Sustainable camel meat preservation using individual and synergistic essential oil bioactive compounds against Listeria monocytogenes

Abstract

Camel meat is an emerging and sustainable meat with a favorable nutritional profile; however, it is highly susceptible to microbial contamination, particularly Listeria monocytogenes. This study assessed the antimicrobial efficacy of four essential oil (EO) bioactive compounds—linalool, eugenol, α-pinene, and α-terpinene—used individually and in binary combinations against L. monocytogenes in vitro and in minced camel meat stored at 4 °C for 12 days. Minimum inhibitory concentration (MIC) assays revealed that eugenol showed the strongest antibacterial activity (1.25 mg/mL), followed by linalool (5 mg/mL), while α-pinene and α-terpinene exhibited weak activity (40 mg/mL). Checkerboard analysis identified four synergistic combinations—linalool + α-pinene (LP), linalool + eugenol (LE), eugenol + α-pinene (EP), and eugenol + α-terpinene (ET)—with fractional inhibitory concentration index (FICI) values ≤ 0.5. Based on FICI findings, individual EO bioactive compounds (1% w/w) and their synergistic combinations (1% and 2% w/w) were incorporated into minced camel meat inoculated with L. monocytogenes. Eugenol achieved the highest individual reduction by day 12 (3.9 log CFU/g), followed by linalool (2.3 log CFU/g), while α-pinene and α-terpinene were ineffective. All 2% combinations completely inhibited L. monocytogenes from day 3 onward, and the LE1 treatment achieved complete inhibition by days 9 and 12. These findings highlight the strong potential of essential oil bioactive components, particularly their synergistic combinations, as promising natural interventions for enhancing the microbial safety of camel meat and warrant further exploration toward multiple strains, different pathogens and industrial application.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-026-49208-5.

Introduction

Camel meat is a nutritious and sustainable red meat with low fat and cholesterol (~ 50 mg/100 g) and high protein and PUFA levels. It also supplies key minerals such as iron and calcium, making it a healthier option compared to beef and lamb^1–4. Its production is expected to expand globally due to its adaptability to harsh climates and increasing global demand for sustainable protein sources⁴.

However, ensuring the microbial safety of camel meat is vital for public health, as eating contaminated meat can increase the incidence of foodborne illnesses⁵. Camel meat and related products harbor a diverse range of microorganisms, raising both food spoilage and public health concerns^5,6. In terms of pathogenic bacteria, previous studies reported the detection of Salmonella spp., Escherichia coli, Staphylococcus aureus, and Listeria monocytogenes^5,6.

L. monocytogenes is extensively distributed in the natural environment, and its presence in meat products is well documented. It has been linked to multiple severe foodborne outbreaks⁷, and causes invasive listeriosis, a severe form of the disease associated with serious clinical symptoms and a high fatality rate of 20–30% globally⁸. Listeriosis ranks as the third most fatal foodborne illness in the United States, resulting in approximately 172 deaths annually⁹. Although data on camel meat are more limited, L. monocytogenes has been reported at a prevalence of 16%, representing the highest incidence among Listeria species isolated from raw samples in Saudi Arabia¹⁰. These findings emphasize the microbial vulnerability of camel meat and highlight the need to develop effective preservation techniques to extend shelf life and ensure food safety.

To mitigate microbial spoilage and pathogen growth, multiple preservation approaches have been investigated for camel meat. These include physical methods such as gamma and electron beam irradiation^11,12, chemical treatments^13–15 such as citric acid¹³, sodium acetate^14,15, and lactic acid¹⁴, natural antimicrobials such as garlic¹⁶, papaya extract¹⁷, Lactobacillus delbrueckii subsp. delbrueckii¹⁸, date seed extract, green tea extract¹⁹, and Mentha spicata essential oil (EO)²⁰, encapsulated bioactive compounds²¹, innovative packaging^22,23, and marination-based interventions^24,25.

Consumer preference is increasingly shifting away from synthetic preservatives because of their potential carcinogenic effects, harm to non-target organisms, and environmental toxicity^26,27. These concerns are supported by evidence showing that sorbic acid, benzoic acid, and their salts can form mutagenic or carcinogenic by-products^28,29, while nitrites and nitrates used in meat are linked to leukemia, colon and bladder cancer, and other health risks^30,31.

As a result, natural preservatives are gaining attention as safer, effective antimicrobial alternatives with lower health risks³². Among these natural agents,

plant-based bioactive compounds of EOs demonstrated notable antibacterial, antifungal, antiviral, insecticidal, and antioxidant effects^33,34, with varied and often synergistic mechanisms that further enhance their effectiveness as natural food preservatives³⁵. Moreover, employing isolated bioactive compounds can reduce the cost, potential toxicity, and undesirable sensory effects associated with using large amounts of EOs in food applications³⁶. It also allows for more consistent and standardized antimicrobial efficacy, as the chemical composition of whole EOs is often influenced by various factors³⁷.

Linalool is an acyclic monoterpene alcohol present in over 200 aromatic plant species as coriander, basil, mint, and cinnamon-rosewood³⁸. It has a number of biological activities including antimicrobial, antioxidant, anti-inflammatory, and anticancer effects³⁹. Previous studies demonstrate its strong antimicrobial potential in food systems as it reduced Shigella sonnei on lettuce⁴⁰ and inhibited S. aureus in fresh beef⁴¹. As an emulsion, it delayed Pseudomonas aeruginosa growth in beef⁴², and when incorporated into an optimized ternary mixture of oxygenated monoterpenes, it suppressed S. aureus, Listeria monocytogenes, Salmonella enterica Typhimurium, and E. coli in raw minced chicken⁴³. Similarly, linalool applied in a plum-sour-based marinade reduced total viable counts and Pseudomonas spp. on chicken fillets⁴⁴.

