Essential Oils of Dill and Nettle as a Natural Alternative to Reduce Pathogenic Bacteria on Dairy Production Surfaces

Abstract

Essential oils (EOs) have emerged as promising natural antimicrobials for food safety applications. However, their direct use on food-contact surfaces—such as wood and plastic, commonly employed in artisanal cheese production—has been scarcely explored. This study aimed to evaluate the antibacterial effects of dill (Anethum graveolens) and nettle (Urtica dioica) essential oils against Listeria monocytogenes and Escherichia coli, both in culture media and on inert surfaces. EOs were extracted via steam distillation and characterized by gas chromatography–mass spectrometry (GC-MS). Antimicrobial activity was assessed using agar diffusion and minimum inhibitory concentration (MIC) assays. In addition, bacterial reduction was quantified following EO application to contaminated wooden and plastic surfaces for 40 min. Dill EO exhibited a high anethole content (63.66%), while nettle EO was dominated by limonene (38.73%). Dill EO produced larger inhibition zones against E. coli (13.7 ± 1.5 mm) and L. monocytogenes (12.3 ± 1.5 mm) compared to nettle EO (6.3 ± 0.6 mm and 8.0 ± 1.7 mm, respectively). On plastic, both EOs achieved complete inhibition of E. coli (100%) and greater than 92% reduction in L. monocytogenes. On wood, dill EO maintained high efficacy (up to 87.9%), whereas nettle EO showed limited reduction (29.3%) against L. monocytogenes. These results demonstrate that EO efficacy is influenced by both surface type and target microorganism, supporting the potential of dill EO as a natural antimicrobial agent for surface sanitation in artisanal cheese production.

1. Introduction

Dairy products, due to their high moisture content and nutritional components such as proteins, lipids, and minerals, constitute an excellent medium for microbial growth [1]. This composition favors not only the development of beneficial lactic acid bacteria but also the proliferation of spoilage and pathogenic microorganisms, posing a significant risk to food safety—particularly in regions where production and storage practices do not always meet adequate hygienic standards [2]. Among the most relevant pathogens in the dairy industry, Listeria monocytogenes stands out as a serious public health concern due to its ability to adhere to inert surfaces and grow under refrigeration conditions, whereas Escherichia coli is considered a key indicator of fecal contamination [3,4].

These microorganisms have been frequently reported in fresh, artisanal cheeses, which represent a significant segment of the traditional dairy market [5]. These cheeses are typically produced using materials such as wood and plastic, particularly during pressing and molding stages, respectively. Wood is valued not only for its structural function but also for its ability to harbor beneficial microbial communities, which can contribute to the organoleptic and microbiological properties of cheese. Several studies have documented the presence of specialized microbiota [6,7] and complex biofilms on wooden surfaces used during cheese ripening [8]. However, various food safety guidelines recommend the use of smooth, non-porous surfaces such as stainless steel in food processing environments to facilitate cleaning and minimize contamination risks [9,10,11].

In response to these challenges, essential oils (EOs) have been proposed as natural antimicrobial agents due to their proven efficacy against a variety of pathogens and their lower environmental impact [12,13]. Numerous studies have documented the antibacterial and antifungal properties of volatile compounds present in plant extracts, including esters, ketones, terpenes, aldehydes, and carvone. These compounds, which are especially abundant in essential oils, act on microbial structures by disrupting cell membranes and interfering with metabolic processes [14,15].

In recent years, several research groups have investigated the use of essential oils as natural antimicrobial agents for the control of foodborne pathogens, both in food matrices and on food-contact surfaces used in the dairy industry. Essential oils derived from oregano (Origanum vulgare), thyme (Thymus spp.), clove (Syzygium aromaticum), cinnamon (Cinnamomum spp.), rosemary (Rosmarinus officinalis), and citrus species have shown marked antibacterial activity against Listeria monocytogenes, Escherichia coli, and other microorganisms of relevance to food safety [12,13,15,16].

