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FEMS Microbiology Letters logoLink to FEMS Microbiology Letters
. 2026 Mar 18;373:fnaf144. doi: 10.1093/femsle/fnaf144

A comparative study on the effects of emulsification methods on the antimicrobial activity of essential oil emulsions

Paul G Kamau 1, Paola Alzate 2, Malco Cruz-Romero 3, Michael A Morris 4, Joe P Kerry 5,✉
Editor: Diane Rip
PMCID: PMC13017692  PMID: 41848567

Abstract

A 3 × 3 factorial design was used to systematically evaluate the impact of three widely used emulsification methods [ultrasonication (UT), high-shear homogenization (HSH), or vortex mixing (VM)] at three treatment times (30, 60, or 120 s) on the antimicrobial efficacy of sage (SEO) and garlic (GEO) essential oil emulsions. Independent of essential oil used, antimicrobial activity was significantly (P < 0.05) influenced by both emulsification method and treatment time, with Gram-negative bacteria showing greater resistance. From the tested methods, emulsions prepared by UT or HSH for at least 60 s exhibited significantly (P < 0.05) higher antimicrobial activity, which might be associated with their significantly (P < 0.05) smaller particle size and polydispersity index. Additionally, UT and HSH produced more stable SEO emulsions, while GEO required ≥60 s treatment for stability and VM led to rapid destabilization regardless of oil or time. This study highlights the importance of selecting an appropriate emulsification method for producing essential oil emulsions with enhanced antimicrobial activity, which is important not only for antimicrobial testing but also for the overall functional performance of the emulsion.

Keywords: Antimicrobial activity, Essential oil emulsions, Ultrasonication, High-shear homogenization

Selecting the right emulsification method and time is crucial, as these factors greatly influence essential oils' antimicrobial potency and allow effective bacterial control with lower doses.

Introduction

Essential oils (EOs), which are plant-derived volatile compounds, have gained significant attention as sustainable antimicrobial agents due to their potent bioactivity against a wide range of foodborne pathogens and spoilage microorganisms (Karoui and Hassoun 2017, Falleh et al. 2020, McClements et al. 2021, Kamau et al. 2025). EOs consist of a complex composition with 20–60 components at different concentrations (Speranza et al. 2023), with their antimicrobial efficacy being closely related to their major constituents (e.g. thymol, carvacrol, linalool, citronellal, menthol, geraniol, eugenol, and cinnamaldehyde) (Hassoun and Emir Çoban 2017, Ebrahimzadeh et al. 2023). Their antimicrobial effects primarily stem from interactions with bacterial membranes, disrupting fatty acids, phospholipids, and polysaccharides, ultimately leading to leakage of cellular contents and cell death (Fang et al. 2017, Zubair et al. 2022).

Among the common EOs is sage (Salvia officinalis), which represents the largest genus of the Lamiaceae family (Ghorbani and Esmaeilizadeh 2017). Sage is rich in terpenoid compounds, flavonoids, and phenolic compounds (Cutillas et al. 2017, Yilar et al. 2018). Antimicrobial activity of sage EO against both Gram-positive and Gram-negative bacteria has been reported (Císarová et al. 2018, Sharifi-Rad et al. 2018, Yazgan 2020). Similarly, garlic (Allium sativum L.), which belongs to the Liliaceae family, possesses a pale-yellow oily liquid with strong antimicrobial properties. Garlic EO stands out for its richness in bioactive sulphur compounds, such as diallyl sulfide, allyl methyl disulfide, allyl methyl trisulfide, diallyl disulfide, and dially trisulfide, all of which are attributed to its antimicrobial properties (Yang et al. 2009, Verma et al. 2023, Sarangi et al. 2024).

However, the antimicrobial activity of EOs is challenged by their low water solubility, instability in aqueous environments, and rapid volatility, which limits their bioavailability in hydrophilic food formulations (Barradas and de Holanda e Silva 2020). To overcome these limitations, emulsification has been widely employed to improve EO solubility, maintain functional properties, and mitigate undesirable sensory impacts in food products (Liew et al. 2020, Yazgan 2022, Torres Neto et al. 2024). Emulsification methods such as ultrasonication (UT), vortex mixing (VM), and high-shear homogenization (HSH) are commonly used, with each method exhibiting distinct mechanisms and efficiencies (Jiang et al. 2020). UT is reported to have a rapid and consistent emulsification capability (Ren et al. 2019, Jiang et al. 2020, McClements et al. 2021, Krishnan et al. 2023) with a demonstrated enhanced antimicrobial effectiveness of emulsified EOs, potentially due to changes in interfacial composition and redistribution of active compounds (O’Sullivan et al. 2017, Liu et al. 2022). Conversely, VM and HSH are simpler methods frequently used in small-scale or laboratory environments to produce emulsions, valued for their robustness and scalability (Chen et al. 2014, Ren et al. 2019, Zafirah Fahizan et al. 2025). However, these methods do not generate the intense mechanical forces characteristic of high-energy methods such as UT, which can influence the incorporation and bioavailability of EO bioactive compounds (Koshani and Jafari 2019).

