Essential Oils from Wild Albanian Lamiaceae: GC-MS Profiling, Biological Activity, and Enhanced Delivery via Nanoencapsulation

1. Introduction

The demand for natural preservatives that can replace artificial additives in food products is increasing due to growing focus over food safety and shelf-life enhancement [1,2]. Although synthetic preservatives are effective, they often provoke health and toxicity concerns, leading customers to prefer more natural food additives. This has led to a renewed interest in plant-based natural preservatives, especially essential oils (EOs) [2]. Essential oils are complex mixtures of volatile organic compounds that are extracted from plants and possess a variety of bioactive qualities, such as antioxidant, antimicrobial, and anticarcinogenic effects [3]. Among the many plant families, the Lamiaceae family which includes medicinal and aromatic plants (MAPs) like Origanum, Thymus, and Satureja has been found to contain a substantial amount of essential oils with strong biological activity, which suggests that they could be used as natural food preservatives [4].

Carvacrol and thymol, two phenolic compounds possessing significant antibacterial and antioxidant properties, are prevalent in essential oils derived from Lamiaceae species, including O. vulgare, T. capitatus (syn. Thymbra capitata), and S. montana [5]. These compounds are suitable for food preservation applications, as they have been shown to inhibit lipid oxidation and microbiological proliferation [6].

One of the primary functions of EOs in food preservation is their antioxidant action, preventing oxidation of lipids and other food constituents, which can lead to rancidity and nutrient degradation. Phenolic substances in EOs, which can act as hydrogen donors and neutralize free radicals, are mainly responsible for their antioxidant properties [3]. EOs from Origanum, Thymus, and Satureja species have been demonstrated in multiple studies to possess antioxidant properties, effectively scavenging free radicals and protecting food from oxidative damage [5,7]. The robust antioxidant capacity of T. capitatus and O. vulgare essential oils has been evidenced by their notable DPPH and ABTS radical scavenging activities. [8]. Likewise, S. montana essential oils have shown antioxidant properties comparable to synthetic antioxidants such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) [9]. The antioxidant qualities of Origanum and Thymus species are largely attributed to their high concentrations of carvacrol and thymol, with carvacrol being especially potent because of its capacity to donate hydrogen atoms in order to neutralize free radicals [10].

Additionally, EOs have well-established antimicrobial profile and have been studied for their ability to stop the growth of bacteria and fungi. It is thought that EOs antimicrobial properties stem from their capacity to denature proteins, damage cell membranes, and obstruct microbial metabolism [11]. Strong activity against a variety of foodborne pathogens, such as S. Enteritidis, E. coli, P. aeruginosa, and C. albicans, has been shown by essential oils derived from Lamiaceae species, especially O. vulgare and T. capitatus [12,13]. In similar fashion as for the antioxidant properties, the principal bioactive compounds in Origanum and Thymus essential oils responsible for their antibacterial effects are thymol and carvacrol. Thymol has been shown to possess bactericidal properties against both Gram-negative and Gram-positive bacteria, including Salmonella enterica and Listeria monocytogenes [14]. Thymol is a multipurpose substance for food preservation because it has antifungal as well as antibacterial qualities [15]. These EOs broad-spectrum antimicrobial activity has prompted research into using them as natural food preservatives because they can control the growth of pathogens and spoilage microorganisms that cause foodborne illnesses.

Even though EOs show significant potential as natural preservatives, their application in food systems is usually limited by their volatility, inadequate solubility, and low stability under various environmental conditions, such as temperature and light. Encapsulation has been proposed and explored to enhance the stability, bioavailability, and controlled release of essential oils in food matrices in order to overcome these challenges. By adding active ingredients to nanoscale delivery systems like nanoemulsions, preservation of essential oils from deterioration and their functional qualities are enhanced [16].

Oil-in-water nanoemulsions have emerged as an effective delivery system for essential oils, as they can encapsulate hydrophobic compounds like EOs, protecting them from oxidation and enhancing their stability during storage [17]. Since the smaller droplet sizes improve the dispersion of essential oils in food products, fostering better interaction with microorganisms and enhancing their antimicrobial and antioxidant effects, nanoemulsions not only offer stability but also the benefit of increased bioavailability [18]. Origanum, Thymus, and Satureja essential oils have been successfully encapsulated in nanoemulsions in a number of studies, indicating their potential as useful food preservation ingredients [19,20,21].