Eugenol is a phenolic compound obtained from various plant sources, including nutmeg oil, cinnamon extract, and clove oil⁴⁵. It exhibits multiple pharmacological effects, such as anesthetic, antioxidant, antimicrobial, anti-inflammatory, anticancer, and antidiabetic activities⁴⁵. In food studies, eugenol inhibited S. aureus in fresh beef⁴⁶, reduced Campylobacter jejuni in raw chicken breast⁴⁷, and when incorporated into yogurt- or pickle-based marinades, decreased L. monocytogenes, Salmonella spp., E. coli O157, total aerobic mesophilic bacteria, lactic acid bacteria, and Pseudomonas spp. in meat products⁴⁸.

α-Pinene is a monoterpene compound that is found abundantly in conifers such as junipers and pines⁴⁹. It has been employed for centuries in respiratory tract infections therapy and serves an important role in the fragrance and flavor industries. Its biological activities have been widely investigated, including its antibacterial, antifungal, anti-inflammatory, antioxidant, and neuroprotective effects⁴⁹. Nevertheless, research regarding the application of α-pinene in food matrices is limited. In broiler chickens, (−)-α-pinene showed minimal effect against C. jejuni when given in drinking water, but a controlled oral dose via gavage significantly reduced fluoroquinolone-susceptible C. jejuni, with no effect on resistant strains⁵⁰.

α-Terpinene is a monoterpene present in the EOs of many foods and aromatic plants, including Mentha piperita⁵¹, Melaleuca alternifolia⁵² and oregano⁵³. α-Terpinene acts as a genuine antioxidant because it undergoes rapid autoxidation, which helps protect other compounds from degradation⁵⁴. To date, no studies have reported the antimicrobial efficacy of α-terpinene in food-matrix systems, highlighting a notable gap in its evaluation within meat preservation applications.

However, studies specifically evaluating the antimicrobial effects of linalool, α-pinene, α-terpinene, and eugenol against L. monocytogenes in camel meat are lacking. Although eugenol has been examined in marinated camel meat for its activity against L. monocytogenes²⁴, the individual effects and synergistic potential of these natural compounds in combination remain unexplored in minced meat.

Therefore, this study aims to (i) compare the antimicrobial activity of individual bioactive compounds (linalool, α-pinene, α-terpinene, and eugenol) against L. monocytogenes, (ii) identify and characterize synergistic combinations of these compounds that enhance anti-Listeria activity relative to their individual effect; and (iii) evaluate and compare the efficacy of the 4 individual compounds to their synergistic combinations in controlling L. monocytogenes in minced camel during refrigerated storage.

Materials and methods

Preparation of EO bioactive compounds

The EO bioactive compounds employed in this study include linalool (≥ 97%, CAS number 78-70-6)), α-pinene (≥ 98%, CAS number 7785-26-4), α-terpinene (≥ 89%, CAS number 99-86-5), and eugenol (≥ 98%, CAS number 97-53-0), all sourced from Sigma-Aldrich, France. Dimethyl sulfoxide (DMSO; was also sourced from Sigma-Aldrich, France.

Bacterial strain preparation

The bacterial strain used was L. monocytogenes ATCC 7644, acquired from the Ministry of Public Health (MOPH) in Doha, Qatar. This strain was preserved in micro-vials (Microbank^® microbial storage, Pro-Lab Diagnostics, Richmond Hill, ON, Canada) at −80 °C. Prior to experimental use, the bacteria were pre-cultured by inoculating 100 µL of the frozen culture into 5 mL of Tryptic Soy Broth (TSB; Liofilchem^®, Roseto degli Abruzzi, Italy) and incubating for 24 h at 37 °C. Subsequently, 100 µL of this pre-culture was transferred to 50 mL of TSB in sterile 500 mL flasks, where it was incubated for 16 h at 37 °C under shaking conditions at 190 rpm to prepare the bacterial culture. Following incubation, the cells were harvested by centrifugation at 5000 × g for 5 min. The bacterial pellet was washed twice with 20 mL of phosphate-buffered saline (PBS; Atom Scientific, England) and then re-suspended in PBS to create a stock suspension. This stock was adjusted to an optical density at 600 nm (OD₆₀₀) corresponding to ~ 10⁸ CFU/mL. From this stock, the inoculum was diluted to a final concentration of ~ 10⁶ CFU/mL using Mueller-Hinton Broth (MHB; VWR Chemicals, Leuven, Belgium) for MIC and checkerboard assays, and in Buffered Peptone Water (BPW; Liofilchem, Via Scozia, Italy) to ~ 10⁷ CFU/mL for the meat challenge study.

Minimum inhibitory concentration (MIC)

The MIC values of the four EO bioactive compounds (linalool, α-pinene, α-terpinene, and eugenol) were assessed against the L. monocytogenes ATCC 7644 strain using Mueller-Hinton Broth (MHB; VWR Chemicals, Leuven, Belgium)⁵⁵. This evaluation was performed using a microdilution growth inhibition assay facilitated by a Biotek Synergy LX Multi-Mode Reader (Biotek, USA). In brief, 100 µL of double serial dilutions of EO bioactive compounds (ranging from 40 to 0.156 mg/mL) were added to 96-well microdilution plates. Subsequently, 100 µL of bacterial strain (10⁶ CFU/mL) was introduced to the wells. Negative control wells contained no bacteria, while positive control wells included DMSO at a final concentration of 2% without any antimicrobial agents. The plates were incubated at 37 °C with continuous shaking, and optical density (OD) was recorded at 600 nm. The MIC was determined as the lowest concentration of the antimicrobial agent that effectively inhibited visible bacterial growth in the microdilution wells after incubation.

