lead to significant droplet growth at high temperatures, and these large new droplets may aggregate. Droplet flocculation occurs whenever the net attractive force of the dispersed phase exceeds its interfacial tension (Ben Jema et al., 2018). Therefore, it is advisable to store these formulated food-grade nanoemulsions at low or ambient temperatures to reduce the risk of nanoemulsion degradation (Falleh et al., 2021).

The stability of nanoemulsions under acid-base conditions is also one of the important indicators to be considered because of the wide pH range of beverages and foods. An acid-base buffer was prepared instead of deionized water to test the tolerance of the microemulsions under different pH conditions of acidity and alkalinity, and the results are shown in Figure 7. The pH increased from 2.0 to 5.0, the mean particle size of G-NE decreased, and the geraniol content increased. pH increased from 5.0 to 9.0, the mean particle size of G-NE increased and there was no significant difference in the geraniol content. pH values in the range of 2.0–9.0 showed a concentrated particle size distribution and no stratification was observed. When pH = 5.0, the mean particle size of G-NE was the smallest and the geraniol content was relatively high. In summary, G-NE was quite stable in the pH range of 2.0–9.0 and showed the best physical stability at pH = 5.0 (Qian et al., 2012).

Freezing is a common method used in food processing to retain the quality and nutritional properties of foods and effectively reduce the growth and propagation of microorganisms, so the freeze-thaw stability of the emulsions needs to be investigated. The effect of freeze-thaw cycle treatment on G-NE is shown in Figure 8. After three freeze-thaw cycles, the mean particle size of G-NE increased from 90.33 ± 5.23 to 17,266.7 ± 636.67 nm, the particle size distribution was not concentrated, the content of geraniol decreased from 2.41 to 2.13%, and oil-water separation occurred, which may be due to the crystallization during the thawing process and resulted in the rupture of the interfacial film, the droplet aggregation and oil-water separation. In summary, the freeze-thaw stability of G-NE is poor (Khan et al., 2015).

The inhibition ability of G-NE was determined by punching method, and it is shown in Figure 9 that G-NE has growth inhibition effect on S. aureus, E. coli, S. typhimurium and L. monocytogenes. The results of the BIC diameter are shown in Table 4, which showed that the BIC size of S. aureus was 9.71 ± 0.14 mm, E. coli was 11.20 ± 0.24 mm, S. typhimurium was 11.97 ± 0.58 mm, and L. monocytogenes was 15.80 ± 0.53 mm. The experiment proved that geraniol retained its excellent inhibition ability after being encapsulated into G-NE.

The MIC of G-NE was determined as shown in Table 5. The MIC of G-NE was 7.81 μl/ml for S. aureus, 3.91 μl/ml for E. coli, 3.91 μl/ml for S. typhimurium, and 7.81 μl/ml for L. monocytogenes. It can be seen that the preparation of geraniol into G-NE still has antibacterial effect and has lower MIC compared with pure geraniol.

As shown in Figure 10A, the results showed that S. aureus in the control group reached the exponential growth phase at 4 h and entered the stable phase at 12 h. After treatment with 0.5 MIC of G-NE, S. aureus entered the exponential growth phase after 4 h and entered the stable phase after 10 h. The growth curve was 2 h earlier than that of the control group; G-NE with 1 MIC entered the exponential growth period after 8 h and did not reach the stable phase at 12 h. The absorbance of 2 MIC did not change during 12 h, which indicated that S.aureus was very sensitive to 2 MIC concentration; different concentrations of G-NE inhibited S.aureus during 12 h.

As shown in Figure 10B, the growth of E. coli in control group reached the exponential growth period at 6 h of incubation and entered the stabilization period at 12 h. E. coli entered the exponential growth period at 8 h after G-NE treatment with 0.5 MIC and did not enter the stabilization period at 12 h. The absorbance of E. coli in 12 h after G-NE treatment with 1 MIC and 2 MIC did not change. This indicates that E. coli is very sensitive to the concentration of 1 MIC and 2 MIC; different concentrations of G-NE inhibited E. coli during 12 h.

Salmonella typhimurium in the control group reached the exponential growth phase at 4 h of incubation and entered the stable phase at 8 h demonstrated in Figure 10C. After G-NE treatment with 0.5 MIC, S. typhimurium entered the exponential growth phase after 4 h and entered the stable phase after 10 h. The growth curve lagged 2 h behind that of the control group; after G-NE treatment with 1 MIC, S. typhimurium entered the exponential growth phase after 8 h and had not reached the stable phase at 12 h. S. typhimurium entered the exponential growth period after 8 h and did not reach the stable phase at 12 h. The absorbance of S. typhimurium did not change in 12 h after G-NE treatment with 2 MIC, which indicated that S. typhimurium was very sensitive at 2 MIC concentration; different concentrations of G-NE inhibited the growth of S. typhimurium during 12 h.

As shown in Figure 10D, L. monocytogenes in the control group reached the exponential growth phase at 4 h of incubation and entered the stable phase at 8 h. After G-NE treatment with 0.5 MIC, L. monocytogenes entered the exponential growth phase after 4 h and entered the stable phase after 8 h. The absorbance of L. monocytogenes did not change within 12 h, which indicated that L. monocytogenes was very sensitive at 1 MIC and 2 MIC concentrations; G-NE at 1 MIC and 2 MIC concentrations had an inhibitory effect on L. monocytogenes within 12 h.

The antibacterial activity of essential oil nanoemulsions depends mainly on the concentration of essential oil in the emulsion, the particle size, and the release rate of the essential oil from the emulsion. As expected, higher G-NE concentrations correspond to better antibacterial activity (Noori et al., 2018). The sustained release of geraniol from the nanoemulsions over time prolonged the action of the essential oils on microorganisms, and the experiments demonstrated that G-NE maintained its inhibitory effect on four food-borne pathogenic bacteria within 12 h.