Because of its varied climate and flora, the territory of Albania is very rich in different MAPs. The country's natural landscapes host extensive wild populations of Origanum, Thymus, and Satureja species, traditionally utilized for culinary and medicinal applications. Albanian Lamiaceae species are an appealing subject for investigation owing to their potential in food preservation, with their biodiversity and optimal growing conditions [22]. Despite the growing number of studies on the biological activities of these plants, the literature still lacks information regarding the distinctive chemical profiles, antioxidant, and antibacterial properties of Albanian varieties of Origanum, Thymus, and Satureja. This work aims to fill this knowledge gap by examining the chemical composition of EOs from these species sourced from various regions of Albania, assessing their bioactive properties, and exploring the viability of their nanoencapsulation for enhanced stability and efficacy in food systems and potential use as natural food preservatives.

2. Results and discussion

2.1. Yield of extracted EOs

EO yield (Table 1), expressed as a percentage of dry plant material, is a critical parameter in assessing a plant's industrial potential for EO production. O. vulgaris subsp. hirtum, commonly known as Greek oregano, demonstrated the highest EO yields among the studied species. The essential oil of O. vulgare subsp. hirtum had a pale-yellow color with a medium yield of 3.945 % (v/w), a medium percentage compared to other studies of wild populations from Albania [23,24]. These results are consistent with previous studies confirming O. vulgare subsp. hirtum has superior EO yields compared to other subspecies and the yield of the population taken in study, if cultivated, could be improved with appropiate agronomical practices [25,26,27]. High yields in this species are often associated with rich contents of carvacrol and thymol, compounds known for their strong antimicrobial activity [28]. The high yield makes this plant a promising candidate for cultivation and commercial applications in food preservation, pharmaceuticals, and aromatherapy.

While T. capitatus produced moderate yields with a medium of 1.205 % (v/w), and sample TC-M nearly doubled the yield of TC-L. EOs resulted in a pale-yellow color. The variability in EO percentage is commonly attributed to edaphoclimatic factors, as well as plant age and genotype [29]. The authors couldn’t identify reports of Thymus capitatus EO yield from Albania. Similar yields have been reported in North African regions, typically ranging from 0.5% to 2.0% [30]. While the yield is lower than O. vulgaris, T. capitatus oils are often rich in phenolic monoterpenes like thymol and carvacrol, providing potent antimicrobial and antioxidant effects that justify its use despite moderate yields [31].

S. montana samples displayed the lowest EO yields in this study, with a medium yield of 0.56% (v/w) where the essential oil had a pale-yellow color. SM-S had a considerably higher EO yield compared to SM-B which would make it more promising for industrial applications. However, these values fall within the range reported in prior reports from Albania and other countries where EO yields for this species are typically between 0.25% and 1.69%, depending on location and genotype [22,32,33,34]. Despite the lower oil output, S. montana essential oil is often high in components associated with strong biological activities [35]. Therefore, despite lower yields, the species might hold significance in specialised applications where quality outweighs quantity.

2.2. Quantitative and qualitative analysis of the EOs

GC-MS analysis of the O. vulgare subsp. hirtum EOs identified in total 26 different components, counting for 96.68% and 97.37% of the EOs of OV-P and OV-L samples respectively. The most abundant compound was Carvacrol, followed by γ-Terpinene, p-Cymene, and Thymol counting on average for 77.2%, 5.6%, 3.42% and 1.28% respectively (Table 2). These results are in line with another report of O. vulgare subsp. hirtum from south Albania where carvacrol composed 79.8% of the EOs [24]. The EOs of O. vulgaris subsp. hirtum is clearly a carvacrol-type, which is consistent with previous chemotaxonomic classifications of Greek oregano [36]. Carvacrol is well known for strong antioxidant, antimicrobial and also anti-inflammatory [37]

Both oils are very similar in chemical composition with minor differences which are unlikely to significantly alter the overall bioactivity profile.

GC-MS analysis of the T. capitatus EOs identified in total 19 different components, counting for 93.65% and 91.6% of the EOs of TC-M and TC-L samples respectively. The most abundant compound was Carvacrol, followed by p-Cymene, and γ-Terpinene, counting on average for 75.25%, 4.48% and 3.37% respectively (Table 3). Consistency in major components indicates genetic stability, despite environmental variability of these two populations.

