Ultrasonic-assisted food grade nanoemulsion preparation from clove bud essential oil and evaluation of its antioxidant and antibacterial activity (2025)

2.1 EO extraction and gas chromatography mass spectrometry analysis

The grounded clove buds 100 g were hydrodistilled at 60°C for 5 h using Clevenger-type equipment. The obtained EO was dried with Na₂SO₄ and filtered. The filtered oil was stored at 4°C, and the yield was determined using the following formula:

(1) EO ( % ) = wEO ( g ) DwS × 100

where wEO is the weight of the EO and DwS is the dry weight of the sample.

Gas chromatography mass spectrometry (GCMS) (Model 6890 GC and model 5973 N MS detector, Agilent Technologies, Santa Clara, CA, USA) was used to examine the bioactive chemicals in the EO. The GC conditions were as follows: the samples were injected with a split ratio of 1:10, and helium was used as the carrier gas at a flow rate of 1.0 mL·min⁻¹. The detector temperature was 280°C, whereas the injector was 90°C. The spectra were collected at the mass number-to-ion charge number (m/z) ratio of 20–550 at 2 scans·s⁻¹. By contrasting their retention time, the bioactive component of the EO was identified. Further identifications were performed by matching the obtained mass spectra data with the NIST MS search version on the Wiley Online Library.

2.2 Total phenolic content of EO

The EO total phenolic content (TPC) was determined using the Foli-Ciocalteu colorimetric method [16]. Briefly, the assay mixture was made by adding 100 µL of methanolic extract 200 µL of 50% Foli-Ciocalteu reagent and double distilled water (2 mL) and incubating for 3 min. After incubation, Na₂CO₃ solution (20%) was added to the reaction mixture and kept under dark conditions at 37°C for 1 h. The absorbance of the sample was recorded at λ _max = 765 nm. Gallic acid standards were used to calibrate the total phenol concentration, and the results were represented as mg gallic acid equivalents per gram of dry weight.

2.3 EO nanoemulsion preparation

Nanoemulsion was prepared using an EO, Tween80, and water. Tween80 is a nonionic surfactant with a high balance between lipophilicity and hydrophilicity. The nonionic surfactant nature stabilizes the prepared droplets in the emulsion via steric stabilization. The surfactant physicochemical properties, like low molecular weight, enhance the minimization of the droplets than the other polymeric surfactant. Fixed concentrations of the EO (6% v/v) were used to prepare the nanoemulsion. Initially, course emulsions were prepared at different proportions by mixing EO:Tween80 (v/v) 1:1, 1:2, 1:3 along with water. Ultrasonic emulsification processes were performed for the prepared course emulsion using Sonics (20 kHz) sonicator (Vibra-Cell, USA) with a maximum power output of 750 W. The device sonotrode consists of a piezoelectric crystal with a diameter of 13 mm. Then, we provided energy input as disruptive pressures to reduce the droplet diameter after dipping the device symmetrically into the prepared coarse emulsion. To minimize the temperature during the emulsification process, we placed the sample container in a larger, ice-filled beaker throughout the emulsification process. The prepared nanoemulsion’s droplet size and polydispersity index (PDI) were analyzed using a particle size analyzer (Particulate system-Nanoplus, USA). The light scattering technique determined the size of droplets.

2.4 Antimicrobial activity of nanoemulsion

Bacterial pathogens such as Listeria monocytogenes, Salmonella typhi, Pseudomonas aeruginosa, Serratia marcescens, Bacillus cereus, and Staphylococcus epidermidis were isolated from frozen, chilled foods and their packing materials, and all the isolates were identified by 16 s rRNA sequencing. Bacterial control strains were Escherichia coli (ATCC 25922) and S. aureus (ATCC 29213). The microtiter plate method determined the antibacterial activity of nanoemulsion with minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC). The nanoemulsion and streptomycin (control drugs) were diluted to reach a final concentration ranging from 0.03 to 62.5 µg·mL⁻¹ by mixing with the MH broth, and up to 170 µL of the solution was transferred to a 96-microwell plate. Then 20 µL of overnight bacterial inoculum (10⁴ CFU·mL⁻¹) and 10 µL of 0.5% 2,3,5-triphenyl tetrazolium chloride (TTC) were added and mixed thoroughly. All the 96-microwell plates were incubated at 37°C for 24 h. Following incubation, the presence of pink coloration caused by TTC indicated the bacterial growth, whereas the absence of color demonstrated that bacterial growth had been inhibited. The first well of the microplate, which held no nanoemulsion, was used as a control. The concentration in the wells in which no color appeared was taken to be the MIC. Bacteria from the well were subcultured on an MH agar plate. The dilution at which no bacteria grew on the MH agar plate was considered the MBC.