To the best of our knowledge, investigations into the influence of different emulsification methods on the antimicrobial activity of EOs emulsions remain scanty, with most existing studies focusing on comparisons between pure EOs and emulsions prepared by either HSH (van der Schaaf and Karbstein 2018, Mazarei and Rafati 2019, da Silva et al. 2023) or UT (Moghimi et al. 2016, Guo et al. 2020, Long et al. 2020, Yazgan 2020, He et al. 2022, Liu et al. 2022). At the same time, literature reports variable antimicrobial activity for the same EOs against similar bacterial strains, which may be attributed to differences in emulsification methods used across studies. For example, thyme EO emulsions prepared via UT exhibited minimum inhibitory concentrations (MICs) of 7.8 × 10⁻⁵ and 3.9 × 10⁻⁵ mg/ml against Staphylococcus aureus and Escherichia coli, respectively (Al-Asmari et al. 2024), compared to emulsions produced by HSH that showed markedly higher MICs of 0.31 mg/ml for E. coli and 1.25 mg/ml for S. aureus (da Silva et al. 2023), while VM emulsions demonstrated substantially lower antimicrobial efficacy, with MICs of 39 and 78 mg/ml against E. coli and S. aureus, respectively (Thielmann et al. 2019). Similar results have been reported for oregano EO emulsions (Boskovic et al. 2015, Hao et al. 2021, Milagres de Almeida et al. 2023, Tahric et al. 2023, Pinto et al. 2024, Tejada-Muñoz et al. 2024). While authors attributed differences in the antimicrobial activity to the intrinsic compositional variability of EOs (Cutillas et al. 2017, Vlčko et al. 2022, Al-Asmari et al. 2024), the potential impact of the emulsification method used to prepare emulsions before the antimicrobial activity test was not considered and this might have significant effects on the reported antimicrobial activity differences. Therefore, the objective of this study was to assess comparatively the effects of the emulsification methods (UT, HSH, or VM) used to make emulsions of sage EOs (SEOs) and garlic EOs (GEOs) on the antimicrobial activity against pure strains of S. aureus, Listeria innocua, E. coli, and Pseudomonas fluorescens.

Materials and methods

Materials

GEOs and SEOs were obtained from Lionel Hitchen Ltd (Barton Stacey, Hampshire, UK). Pure methanol, ethanol (≥99.5%), dyes (Nile Blue and Nile Red), glycerol, and Tween 80® were all purchased from Sigma–Aldrich (St. Louis, MO, USA). Media for microbiological analysis, including plate count agar (PCA), Mueller–Hinton Broth (MHB), and Maximum Recovery Diluent (MRD), were purchased from Oxoid (Basingstoke, Hampshire, UK). Sterile deionized water was used in all experiments.

Preparation and characterization of sage and garlic EO emulsions

Preparation of EO emulsions

Emulsions were prepared by dissolving 0.5% (v/v) SEO or GEO in 50% (v/v) ethanol to which Tween 80® (non-ionic) surfactant was added at an oil-to-surfactant ratio of 10:1 (v/v), and emulsions of these solutions were prepared using the following methods:

  1. VM: Emulsions were prepared using VM as previously described by Zafirah Fahizan et al. (2025) with some modifications. The EO/Tween 80® in 50% (v/v) ethanol solution was vortexed (Stuart vortex mixer SA 8) at 2500 rpm for 30, 60, or 120 s.

  2. UT: Emulsions were prepared using UT as previously described by Liu et al. (2022) with some modifications. The EO/Tween 80® in 50% (v/v) ethanol solution was sonicated using a probe sonicator (EpiShear™ Probe Sonicator, Active Motif, UK) equipped with a 2 mm probe, operated at 20 kHz frequency and 80% amplitude. Sonication was performed for a total of 30, 60, or 120 s in 30-s bursts interspersed with 10-s rest periods. To prevent the temperature increase of the samples, tubes were placed on ice during sonication.