T. capitatus samples clearly fits the carvacrol chemotype, similar to Origanum oils, and confirms its high bioactivity profile effective against Gram-positive, Gram-negative bacteria [38] and fungi [39]. The high carvacrol levels exceed many commercial standards, placing these oils among the most potent natural antimicrobial agents in the Lamiaceae family.

GC-MS analysis of the S. montana EOs identified in total 47 different components, counting for 98.33% and 80.83% of the EOs of SM-B and SM-S samples respectively. The most abundant compound was Thymol, followed by, p-Cymene, Carvacrol methyl ether and γ-Terpinene counting on average for 40.65%, 10.35%, 5.35% and 5.05% respectively (Table 4). Thymol was markedly higher in SM-B sample with almost the double of SM-S concentration.

S. montana oils display thymol chemotype, particularly in SM-B. This population presents the highest thymol concentration compared to other reports from Albania [22,32,40] making it a valuable candidate for cultivation and industrial applications. Thymol is associated with potent antiseptic and antibacterial properties [41] which could be suitable for food preservation. The higher thymol content in SM-B indicates a stronger bioactivity potential compared to SM-S. However, SM-S shows greater chemical diversity, possibly due to genetic biodiversity from ecological and geographic variability [22].

All three species contain biosynthetic precursors (γ-terpinene, p-cymene), showing enzymatic direction toward either carvacrol or thymol pathways [42]. Based on carvacrol/thymol content, O. vulgaris subsp. hirtum and T. capitatus are stronger antimicrobials, while S. montana shows slightly broader compositional complexity but lower potency. Oils rich in carvacrol (above 70%) are ideal for pharmaceutical and preservative uses. S. montana could appeal to niche markets that favor thymol-based formulations with milder olfactory characteristics.

2.3. Antioxidant activity

Antioxidant activity of the EOs expressed as IC₅₀ for DPPH and ABTS assays are reported in table 5. Lower IC₅₀ values (μg/mL) indicate stronger antioxidant activity.

Table 5. Antioxidant activity of EOs expressed as IC50 values (μg/mL). .

EOs FROM SAMPLES	DPPH μg/mL	ABTS μg/mL
OV-L	530 ± 8	110 ± 10
OV-P	600 ± 12	120 ± 8
TC-M	530 ± 9	180 ± 9
TC-L	570 ± 8	220 ±13
SM-B	1200 ± 5	460 ± 27
SM-S	820 ± 4	500 ± 14

Table 5. Antioxidant activity of EOs expressed as IC50 values (μg/mL). .

EOs FROM SAMPLES	DPPH μg/mL	ABTS μg/mL
OV-L	530 ± 8	110 ± 10
OV-P	600 ± 12	120 ± 8
TC-M	530 ± 9	180 ± 9
TC-L	570 ± 8	220 ±13
SM-B	1200 ± 5	460 ± 27
SM-S	820 ± 4	500 ± 14

OV-L and OV-P EOs exhibited the lowest IC₅₀ values, especially in the ABTS assay, indicating the strongest antioxidant capacity among all tested oils. This correlates strongly with their high carvacrol content (74.6–79.8%), a phenolic monoterpene known for effective free radical scavenging, hydrogen donation ability due to its hydroxyl group and lipophilic character enhancing membrane interaction [43]. T. capitatus EOs, also rich in carvacrol (72.8–77.7%), showed similarly high antioxidant performance, although slightly less potent than O. vulgaris, possibly due to marginally lower carvacrol levels and total phenolics.

S. montana EOs demonstrated weaker antioxidant activity, especially SM-B with the highest DPPH IC₅₀ (1200 μg/mL). Despite having high thymol content (52.8% in SM-B, 28.5% in SM-S), the antioxidant potency was significantly lower than carvacrol-rich oils. Although thymol is also a phenolic compound, it is generally less effective than carvacrol in radical scavenging due to differences in redox potential, steric hindrance from methyl groups [44], electronic resonance stabilization of the phenoxyl radical.

The ABTS assay proved more sensitive across all oils, in accordance with what is generally reported in other works [45].

