Nanoemulsion containing Lippia origanoides essential oil: characterization, chemical composition, antibacterial activities

Nanoemulsão contendo óleo essencial de Lippia origanoides: caracterização, composição química e atividade antibacteriana

Abstract

Essential oils are vital for controlling microbial growth due to their antimicrobial properties, which disrupt bacterial cell membranes and inhibit metabolic processes, thereby preventing biofilm formation. This study assessed the antimicrobial efficacy of Lippia origanoides essential oil and its chemical composition in the development of a nanoemulsion. The essential oil predominantly comprises carvacrol (41.56%), thymol (12.56%), and p-cymene (12.49%), yielding a total of 2.13%. The nanoemulsion was characterized by a droplet size of 30.00 ± 1.70 nm, a polydispersity index of 0.25 ± 0.03%, Zeta potential -23,96 ± 3,61 mV, Whiteness index 27,93 ± 1,17, Refractive index 1,34 ± 0,00. The minimum inhibitory concentration (MIC) for the unencapsulated essential oil (OELO) ranged from 1,360 µg/mL to 5,460 µg/mL, while the nanoemulsion (NE-OELO) demonstrated significantly lower MIC values, ranging from 156 µg/mL to 1,250 µg/mL. These findings indicate that NE-OELO is more effective than OELO, emphasizing the benefits of nanoemulsions in enhancing the antimicrobial activity of essential oils. This research could pave the way for innovative products that improve food safety by effectively targeting harmful microorganisms.

Keywords:
nanoemulsion; antimicrobial; carvacrol; nanotecnology

Resumo

Os óleos essenciais são promissores para o controle do crescimento microbiano devido às suas propriedades antimicrobianas, incluindo o rompimento das membranas celulares bacterianas e inibição de processos metabólicos, prevenindo assim a formação de biofilmes. Este estudo avaliou a eficácia antimicrobiana do óleo essencial de Lippia origanoides e sua composição química no desenvolvimento de uma nanoemulsão. O óleo essencial é composto predominantemente por carvacrol (41,56%), timol (12,56%) e p-cimeno (12,49%), com rendimento de extração de 2,13%. A nanoemulsão foi caracterizada por um tamanho de gota de 30,00 ± 1,70 nm, um índice de polidispersão de 0,25 ± 0,03%, potencial zeta -23,96 ± 3,61 mV, índice de brancura 27,93 ± 1,17 e índice de refração 1,34 ± 0,00. A concentração inibitória mínima (CIM) para o óleo essencial não encapsulado (OELO) variou de 1.360 µg/mL a 5.460 µg/mL, enquanto a nanoemulsão (NE-OELO) apresentou valores de CIM significativamente menores, variando de 156 µg/mL a 1.250 µg/mL. Esses resultados indicam que a NE-OELO é mais eficaz que o OELO, enfatizando os benefícios das nanoemulsões no aumento da atividade antimicrobiana dos óleos essenciais. Esta pesquisa pode abrir caminho para produtos inovadores que melhorem a segurança alimentar, atuando eficazmente contra microrganismos nocivos.

Palavras-chave:
nanoemulsão; antimicrobiana; carvacrol; nanotecnologia

1. Introduction

The utilization of plants with therapeutic properties is a prevalent practice, especially among communities with limited access to conventional medical treatments (Malik et al., 2022). The therapeutic potential of plants is linked to their secondary metabolism, which produces bioactive compounds like essential oils (EOs). In their 2025 literature review, Khwaza and Aderibigbe discribed the antimicrobial, anti-inflammatory, and antioxidant properties of EOs (Khwaza and Aderibigbe, 2025).

Bordón et al. (2025) found that certain essential oil components can penetrate bacterial membranes, altering their structure and increasing fluidity and permeability. This disruption undermines the membrane's barrier function, leading to bacterial cell death.

Essential oils offer beneficial biological properties but also face challenges, including instability from light, heat, and oxidation; high volatility; potential cytotoxicity at high doses; and low solubility in water. These issues can limit their technological applications (Taher et al., 2024).

To overcome these challenges, nanoemulsions have emerged as an effective solution. They can encapsulate essential oils within nanometric matrices, enhancing stability, enabling controlled release, and improving therapeutic efficacy (Taher et al., 2024).