Checkerboard assay

The checkerboard assay was utilized to investigate the interactions and combined effects of each pair of the four compounds (linalool, α-pinene, α-terpinene, and eugenol), against the L. monocytogenes ATCC 7644 strain. This method involved the use of 96-well microtitre plates, as previously described⁵⁶ to determine the Fractional Inhibitory Concentration (FIC) index. Serial dilutions of each compound were prepared as described above in a range of concentrations to include the MIC values for each. The arrangement of the microplate assay involved diluting EO bioactive compound A two-fold along the x-axis and EO bioactive compound B two-fold along the y-axis. Each well contained a final volume of 200 µL, consisting of 100 µL of each EO bioactive compound dilution. Following this, 100 µL of media containing 10⁶ CFU/mL of the L. monocytogenes ATCC 7644 strain was added to all wells. The plates were incubated at 37 °C for 18 h, after which the optical density (OD) of each well was measured at 620 nm to assess the interactions between the compounds. The FIC indices were calculated as the sum of FIC A and FIC B, where FIC A and FIC B represent the minimum concentrations that inhibited bacterial growth for EO bioactive compounds A and B, respectively. The formulas used were:

Results were categorized into synergy (FIC ≤ 0.5), additive (0.5 < FIC ≤ 1), indifference (1 < FIC ≤ 4), or antagonism (FIC > 4)^57–60. All experiments were conducted in triplicate.

Camel meat preparation

Fresh camel meat from the shoulder of two-year-old animals (pH 6.41) was sourced from a local slaughterhouse company (Widam Food Company, Doha, Qatar). A single homogeneous batch was used for all experiments and the meat was minced using cleaned and sanitized equipment to ensure strict hygienic conditions and was subsequently packed in food grade polyethylene bags to be transported to the Research Complex at Qatar University within two hours of slaughter. To ensure proper refrigeration during transport, the meat was placed in an icebox filled with ice. Once at the research facility, the minced meat was stored in a refrigerator.

Before the experiment, the camel meat was screened for L. monocytogenes using the standard ISO 11290-1⁶¹ protocol. All tested samples were confirmed free of the pathogen.

Preparation of treatments and samples

The minced camel meat samples were inoculated with the prepared suspension of L. monocytogenes to achieve a final inoculum dose of approximately 5 log CFU/g (1 mL of bacterial suspension of 10⁷ CFU/ml for every 100 g of meat). Initially, the inoculated samples were hand-massaged for 1 min and then thoroughly mixed using a Stomacher (Stomacher^® 400 Circulator, Seward, England) for 2 min to ensure even distribution of the pathogenic bacteria^20,62,63. The inoculated meat was subsequently divided into several groups for various treatments. EO bioactive compounds were applied either individually or in combination, and the treated samples were again processed in the stomacher for an additional 2 min to ensure uniform distribution of the EO bioactive compounds^20,63,64. Based on the in vitro checkerboard assay, which identified combinations exhibiting synergistic antimicrobial activity against L. monocytogenes, four combinations of EO bioactive compounds were selected for application in minced camel meat. This screening step was used to reduce the number of experimental combinations and to focus on those with the highest antimicrobial potential, rather than evaluating all possible mixtures. Twelve antimicrobial treatments of the inoculated meat were utilized in this study: Linalool 1% (w/w) (L1), α-Pinene 1% (w/w) (P1), α-Terpinene 1% (w/w) (T1), Eugenol 1% (w/w) (E1), Linalool 0.5% (w/w) + α-Pinene 0.5% (w/w) (LP1), Linalool 0.5% (w/w) + Eugenol 0.5% (w/w) (LE1), Eugenol 0.5% (w/w) + α-Pinene 0.5% (w/w) (EP1), Eugenol 0.5% (w/w) + α-Terpinene 0.5% (w/w) (ET1), Linalool 1% (w/w) + α-Pinene 1% (w/w) (LP2), Linalool 1% (w/w) + Eugenol 1% (w/w) (LE2), Eugenol 1% (w/w) + α-Pinene 1% (w/w) (EP2), and Eugenol 1% (w/w) + α-Terpinene 1% (w/w) (ET2). To allow meaningful comparison between individual compounds and their combinations, concentrations were standardized across treatments. The selected concentrations (1% and 2% w/w) were based on preliminary trials conducted to ensure observable antimicrobial effects in the meat matrix, as well as commonly reported concentration ranges in the literature for food applications^24,65,66. These levels were also selected to remain within practical limits, as higher concentrations of essential oil components are often associated with undesirable sensory effects^36,67.

Samples inoculated with the bacterial strain without any antimicrobial served as the control. Following treatment, the samples were stored in sterile containers and analyzed in duplicate at 0, 3, 6, 9, and 12-days during storage at 4 °C.