More specifically, essential oils obtained from dill (Anethum graveolens L.) and nettle (Urtica dioica L.) have demonstrated promising effects against foodborne pathogenic bacteria, mainly attributable to their content of monoterpenes, phenolic compounds, and oxygenated terpenoids. Recent studies have reported the antibacterial activity of A. graveolens essential oil against L. monocytogenes, E. coli, and Staphylococcus aureus, linking its effects to disruption of cell membrane integrity and interference with essential metabolic processes [14,16,17]. Similarly, extracts and essential oils derived from U. dioica have exhibited antibacterial activity against both Gram-positive and Gram-negative bacteria, particularly in food-related contexts and meat or dairy matrices [18,19,20].

These essential oils have therefore been proposed as natural alternatives to conventional chemical antimicrobial agents and other synthetic compounds used for microbial control in food-processing environments [12,21,22].

However, despite extensive research on essential oils as natural antimicrobial agents, little attention has been given to their direct application on specific inert surfaces, such as wood and plastic, commonly used in the production of fresh artisanal cheeses. These surfaces represent critical points of contamination due to repeated use and constant exposure to curd, water, and organic matter. Wood, due to its porosity and roughness, facilitates biofilm formation and microbial retention, while plastic, although less absorbent, often shows cracks, residues, and bacterial adhesion when hygiene protocols are inadequate [10,23]. Nevertheless, the effectiveness of essential oils in reducing bacterial loads on these specific materials remains insufficiently studied, limiting their practical implementation.

In this context, the present study aimed to evaluate the effectiveness of dill and nettle essential oils against L. monocytogenes and E. coli. The evaluation was conducted through antimicrobial sensitivity tests and by applying the oils directly onto inert surfaces commonly used in the production of fresh artisanal cheeses, specifically wood and plastic.

2. Materials and Methods

2.1. Extraction and Physical Analysis

Essential oils of dill and nettle were obtained from fresh leaves and stems of the plants through steam distillation, using a 1:2 ratio of water to plant material. The process was carried out at an average temperature of 92 ± 5 °C for 6 h, yielding approximately 0.3% essential oil per extraction. Distillation was performed in batches of 1000 g of plant material until approximately 3 mL of essential oil was obtained [24].

Subsequently, the density was measured using a pycnometer and a four-digit analytical balance (Mettler Toledo, Mettler-Toledo International Inc., Greifensee, Switzerland), and the refractive index was determined using a calibrated refractometer (Atago, Tokyo, Japan), under laboratory conditions at room temperature.

2.2. Phytochemical Characterization by GC-MS

The characterization of phytoconstituents was carried out using gas chromatography coupled with mass spectrometry (GC/MS), employing high-purity helium gas (5.0) as the carrier gas at a flow rate of 1.0 mL/min. The system used was an Agilent 6890 N (Agilent Technologies, Santa Clara, CA, USA), equipped with a capillary column (30 m × 0.25 mm × 0.25 µm). The identification of essential oil components was performed using the Wiley 275.L library available at the CILAB Salud Laboratory at Universidad Andina Simón Bolívar.

The chromatographic method used for both essential oils consisted of the following oven temperature program: an initial temperature of 70 °C held for 2 min, then increased to 150 °C at a rate of 15 °C/min, and finally raised to 200 °C at a rate of 25 °C/min. The injector temperature was set at 250 °C, the auxiliary temperature at 280 °C, the ion source at 230 °C, and the quadrupole at 150 °C. The injection volume was 3 µL in SCAN mode, with a mass range of 45 to 500 m/z [25,26]. The GC/MS system underwent calibration procedures prior to sample analysis.

Prior to injection, the essential oils were diluted in hexane to prevent system saturation, reaching final concentrations of 0.125% (v/v) for dill oil and 0.5% (v/v) for nettle oil. Samples were stored in 2 mL vials until GC-MS analysis.

2.3. Preparation of Bacterial Inocula

The bacterial strains used in this study were L. monocytogenes (ATCC 19114) and E. coli (ATCC 8739) obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Both were activated using appropriate selective media. For L. monocytogenes, a pre-enrichment step was carried out in Tryptone Soy Broth (TSB) (Neogen, Lansing, MI, USA) for 16 h at 35 ± 2 °C, followed by incubation on Oxford Listeria Agar (Merck, Darmstadt, Germany) supplemented with Listeria selective agents, for 24 h at the same temperature.