  3. HSH: Emulsions were prepared using HSH method as previously described by da Silva et al. (2023) with some modifications. The EO/Tween 80® in 50% (v/v) ethanol solution was homogenized using an IKA Ultra-Turrax™ homogenizer (IKA Werke, Janke & Kunkel GmbH & Co KG, Staufen, Germany) at 12 000 rpm for 30, 60, or 120 s. The Ultra-Turrax homogenization speed of 12 000 rpm was determined on a preliminary trial at which the solutions were homogenized at 8000, 10 000, and 12 000 rpm for 60 s, with 12 000 rpm yielding the most stable and homogeneous emulsion. To control temperature increase of the solutions during homogenization treatment, tubes were kept on ice.

Characterization of emulsions

Particle size and polydispersity index

Particle size analysis and polydispersity index (PDI) of GEO and SEO emulsions prepared using different emulsification methods were determined using a dynamic light scattering analyser (Malvern, UK). To minimize multiple scattering effects, prior to measurement, samples were 10-fold diluted with 50% (v/v) ethanol. The diluted emulsions were loaded into disposable cuvettes (ZEN0040) and analysed at 25°C using a back scattering (BS) angle of 173° and 1.5 refractive index. Each EO emulsion was analysed in duplicate in three independent experiments. Particle size distributions were calculated using the Mark–Houwink and Smoluchowski models.

Confocal laser scanning microscopy

For microstructural characterization of GEO and SEO emulsions prepared using different emulsification methods, emulsions were stained by adding 20 μl of Nile Red and/or Nile Blue dye (0.5 mg/ml in methanol) to 200 μl of emulsion. Samples were incubated in the dark for 20 min. After staining, 10 μl of each sample was placed on an eight-well glass-bottom dish (Sarstedt Inc., NC, USA). Imaging was performed using a confocal laser scanning microscope (Olympus Corporation, Tokyo, Japan) equipped with a 63X oil immersion objective. The excitation wavelength was set to 488 nm, with emission collected at 515 nm for Nile Red and at 633 nm for Nile Blue. In the resulting micrographs, oil particles appeared red, while the aqueous phase remained dark.

Stability test

The stability of GEO and SEO emulsions prepared using different emulsification methods was further studied by the Turbiscan LAB™ stability analyser (Malvern, UK) following the procedure described by Zheng et al. (2018). This instrument was equipped with a He–Ne laser (λ = 633 nm) as a light source and two synchronous detectors: BS and transmission (T). Briefly, 20 ml of freshly prepared emulsions were transferred into a cylindrical glass cell and monitored at 25°C for a period of up to 24 h. During analysis, an optical reading head scanned the length of each sample (over the total sample height), recording T and BS data every 40 μm (Zheng et al. 2018). Since the EO emulsion was not transparent, only backscattered light was considered and transmitted light was disregarded (Kang et al. 2011). To quantify creaming and other destabilization phenomena, the Turbiscan stability index (TSI) was calculated using Turbiscan EasySoft (Formulaction, Toulouse, France). TSI provides a single value summarizing all variations in each sample, reflecting the extent of destabilization, and is determined as follows:

graphic file with name TM0001.gif

where, n is the number of scans, xi is the average BS for each min of measurement, and xBS is the average xi (Zhu et al. 2015, Zheng et al. 2018).

Antimicrobial activity

Preparation of bacterial inoculum

In this study, the antimicrobial activity of GEO and SEO emulsions prepared using different emulsification methods against pure strains of Gram-positive bacteria [S. aureus (NCDO 949) and L. innocua (FPL 012)] and Gram-negative bacteria [E. coli (NCIMB 11943) and P. fluorescens (NCIMB 9046)] were assessed. All strains were maintained on PCA slants at 4°C until use. To achieve cultures in the logarithmic growth phase, a loop was inoculated into 10 ml of sterile double strength MHB in a sterile glass tube and incubated on an orbital shaker (New Brunswick™ Innova® 2300, Germany) at 170 rpm for 18 h at 37°C (E. coli, S. aureus, and L. innocua) or 30°C (P. fluorescens) followed by an additional hour of incubation in 10 ml of sterile MHB at the same respective temperatures.