Microbial enumeration

At each testing day, individual samples (10 g) were aseptically placed into sterile stomacher bags, to which 90 mL of buffered peptone water was added. The samples were then homogenized using a Stomacher for 2 min. Following homogenization, 0.1 mL of the appropriate decimal dilutions were plated in duplicate onto Listeria Ottaviani and Agosti selective agar plates (Neogen^® Culture Media, Harlequin^®, US). The plates were incubated at 37 °C for 24 h. Colonies within the range of 25 to 250 CFU were enumerated using a colony counter (Stuart Scientific, UK), and the data were converted to log CFU/g.

Statistical analysis

All treatments were conducted in duplicates, with two replicates for microbiological testing. The results’ statistical significance was analyzed using GraphPad Prism 10.0 software (GraphPad Software, San Diego, CA, USA). For the 12-day study on the antimicrobial treatment of camel samples, one-way ANOVA was utilized, followed by Tukey’s multiple comparison tests, to determine differences in mean log CFU/g among treatment groups on each day and to compare means across different treatments throughout the study period. A p-value of less than 0.05 was considered statistically significant.

Results and discussion

Antibacterial activity of individual active EO bioactive compounds in vitro

The antimicrobial activity of EO bioactive compounds largely depend on their chemical structure and the susceptibility of the target microorganism. In our study, four EO bioactive compounds eugenol, linalool, α-pinene, and α-terpinene were evaluated against L. monocytogenes. The MIC results indicated that eugenol exhibited the strongest antibacterial effect (1.25 mg/mL), followed by linalool (5 mg/mL), while α-pinene and α-terpinene showed substantially lower activity (40 mg/mL each) (Table 1). These findings confirm that phenolic compounds, such as eugenol, generally possess strong antimicrobial activity against L. monocytogenes, consistent with previously reported results⁶⁸.

Table 1.

Minimum inhibitory concentration (MIC) and fractional inhibitory concentration index (FICI) of eugenol, linalool, α-terpinene, and α-pinene against L. monocytogenes.

Combinations	Active compounds	MIC1 (mg/mL)		FIC2	FICI3	Results
		Alone	Combined
Eugenol + α-terpinene	Eugenol α-terpinene	1.25 40	0.31 1.25	0.25 0.03	0.28	Synergistic
Eugenol + α-pinene	Eugenol α-pinene	1.25 40	0.16 5	0.13 0.13	0.25	Synergistic
α-pinene + α-terpinene	α-pinene α-terpinene	40 40	20 20	0.50 0.50	1.00	Indifferent
Linalool + α-pinene	Linalool α-pinene	5 40	0.31 0.63	0.06 0.02	0.08	Synergistic
Linalool + α-terpinene	Linalool α-terpinene	5 40	2.5 2.5	0.50 0.06	0.56	Additive
Linalool + Eugenol	Linalool Eugenol	5 1.25	0.63 0.08	0.13 0.06	0.19	Synergistic

¹MIC: Minimum inhibitory concentration (mg/mL).

²FIC: Fractional inhibitory concentration, calculated as the MIC of each compound in combination divided by its MIC when tested alone¹⁰⁸.

³FICI: Fractional inhibitory concentration index, obtained as the sum of the FIC values of the two combined compounds (FICI = FIC of the first bioactive compound + FIC of the second bioactive compound)¹⁰⁸.

Interpretation of FICI values: Synergistic (FICI ≤ 0.5), additive (0.5 < FICI ≤ 1.0), indifferent (1.0 < FICI ≤ 4.0), and antagonistic (FICI > 4.0)^57–60.

The high effectiveness of eugenol observed in this study aligns with numerous reports describing its potent antimicrobial activity against a variety of pathogens. Qian et al. (2019) reported a MIC of 0.2 mg/mL against carbapenem-resistant Klebsiella pneumoniae, while Devi et al. (2010) found that eugenol completely inhibited Salmonella typhi growth within 60 min at 0.0125% (0.125 mg/mL)^69,70. Against L. monocytogenes, eugenol MICs ranging from 0.625 mg/mL to 1.28 mg/mL have been reported⁷¹, which aligns with our findings. Interestingly, clove EO, which is rich in eugenol, has demonstrated MICs as low as 0.05 mg/mL against L. monocytogenes⁷². Linalool showed moderate antibacterial activity in our study, with a MIC of 5 mg/mL. Comparable findings by several studies have confirmed the antibacterial activity of linalool against L. monocytogenes, reporting MIC values of 1.29 mg/mL⁷³, 4.3 mg/mL⁷⁴, and 10.75 mg/mL⁷⁵. Furthermore, another study reported a MIC value of 1.25 µL/mL (1.07 mg/mL) against P. fluorescens⁷⁵.

In contrast, α-pinene and α-terpinene exhibited the weakest inhibitory effects against L. monocytogenes (MIC = 40 mg/mL). To the best of our knowledge, only two previous research studies reporting MIC values of α-pinene against L. monocytogenes were identified. Previous studies have shown that α-pinene exhibits strong anti-L. monocytogenes activity, with MIC values reported at 0.019% (0.19 mg/mL)⁷⁶ and as low as 0.1 µL/100 mL (0.00086 mg/mL)⁷⁷. While Hartman and colleagues observed a MIC of 124.95 mg/mL for (−)-α-pinene against methicillin-resistant S. aureus⁷⁸. Another study also demonstrated α-pinene activity against common foodborne bacteria, with MICs ranging from 0.42 to 0.686 mg/mL against E. coli, S. enterica, and S. aureus⁷⁹. Such variations in MICs likely reflect differences in bacterial strains, growth media, and testing methodologies⁸⁰.