For E. coli, the pre-enrichment was performed using Lactose Broth (Difco, Le Pont de Claix, France) for 16 h at 35 ± 2 °C, followed by incubation on Eosin Methylene Blue (EMB) agar (Himedia, Thane, India) for 24 h under the same conditions.

2.4. Minimum Inhibitory Concentration (MIC)

The methodology was based on a previously described protocol [27], with specific modifications for the evaluation of EOs, following recommended procedures for dilution assays using hydrophobic compounds. Tryptone Soy Broth (TSB) was used as the culture medium. As a positive control, TSB supplemented with an antibiotic was used, and TSB without additives served as the negative control.

EOs were previously dissolved in sterile dimethyl sulfoxide (DMSO) to facilitate handling, resulting in a final DMSO concentration of 1% (v/v) in all assays, a level reported as non-inhibitory for bacterial growth according to CLSI-based microdilution protocols [28,29]. Serial 1:2 dilutions were prepared in sterile 2 mL Eppendorf tubes. In the first tube, 1000 μL of 100% EO (dissolved in DMSO) was added. The remaining nine tubes each contained 500 μL of TSB. Then, 500 μL from the first tube was transferred to the second and thoroughly mixed; this procedure was repeated successively until the ninth tube. The excess 500 μL from the final tube was discarded.

Each tube was then inoculated with 500 μL of a bacterial suspension adjusted to 0.5 McFarland standard (~1.5 × 10⁸ CFU/mL), yielding a final volume of 1000 μL per tube. The tenth tube, corresponding to the positive control, contained 500 μL of TSB with antibiotic and 500 μL of the bacterial suspension.

Tubes were incubated for 24 h at 35 °C. Bacterial growth inhibition was assessed visually based on the absence of turbidity and confirmed by measuring absorbance at 550 nm using a UV spectrophotometer (Genesis 20.0) (Thermo Spectronic, Rochester, NY, USA). The MIC was defined as the lowest EO concentration at which no visible turbidity was observed.

EO concentrations were expressed as percentages (v/v), and the equivalent concentrations in mg/mL were also calculated using the experimentally determined densities.

2.5. Antimicrobial Sensitivity Test (AST)

Inocula of each bacterial strain were prepared by adjusting the turbidity to the 0.5 McFarland standard in 5 mL of sterile water, both for L. monocytogenes and E. coli. Subsequently, 0.1 mL of the inoculum was spread on Mueller-Hinton (MH) agar plates (Merck, Darmstadt, Germany) using the surface spreading method, in accordance with reference standards [29].

Agar wells were manually perforated using a sterile 6 mm-diameter glass tube. Then, 50 μL of each essential oil, at its original concentration obtained by steam distillation (undiluted), was added to each well. Antibiotic disks were used as positive controls to validate assay sensitivity: gentamicin (10 µg) for E. coli and ampicillin (10 µg) for L. monocytogenes, following standard agar diffusion protocols. Sterile dimethyl sulfoxide (DMSO) was used as a negative control [28].

All plates were incubated at 35 ± 1 °C for 16 h, a time period selected in accordance with CLSI recommendations for antimicrobial susceptibility testing and to ensure adequate bacterial growth while minimizing potential volatilization and diffusion effects of essential oils that may occur during longer incubation periods. Antibacterial activity was determined by measuring the diameter of the inhibition zones in millimeters (mm) using a ruler [29]. Each experiment was performed in triplicate to ensure reproducibility and reliability of the results.

2.6. Description of Inert Surfaces

For this assay, materials with at least one year of use in traditional dairy production were selected, specifically a plastic cheese mold and a wooden board used for pressing cheese [8,23,30]. The wooden boards were cut into pieces measuring approximately 15–20 cm to facilitate handling in trays.

Prior to use, all materials were washed three times with distilled water. Surfaces were not autoclaved in order to preserve the original resident microbiota from the cheese-making environment, thereby simulating real conditions commonly found in artisanal dairy production [8,23,31]. In parallel, sterilized control surfaces (autoclaved at 121 °C for 15 min) were included as methodological controls, following approaches reported in previous studies evaluating antimicrobial activity on food-contact materials [23].

Preliminary assays performed on sterilized surfaces showed comparable trends in bacterial reduction, confirming that the presence of resident microbiota did not interfere with the evaluation of the antibacterial efficacy of the essential oils.