Minimum inhibitory concentration

The antimicrobial activity of GEO and SEO emulsions was assessed by determining their minimum inhibitory concentration (MIC) against the target microorganisms using 96 well flat bottom tissue culture microplates (Sarstedt Inc., NC, USA), following the NCCLS (2000) broth microdilution protocol as described by measured by Cruz-Romero et al. (2013). Bacterial strains were cultured overnight at an appropriate temperature and adjusted to a final density of 105 CFU ml−1 using MRD and the inoculum used within 15 min of preparation. Briefly, 100 μl of double-strength MHB (2XMHB) was added to each well in rows A–F, 200 μl of adjusted bacterial culture suspension was added to row H in columns 1–11, and 200 μl of sterile 2XMHB was added to column 12. In each well of row G, 200 μl of GEO or SEO emulsions were dispensed and a two-fold serial dilution was performed by transferring 100 μl of antimicrobial solutions from row G into the corresponding wells of row F through to row B. After mixing, 100 μl of the resultant mixture in row B was discarded. Finally, using a 12-channel electronic pipette (Model EDP3-Plus, Rainin Instrument LLC, Oakland, California, USA), 15 μl of the tested microorganisms was pipetted from each well in row H into the corresponding wells in row A followed by rows B–G. Positive (row A) and negative growth controls (column 12) were included in each assay plate.

The inoculated plates were incubated in a wet chamber for 24 h at 37°C (E. coli, S. aureus, and L. innocua) or 30°C (P. fluorescens). Resazurin (0.015%) was added to wells A–F (30 μl per well) and further incubated for 2 h. Wells that showed no colour change (resazurin remained blue, indicating no microbial growth) were scored as above the MIC value. The assay was independently repeated three times.

Statistical analysis

Statistical analysis was performed using the software STATGRAPHICS® Centurion XV (Statpoint, Inc., USA). The results of particle size, PDI, stability, and antimicrobial activity against Gram-positive and Gram-negative bacteria were compared using multifactor analysis of variance with emulsification method and treatment time as factors. Interaction between factors, treatment means, and least significant difference values are presented (P < 0.05).

Results and discussion

Effect of different emulsification methods on the particle size, particle size distribution, PDI, and stability of EO emulsions

Particle size and PDI

The effects of different emulsification methods and time on mean particle size, PDI, and emulsion stability of GEO and SEO emulsions are shown in Tables 1 and 2, respectively. As highlighted by He et al. (2022), both particle size and PDI are critical parameters for assessing emulsification methods, as EOs vary in particle size largely due to differences in their chemical composition and extraction method. Regardless of the emulsification method, GEO generally produced larger particle sizes, likely due to its predominant sulphur-containing compounds such as dimethyl trisulfide and diallyl disulfide, which increase viscosity and promote the formation of larger particles, typically ranging from 1 to 3 mm on non-emulsified EO, depending on the extraction method (Kumar Gupta et al. 2021). In contrast, SEO exhibited comparatively smaller particle sizes (<1 mm), consistent with literature reports, with variations depending on extraction technique and attributed to its major constituents, including thujone, camphor. and 1,8-cineole, which are smaller and more volatile compounds that favour the formation of finer particles (Đurović et al. 2022).

Table 1.

The influence of different preparation methods (HSH, UT, or VM) and time (s) on the mean particle size (nm), PDI, and emulsion stability (TSI) of GEO emulsions.

Particle size (nm) PDI Stability (TSI)
A: method
HSH 469.03a 0.24a 1.11a
UT 307.80b 0.13b 0.57b
VM 2561.73c 0.47c 4.24c
SL 0.00 0.00 0.00
B: time
30 1194.87a 0.37a 3.03a
60 1076.27b 0.23b 1.66b
120 1067.43b 0.24b 1.23c
SL 0.00 0.00 0.00
Interactions A×B
SL 0.00 NS 0.00
Samples
HSH/30 574.30 0.30 2.80
HSH/60 418.80 0.20 0.33
HSH/120 413.99 0.21 0.20
UT/30 343.30 0.20 1.17
UT/60 293.10 0.10 0.30
UT/120 287.00 0.10 0.23
VM/30 2667.00 0.60 5.13
VM/60 2516.90 0.40 4.33
VM/120 2501.30 0.40 3.27
LSD 44.38 0.13 0.32
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SL, significant level; NS, not significant; and LSD, least significant difference.

a, b, c different superscripts in the same column indicate significant differences (P<0.05).

Table 2.

The influence of different emulsification methods (HSH, UT, or VM) and time (s) on the mean particle size (nm), PDI, and emulsion stability (TSI) of SEO emulsions.