Nanoemulsions are colloidal systems formed by combining two immiscible liquids, such as oil and water, using a surfactant. These nanometric droplets enhance the kinetic stability and solubility of essential oils while preserving their chemical composition (Quintão et al., 2013; Chinnaiyan et al., 2022). This leads to improved bioavailability and effective release of active compounds at targeted sites, ensuring desired cellular effects (Damodharan, 2021). Furthermore, this method reduces the risk of adverse effects, such as toxicity, thereby increasing safety and therapeutic efficacy (Kaur et al., 2020).

Therefore, this study aimed to explore the chemical composition and antimicrobial activity of the essential oil extracted from Lippia origanoides, a medicinal plant native to the Caatinga biome of Brazil, recognized for its antimicrobial properties. Furthermore, the research sought to develop and characterize a nanoemulsion of Lippia origanoides essential oil to assess whether this formulation could enhance the oil's antimicrobial effectiveness.

2. Material and Methods

The medicinal plant Lippia origanoides was collected in the municipality of Riachão do Jacuípe, situated at coordinates 11.75062° S and 39.48387° W, in May 2024. The collection took place in the morning, between 10:00 and 11:30 a.m., under dry weather conditions. A specialist visually identified the botanical material based on the distinctive characteristics of the leaves and inflorescence. Access to the genetic heritage was authorized following registration in the National System for the Management of Genetic Heritage and Associated Traditional Knowledge (SisGen) under code A4F716D.

The aerial parts of the selected species, including leaves and flowers, were carefully separated and dried at room temperature in a designated area, away from light, for one week. Once dried, the samples were packaged in new bags and dispatched to the Natural Products Laboratory at the State University of Feira de Santana for the extraction of essential oil.

2.1. Essential oil extraction

The essential oil from the aerial parts of Lippia origanoides (OELO) was extracted using the hydrodistillation method, using approximately 200 grams of plant material. The extraction process lasted three hours, beginning from the condensation of the first drop.

At the end of the extraction process, the essential oil volume was measured using the Clevenger apparatus. The oils were stored in amber glass vials, with anhydrous sodium sulfate added to remove any residual water. They were then packaged in 2 mL amber bottles, labeled, and stored in a commercial freezer at -20°C until chemical analysis. The extraction was performed six times, yielding a total of 1,220 grams of dry plant material. The essential oil yield was calculated using the Equation 1:

$$Yield\left(\%\right)=\left(volumeobtained\left(mL\right)/drymass\left(g\right)\right)\times 100$$ (1)

2.1.1. Determination of the chemical composition of the essential oil

The chemical composition of the essential oils was analyzed at the Analytical Center of the Institute of Chemistry at São Paulo University utilizing gas chromatography coupled with mass spectrometry (GC/MS). This analysis was performed on an Agilent 7809A chromatograph (Agilent Technologies), which was paired with an Agilent 5975C triple-axis detector and a ZB-5 capillary column (Phenomenex; dimensions: 30 m × 0.25 mm) featuring a film thickness of 0.25 mm. The injector temperature was set to 250°C, and helium was used as the carrier gas at a flow rate of 1 mL/min. Both the interface temperature and the ionization source temperature were maintained at 250°C. The ionization energy was configured to 70 eV, with an ionization current of 0.7 kV. The oven temperature initially started at 45°C for 1 minute, before being raised to 300°C at a rate of 10°C per minute, where it was held for an additional 5 minutes.

2.2. Nanoemulsion preparation

A nanoemulsion of Lippia origanoides essential oil (NE-OELO) was created using high-energy ultrasonication. The formulation consisted of 1% (w/w) OELO combined with a 1:3 (w/w) ratio of OELO to Tween 80, suspended in deionized water. The preparation process encompassed three key steps: The initial mixture was subjected to sonication in an ultrasonic cleaner (Q3.0/40 - Eco-Sonics) at ambient temperature for 10 minutes. Subsequently, the mixture was stirred with a magnetic stirrer for 30 minutes at 1000 rpm, also without heating, to produce a coarse precursor emulsion (Nie et al., 2023). Finally, the coarse emulsion was sonicated for 10 cycles of 3 minutes each in an ice bath using an ultrasonic instrument (Ultronique – Eco-Sonics), equipped with a 4 mm diameter probe operating at a frequency of 20 kHz and a power output of 550 W. Following these steps, the resulting nanoemulsion was characterized.