2.7. Surface Application Assays

Bacterial cultures of L. monocytogenes and E. coli were adjusted to a turbidity equivalent to 0.5 on the McFarland standard. Then, 0.1 mL of each inoculum was applied directly onto a 5 × 5 cm² area of the surface, spread evenly using a sterile cotton swab, and left to rest for 10 min. Subsequently, 50 µL of the essential oils were applied to the surfaces and left to act for 40 min, adapting the method previously described [8,23,30].

All assays were conducted in a controlled chamber set at 20 °C and 78% relative humidity, measured with a digital thermo-hygrometer (Taylor, Tlalnepantla, Mexico), to simulate typical environmental conditions in artisanal cheese production facilities.

Before applying each essential oil, bacterial counts were performed to confirm the adhesion of microorganisms to both wooden and plastic surfaces [32]. After EO application, microbial counts were repeated to assess the antimicrobial effect. All tests were carried out in triplicate using selective culture media. Results were expressed as colony-forming units per square centimeter (CFU/cm²), reporting the mean values.

2.8. Statistical Analysis

Statistical differences (p < 0.05) were evaluated using Tukey’s multiple comparison test to determine significant differences among treatments. In addition, specific pairwise comparisons were conducted using the independent samples t-test to assess relevant contrasts between treatment combinations. The results of this test are presented by reporting the difference between means (Δ), the t-statistic, and the corresponding p-value. All statistical analyses were performed considering three replicates per treatment (n = 3, with a total of 8 treatments), and the results are expressed as mean ± standard deviation (SD), which reflects the variability among experimental replicates rather than the precision of the mean.

Before applying these tests, the assumptions of normality and homogeneity of variances were verified using the Shapiro–Wilk and Levene’s tests, respectively. Parametric tests were applied only to datasets that met these assumptions, ensuring the validity of the statistical methods employed.

Similar statistical approaches based on ANOVA followed by Tukey’s post hoc test and pairwise t-tests have been widely applied in studies evaluating the antimicrobial activity of essential oils against foodborne microorganisms and contact surfaces [15,33]. One-way ANOVA followed by Tukey’s multiple comparison test (p < 0.05) has also been used to compare the antimicrobial efficacy of essential oils against multiple microbial targets, supporting the suitability of the statistical strategy adopted in the present study [34].

3. Results

3.1. Characterization of Essential Oils

The essential oil of A. graveolens exhibited a mean density of 1.1567 ± 0.0007 g/cm³ and a refractive index of 1.507 ± 0.0007, while the EO of U. dioica showed lower values for both parameters, with a density of 1.0685 ± 0.0005 g/cm³ and a refractive index of 1.4590 ± 0.0003, based on the mean of three replicate measurements.

Statistical analysis revealed significant differences (p < 0.05) between the two essential oils in both density and refractive index, confirming clear physicochemical differences likely attributable to their divergent chemical composition, botanical origin, and potential biological activity [35].

The chemical composition of the essential oils extracted from A. graveolens and U. dioica is summarized in

Table 1. Chemical composition and relative abundance (%) of major constituents identified by GC-MS in essential oils of A. graveolens and U. dioica.

Compound	Type	Dill EO (%)	Nettle EO (%)
Anethole	Phenolic ether	63.66 ± 0.31	-
Limonene	Phenolic terpene	6.72 ± 0.04	38.73 ± 0.53
Alpha-Phellandrene	Monoterpene	6.55 ± 0.04	-
Alpha-Thujone	Monoterpene	4.87 ± 0.03	-
Delta-4-Carene	Monoterpene	2.96 ± 0.02	-
Estragole	Phenylpropene	2.9 ± 0.002	-
4-Ethyl-o-xylene	Aromatic hydrocarbon	1.14 ± 0.007	-
Myrcene	Monoterpene	0.93 ± 0.006	1.55 ± 0.02
Exobornyl Acetate	Monoterpene ester	-	11.62 ± 0.16
α-Pinene	Monoterpene	-	5.79 ± 0.02
Camphor	Aromatic ketone	-	3.68 ± 0.05
Linalool	Terpene alcohol	-	3.64 ± 0.01
Dodecane	Saturated hydrocarbon	-	2.43 ± 0.03
Tridecane, 4,8-dimethyl-	Branched alkane	-	2.06 ± 0.03
Pentadecane	Saturated hydrocarbon	-	1.79 ± 0.02
2-Methyltetradecane	Branched alkane	-	1.78 ± 0.02
β-Pinene	Monoterpene	-	1.63 ± 0.02
Triacontane	Saturated hydrocarbon	-	1.37 ± 0.02
Camphene	Bicyclic monoterpene	-	1.34 ± 0.02
n-Tetradecane	Saturated hydrocarbon	-	1.22 ± 0.02
Tetradecane, 3-methyl	Branched alkane	-	1.15 ± 0.02
2-Methylpropanoic acid, 2-ethylhexyl ester	Carboxylic ester	-	1.06 ± 0.01
Sabinene	Bicyclic monoterpene	-	1.05 ± 0.02
Others (<1%)	Various	10.27 ± 0.02	18.06 ± 0.02