Particle size (nm) PDI Stability (TSI)
A: method
HSH 246.5a 0.10a 0.49a
UT 240.23b 0.10a 0.33a
VM 575.70c 0.33b 5.58b
SL 0.00 0.00 0.00
B: time
30 368.57a 0.20a 2.57a
60 348.20b 0.17a 2.02b
120 345.69b 0.17a 1.81c
SL 0.00 NS 0.00
Interactions A×B
SL 0.00 NS 0.00
Samples
HSH/30 260.20 0.10 0.72
HSH/60 241.20 0.10 0.52
HSH/120 238.17 0.10 0.23
UT/30 249.30 0.10 0.50
UT/60 236.70 0.10 0.27
UT/120 234.70 0.10 0.23
VM/30 596.20 0.40 6.50
VM/60 566.70 0.30 5.27
VM/120 564.20 0.30 4.97
LSD 9.69 0.10 0.34
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SL, significant level; NS, not significant; and LSD, least significant difference.

a, b, c different superscripts in the same column indicate significant differences (P<0.05).

Independent of the EO type, the results showed that both the emulsification method and treatment time significantly (P < 0.05) affected the particle size and PDI. Among the methods tested, UT produced the smallest particle sizes and lowest PDIs in both SEO and GEO emulsions compared to HSH or VM methods. This can be attributed to the sonication mechanism, where high-frequency acoustic waves (>20 kHz) generate cavitation microbubbles in the aqueous phase. The subsequent collapse of these bubbles produces intense localized pressure gradients and high-velocity liquid jets, creating internal shear forces that effectively break down coarse oil-in-water emulsions into micro-sized particles (Modarres-Gheisari et al. 2019, McClements et al. 2021). In contrast, VM relies on mechanical stirring with considerably lower energy input, which primarily causes phase agitation without inducing substantial particle size reduction (Ren et al. 2019). Treatment time significantly (P < 0.05) affected particle size; however, no differences (P > 0.05) were observed between 60- and 120-s treatments for either oil. Similarly, PDI values showed no significant variation (P > 0.05) across treatment times for SEO, and for GEO no differences (P > 0.05) were detected between 60 and 120 s (Table 1). Consistent with our findings, Liu et al. (2022) demonstrated that UT at 20 kHz and 500 W significantly reduced the particle size of GEO emulsion from 820.3 to 279.2 nm within 60 s, while also markedly lowering PDI values compared to controls, indicating improved particle uniformity and enhanced emulsion stability achieved through UT emulsification. Likewise, Moghimi et al. (2016) reported a minimum particle size of 222 nm in SEO emulsions after 15 min of UT at 20.5 kHz and 400 W. Similarly, the effectiveness of HSH in producing smaller particles has been confirmed. For instance, carvacrol emulsions have been reported to attain particle sizes between 150 and 350 nm after homogenization at 15 000 rpm for 5 min (Mazarei and Rafati 2019), while thymol emulsions reached as low as 68.47 nm after HSH at 6600 rpm for 9 min (da Silva et al. 2023). Furthermore, da Silva et al. (2023) demonstrated that HSH treatment significantly lowered the PDI, thereby producing more homogeneous emulsions. Peccini et al. (2024) also reported similar differences in oil particle size profile between primary thymol emulsions and those generated under high-shear conditions. In their study, different surfactant concentrations were applied with homogenization times of 2–4 min using VM and 3–6 min using high-intensity UT. The authors found that higher energy input favoured the formation of emulsions with smaller oil particles and enhanced homogeneity. This efficiency is attributed to the rotor–stator principle of HSH, where the emulsion is drawn axially into the dispersion head at high circumferential speeds and forced radially through narrow slots and minimal gaps between rotor and stator, generating powerful mechanical shear and cavitation forces that fragment oil phases into fine particles (Gazolu-Rusanova et al. 2020).

Particle size distribution analysis showed that UT and HSH emulsions exhibited narrow, monomodal peaks, indicating homogeneous particles, whereas VM produced broad, bimodal distributions characteristic of heterogeneity (Fig. 1). These findings correspond with the significant interaction (P < 0.05) observed between emulsification method and treatment time for particle size and stability (Tables 1 and 2). Moreover, PDI values for UT and HSH were <0.3, confirming uniform particle formation, while values >0.5 (VM) are typically associated with heterogeneous dispersions (He et al. 2022, da Silva et al. 2023).

Figure 1.

Figure 1.