Moreover, no previous studies have specifically examined the antibacterial activity of α-terpinene against L. monocytogenes. Previous studies have nevertheless documented its activity against other bacteria. For instance, α-terpinene inhibited C. jejuni at concentrations of 0.125–0.25% (1.25–2.5 mg/mL)⁸¹, while MIC values of 2.5–5 µL/mL (2.1–4.2 mg/mL) were reported against Gardnerella species⁸².

The variation in antimicrobial activity among the tested compounds is closely linked to their chemical structures and mechanisms of action. Eugenol, the most potent compound (MIC = 1.25 mg/mL), owes its strong antibacterial activity to its free hydroxyl group, which enables binding to membrane proteins and interference with enzymatic activity, leading to membrane permeabilization and ion leakage^35,83–87. It also alters fatty acid composition and generates reactive oxygen species that disrupt cell integrity and function^88,89. These multiple targets explain eugenol’s superior efficacy compared with other compounds. Linalool (MIC = 5 mg/mL), an oxygenated monoterpene alcohol, acts mainly by disturbing membrane integrity and reducing membrane potential, causing leakage of intracellular contents and metabolic inhibition^90–93.

α-Pinene (MIC = 40 mg/mL) showed weaker activity probably due to its non-oxygenated hydrocarbon nature, which limits polarity and interaction with membrane components^79,94. Its mechanism involves inhibition of the DnaKJE–ClpB bichaperone system, a key protein quality-control machinery responsible for assisting proper protein folding and refolding of stress-denatured proteins, thereby interfering with protein folding and partially disrupting proteostasis^79,94. The specific mode of action of α-terpinene is less explored; however, as a monoterpene hydrocarbon, it can disrupt the microbial cytoplasmic membrane, reducing its impermeability to protons and larger ions; once membrane integrity is disturbed, its essential functions as a barrier, enzymatic matrix, and energy transducer are compromised^84,95–97.

Antibacterial activity of combined active EO bioactive compounds in vitro

Beyond their individual action, active compounds can interact with another compound in such a way that it causes synergistic, additive, indifferent, or antagonistic effects⁹⁸. Exploring synergistic combinations of EO bioactive compounds offers a promising approach to enhancing food preservation⁹⁸. The interaction analysis using the checkerboard assay revealed that combining EO bioactive compounds significantly enhanced antibacterial activity in several cases. As shown in Table 1, four combinations of compounds demonstrated synergistic effects (FIC ≤ 0.5), indicating a significant reduction in the effective MICs of both EO bioactive compounds. The lowest FICI value was observed for linalool and α-pinene (FICI = 0.08), followed by linalool and eugenol (FICI = 0.19), eugenol with α-pinene (FICI = 0.25), and eugenol with α-terpinene (FICI = 0.28). In contrast, α-pinene combined with α-terpinene exhibited an indifferent effect (FICI = 1.00), suggesting no significant enhancement of activity, whereas linalool with α-terpinene showed an additive effect (FICI = 0.56).

Similar synergistic patterns have also been reported previously. A study observed synergism between eugenol and linalool against L. monocytogenes and P. aeruginosa, as well as between eugenol and carvacrol⁹⁹. Moreover, it was found that eugenol combined with cinnamaldehyde enhanced antibacterial activity against E. coli¹⁰⁰. Similar to our findings, it was demonstrated that oxygenated monoterpenes such as linalool, eucalyptol, and camphor exhibited potent antibacterial and synergistic effects against L. monocytogenes⁴³. Another research supports that linalool enhances the antimicrobial efficacy of other EO bioactive compounds as it exhibited synergistic effect when combined with clove oil against P. aeruginosa and Aspergillus brasiliensis¹⁰¹. In our study, the combination of linalool and eugenol produced a low FICI value with a synergistic effect (FICI = 0.19), Table 1. Similarly, a study reported that combining a monoterpenoid phenol (eugenol) with a monoterpenoid alcohol (linalool or menthol) resulted in the highest synergistic activity, consistent with our findings for eugenol–linalool combinations⁹⁹. Another study also documented that α-pinene exhibited synergistic activity with eugenol, farnesol, and carvacrol, leading to a substantial reduction in its MIC from 124.95 mg/mL (when used alone) to 0.26, 0.87, and 0.96 mg/mL, respectively⁷⁸. Similarly, in our study, the combination of linalool and α-pinene produced a notable synergistic effect, reducing the MIC of α-pinene from 40 mg/mL (alone) to 0.63 mg/mL and resulting in the lowest FICI value (0.08) among all tested combinations (Table 1).

Non-synergistic outcomes have also been observed in previous research, where certain combinations of EO bioactive compounds either failed to enhance activity or produced antagonism. For example, eugenol in combination with cinnamaldehyde exhibited additive antimicrobial activity against L. monocytogenes and E. coli O157:H7¹⁰². Furthermore, the combination of linalool with clove oil showed an additive effect against S. aureus, E. coli, and Candida albicans, while linalool also additively enhanced the antimicrobial activity of thyme oil against P. aeruginosa¹⁰¹. However, an indifferent interaction between eugenol and α-pinene against C. albicans was found¹⁰³. Moreover, a study reported that the combination of eugenol with thymol and eugenol with geraniol showed indifferent effects against S. aureus and E. coli, while both combinations exhibited partial synergistic effects (0.50 < FIC ≤ 0.75) against Bacillus cereus¹⁰⁴. A study observed that combining citral with linalool in a nanoemulsion remarkably reduced antibacterial activity compared to citral alone, indicating an antagonistic interaction⁷⁵. Furthermore, the combination of carvacrol and eugenol was antagonistic against S. aureus, B. cereus, and E. coli¹⁰⁴.