Note: Values are expressed as mean ± standard deviation (n = 3). A total of 24 major constituents were identified across both oils, with notable differences in dominant compounds, compound classes, and relative abundance.

.

The dill EO was characterized by a high concentration of anethole (63.66 ± 0.31%), a phenolic ether with recognized antimicrobial activity. Other major components included limonene (6.72 ± 0.04%), α-felandrene (6.55 ± 0.04%), α-thujone (4.87 ± 0.03%), and Δ-4-carene (2.96 ± 0.02%), all belonging to the monoterpene class. Myrcene (0.93 ± 0.006%) was detected in smaller quantities. Minor compounds (<1%) collectively accounted for 10.27 ± 0.02% of the total oil.

In contrast, the EO from U. dioica had limonene (38.73 ± 0.53%) as the predominant compound, followed by exo-borneol acetate (11.62 ± 0.16%) and α-pinene (5.79 ± 0.02%). Other constituents included camphor (3.68 ± 0.05%), linalool (3.64 ± 0.01%), and various saturated and branched hydrocarbons such as dodecane (2.43 ± 0.03%) and 4,8-dimethyltridecane (2.06 ± 0.03%). Minor constituents (<1%) represented 18.06 ± 0.02% of the total oil content.

These compositional differences suggest distinct functional potentials for each EO. The dill oil demonstrated a phenolic and monoterpene-rich profile, while the nettle oil contained a higher proportion of terpenes and hydrocarbon compounds, which may influence their antimicrobial performance and application scope [27,36,37].

3.2. MIC Assessment Results

The tubes that exhibited no turbidity showed absorbance values close to the blank, confirming the absence of bacterial growth (

Table 2. Minimum inhibitory concentration (MIC) results for dill and nettle essential oils against E. coli and L. monocytogenes.

Tube	EO Concentration (%)	EO Dill—E. coli	EO Dill—L. monocytogenes	EO Nettle—E. coli	EO Nettle—L. monocytogenes
1	100%	−	−	+	+
2	50%	+	+	+	+
3	25%	+	+	+	+
4	12.5%	+	+	+	+
5	6.25%	+	+	+	+
6	3.13%	+	+	+	+
7	1.56%	+	+	+	+
8	0.78%	+	+	+	+
9	0.39%	+	+	+	+
10	Negative control	+	+	+	+

Note: (+) visible growth; (−) absence of growth (inhibition). Concentrations are expressed as % v/v.

).

The undiluted essential oil of A. graveolens (100% v/v, equivalent to 1156.7 mg/mL, calculated based on the experimentally determined oil density) completely inhibited the growth of both E. coli and L. monocytogenes. In contrast, none of the diluted concentrations produced visible inhibition. On the other hand, Urtica dioica essential oil showed no inhibitory effect at any of the concentrations tested (4.2–1068.5 mg/mL).

These findings suggest a dose-dependent antimicrobial effect, particularly for dill essential oil. In the case of nettle oil, the lack of antibacterial activity could be attributed to limitations inherent to the broth dilution method or to specific interactions with the assay matrix, which may not fully reflect the oil’s potential under other application conditions.

3.3. Antimicrobial Sensitivity

The evaluation of the antibacterial activity of the essential oils using the agar diffusion assay revealed statistically significant differences among the treatments applied (p < 0.05) (

Table 3. Inhibition zones (mm) produced by essential oils and controls against E. coli and L. monocytogenes.