Oxidative degradation of food lipids leads to rancidity, loss of flavor, discoloration, and decrease of nutritional value. Essential oils rich in carvacrol (e.g., O. vulgaris, T. capitatus) showed strong antioxidant activity [46], indicating their potential as natural alternatives to synthetic antioxidants like BHT and BHA. Their ability to scavenge both lipophilic (DPPH) and hydrophilic (ABTS) radicals broadens their applicability in emulsions, fats, meat products, and oils. While S. montana EOs were less effective as antioxidants, their aromatic and antimicrobial properties may still be valuable as sensory enhancers and microbial inhibitors, especially in combination with other preservatives.

2.4. Antimicrobial activity

Table 6 shows the antimicrobial activity of the six EOs using the broth microdilution method in accordance with CLSI guidelines [47].

MICs were determined and ranged from 0.15 to 2.5 mg/mL. C. albicans and S. maltophilia were the most susceptible organisms overall, while P. aeruginosa was the most resistant. P. aeruginosa is known for its resistance mechanisms, including low outer membrane permeability and active efflux systems, which limit the efficacy of hydrophobic substances like EOs [48].

O. vulgaris subsp. hirtum EOs showed strong activity (MIC ≤ 0.625 mg/mL) against E. coli, M. luteus, S. maltophilia, and C. albicans. Likely due to high carvacrol content (74.6–79.8%), a phenolic monoterpene known to disrupt bacterial membranes and induce leakage of cytoplasmic contents [13,48]. Slight differences in MIC values between OV-L and OV-P EOs may reflect minor differences in thymol content or minor components.

In T. capitatus sample TC-M showed broad-spectrum activity, with very low MICs against C. albicans (0.156 mg/mL) and E. coli (0.312 mg/mL). TC-L had no inhibitory effect on P. aeruginosa, indicating possible influence of lower carvacrol content (4.9% less) and other minor constituents. Still, their antimicrobial potency, is explained through their high carvacrol content (72.8–77.7%).

S. montana samples demonstrated weakest antimicrobial activity, with the highest MICs levels on all tested strains compared to the other two species taken in study. Lower efficacy is consistent with lower carvacrol content and higher relative content of thymol and monoterpenes with weaker antimicrobial effects (e.g., borneol, p-cymene).

Carvacrol is generally reported to have stronger antimicrobial activity than thymol, although both are potent and structurally similar phenolic compounds [49].

The variation in antimicrobial activity between the essential oils of the same species can be attributed to small differences in chemical composition where the interaction among major and minor compounds could have synergistic or antagonistic interactions which explain the differences in MICs [50].

To our knowledge this is the first report on the activity of the EO from S. montana on the multidrug resistant S. maltophilia bacteria with MIC doses of 1.25 and 0.625 mg/ml, SM-B and SM-S respectively.

EOs are generally recognized as safe (GRAS) when used at appropriate concentrations. In the context of targeting spoilage and pathogenic microorganisms, EOs from O. vulgaris and T. capitatus, due to their low MICs and broad-spectrum efficacy, are suitable for preserving perishable foods such as minced meats and sausages, ready-to-eat salads or dips, dairy products etc. [51,52] . The strong aroma and flavour of carvacrol-rich oils may require dose optimization, combination with other hurdles (e.g., refrigeration, mild heat, or acids) [53] or better incorporation in delivery systems, such as edible films and coatings [54], or nanoemulsions [55] for improved dispersion in aqueous food systems, to enhance efficacy and minimize sensory impacts.

As encapsulation and nanoemulsion technologies can improve stability, solubility, and controlled release, minimizing flavor impact while preserving efficacy, the most promising EOs were tested for incorporation in nanoemulsion delivery system.

2.5. EOs nanoemulsions

To be ideal candidates for incorporation into nanoemulsions, EOs should meet the following: high yield – to ensure feasibility and cost-effectiveness, strong antioxidant activity – to prevent oxidative deterioration, potent antimicrobial activity – to inhibit or eliminate foodborne pathogens, chemical stability and compatibility with emulsifiers and food matrices.