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Effects of different preparation methods (VM, HSH, or UT) on the particle size distribution of SEO emulsions prepared using (A) VM, (B) HSH, or (C) UT, and GEO emulsions prepared by (D) VM, (E) HSH, or (F) UT. Emulsions were prepared at 30, 60, and 120 s.

These results highlight that both emulsification time and method substantially affected distribution uniformity, a critical factor influencing quality, stability, uniformity, and dispersibility of EO emulsions (Dávila-Rodríguez et al. 2020). These factors could also impact other characteristics, such as antimicrobial activity, since particle size and their distribution have been reported to significantly affect the antimicrobial activity of EO emulsions (Clavijo-Romero et al. 2019).

Confocal laser scanning microscopy

As shown in Fig. 2, microstructures of GEO and SEO emulsions corresponded with their respective particle size and PDI values, further highlighting the influence of emulsification method on particle size and dispersion. GEO emulsions prepared by VM (Fig. 2A) contained very large particles, reflecting a heterogeneous size distribution and rapid destabilization, likely due to droplet coalescence associated with the low energy input of VM, which is less effective at generating fine, stable emulsions (van der Schaaf and Karbstein 2018). In contrast, HSH treatment produced smaller particles with improved uniformity (Fig. 2B), while UT yielded the smallest and most evenly distributed particles, in agreement with average size data (Fig. 2, Tables 1 and 2) These results are consistent with Liu et al. (2022), who also observed a significant size reduction in GEO emulsions after UT treatment. A similar trend was observed in SEO emulsions, where UT produced smaller, more uniform particles compared with VM and HSH (Fig. 2D–F). Such improvements are attributed to the high energy input of UT, which enhances droplet disruption and stability by reducing recoalescence (Yiu et al. 2023, Santos et al. 2025).

Figure 2.

Figure 2.

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Confocal laser scanning microscopy images illustrating the particle size and morphology of GEO emulsions prepared using (A) VM, (B) HSH, or (C) UT, and SEO emulsions prepared by (D) VM, (E) HSH, or (F) UT. All emulsions were prepared with a 60-s treatment time. The scale bars represent 20 μm in all panels.

Stability test

Emulsion stability was assessed over a 2.5-h period, a critical timeframe to ensure antimicrobial agents remain stable before activity testing, as stability strongly influences biological efficacy (Santos et al. 2025) and aligns with established protocols (Barnes et al. 2023). The TSI was used as a quantitative measure of destabilization, with values reported here reflecting those at the end of 2.5 h. Higher TSI values indicate increased particle migration phenomena such as creaming, sedimentation, or coalescence, signifying lower stability, whereas values <1 denote a more homogeneous and stable emulsion (Ma et al. 2016). TSI profiles further revealed the effect of preparation method and time on emulsion stability with emulsions (SEO or GEO) prepared by UT and HSH methods for at least 60 s, maintaining high stability over time (TSI < 1). The enhanced stability can be attributed to the UT and HSH methods, which reduce oil particle size and limit coalescence (Soares et al. 2019). Additionally, these methods facilitate rapid emulsifier adsorption at the oil–water interface, thereby stabilizing the particles, preventing aggregation, and maintaining emulsion integrity (Singh and Pulikkal 2022, Peccini et al. 2024). According to Komaiko and Mcclements (2016), achieving more stable emulsions requires oil particle diameters in the range of ∼20–500 nm. The smaller particles have been reported to exhibit increased Brownian motion, minimizing gravitational separation and flocculation, key mechanisms responsible for phase instability (Zheng et al. 2018).

However, emulsions prepared by VM underwent rapid destabilization irrespective of treatment time, indicated by a steep TSI increase within 2.5 h and surpassing a TSI of 1 in under 20 min, marking the onset of particle destabilization (Fig. 3A–F). This instability corresponds to their larger particle size (Tables 1 and 2) and heterogeneous particle distributions. Consistent with our findings, Aslan and Dogan (2018) demonstrated that emulsions formulated with low-energy mechanical homogenizers exhibited phase separation within the first day of storage, while UT-treated emulsions remained stable. Numerous studies have linked the smaller particle size achieved through HSH or UT methods to improved emulsion stability, with higher energy input producing stable, homogeneous systems with uniformly dispersed particles (Dávila-Rodríguez et al. 2020, Yazgan 2020, He et al. 2022, Peccini et al. 2024, Santos et al. 2025).

Figure 3.

Figure 3.