2.5 LIVE and DEAD staining assay

Using the LIVE/DEAD BacLight kit (Invitrogen, USA), the effect of nanoemulsions on bacterial cells was evaluated. Gram-positive and Gram-negative bacterial cells were collected from overnight culture. Nanoemulsion was applied to bacterial inoculum (10⁶ CFU·mL⁻¹) for 30 min, and the pellet was recovered by centrifuging at 1,200 g for 10 min. The recovered pellets were resuspended in PBS after being washed twice with PBS, and then 5 µL of double stains (2.5 µL of SYTO9 and 2.5 µL of propidium iodide (PI)) were added to the suspension and incubated for 5 min at 37°C. PBS was used to wash cells twice to eliminate unbound dyes. PBS was then used to resuspend the collected pellets. A fluorescent microscope (Leica, DM-2500; Wetzlar, Germany) fitted with a Leica-DFC-295 camera and a Leica-Application Suite 3.8 processor was used to capture the images.

2.6 DPPH radical scavenging assay

The ability of nanoemulsion to transform DPPH (2,2′-diphenyl-1-picrylhydrazyl) into a reduced form of DPPH-H was evaluated as described previously [17]. The assay mixture was prepared by adding 1:1 ratio of nanoemulsion at different concentrations (0.03–62.5 µg·mL⁻¹) and 0.1 mM DPPH (dissolved in 95%) solution, followed by vortexing and incubating in the dark at ambient temperature for 2 h. After incubation, the absorbance was detected using a UV-Vis spectrophotometer at 517 nm, and triplicates were used in all experiments. Ascorbic acid (AA) was used as a standard. The percentage of antioxidant activity is calculated using the following formula:

(2) Inhibition = A 0 − A s A 0 × 100

where A ₀ represents the absorbance of DPPH without samples, and A _s represents the absorbance of DPPH with a sample. The appropriate concentration required to scavenge 50% of the free radicles was estimated.

2.7 ABTS⁺ radical cation decolorization assay

The nanoemulsion potential of ABTS radical cation scavenging activity was analyzed using the previously described method with some modifications [18]. Briefly, 7 mM ABTS aqueous solutions and 4.95 mM potassium persulfate solution were mixed (1:1; v/v) and incubated for 16 h at ambient temperature in a darkroom. The experiment was performed using fresh ABTS⁺ solution (3.9 mL) with 0.1 mL nanoemulsion at different concentrations. Trolox was used as a standard. After 6 min, the absorbance decrease was measured at 734 nm. Each experiment was carried out in triplicates.

(3) Inhibition ( % ) = ODc − ODs ODc × 100

where ODc is the absorbance of the control, and ODs is the absorbance of the sample.

2.8 Cell cytotoxicity (MTT) assay

The cell cytotoxicity of nanoemulsion was examined using an MTT assay. Briefly, the human mesenchymal stem cell was seeded at a density of 10³ cells/well in 96-well plates and incubated at 37°C for 24 h in a 5% CO₂ environment. Then nanoemulsions were added to each well at different concentrations ranging from 0.48 to 62.5 µg·mL⁻¹. MTT (5 mg·mL⁻¹) of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide 20 µL was added to each well after 24 h incubation. The treated plates were incubated for 4 h overnight, after which the supernatant was removed by centrifugation. Dimethyl sulfoxide (DMSO 100%) was used to dissolve the insoluble formazan, and a microplate reader was used to detect the optical density at 570 nm and reference filter (630 nm). The percentage viability (relative to survival of control cells) was computed using obtained results.

2.9 Data analysis

The obtained values from the triplicates in each experiment were statistically evaluated by the SPSS software, version 22.0 (Chicago, IL, USA) and Microsoft Excel using one-way analysis of variance. The statistically significant differences were expressed as *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001.