2.3. Nanoemulsion characterization

The OELO nanoemulsion (NE-OELO) was characterized by evaluating its droplet size, polydispersity index (PDI), zeta potential (ZP), whiteness index (WI), and refractive index (RI).

Droplet size (in mm), PDI (in %), and zeta potential (in mV) were measured using the Zetasizer Nano ZS from Malvern Instruments (UK). To minimize multiple dispersion effects, the nanoemulsion was diluted in a 1:100 ratio with deionized water. All measurements were carried out in triplicate. To assess stability, size, and PDI measurements were repeated after 30 days of refrigerated storage. The turbidity of the nanoemulsion was analyzed by measuring its absorbance using a UV-Vis spectrophotometer (K37, KASVI) at a wavelength of 600 nm (Ghosh et al., 2014). The refractive index was determined with a refractometer.

Color measurements were performed using a colorimeter (CR-400, KONICA MINOLTA) that operated under standard illuminant D65. Following the determination of CIE L*, a*, and b* values, the whiteness index (WI) was calculated using Equation 2 (ASTM, 2010):

$$WI=100-\surd \left(\left(100-L*\right)²+\left(a*\right)²+\left(b*\right)²\right)$$ (2)

2.4. Antimicrobial activity assay

Standard strains of food-contaminating microorganisms were sourced from the American Type Culture Collection (ATCC) through the National Institute for Quality Control in Health (INCQS/FIOCRUZ), including Staphylococcus aureus (ATCC 00039), Staphylococcus epidermidis (ATCC 016), Enterococcus faecalis (ATCC 234), Shigella dysenteriae (ATCC 13313), Escherichia coli ETEC (ATCC 11105), Escherichia coli EPEC (CDC 0111 AB), and Salmonella typhimurium (ATCC 14028).

The Minimum Inhibitory Concentration (MIC) was assessed using the broth microdilution technique in 96-well plates, following the methodology outlined in the Clinical and Laboratory Standards Institute Manual (CLSI, 2018), with minor modifications. For the Minimum Inhibitory Concentration (MIC) assay, OELO was diluted in 10% Tween80 to an initial concentration of 175 mg/mL, while NE-OELO was prepared at 10 mg/mL. Both solutions were sterilized with 0.22 μm filters.

A serial dilution resulted in concentrations of OELO from 175 mg/mL to 0.04 mg/mL and NE-OELO from 10 mg/mL to 0.0024 mg/mL. Bacterial strains were cultured on nutrient agar for 20 hours, then homogenized in sterile saline to match a 0.5 McFarland standard (approximately 1.5 x 10⁸ CFU/mL). Spectrophotometric readings at 625 nm confirmed the concentration, adjusting absorbance to 0.08-0.1.

A 1:100 dilution in sterile saline yielded a suspension of 1.0 x 10⁶ CFU/mL for the assays. Viability controls for the microorganisms and sterility controls for the culture medium, oil, solvent, and nanoemulsion were included. The microplates were incubated at 37°C for 24 hours, with tests conducted in triplicate.

After the incubation period, 30 μL of 0.01% resazurin was added to each well, and the plates were incubated again for 3 hours to facilitate colorimetric analysis of microbial growth. The MIC was defined as the last dilution that showed no microbial growth.

The Minimum Bactericidal Concentration (MBC) determination test was performed on all wells where the MIC test showed inhibition of bacterial growth. To conduct the test, five microliters of the contents from each well were inoculated onto Müller-Hinton agar plates, which were subsequently incubated at 37°C for 24 hours. If the inoculum did not produce any microbial growth, the minimum inhibitory concentration of the sampled well was deemed bactericidal. Conversely, if microbial growth occurred, the MIC of the sampled well was classified as bacteriostatic.

3. Results

The chemical composition of the essential oil obtained from Lippia origanoides is comprehensively outlined in Table 1, which enumerates the chemical constituents along with their respective percentages. A total of twenty distinct compounds have been identified within the oil, with the dominant constituents being carvacrol at 41.56%, thymol at 12.56%, and p-cymene at 12.49%. Additionally, β-caryophyllene and γ-terpinene were also notable components, accounting for 6.61% and 6.27%, respectively. Furthermore, the extraction process yielded a total of 2.13% of essential oil, highlighting the efficiency of the extraction method used for this aromatic and medicinal plant.