In line with these observations, the current findings confirm that eugenol and linalool are the most potent individual inhibitors of L. monocytogenes, and the synergistic interactions observed for eugenol with α-terpinene and α-pinene, and for linalool with α-pinene and eugenol, can further enhance antibacterial performance. These interactions can be explained by the different chemical properties and mechanisms of the compounds involved. Synergistic effects often occur when one compound, such as α-pinene or α-terpinene, increases membrane permeability, facilitating the entry of more active molecules like eugenol that subsequently disrupt intracellular proteins and enzymatic activity¹⁰⁰. Moreover, eugenol or linalool tend to produce synergistic interactions, likely due to complementary mechanisms of action such as membrane permeabilization and protein denaturation¹⁰⁵. Additive effects, on the other hand, typically arise when compounds share similar chemical structures and mechanisms of action, resulting in a combined effect equal to the sum of their individual activities¹⁰⁶. In contrast, antagonistic or indifferent interactions may occur when compounds compete for the same target site or when non-polar hydrocarbons reduce the solubility and bioavailability of more active oxygenated compounds⁹⁸. Collectively, these findings reinforce that the type of interaction, whether synergistic, additive, or antagonistic, largely governed by molecular structure and compatibility among EO constituents, determining their overall antimicrobial efficacy^35,107.

Antibacterial activity of active EO bioactive compounds used alone or in combination in minced camel meat

The antimicrobial efficacy of individual and combined EO bioactive compound treatments against L. monocytogenes in minced camel meat was monitored over a 12-day storage period (Fig. 1, Table S1). Overall, individual L1 and E1 and all combination treatments, showed significantly lower bacterial counts compared to the control (p < 0.05). In contrast, the individual P1 and T1 treatments exhibited no significant differences from the control across all storage days (p > 0.05), confirming their limited or negligible antibacterial activity in the meat matrix, consistent with their weak performance observed in culture MIC assays.

Fig. 1.

Effects of individual and combined treatments of linalool, eugenol, α-pinene, and α-terpinene on L. monocytogenes count (log CFU/g ± standard deviation) in minced camel meat stored at 4 °C for 0, 3, 6, 9, and 12 days. L1, Linalool 1%; P1, α-Pinene 1%; T1, α-Terpinene 1%; E1, Eugenol 1%; LP1, Linalool 0.5% + α-Pinene 0.5%; LE1, Linalool 0.5% + Eugenol 0.5%; EP1, Eugenol 0.5% + α-Pinene 0.5%; ET1, Eugenol 0.5% + α-Terpinene 0.5%; LP2, Linalool 1% + α-Pinene 1%; LE2, Linalool 1% + Eugenol 1%; EP2, Eugenol 1% + α-Pinene 1%; ET2, Eugenol 1% + α-Terpinene 1%. The horizontal dashed line indicates the method’s detection limit (2 Log CFU/g).In this study, distinct differences were observed in the antimicrobial efficacy of individual and combined EO bioactive compounds against L. monocytogenes in a minced camel meat matrix. Neither α-terpinene nor α-pinene, when applied individually, produced a significant reduction in L. monocytogenes counts in minced camel meat, and their bacterial levels increased over time in a pattern similar to the control. This indicates that both compounds have limited antibacterial activity in this food matrix.

Bactericidal effects were detectable from day 0. The 2% combination treatments with E1 achieved the strongest impact, reducing counts by 2.5–2.8 log CFU/g (p < 0.05), followed by lower reductions of 0.5–1.6 log CFU/g for the 1% combinations and L1. In contrast, the P1 and T1 treatments did not show significant log reduction from the control (p > 0.05).

By day 3, all four 2% combination treatments completely inhibited L. monocytogenes and maintained undetectable counts for the remainder of storage (counts under the detection limit 2 log CFU/g). It is important to note that the improved antimicrobial efficacy observed at 2% reflects both increased concentration and combinatory effects. Therefore, the enhanced performance of combination treatments at higher concentrations cannot be attributed solely to synergistic interactions. The identification of synergistic combinations in this study provides a foundation for future work aimed at optimizing these formulations at lower concentrations, with the goal of reducing potential sensory impacts while maintaining antimicrobial efficacy.

The LP1, EP1, ET1, and the individual L1 treatment all achieved comparable reductions of 1.9–2.3 log CFU/g, as compared to the control (p < 0.05) at days 3 and 6. The LE1 combination treatment performed better, with a 2.8 log reduction, while E1 achieved higher pronounced effect at day 3 with a 3.7 log reduction. However, by day 6, both E1 and LE1 achieved 3.7 log reductions, with no significant difference among those 2 treatments (p > 0.05).

By Days 9 and 12, LP1 recorded the lowest log reductions of 1.3–1.4 log CFU/g, compared to other treatments (p < 0.05), although Listeria counts remained significantly lower than the control (p < 0.05). This was followed by EP1 and ET1 combinations and individual L1 treatment that demonstrated similar reductions of approximately 2.3–2.7 log CFU/g, with no significant differences among them (p > 0.05). Consistent with earlier observations, E1 maintained significantly stronger activity than the former treatments (p < 0.05), achieving 3.8–3.9 log reductions (compared to control). However, its effect at those later storage days became significantly lower than that of LE1 (p < 0.05), which outperformed all treatments and achieved complete inhibition, similar to the four 2% combinations.