Bacteria	Treatment	Inhibition Zone (mm) ± SD	p < 0.05
E. coli	Positive control	25.3 ± 0.6	a
	Dill (A. graveolens)	13.7 ± 1.5	b
	Nettle (U. dioica)	6.3 ± 0.6	c
	Negative control	<1	d
L. monocytogenes	Positive control	14.0 ± 1.7	a
	Dill (A. graveolens)	12.3 ± 1.5	a
	Nettle (U. dioica)	8.0 ± 1.7	b
	Negative control	<1	c

Note: Values are mean ± SD (n = 3). <1 indicates no visible inhibition. Different letters (a–d) within each column indicate statistically significant differences (Tukey’s test, p < 0.05).

).

For E. coli, the largest inhibition zone was observed with the positive control (gentamicin), with an average of 25.3 ± 0.6 mm, followed by A. graveolens essential oil (13.7 ± 1.5 mm). U. dioica oil showed moderate inhibition (6.3 ± 0.6 mm), while the negative control (DMSO) exhibited no detectable inhibition zone (<1 mm).

In the case of L. monocytogenes, no statistically significant differences were found between the positive control (ampicillin, 14.0 ± 1.7 mm) and A. graveolens essential oil (12.3 ± 1.5 mm) (p > 0.05), indicating comparable activity. Conversely, U. dioica essential oil produced a significantly smaller inhibition zone (8.0 ± 1.7 mm) compared to the aforementioned treatments (p < 0.05), and the negative control (DMSO) did not exhibit antimicrobial activity (<1 mm), validating its use as a negative control.

For both microorganisms, the results confirmed the strong efficacy of dill essential oil, whereas nettle oil demonstrated more limited antibacterial activity.

3.4. Antimicrobial Efficacy on Inert Surfaces

Antimicrobial efficacy showed contrasting patterns between E. coli and L. monocytogenes (

Table 4. Bacterial load (CFU/cm²) before and after treatment with essential oils on wood and plastic surfaces.

Oil	Surface	Bacteria	CFU/cm2 Before Applying OE	CFU/cm2 After Applying OE	Reduction (%) ± SD
Dill OE	Wood	E. coli	6.51	1.13	82.2 ± 6.3
		L. monocytogenes	1.95	0.31	87.9 ± 16.7
	Plastic	E. coli	1.70	0.00	100 ± 0.0
		L. monocytogenes	2.90	0.20	92.7 ± 6.8
Nettle EO	Wood	E. coli	5.00	0.00	100 ± 0.0
		L. monocytogenes	2.60	1.80	29.3 ± 0.3
	Plastic	E. coli	1.10	0.00	100 ± 0.0
		L. monocytogenes	3.40	0.90	93.2 ± 7.5

Note: Values are expressed as mean ± SD (n = 3). Reduction percentage calculated as: [(Initial CFU − Final CFU)/Initial CFU] × 100.

), revealing a strong dependence on the type of essential oil and the contact surface.

In the case of E. coli, both essential oils—dill and nettle—achieved complete reductions (100%) on plastic surfaces, and values ranging from 82.2% to 100% on wood. This behavior is consistent with previous studies reporting high susceptibility of Gram-negative bacteria to phenolic and terpenoid compounds present in essential oils rich in carvone, limonene, or anethole [21,38]. The lower variation observed between replicates suggests a consistent response, likely linked to the disruption of the outer membrane and destabilization of lipopolysaccharides—mechanisms widely described for this bacterial group [15].

In contrast, L. monocytogenes showed a more heterogeneous response, strongly influenced by the type of surface. Dill EO maintained high bacterial reduction on both wood (87.9%) and plastic (92.7%), which aligns with previous reports demonstrating its high content of bactericidal monoterpenes effective against Gram-positive bacteria [39,40]. However, Nettle EO showed a marked decrease in efficacy when applied to wood, achieving only 29.3% reduction, while regaining high effectiveness on plastic (93.2%).