Table 1
Chemical composition of the essential oil obtained from Lippia origanoides.

Peak	RT (min)	Compound	GC-MS (%)
1	5,548	α-thujene	1.23
2	5,707	α-pinene	0.49
3	7,137	myrcene	2.18
4	7,802	γ-terpinene	1.43
5	8,021	p-cymene	12.49
6	8,175	limonene + eucalyptol	3.23
7	8,954	γ-terpinene	6.27
8	9,215	Sabinene hydrate	0.34
9	10,145	linalool	0.59
10	12,324	terpinene-4-ol	0.69
11	13,897	Thymol, methyl ether	5.09
12	15,727	thymol	12.56
13	16,009	carvacrol	41.56
14	16,677	γ-elemene	0.48
15	18,845	(E)-caryophyllene	6.61
16	19,336	aromandendrene	0.86
17	20,745	viridiflorene	1.85
18	20,840	NI	0.64
19	22,749	spatulenol	0.98
10	22,836	Caryophyllene oxide	0.43
Total			100

• RT: Retention Time; GC-MS: Gas Chromatography – Mass Spectrometry.

The nanoemulsion was characterized by a droplet size of 30.00 ± 1.70 nm, which indicates the uniformity and stability of the emulsion; a polydispersity index of 0.25 ± 0.03%, that reflects the distribution of droplet sizes; Zeta potential -23,96 ± 3,61 mV, which provides insight into the electrostatic stability of the emulsion; Whiteness index 27,93 ± 1,17, which assesses the color quality; Refractive index 1,34 ± 0,00, which serves as a measure of how light propagates through the nanoemulsion. Together, these parameters offer valuable information about the physicochemical characteristics of the nanoemulsion.

Minimum inhibitory concentration (MIC) tests conclusively demonstrated that the essential oil derived from Lippia origanoides (OELO) and its nanoemulsion (NE-OELO) displayed significant antimicrobial activity against all standard strains evaluated, as summarized in Table 2. The control samples, comprising a 10% Tween solution and a nanoemulsion lacking the active essential oil, showed no inhibitory effects, underscoring the efficacy of the tested formulations.

Table 2
Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of essential oil and nanoemulsion of Lippia origanoides against different standard strains of food contaminating microorganisms.

Microorganism (ATCC)	MIC (µg/mL-1)		MBC (µg/mL-1)
	OELO	NE-OELO	OELO	NE-OELO
Staphylococcus aureus (00039)	1,360	625	43,700	2,500
Staphylococcus epidermidis (016)	5,460	312	21,870	1,250
Enterococcus faecalis (234)	5,460	1,250	21,870	NA
Shigella dysenteriae (13313)	5,460	156	21,870	1,250
Salmonella typhimurium (14028)	680	156	5,460	625
Escherichia coli ETEC (11105)	5,460	625	21,870	2,500
Escherichia coli EPEC (cdc 0111 ab)	2,730	625	21,870	2,500

The MIC values for OELO exhibited a range from 1,360 µg/mL to 5,460 µg/mL across the various standard strains, indicating its antimicrobial potential. In contrast, the MIC values for NE-OELO fell within a considerably lower range of 156 µg/mL to 1,250 µg/mL for the same strains. This consistent pattern highlights that NE-OELO not only surpassed OELO in effectiveness but also emphasizes the advantages of employing nanoemulsions as a superior strategy for combating microbial pathogens.

4. Discussion

The nanoemulsion derived from Lippia origanoides essential oil (NE-OELO) showcased not only an optimal particle size but also remarkable stability, making it a promising candidate for various applications. Comprehensive antimicrobial activity tests conducted against a range of standard strains of food-contaminating microorganisms revealed that this nanoemulsion exhibited significantly lower minimum inhibitory concentrations (MICs) in comparison to the OELO.

In this study, the extraction yield of essential oil from Lippia origanoides was found to be 2.13%. Analyzing the extraction yield of essential oils (EOs) provides critical insights for biotechnological applications. Several factors can affect the extraction yield and chemical composition of EOs, including the time of harvest, geographical location, the specific part of the plant used, the treatment of the harvested material, the solvent chosen, and the extraction method applied (Kamal et al., 2023). Additional influences may include circadian rhythms and seasonal variations (Ribeiro et al., 2021).