Across all storage days, the control, P1 and T1 treatments exhibited significant microbial growth over time (p < 0.05). In contrast, L1, E1, and the three 1% combinations (LP1, EP1, ET1) maintained the initial reductions achieved at day 0, with no significant changes at the end of the storage (p > 0.05). The LE1 and all 2% combinations produced significant decreases in L. monocytogenes counts during storage (p < 0.05). Despite both E1 and LE1 reaching the same log-reduction value by day 6, E1 maintained a stable bacterial count throughout storage, whereas LE1 exhibited a significant gradual decrease across all storage days (p < 0.05).

In summary, these findings indicate that the 2% active EO bioactive compound combinations were the most effective treatments, achieving complete inhibition of L. monocytogenes throughout storage. Among the individual compounds, eugenol showed the strongest antibacterial activity, while its 1% combination with linalool also produced a strong and sustained inhibitory effect—matching the performance of the four 2% combinations at days 9 and 12. Additionally, the two combinations containing eugenol at 1% performed comparably to linalool alone and surpassed the linalool–α-pinene 1% combination at days 9 and 12. In contrast, α-pinene (P1) and α-terpinene (T1) exhibited negligible antimicrobial activity across all storage days.

Literature also supports the limited antimicrobial potential of α-terpinene, as no studies have evaluated it in food systems and previous reports describe generally weak activity^109[,110. Similarly, α-pinene has demonstrated poor direct antibacterial efficacy. For example, (-)-α-pinene showed only a slight and non-significant reduction of C. jejuni when administered via drinking water in broiler chickens, whereas a significant reduction of 0.8 log10 CFU/g was achieved only when it was delivered by direct oral gavage, and only against fluoroquinolone-susceptible strains⁵⁰. This highlights that the antimicrobial effect of α-pinene is highly dependent on both the route of administration and the target organism. Accordingly, direct application of α-pinene to camel meat did not decrease L. monocytogenes counts, and bacterial numbers increased similarly to the untreated control, suggesting that its activity observed in animal models does not necessarily translate to food matrices. In contrast, all binary combinations containing α-terpinene or α-pinene produced significant microbial inhibition in camel meat. This shift from minimal individual activity to strong combination effects suggests that these compounds may contribute primarily through synergistic interactions rather than acting as effective antimicrobial agents on their own.

Among the individual compounds, linalool and eugenol showed significant antimicrobial effects. Linalool has previously been reported to exhibit moderate bactericidal activity in a variety of food systems: it achieved at 2×MIC (3.0 mg/mL) a 4.33 log CFU/cm² reduction of S. sonnei on lettuce within 30 min⁴⁰, and produced 2.07 log reduction at 2×MIC in fresh beef after 9 days at 4 °C⁴¹. Similarly, linalool (0.15%) combined with plum-sour-based marination of chicken breast led to reductions of ~ 2.183 log CFU/g in total viable count and 1.967 log CFU/g in Pseudomonas spp. on day 9 at 4 °C⁴⁴. These values are comparable to the moderate reductions observed for linalool alone in our camel meat model, confirming that linalool can exert appreciable, yet seldom complete, antimicrobial effects when used individually in real food systems.

Eugenol treatment produced the greatest antibacterial activity among the individual compounds in our study, achieving up to 3.88 log CFU/g reduction of L. monocytogenes in minced camel meat. This is consistent with previous work showing strong anti-Listeria activity of eugenol and related phenolics in food matrices. For example, eugenol has produced reductions of approximately 2–3 log CFU/g in fresh beef against S. aureus after 7 days at 25 °C⁴⁶, and dose-dependent reductions up to 4.5 log CFU/g in chicken breast contaminated with C. jejuni⁴⁷. In camel meat specifically, eugenol 1% in a yogurt-based marinade reduced L. monocytogenes by 1.9 log CFU/g at 10 °C²⁴.

All 2% combinations (LP2, LE2, EP2, ET2) produced complete inhibition of L. monocytogenes beginning on day 3 and maintained suppression throughout refrigerated storage, while the LE1 treatment also exhibited a progressive inhibitory effect, ultimately achieving full suppression at later storage times. This pattern, where synergistic combinations provide stronger and more sustained control than single compounds, is consistent with previous research that first screened for synergy in vitro and then applied selected combinations in food systems. For instance, benzyl isothiocyanate (BITC) and resveratrol showed synergy against L. monocytogenes in checkerboard assays and, when applied to chicken meat at ½ MIC (0.00093% BITC + 0.01997% resveratrol), achieved a 1.08 log CFU/g reduction after 72 h at 25 °C, whereas neither compound alone at the same level produced a significant effect (p > 0.05)¹¹¹.