From the plant species taken in our study, O. vulgaris subps. hirtum and T. capitatus resulted the most prominent, respectively represented by EOs of samples OV-L and TC-M which performed better. Specifically, OV-L had a slightly higher yield, and slightly stronger antioxidant and antimicrobial activity compared to OV-P. TC-M, in general performed better then TC-L sample (Table 7). Nevertheless, high EO yields in O. vulgaris subps. hirtum (3.8–4.1%) ensure scalability and economic viability. Although yield is lower in T. capitatus samples, its chemical richness in carvacrol, potent antifungal and antibacterial activity, and strong antioxidant action justify its use in targeted nanoemulsion formulations, especially for high-value foods.

Results from DLS (Dynamic Light Scattering) of nanoemulsions incorporating OV-L and TC-M EOs, for the particle size and Z-potential which are key parameters influencing the stability, bioavailability, and functionality of EO nanoemulsions in food systems are shown in Table 8.

According to literature, emulsion droplets with size <200 nm are ideal for improved optical clarity, enhanced bioavailability and penetration into microbial membranes and greater colloidal stability due to reduced gravitational separation [56]. OV-L EOs nanoemulsion particle size was in optimal nano-range (<200 nm) with a mean size of 132.5 nm which indicates well-dispersed and finely emulsified droplets, while also TC-M EOs nanoemulsion was in nano-range even though at the upper end limit.

Zeta potential may act as a partial indicator of the physical stability of the emulsion created. The higher the absolute value of Zeta potential, the more stable is an emulsion. To ensure the formation of a robust energy barrier against the coalescence of dispersed droplets, it has been advised to attain high absolute zeta potential values (exceeding ±30 mV) in most prepared emulsions [57]. Nevertheless, analyzing zeta potential for a nanoemulsion stabilized with Tween 80 is not always essential for understanding stability — and its interpretive value is limited — because Tween 80 provides primarily steric rather than electrostatic stabilization by forming a hydrated, bulky layer around droplets, preventing coalescence through physical hindrance rather than electrostatic repulsion. For example, Tan, T.B. et al. [58] reported that no increase in particle size was seen for the Tween 80-stabilized nanodispersion, even when zeta potentials were brought near 0 mV, due to the non-ionic characteristics of the Tween 80 emulsifier, which imparts stability via steric hindrance.

In summary it can be deduced that both nanoemulsion formulations were good but might need co-stabilizers for an extended shelf life. OV-L EO nanoemulsion offer the best size characteristics for transparent or colloidally stable food systems (e.g., beverages, edible films), though stabilization can be further optimised. TC-M EO emulsions are adequate for use in short-term preservation, active packaging, or surface treatments where long-term droplet stability is less critical.

3. Materials and Methods

3.1. Plant material

Six plants, two of each concerned species, O. vulgaris subsp. hirtum, T. capitatus and S. montana were collected in August 2023, as they are known to accumulate higher amounts of essential oil when exposed to high radiation and temperature [29,59,60].

Information on the collection place of these populations is shown in Table 9.

The species identification for each population was carried out by the Genetics and Plant Breeding Laboratory, Agricultural University of Tirana, Albania, and the herbarium specimen vouchers were deposited in the same laboratory. The plant materials were dried in a well ventilated, shaded area, at room temperature about 25 °C and relative humidity around 50%. Once dried, stems were separated from leaves and flowers.

3.2. EOs extraction

The EOs were extracted by hydrodistillation using a Clevenger apparatus [61]. One hundred grams of dried plant material were minced finely and put into a one-liter flask with 500 mL of distilled water. Distillation went on for three hours at a rate of three millilitres per minute. The oil yield was calculated as a percentage of volume by weight (% v/w) relative to the dry weight of the plant material. The obtained EOs were stored at 4 °C prior to analysis.

3.3. Reagents and microbial strains

Antioxidant radical screening reagents 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS)] were purchased from Alfa Aesar (Massachusetts, United States). Methanol was secured from VWR International (Fontenay-sous-Bois, France) and ethanol from Merk KGaA (Darmstadt, Germany).

The bacterial American type culture collection strains S. enterica serovar Enteritidis (ATCC:49223), E. coli (ATCC:10535), P. aeruginosa (ATCC:9027), S. maltophilia (ATCC:13637), M. luteus (ATCC:10240), along with one fungal isolate C. albicans (ATCC:10231) were procured from Microbiologics, Inc., (Minnesota United States), 96-well plates were secured from Corning Inc. (New York, United States).Blood agar medium and Muller Hinton Broth were procured from Remel Inc, (California, United States), 0.5 Polymer McFarland Standard from Thermo Fisher Scientific (Massachusetts, United States) and Dimethylsulfoxide (DMSO) from Sigma-Aldrich (Missouri, United States).