3.1 Clove bud EO compositions

The hydro-distillation of 100 g of clove bud yielded 11.06 ± 1.50% (v/w) of oil. Using gallic acid as a quantitative standard, the TPC of the obtained clove bud EO was determined. The TPC of the EO was 38.23 ± 1.19 mg·g⁻¹ (DW ± SD). Table 1 presents the bioactive constituents of EO analyzed by GCMS (Figure 1). GCMS analysis shows that clove bud EO consists of compounds from various chemical groups such as alcohol, aldehyde, esters, benzenes, carboxylic acid, esters, fatty acids, and nitrogen compounds. In total, 59 compounds from various chemical groups were identified, representing 100% EO. The main components are 1,2-dimethoxy-4-(2-propenyl)-benzene (26.58%), 2-tert-butyl-6-(3-tert-butyl-2-methoxy-5-methylbenzyl)-4-methylphenol (13.29%), 9,12-octadecadienoic acid (Z,Z)-, methyl ester (11.31%), 9-octadecenoic acid (Z)-, methyl ester (9.20%), hexadecanoic acid, methyl ester (7.08%), octadecanoic acid, methyl ester (2.20%), phthalic acid, di(3-methylphenyl) ester (2.15), caryophyllene (1.78%), γ-sitosterol (1.63%), butylated hydroxytoluene (1.39%), caryophyllene oxide (1.08%), and eugenol (0.79%). 1,2-Dimethoxy-4-(2-propenyl)-benzene, a methyl eugenol of phenylpropanoid compound, showed potent antibacterial and antifungal activities. Also, eugenol, β-tocopherol, benzenepropanoic acid, 3,5-bis(1,1-dimethylethyl)-4-hydroxy (hydroxycinnamic acid), butanoic acid and 1-(4-nitrophenyl)piperazine demonstrated strong antibacterial activity against extensive bacterial pathogens, including drug-resistant strains [19,20,21,22,23]. The United Nation Food and Agriculture Organization (FAO) listed fumaric acid from a natural source as a food additive for coffee and its substitutes, cooked and fried fish products, milk-based products, dried fruits, and cooked or fried vegetables under good manufacturing practices (GMP) [24]. Also, 2-tert-butyl-6-(3-tert-butyl-2-methoxy-5-methylbenzyl)-4-methylphenol reported as food additives [25]. Octanoic acid and benzothiazole are chemotherapeutic applications [26,27]. Our study results found that clove bud EO consists of biologically active compounds with nutraceutical values and qualify as dietary supplements and food additives suitable for food-based applications.