This differential behavior can be explained by several mechanisms. Wood is a porous material that can partially or completely absorb the essential oil, reducing its availability at the interface with the pathogen [10,31]. Moreover, the structural properties of wood promote bacterial cell retention and initial microcolony formation, decreasing the effectiveness of contact-based antimicrobial agents [38,41]. For oils such as Nettle EO, whose active compounds are more susceptible to surface oxidation or degradation, this phenomenon is especially pronounced, which could explain the drastic reduction observed on wood and the increased variability among replicates.

Pairwise comparisons between specific treatment combinations revealed statistically and microbiologically relevant differences (

Table 5. Significant multiple comparisons between treatments combining essential oil, surface, and microorganism.

Comparison	Difference (%)	t	p-Value
Dill EO, Wood, E. coli vs. Dill EO, Plastic, E. coli	−17.8	−4.92	3.8 × 10−2 *
Dill EO, Wood, L. monocytogenes vs. Dill EO, Plastic, L. monocytogenes	−4.77	−0.54	6.2 × 10−1 NS
Nettle EO, Wood, L. monocytogenes vs. Nettle EO, Plastic, L. monocytogenes	−63.87	−14.1	<1.0 × 10−3 **
Dill EO, Wood, E. coli vs. Nettle EO, Wood, E. coli	−17.8	−4.92	3.8 × 10−2 *
Dill EO, Wood, L. monocytogenes vs. Nettle EO, Wood, L. monocytogenes	58.6	7.67	1.7 × 10−2 *

Note: Mean differences are reported with exact p-values and significance indicators: * (p < 0.05) for significant differences, ** (p < 0.001) for highly substantial differences, and NS (p > 0.05) for non-significant results.

).

In the case of E. coli, the comparison between dill essential oil (Dill EO; A. graveolens) applied on wood versus plastic showed a significant difference in efficacy, with a lower mean reduction on wood (82.2%) compared to plastic (100%). This difference was statistically significant (Δ = −17.8 percentage points, t = −4.92, p = 3.8 × 10⁻²), indicating greater effectiveness of the oil on plastic surfaces (p < 0.05). This result suggests that although E. coli is highly susceptible to the bioactive compounds in the oil, the porosity of the material influences the ability of the antimicrobial agent to achieve maximum bacterial reduction [7,33].

An opposite trend was observed when comparing Dill EO and nettle essential oil (Nettle EO; Urtica dioica). applied on wood against E. coli. In this case, Nettle EO achieved complete efficacy (100%), significantly outperforming Dill EO (82.2%) with a difference of −17.8 percentage points (t = −4.92, p = 3.8 × 10⁻²). These findings indicate that although both oils were effective against E. coli, the specific combination of Nettle EO on wood was more efficient than Dill EO under the same condition, suggesting enhanced sensitivity of this bacterium to the phenolic, terpenoid, and flavonoid compounds present in Urtica dioica [19,42].

For L. monocytogenes, the comparisons revealed greater variability in antimicrobial response. Dill EO showed high and stable efficacy on both wood (87.9%) and plastic (92.7%) surfaces, with no statistically significant difference (Δ = −4.77, t = −0.54, p = 6.2 × 10⁻¹). In contrast, Nettle EO’s efficacy was markedly affected by the surface type: its application on wood resulted in a mean reduction of only 29.3%, while on plastic, it reached 93.2%. This difference was highly significant (Δ = −63.87, t = −14.1, p < 1.0 × 10⁻³), representing the least effective condition in the study and the only one that failed to exceed the 30% reduction threshold.

Additionally, the comparison between both oils applied on wood against L. monocytogenes showed that Dill EO was significantly more effective than Nettle EO (87.9% vs. 29.3%), with a difference of +58.6 percentage points (t = 7.67, p = 1.7 × 10⁻²). This result confirms that Nettle EO, when in contact with porous surfaces, is not suitable for effective control of L. monocytogenes, likely due to lower bioavailability of its active compounds or unfavorable interactions with the lignocellulosic plant substrate [38,43].

3.5. Electron Microscopy

Bacterial strains present on inert surfaces were observed using scanning electron microscopy (SEM) (Jeol IT300, JEOL, Tokyo, Japan), employing secondary and backscattered electron detectors under moderate vacuum conditions (Figure 1). The images correspond to L. monocytogenes and E. coli observed on plastic surfaces treated with dill essential oil. This procedure was not performed on wooden surfaces due to their internal roughness, which hindered the identification of bacterial cells. Similarly, no images are shown for surfaces treated with nettle essential oil, as the visual quality of the micrographs was insufficient for analysis.