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Effects of different preparation methods (VM, HSH, or UT) on TSI stability profiles after 2.5 h for SEO emulsions prepared using (A) UT, (B) HSH, or (C) VM, and GEO emulsions prepared by (D) UT, (E) HSH, or (F) VM. Emulsions were prepared at 30, 60, and 120 s.

Antimicrobial activity

The effects of different emulsification methods and time on antimicrobial activity are shown in Table 3 for GEO emulsions and in Table 4 for SEO emulsions. Regardless of emulsion type (SEO or GEO), a statistically significant interaction (P < 0.05) was observed between the emulsification method and treatment time, both of which influenced antimicrobial efficacy against Gram-positive and Gram-negative bacteria. Across all bacteria tested, emulsions prepared with either the UT or HSH method for at least 60 s showed significantly (P < 0.05) higher antimicrobial activity compared to the VM method or emulsification lasting only 30 s, regardless of the method applied. This enhanced activity might be associated with reductions in particle size, improved emulsion stability (Tables 1 and 2), and increased homogeneity (Fig. 1) for both SEO and GEO emulsions treated by UT and HSH for at least 60 s. These factors likely enhance the penetration of emulsions into bacterial cells, facilitating interactions with bacterial membranes, especially the lipid components, thereby maximizing antimicrobial potential. Numerous studies have similarly linked the particle size of EO emulsions to their antimicrobial efficacy (Ozogul et al. 2020, Pongsumpun et al. 2020, McClements et al. 2021, Mahmud et al. 2024). For instance, Moghimi et al. (2016) reported that a sage EO emulsion prepared via UT with a mean particle diameter of 222 nm exhibited a four-fold reduction in MIC against S. typhi and E. coli compared to pure sage EO. Similarly, El-Sayed et al. (2017) observed superior antibacterial activity of GEO emulsions compared to their coarse counterparts at equivalent concentrations. Liu et al. (2022) further demonstrated that GEO emulsions produced by UT with an average particle diameter of ∼215 nm showed enhanced antimicrobial activity against methicillin-resistant S. aureus relative to coarse emulsions under identical concentration and exposure conditions. Comparable trends have also been documented for other EO emulsions, such as HSH-prepared thymol oil emulsions (da Silva et al. 2023) and UT-treated cinnamon, oregano, and rosemary EO emulsions (Dávila-Rodríguez et al. 2020). These authors attributed improved antimicrobial activity to reduced particle size, which in turn increased surface area and thus facilitating better dispersion of EO molecules within the continuous phase. This enhanced dispersion promotes more efficient interactions between EO molecules and microbial cells, supporting increased permeation of EO constituents into bacterial membranes and ultimately augmenting antimicrobial efficacy (Van de Vel et al. 2019). Conversely, emulsions prepared by the VM method for both SEO and GEO exhibited lower antimicrobial performance, likely due to their larger particle size and less effective oil dispersion, factors that reportedly reduce the bioavailability of the EOs (Soares et al. 2019).

Table 3.

Effect of different preparation methods (HSH, UT, or VM) and time (s) on the antimicrobial activity (MIC, mg/ml) of GEO emulsions against Gram-positive and Gram-negative bacteria.

Gram positive* Gram negative**
A: method
HSH 1.25a 2.50a
UT 1.25a 2.50a
VM 3.13b 6.25b
SL 0.00 0.00
B: time
30 3.13a 6.25a
60 1.25b 2.50b
120 1.25b 2.50b
SL 0.00 0.00
Interactions A×B
SL 0.00 0.00
Samples
HSH/30 3.13 6.25
HSH/60 0.31 0.63
HSH/120 0.31 0.63
UT/30 3.13 6.25
UT/60 0.31 0.63
UT/120 0.31 0.63
VM/30 3.13 6.25
VM/60 3.13 6.25
VM/120 3.13 6.25
LSD 0.00 0.00
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SL, significant level; NS, not significant; and LSD, least significant difference.

a, b different superscripts in the same column indicate significant differences (P<0.05).

*

Gram positive (S. aureus, L. innocua).

**

Gram negative (E. coli, P. fluorescens).

Table 4.

Effect of different preparation methods (HSH, UT, or VM) and time (s) on the antimicrobial activity (MIC, mg/ml) of SEO emulsions against Gram-positive and Gram-negative bacteria.