3.2 Characterization of nanoemulsion

The size distribution of nanoemulsion droplets was measured by the laser diffraction method from NanoPlus zet/nanoparticle analyzer. The size distribution of nanoemulsion droplets was based on the best fit between experimental findings and the Mie theory. The refractive index (RI) value of 1.3348 was used during measurements. Nanoemulsions were diluted with distilled water, followed by probe sonication to avoid agglomeration of droplets before measurements. The experimental condition was set as the temperature at 25°C and the viscosity of 0.8878 cP. The droplet diameter from the Z-average value was observed and calculated in mean hydrodynamic diameter according to the International Standard on dynamic light scattering ISO 13321. Figure 2a–d show droplets distribution intensity, volume, and number percentage of prepared nanoemulsion properties. Oil and surfactant ratio at 1:1 (v/v) found highest droplet z-average diameter 176.2 nm, PDI 0.192, and diffusion constant 2.792 × 10⁻⁸ cm²·s⁻¹ including distribution intensity (214 ± 106%), volume (118.4 ± 50.1%), and number (88.2 ± 24.1%) (Figure 2a1–a3). At 1:2, the reduction was observed in the droplet size (160.7 nm in diameter), volume (84.3 ± 43.9%), and number distribution (59.3 ± 16.8%) (Figure 2b2 and b3). Also, a mild increase in intensity distribution (219.8 ± 146.1%) (Figure 2b1) was observed. However, the PDI and diffusion constant were increased to 0.276 and 3.062 × 10⁻⁸ cm²·s⁻¹, respectively. In our study, we used surfactant Tween80, which has hydrophilic–lipophilic balance-15 (HLB) to prepare nanoemulsion [28]. The increased concentration of surfactant (1:3 v/v ratio of oil and surfactant) efficiently reduced the droplet diameter to 77.7 nm, distribution intensity (117.6 ± 95.3%), volume (40.3 ± 20.6%), and number (29.1 ± 7.9%) (Figure 2c1–c3). Table 2 presents the results of the droplet size distribution of nanoemulsion prepared by a different ratio of surfactant. The HLB properties of the surfactant lowered interfacial tension at the oil/water interface, hence altering the droplet properties of nanoemulsion at different ratios of surfactant [15]. The present study results follow earlier studies that can reduce diameter of droplets via low oil surfactant ratio [29]. According to the reports, small particle size in the nanoemulsion greatly enhanced the release of core material and intracellular absorption [30]. Hence, we selected the concentration of 1:3 for further studies. However, intracellular absorption is initiated by attachment to the cell membrane and internalization phenomena [31]. Diffusion constants and surface charges enhance these processes, resulting in a considerable penetration of nanodroplets into bacterial cells and exerting antimicrobial effects [32]. The current formulated clove bud EO emulsion is an oil-in-water-based emulsion containing nanodroplets (77.7 nm) that are relatively small nanometer in size. The low nanometer droplets are stable for breakdown through gravitational separations and droplet aggregation mechanisms. In addition, droplets’ small size can greatly increase encapsulated lipophilic substance bioavailability. Nanoemulsion may have applications in the food and pharmaceutical industries as the delivery system.

3.3 Antibacterial activity

Evaluated in vitro antibacterial activity of nanoemulsion against food-borne pathogenic strains isolated from frozen food and their packing materials. Table 3 presents the obtained MIC and MBC values of the nanoemulsion and streptomycin control drug. The study results show that E. coli, B. cereus, L. monocytogenes, S. epidermidis, and S. aureus were more sensitive to nanoemulsion with MIC at 0.24 µg·mL⁻¹. The growth of gram-negative pathogens S. typhi, P. aeruginosa, and S. marcescens was inhibited at 0.48 µg·mL⁻¹. The obtained results coincide with several other studies that nanoemulsions are more sensitive to Gram-positive pathogens than Gram-negative ones [33,34,35]. However, the nanoemulsion bactericidal action (MBC) range was 0.97–1.95 µg·mL⁻¹ in both Gram-positive and Gram-negative bacteria. The control drug, streptomycin, exhibits MIC from 1.95 to 7.81 µg·mL⁻¹ and MBC from 3.90 to 15.62 µg·mL⁻¹. The GCMS analysis showed the presence of numerous bioactive compounds with a high quantity of total polyphenols present in the clove bud EO; hence, the study results show that the present nanoemulsion exhibit antibacterial activity than the control drug streptomycin at sixfold to tenfold less concentration. The application of nanoemulsions as control agents of food pathogens is a new and promising innovation of antimicrobials [36,37]. The current problem of developing antimicrobial-resistant strains due to the widespread use of synthetic antimicrobials and disinfectants prompted the scientific community to further research and development of new antimicrobial agents targeting specific pathogens while being without side effects. Since the action of nanoemulsion from EO with numerous bioactive compounds and hydrophobicity/hydrophilicity appears to be the nonspecific disruption of bacterial cell membrane causing leakage of ions and other cell components, cell death would not result in the development of resistant strains [38]. The physicochemical properties of nanoemulsion can be further diluted in an aqueous solution and stored in different environmental conditions even up to years. The studies reported that nanoemulsion has extensive bactericidal and virucidal potentials, and biocidal concentrations are nontoxic to the mucous membrane and the gastrointestinal tract [39,40]. Some other studies reported in food applications that EO can be applied as a preservative to control the bacterial growth, in cheese and beef burgers [41,42]. According to the present study, clove bud EO nanoemulsion with droplets of mean diameter 77.7 nm showed remarkable inhibitory effects on Gram-positive and Gram-negative bacterial food pathogens at very high dilutions.