3.4. Gas Chromatography-Mass Spectrometry

Gas Chromatography-Mass Spectrometry EOs analyses were performed on a Shimadzu GC-2010-GCMSQP2010 system operating at 70 eV. The temperature program was from 60 ^oC to 250 ^oC, at a rate of 5 ^oC/min. Helium was used as a carrier gas at a flow rate of 1.0 ml/min. The injection volume of each sample was 1 μL. Retention times for all compounds were determined according to Van den Dool and Kratz, 1963 [62], using n-alkanes as standards. The identification of the components was based on comparison of their mass spectra with those of NIST21 and NIST107 (National Institute of Standards and Technology, USA), Massada, 1976 [63] and by comparison of their retention indices with literature data Adams, 2007 [64]. Component relative concentrations were calculated based on GC peak areas without using correction factors. EOs were often subjected to co-chromatography with authentic compounds (Fluka, Sigma).

3.5. Free radical scavenging activity

The free radical scavenging activity of the essential oils was measured in vitro using DPPH and ABTS assays according to Brand-Williams et al., (1995) [65] and to Re et al., (1998) [66] respectively. The stock solution for the DPPH assay was prepared by dissolving 24 mg of DPPH in 100 mL of methanol and stored at 20°C. The working solution was obtained by diluting the DPPH stock solution with methanol to achieve an absorbance of about 0.98±0.02 at 517 nm using a spectrophotometer (Biochrom Ltd. Libra S22). The stock solution for the ABTS assay was prepared by dissolving ABTS in water at a concentration of 7 mM. The ABTS radical cation (ABTS•⁺) was generated by mixing the ABTS stock solution with potassium persulfate at a final concentration of 2.45 mM, followed by incubation in the dark at room temperature for 12–16 h. The working solution was then prepared by diluting the ABTS stock with ethanol until the absorbance reached approximately 0.70 ± 0.02 at 734 nm, as determined using the spectrophotometer.

Three mL aliquots of each solution were combined with 77 μL of the sample at 6 concentrations (50, 100, 200, 5000, 1000, 2000 μg/mL), then thoroughly mixed. For the DPPH assay, mixtures were incubated in darkness at room temperature for 30 min., then the absorbance was measured at 517 nm. For the ABTS assay the absorbance was recorded at 734 nm after 5-6 minutes of reaction in similar conditions. The controls were prepared as above but without essential oil. The activity was evaluated based on the percentage of the DPPH and ABTS radicals removed as the following equation:

Scavenging activity in % = {(absorbance of control - absorbance of sample) / (absorbance of control)} x 100. The result was calculated as the concentration of essential oil that inhibits 50% of the free radical (Inhibition Concentration - IC₅₀).

3.6. Evaluation of antimicrobial activity by Micro-dilution Broth method

The antimicrobial activities of the EOs were evaluated against a panel of clinical and food-borne pathogens using standard American Type Culture Collection strains (ATCC), five bacteria along with one fungal isolate. Stock cultures were maintained at 4 °C and subcultured immediately before use. Prior to EO treatment, bacterial strains were incubated at 37 °C and the fungal isolate at 28 °C, each for 18–20 h on blood agar, ensuring cultures were in optimal growth phase.