4. Discussion

The essential oils of A. graveolens and U. dioica exhibited notable differences in chemical composition, density, and refractive index, which may influence their biological behavior, as previously reported for other plant species [35,44]. These findings also highlight their potential as natural antimicrobial agents against L. monocytogenes and E. coli, two of the most relevant pathogens in the dairy industry [2,3,45]. However, the concentration and efficacy of bioactive compounds in EOs may vary depending on factors such as the extraction method [24], environmental conditions, and the specific plant parts used [46].

Although different essential oils (eucalyptus, lemon, and mandarin) were evaluated, their phytochemical profiles displayed similar antimicrobial behaviors against E. coli and S. aureus, suggesting that common constituents such as terpenes and phenolic compounds may be responsible for these effects [47].

The dill EO exhibited significantly higher inhibitory activity against L. monocytogenes, which may be attributed to the structural characteristics of Gram-positive bacterial cell walls, making them more susceptible to lipophilic compounds [17,28]. These results are consistent with previous studies showing the antimicrobial effectiveness of A. graveolens EO in both in vitro assays and real-food matrices such as fish and dairy products [22,24].

In contrast, the nettle EO displayed lower but not negligible activity, particularly on plastic surfaces contaminated with E. coli. While its antimicrobial effect did not surpass that of dill EO, its composition, rich in oxygenated terpenes, suggests potential interest as a complementary antimicrobial, especially when combined with technologies such as nanoencapsulation to improve bioavailability and stability [9,20,48].

A noteworthy finding was the high efficacy of the EOs when applied directly to inert surfaces commonly used in artisanal cheese production. Dill EO achieved complete bacterial reduction (100%) on both plastic and wood surfaces, even surpassing its performance in agar diffusion tests. This enhanced activity may be related to its higher viscosity, allowing prolonged adhesion and contact time [24]. Differences in efficacy between surfaces also emphasize the influence of physical characteristics such as roughness and porosity, with wood being more prone to retain bacteria and facilitate L. monocytogenes microcolony formation [10,31].

Moreover, the interaction between bacterial structural features and surface types revealed the importance of the physical context in antimicrobial performance. Dill EO, in particular, may be considered not only for its immediate bactericidal activity but also for its potential anti-biofilm effect, as suggested by recent studies on quorum sensing disruption and membrane damage [13,17,22].

Despite the promising results, this study has several limitations. Only two bacterial strains of food safety interest were evaluated under controlled laboratory conditions, which may not fully reflect the microbial complexity or physicochemical challenges present in real industrial environments. Factors such as residual organic matter, relative humidity, or temperature fluctuations, which could affect EO efficacy, were not included. Additionally, the potential sensory impact of EOs on food products was not assessed, which is essential for their commercial application.

Nonetheless, the findings reinforce the potential of essential oils—particularly dill—as a natural alternative for microbial reduction on surfaces commonly used in artisanal food production. These results provide a foundation for future research focused on the development of more stable and effective formulations, through approaches such as nanoencapsulation or incorporation into biodegradable matrices, and their validation under real processing conditions.

5. Conclusions

The essential oils of A. graveolens and U. dioica exhibited differential antibacterial activity against L. monocytogenes and E. coli, with dill essential oil demonstrating greater efficacy, both in culture media and on plastic and wooden surfaces. Its composition, rich in phenolic and terpenoid compounds, supports its potential as a natural antimicrobial agent, particularly against Gram-positive bacteria.

The effectiveness of these oils on inert surfaces commonly used in artisanal cheese production suggests their feasibility as natural alternatives to synthetic compounds, contributing to more sustainable hygiene practices in the food industry.

These findings lay the groundwork for future research focused on the development of more stable and efficient formulations under real processing conditions, including the assessment of multidrug-resistant strains and the evaluation of potential sensory impacts on treated food products.

From a food safety perspective, the present results highlight the potential application of dill essential oil as a natural antimicrobial approach for reducing bacterial contamination on food-contact surfaces in dairy-processing environments, thereby contributing to improved hygiene management and public health protection.