3.4 LIVE and DEAD staining

The antibacterial effect of nanoemulsion on Gram-positive and Gram-negative bacterial cells stained with LIVE/DEAD BacLight kit is shown in Figure 3. The live cell that consists of an intact cell membrane allows SYTO9 and appears green; hence, untreated (control) cells appear green florescent (Figure 3a1,b1). All the bacterial cells treated with nanoemulsion appear red fluorescent (Figure 3a2,b2) due to the antibacterial action of nanoemulsion that damages the bacterial cell membrane and results in cell death. These cells allow PI to stain and appear red. Hence, the results confirmed that the present nanoemulsion potentially acts on cell membranes and causes cell death.

3.5 Antioxidant activity

We evaluated the free radical scavenging ability of nanoemulsions using two different screening assays. DPPH and ABTS free radical scavenging assays are the most commonly used screening methods due to their excellent reproducibility under certain conditions [43]. The percentage inhibition of DPPH results is shown in Figure 4. The results showed that the concentration-dependent free radical scavenging activity and DPPH inhibition percentage indicate that clove bud EO nanoemulsion has a substantial potential to scavenge free radicals. The IC₅₀ value for scavenging the DPPH free radical was 13.46 ± 0.32 and 10.51 ± 0.32 µg·mL⁻¹ for nanoemulsion and AA, respectively. As shown in Figure 2, the hydrogen donating ability of the nanoemulsion significantly (p < 0.05) was high at the concentration of 0.48–7.81, 31.25, and 62.5 µg·mL⁻¹ than the AA standard. The antioxidant study of clove bud EO shows IC₅₀ at 40 µg·mL⁻¹ [44]. The present nanoemulsion IC₅₀ concentration was lower than the clove bud EO. The higher efficiency of EO nanoemulsion was linked with the synergic interaction of nanodroplets with phenolic compounds and other secondary metabolites. The study results found that a higher degree of inhibition percentage of DPPH by nanoemulsion showed higher efficiency of polyphenolic bioactive compounds in the oil to scavenge the free radicals [45]. The nanoemulsion antioxidant effects are linked with phenolic compounds in nanodroplets. However, the higher the number of hydroxyl groups in the phenolic compounds, the higher the antioxidant potential [46]. ABTS cationic radical scavenging activity was positively correlated with nanoemulsion at different concentrations (Figure 5). The more increased activity was observed in the nanoemulsion than in the standard Trolox, and all the obtained values were statistically highly significant at different levels (**p < 0.01 and ***p < 0.001). The nanoemulsion and Trolox exhibit IC₅₀ at the concentrations of 5.46 ± 0.28 and 27.17 ± 0.21 µg·mL⁻¹, respectively. The EO nanoemulsion highly interacted with pregenerated ABTS⁺ radical cation due to the hydrophobic and hydrophilic nature of nanodroplets that consist of bioactive components; hence, the IC₅₀ concentration nanoemulsion was fourfold less than the Trolox standard. These antioxidant properties of nanoemulsion prevent the reaction of various food constituents from oxygen and can provide protective effects from oxidation and food pathogenic microbes. Hence, clove bud EO nanoemulsion food-grade nanostructures would offer a wide range of natural preservatives and meet the current demand for natural food preservatives in the food industry [30].

3.6 Cytotoxicity effects

All the secondary plant metabolites, especially polyphenols produced by plants, are nontoxic; some phenols may be toxic to human cells at particular concentrations. The toxicity of phenols is closely related to their structures and concentrations [47]. Hence, we investigated the cytotoxicity of EO nanoemulsion on human mesenchymal stem cells. Figure 6 shows the obtained results of the percentage of toxicity/cell viability against concentration in 24 and 48 h. It can be observed in the cytotoxicity assay of the nanoemulsion that the survival rate ranged from 81.41 ± 1.26% to 106.24 ± 4.20% in 24 h and 75.05 ± 0.81 to 112.51 ± 1.66 in 48 h. The cell viability in relation to control demonstrated no cytotoxicity in the concentration conditions addressed. However, at low concentrations ranging from 3.5 to 0.48 µg·mL⁻¹, nanoemulsion exhibits low stem cell proliferation potentials in 24 and 48 h. Hence, these results suggest that the tested EO nanoemulsion concentration is nontoxic to human cells.