Gram positive* Gram negative**
A: method
HSH 0.73a 1.46a
UT 0.73a 1.46a
VM 1.56b 3.13b
SL 0.00 0.00
B: time
30 1.56a 3.13a
60 0.73b 1.46b
120 0.73b 1.46b
SL 0.00 0.00
Interactions A×B
SL 0.00 0.00
Samples
HSH/30 1.56 3.13
HSH/60 0.31 0.63
HSH/120 0.31 0.63
UT/30 1.56 3.13
UT/60 0.31 0.63
UT/120 0.31 0.63
VM/30 1.56 3.13
VM/60 1.56 3.13
VM/120 1.56 3.13
LSD 0.00 0.00
Open in a new tab

SL, significant level; NS, not significant; and LSD, least significant difference.

a, b different superscripts in the same column indicate significant differences (P<0.05).

*

Gram positive (S. aureus, L. innocua).

**

Gram negative (E. coli, P. fluorescens).

Overall, both SEO and GEO showed stronger antimicrobial activity against Gram-positive bacteria (S. aureus and L. innocua) compared to Gram-negative bacteria (P. fluorescens and E. coli), irrespective of the emulsification method, which aligns with earlier findings reported by Santos et al. (2025). The difference is likely due to the extra lipopolysaccharide (LPS) layer present in Gram-negative bacteria, which serves as a robust barrier that restricts hydrophobic EO molecules from entering (Angane et al. 2022). Additionally, the LPS provides Gram-negative bacteria with structural and functional mechanisms that help expel toxic substances and resist harsh conditions (Romano and Hung 2023). In contrast, Gram-positive bacteria are more susceptible to EOs, as their membrane structure, characterized by lipophilic ends and lipoteichoic acids, facilitates oil penetration (da Silva et al. 2021, Zubair et al. 2022, Kamau et al. 2025). This greater permeability enables the EOs to disrupt vital cell wall components, leading to leakage of intracellular contents, including proteins, DNA, and RNA, ultimately decreasing ATP production and resulting in bacterial inactivation or cell death (He et al. 2022, Verma et al. 2022).

This study provides deeper insight into how the emulsification method influences antimicrobial activity, particularly when physicochemical characteristics are precisely controlled using methods such as UT and HSH. Moreover, since the energy applied during emulsion formation often surpasses the oil particle surface energy, with much of this energy dissipated as heat or hydrodynamic forces, prolonged processing can lead to the evaporation of volatile compounds (Santos et al. 2025). Therefore, selecting an optimal emulsification time of 60 s for these methods is vital to preserve the integrity of encapsulated bioactive compounds and maintain consistent antimicrobial efficacy in practical applications.

Conclusion

This study demonstrates that both emulsification method and treatment duration critically influence the antimicrobial activity of SEO and GEO emulsions. Emulsions prepared using HSH and UT for at least 60 s showed similar antimicrobial effectiveness, which was significantly (P < 0.05) higher than that achieved by the VM method. Across all treatments, Gram-positive bacteria were more susceptible than Gram-negative bacteria, irrespective of the EO used. The enhanced antimicrobial potency of HSH and UT emulsions was closely linked to their significantly (P < 0.05) smaller particle sizes, PDI, and more uniform particle distributions. Additionally, the greater energy input of UT and HSH was shown to suppress recoalescence, thereby supporting particle stability and ensuring sustained bioactivity over 2.5 h of storage. Importantly, these insights highlight that selecting the appropriate emulsification method and treatment time is essential not only for reliable antimicrobial testing but also for producing stable and effective EO emulsions, directly impacting their functional performance in food industry applications.

Contributor Information

Paul G Kamau, Food Packaging and Materials Science Group, School of Food and Nutritional Sciences, University College Cork, Cork, T12 Y337, Ireland.

Paola Alzate, Food Packaging and Materials Science Group, School of Food and Nutritional Sciences, University College Cork, Cork, T12 Y337, Ireland.

Malco Cruz-Romero, Food Packaging and Materials Science Group, School of Food and Nutritional Sciences, University College Cork, Cork, T12 Y337, Ireland.

Michael A Morris, AMBER and the School of Chemistry, Trinity College Dublin, Dublin 2, Ireland.

Joe P Kerry, Food Packaging and Materials Science Group, School of Food and Nutritional Sciences, University College Cork, Cork, T12 Y337, Ireland.

Conflict of interest

The authors hereby declare that no conflict of interest is present in this paper.

Funding

This research received funds from the Food Institutional Research Measure (FIRM), grant number 2021R412, administered by the Department of Agriculture, Food and the Marine, Ireland.

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