For example, Alonso et al. (2021) reported on the essential oil yield of Lippia origanoides across different seasons in the Brazilian Cerrado region. They discovered that collections taken at 10:00 a.m. yielded a greater percentage of essential oil when the plant was harvested in August (3.21%), while in February, the yield was recorded at 2.45%, a figure close to that observed in the current study. De Melo et al. (2020) similarly found that the essential oil yield from L. origanoides was higher in the morning, suggesting that 10:00 a.m. is the optimal time for harvesting.

A comprehensive understanding of chemical composition is vital for evaluating the antimicrobial potential of various substances. According to Nocchi et al. (2017), the production of secondary metabolites in plants peaks under high levels of solar radiation, facilitating the biosynthetic reactions driven by photosynthesis.

The essential oil of L. origanoides can be classified into three distinct chemotypes based on their major chemical components. Chemotype A is characterized by p-cymene as the predominant compound. Chemotype B is identified primarily by carvacrol, whereas Chemotype C is defined by thymol as its main constituent (Stashenko et al., 2010). In this study, the essential oil examined falls under chemotype B, as it contains 41.56% carvacrol in its composition.

Santos Filho et al. (2023) investigated Lippia species and reported that the primary constituents of L. origanoides oil included thymol (47.2%), p-cymene (16.0%), and E-caryophyllene (11.3%). Furthermore, studies by Menezes et al. (2018) identified carvacrol as the major compound, comprising 56% of the essential oil's chemical composition.

The variations noted between the studies can be attributed to several factors that influence the chemical constituents of essential oils. These factors encompass climate conditions, water availability, altitude, soil characteristics, nutrient levels, circadian rhythms, cultivation practices, harvesting techniques, processing methods, and the genetic diversity of the plants (Mugao, 2004).

Investigating the antimicrobial properties of essential oils is vital for the development of natural alternatives to combat pathogenic microorganisms. These oils are rich in a variety of bioactive compounds that demonstrate effectiveness against infectious agents. This study explored the antimicrobial properties of essential oil extracted from Lippia origanoides, focusing on its effectiveness against various microorganisms that pose a risk of contaminating food products, highlighting the potential application of this natural extract in enhancing food safety.

Minimum inhibitory concentration (MIC) values for essential oils that fall below 2,500 µg/mL are indicative of their potential antimicrobial activity. Conversely, MIC values exceeding 2,500 µg/mL suggest that these oils may not be suitable for further investigation or practical application (Scapinello et al., 2023). Given this reference point, the results presented indicate that the essential oil under study has significant biotechnological potential for the development of antimicrobial products.

In a related study, Gallardo et al. (2022) evaluated the antimicrobial efficacy of oregano essential oil in inhibiting the growth of resistant strains of E. coli and S. aureus isolated from food. The authors reported minimum inhibitory concentrations of 2,000 µg/mL and minimum bactericidal concentrations of 6,000 µg/mL, respectively.

In a study by Pinheiro et al. (2022) explored the antimicrobial activity of the essential oil of L. origanoides (OELO) against Gram-negative bacteria. Their findings revealed that the MIC of OELO was 625 µg/mL for both E. coli and Klebsiella pneumoniae, and 2,500 µg/mL for Pseudomonas aeruginosa. Notably, the minimum bactericidal concentration (MBC) values in their study were lower than the MIC values, contrasting with the current study, where the MBC values were found to be higher than the MIC.

The diversity and concentration of metabolites play a crucial role in determining the biological activity of essential oils (EOs), which accounts for the varying outcomes observed in similar studies. Essential oils (EOs) consist of chemical compounds that are highly lipophilic, giving them a strong affinity for plasma membranes. This affinity can lead to disruptions in membrane integrity (Man et al., 2019).

These compounds engage with the hydrophobic core of lipid bilayers, resulting in alterations to the physicochemical properties of cell membranes. Such interactions can cause membrane ruptures, leakage of cellular contents, deformation, and cell swelling, indicating a process recognized as membrane permeabilization (Bordón et al., 2025). A study investigating the effects of EOs utilized electron microscopy to reveal a complete loss of integrity in both the plasma membrane and the cell wall of E. coli bacteria (Gallardo et al., 2022).

Essential oils demonstrate promising antimicrobial properties; however, their application faces significant challenges, including high volatility, low water solubility, and instability within food systems. These limitations hinder their effectiveness (Marín et al., 2024). To overcome these obstacles, the development of nanoformulations containing bioactive compounds derived from essential oils may offer a practical solution.