The MIC (Minimum Inhibitory Concentration) of each EO was determined using a broth microdilution method in 96-well plate following the Clinical & Laboratory Standards Institute (CLSI) protocols (47). In brief, bacterial suspensions were adjusted to a final concentration of 10⁵ CFU/mL cells standardized by 0.5 McFarland in Muller Hinton Broth (MHB) media. EO stock solutions were prepared at 100 mg/ml of by dissolving them in DMSO. From this stock, a working solution of 5 mg/ml was subsequently diluted in MHB media, ensuring the final DMSO concentration is less than 5%. Serial two-fold dilutions were performed to achieve EO concentrations ranging from 5.0 to 0.0097 mg/mL in the microplate wells. Finally, 100 µL of the bacterial suspension was added to each well. The plate setup included the 11^th column as the media control (negative control), and the 12^th column containing bacteria and media (positive control). Additionally, rows D and E were designated for solvent controls, with row D with only EO (solvent control) and row E for DMSO (concentration used to dissolve EO) and bacteria served as DMSO control. The plates were incubated at 37 °C for 24 h. MIC was determined using Tecan I-control software (Infinite M Plex TECAN) to measure the Optical Density (OD) at 600nm compared with the positive control. Everything was kept constant for determining antifungal activity except incubation, which was done for 45-48h, with OD determined at 530nm. MIC was determined as the lowest concentration of EO that inhibited the visible growth of the tested microorganism [67].

3.7. Encapsulation of EOs

The oregano and thyme EOs nanoemulsions were prepared by using the method previously described by Bodea, Cătunescu, Palop, Fernandez, and Garre, (2023) [68] with some modifications. Specifically, a total oil phase was formed with 2 mL oregano EO and 1.35 mL sunflower oil as carrier oil. A coarse emulsion was first produced by mixing the oil phase and 2.5 mL Tween 80, followed by combining with deionised water added dropwise under continuous stirring at room temperature. The emulsion formed after 4.15 mL of water was added. For the thyme EO nanoemulsion the constituents were in slight different concentrations: 1 mL of EO, 1.0125 mL of sunflower oil, and 1.875 mL of Tween 80. The coarse emulsion was homogenised by ultrasonification (USF) for 15 minutes at 100% amplitude to obtain the functionalized nanoemulsion. The maximum temperature of the samples during sonication was kept at 25ºC.

Nanoemulsions were formed using water, sunflower oil, which are all recognised as GRAS, and Tween 80, a permitted food additive, by the Food and Drug Administration of USA. Due to its safety profile, Tween 80 (T80), sometimes referred to as Polysorbate 80, is widely used and has been approved by the European Food Safety Authority (EFSA) for use in food items [69]. Nanoemulsion were immediately stored at refrigeration and analysed after two weeks.

The average droplet size and zeta potential were determined by dynamic light scattering (DLS) (Zetasizer Nano, Malvern).

3.8. Statistical analysis

All experiments were performed in triplicate and the standard deviation calculated. The IC₅₀ value was determined by simple linear regression, using the percentage inhibition data over the linear dose range. The standard deviation of the IC₅₀ was calculated by error propagation, based on the standard errors of the slope and intercept. The analyses were performed with GraphPad Prism software 10.5.0.

4. Conclusions

This work investigated on the potential of essential oils (EOs) from wild Albanian Lamiaceae species, O. vulgare subsp. hirtum, T. capitatus, and S. Montana, as natural preservatives for food applications. The main bioactive compounds in these oils were carvacrol and thymol, both of which are wildly known for their strong antioxidant and antimicrobial activities. The extracted EOs exhibited high antioxidant activity, as evidenced by their low IC₅₀ values in the DPPH and ABTS assays, confirming their ability to scavenge free radicals and prevent oxidative damage. Additionally, the antimicrobial efficacy of the EOs against six foodborne pathogens indicated that the oils possessed broad-spectrum antimicrobial activity, including Gram-negative, Gram-positive bacteria, and fungi. These findings underline the potential of these EOs as natural preservatives.

Oil-in-water nanoemulsions were formed to enhance the stability and delivery of the EOs. The formed nanoemulsions exhibited promising physical characteristics, including small droplet sizes, indicating the potential for prolonged release of the bioactive compounds. However, further investigation is required to assess their long-term stability and practicality in food applications. The commercial viability of these encapsulated oils will depend on the ability to comprehend how they behave at various temperatures, storage conditions, and during food processing.

In conclusion, the results of this study indicate the high potential of EOs from Albanian O. vulgare subsp. hirtum, and T. capitatus as natural preservatives in the food industry. Considering their strong antioxidant and antimicrobial activities as well as their successful nanoencapsulation, they can be effective substitutes for synthetic preservatives which are increasingly viewed as undesirable by consumers. Additionally, the nanoemulsions obtained in this study can be utilized as functional ingredients in food formulations, providing a practical way to add essential oils to foods without changing the organoleptic characteristics.