Research suggests that nanoemulsions of essential oils enhance the solubility of these compounds, thereby improving their bioavailability and antimicrobial efficacy, particularly for essential oils that are rich in lipophilic compounds such as carvacrol (Zhang et al., 2024). Therefore, to enhance essential oil efficacy and bioavailability, a nanoemulsion of the essential oil was developed, tested and characterized using droplet size, polydispersity index, zeta potential (PZ), whiteness index (BI), refractive index (RI).

The findings demonstrate that the high-energy emulsification technique, specifically utilizing an ultrasonic homogenizer, was effective in the production of essential oil nanoparticles, as indicated by the average droplet size (30,00 ± 1,70 nm) of the developed nanoemulsion (NE-OELO). The size of the droplets is crucial for the bioactivity, absorption, and stability of nanoemulsions (Bolgen et al., 2025).

The reduction in droplet diameter can be attributed to the essential role of emulsifiers, which can be understood through the principle of excess surface area. The emulsifying layer that surrounds the droplets acts as a barrier to coalescence, effectively lowering the interfacial tension between oil and water. Furthermore, the application of high-energy ultrasonic homogenization enhances the fragmentation of the dispersed phases, increasing the available surface area. As a result, emulsions become not only highly stable but also exhibit a more uniform distribution of droplet sizes (Chouaibi, 2022).

Ultrasonic homogenization is a static process in which a probe emits acoustic energy into a precursor emulsion, acting as a medium for transmitting mechanical vibrations. These vibrations lead to the formation and collapse of air or gas microbubbles through a phenomenon known as cavitation. This process creates intense turbulence and shear forces, which facilitate the breakdown of the dispersed phase (Wang et al., 2015).

The polydispersity index (PDI) serves as an indicator of the homogeneity of the developed formulation by representing the uniform distribution of droplet sizes, which fall within the range of 0.0 to 1.0 (Kayiran et al., 2025). PDI values of ≤0.7 indicate the presence of a monodisperse nanoemulsion, suggesting a uniform nanoemulsion (NE). A PDI value of less than 0.25 signifies a narrow size distribution within the system (Ozogul et al., 2025), which is ideal for formulation applications. The more uniform the particle size, the more predictable the action of the compounds becomes. Consequently, the PDI results from the present study (0.25 ± 0.03%) confirm the uniformity of the droplet size distribution.

Benitez-Llano et al. (2023) developed a novel NE-OELO and successfully obtained particle diameters smaller than 200 nm, with a polydispersity index of under 0.3. This finding aligns with the results of the present research. Similarly, Silva et al. (2023) reported polydispersity index values for carvacrol nanoemulsion below 0.3, demonstrating a narrow size distribution and a high level of uniformity in the formulation.

An essential parameter in the characterization of the nanoemulsion is the zeta potential, which reflects the electrostatic charge between particles and serves as an indicator of the nanoemulsion's stability. The results of this study revealed that the nanoemulsion exhibited a negative zeta potential value, attributable to the presence of the nonionic surfactant Tween 80 and the chemical composition of the essential oil incorporated into the formulation.

Negative zeta potential values can be explained by the ionization of hydroxyl groups in Tween 80 during dispersion in the medium, along with the presence of terpenes in essential oils (Tinh et al., 2025). The surfactant influences the size and characteristics of the nanoemulsion. For instance, Singh and Pulikkal (2022) observed that nanoemulsions prepared with Tween 80 had a mean droplet diameter of 40.9 nm and a negative zeta potential of -25.10 mV.

According to Showkat et al. (2025), the addition of essential oils to the nanoemulsion can influence the charge on the surface of the emulsion droplets. Similarly, Kotwiski et al. (2024) reported negative zeta potential values in their research on a nanoemulsion of Lippia origanoides essential oil, which is consistent with the findings presented in the current study.

It is widely acknowledged that nanoparticles exhibiting diameters smaller than 100 nm, zeta potential values around ±30 mV, and a polydispersity index (PDI) below 1.0 are indicative of high physicochemical stability. These measurements, which reflect the uniformity and charge of the nanoparticles, align with the findings of the current study. Furthermore, such properties are believed to significantly enhance the bioavailability of the active ingredient they carry, thereby improving its effectiveness in various applications (Liu and Liu, 2020).

The refractive index determined in this study was 1.34 ± 0.00, indicating the translucency of the sample and illustrating the high homogeneity of the developed nanoemulsion. Similar values were observed by Kotwiski et al. (2024) when examining various OELO nanoemulsions, which displayed refractive index values ranging from 1.361 ± 0.032 to 1.372 ± 0.030. Additionally, the low whiteness index values of the nanoemulsions further confirm their translucent physical characteristics. This translucency can be attributed to the small droplet size, which scatters light less than larger particles.

The characteristics of essential oil nanoemulsions are shaped by several factors, including the chemical composition of the essential oil, its molecular structure, the surface tension of the surfactant used, and the synthesis method employed (Singh and Pulikkal, 2022). For example, in research conducted by Das et al. (2022) on the development and characterization of cardamom essential oil nanoemulsion, the authors reported a whiteness index value of 86.32 ± 0.34, which markedly contrasts with the value of 1.34 found in the current study.

Having fully characterized the OELO nanoemulsion, the next step was to compare the antimicrobial activities of both the essential oil and its nanoemulsion. This comparison aimed to evaluate whether the nanoemulsion formulation demonstrates enhanced effectiveness in inhibiting microorganisms, thereby providing a more efficient alternative for their control.

This study revealed that the OELO nanoemulsion demonstrated minimum inhibitory concentrations (MICs) ranging from 156 µg/mL to 1,250 µg/mL. Compared to the results obtained for OELO, these findings suggest that nanotechnology significantly enhances the antimicrobial activity of essential oils. To date, there have been no similar studies that developed and evaluated NE-OELO against standard bacterial strains contaminating the tested food matrices, making direct comparisons of results unfeasible.

Zaharioudakis et al. (2024) conducted an analysis of essential oils (EO) and nanoemulsions high in carvacrol. Their findings indicated that the nanoemulsion demonstrated significantly greater antibacterial activity against E. coli and Listeria monocytogenes compared to the derivatives of free essential oils. In another study, Kotwiski et al. (2024) assessed the antifungal activity of a nanoemulsion of essential oil from Lippia origanoides (NE-OELO) that was rich in carvacrol. They reported a minimum inhibitory concentration between 234.4 and 156.3 μg/mL against the tested dermatophytes.

The findings of this research highlight the significant antimicrobial properties of NE-OELO, a nanoemulsion characterized by its high content of the natural compound carvacrol. Recent research by Cirino et al. (2023) has demonstrated that the chemical compounds carvacrol and thymol can disrupt protein synthesis, ultimately contributing to bacterial cell degradation.

The results of this study indicate that nanoemulsions enhance the antimicrobial activity of essential oils compared to their unencapsulated forms. This enhancement is likely attributable to the improved dispersion of the oil in aqueous media when encapsulated, which increases bioavailability and enhances interaction with the cell membranes of microorganisms.

The literature suggests that nanoemulsions significantly enhance stability by safeguarding active compounds from oxidative degradation. They reduce the surface area of these compounds and allow for controlled release, which facilitates their movement through the cell walls of microorganisms, thereby enhancing antimicrobial activity (Barradas and Silva, 2020; Low et al., 2020). Consequently, the use of nanoemulsions emerges as a promising strategy for formulating more effective and sustainable solutions. This approach expands the applications of essential oils across various sectors, including healthcare, the food industry, and agriculture.

This study aims to provide compelling evidence of the biotechnological potential inherent in Lippia origanoides essential oil, particularly in its nanoemulsified form, within the field of food science. The findings from this research could pave the way for the development of innovative products endowed with potent antimicrobial properties, capable of effectively inhibiting food-contaminating microorganisms. Ultimately, this advancement holds great promise for enhancing food safety and protecting consumers from foodborne illnesses.

Acknowledgements

This work was supported by the Fundação de Amparo à Pesquisa do Estado da Bahia [BioproFAR-BA PIE0001/2024]; and was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Brazil).

• Data Availability Statement
  All the data supporting this study's findings is included in the article.

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Edited by

• Editor:
  Takako Matsumura Tundisi

Data availability

All the data supporting this study's findings is included in the article.

Publication Dates

• Publication in this collection
  16 Mar 2026
• Date of issue
  2026

History

• Received
  27 Oct 2025
• Accepted
  20 Jan 2026