Previous research on camel meat preservation consistently shows that natural plant-based additives can significantly reduce spoilage and pathogenic microorganisms. For example, Mentha spicata EO at 0.5–1.5% lowered microbial counts by 1–4 log CFU/g over 12 days at 4 °C, improving shelf life²⁰. These reductions are similar to most treatments in our study but remain lower than the complete inhibition of L. monocytogenes achieved by LE1 and all 2% EO combinations. This total suppression underscores the strong antibacterial potential of optimized active EO blends in meat preservation. Comparable findings were reported with garlic segments (15–20%), which fully prevented total aerobic counts growth under various conditions¹⁶. Other studies using EO-based marinades achieved reductions of 0.9–4.8 log CFU/g for pathogens and spoilage organisms^24[,25. Similarly, Spirulina and olive leaf extract–nisin interventions reduced psychrotrophs by up to 3.2 log CFU/g^23,112. Conventional chemical preservatives like citric acid, sodium lactate, and chitosan improved shelf life but mainly acted through pH reduction and did not fully suppress pathogens^113[,114. Advanced delivery systems such as nanocomposite films and encapsulation enhanced EO activity, achieving 1–4 log CFU/g reductions^115[,116. Moreover, in vacuum-packed camel meat slices, combining 1% or 2% Citrox with 1% chitosan produced reductions of 3.5–4.0 log CFU/g, exceeding the effect of Citrox alone¹¹⁷. While this study used different natural compounds and targeted pathogenic microorganisms, they collectively reinforce the principle that synergistic interactions between natural antimicrobials can enhance efficacy within camel meat matrices, a trend also evident in the present study. Collectively, these comparisons highlight that rationally designed combinations of EO bioactive compounds can outperform traditional and advanced preservation strategies, offering strong and sometimes total pathogen inhibition under simple refrigerated storage.

Our study demonstrated that four active EO bioactive compounds combinations exhibited synergistic effects in vitro; however, their performance in food matrices varied at the lower concentration of 1%. The strongest effect was observed for linalool–eugenol combinations, followed by two blends containing eugenol with either α-pinene or α-terpinene, while the weakest performance was recorded for linalool–α-pinene. Interestingly, previous research reported contrasting interactions between linalool and eugenol, as well as discrepancies between in vitro results and food applications. In vacuum-packed beef marinated with a pickle-based system, linalool or eugenol alone (0.2%) reduced total aerobic mesophilic bacteria by 1.56 and 1.46 log CFU/g, Pseudomonas spp. by 1.30 and 1.08 log CFU/g, and lactic acid bacteria by 0.32 and 0.36 log CFU/g, respectively. Surprisingly, their combination (1:1 ratio) was less effective, achieving only 1.03 log CFU/g reduction for mesophilic bacteria, 1.08 log CFU/g for Pseudomonas spp., and 0.28 log CFU/g for lactic acid bacteria, consistently lower than individual treatments⁴⁸. The fact that linalool and eugenol combinations in our study produced stronger inhibition than either compound alone, may be explained by differences in the type of target microorganisms and food matrices. Furthermore, several studies have reported that antimicrobial activity of EO bioactive compounds is often reduced in food applications compared to in vitro conditions. This reduction is largely attributed to interactions with food components such as fats, proteins, and carbohydrates, which can bind EO bioactive compounds and limit their availability, thereby diminishing their ability to disrupt bacterial cells^{35[,118–120[,121}. Additionally, the higher nutrient content in food systems, compared to laboratory media, facilitates faster recovery and growth of bacterial populations^122[,123. For example, oregano and thyme, despite exhibiting strong in-vitro inhibition, achieved only modest reductions in minced pork, approximately 0.22–0.51 log CFU/g, even at MIC and 2× MIC levels¹²⁴. Similarly, EOs such as thyme and garlic, which were highly inhibitory to L. monocytogenes in broth assays, showed minimal or no antimicrobial effect in cooked ham, even at concentrations that were strongly effective in vitro¹²⁵. These discrepancies highlight that reduced synergy in food systems may result from both differences in target microorganisms and stronger EO bioactive compounds –matrix interactions. In complex food matrices, these interactions can limit antimicrobial activity, as lipids may sequester hydrophobic compounds and proteins may bind active components, reducing their availability to act against microorganisms^126[,127. Overall, leveraging synergistic interactions among EO bioactive compounds in real food matrices offers a promising strategy to achieve high microbial control at lower concentrations, which can reduce formulation costs and minimize sensory impact.

While this study provides clear evidence of the antimicrobial potential of individual and combined EO bioactive components against L. monocytogenes ATCC 7644, further validation using multiple strains, particularly diverse clinical and food isolates, is essential. Such work would help account for the well-documented strain-dependent variability in antimicrobial susceptibility and strengthen the generalizability of the findings across real-world food systems and broader pathogen targets. It is important to consider that the concentrations required to achieve complete inhibition in this study may raise challenges related to sensory acceptability, regulatory limits, and industrial feasibility. However, the strong synergistic interactions observed, particularly in combinations containing eugenol and linalool, demonstrate that effective antimicrobial activity can be achieved at reduced concentrations compared to individual compounds. In addition, because this study employed relatively high inoculum levels typical of challenge‑test designs, and natural contamination levels in real processing environments are expected to be substantially lower, it is likely that lower EO concentrations would be sufficient under practical conditions. This further supports the relevance of combination strategies to mitigate the limitations associated with high EO levels while maintaining efficacy. Nevertheless, further studies are required to evaluate sensory impact and regulatory compliance under practical application conditions.

Conclusion

This study provides a comprehensive evaluation of the antimicrobial activity of linalool, eugenol, α-pinene, and α-terpinene, individually and in combination, against L. monocytogenes in minced camel meat. Among the tested compounds, eugenol and linalool showed the strongest individual effects, while several binary combinations demonstrated clear synergistic interactions in vitro and enhanced antimicrobial efficacy in the meat system. These findings highlight the potential of synergistic combinations to improve antimicrobial performance and reduce the concentrations required compared to individual compounds. However, the translation of in vitro synergy to food systems was influenced by matrix-related factors, emphasizing the need for validation under real food conditions.

While the results demonstrate promising antimicrobial potential, their practical application should be interpreted with caution, as sensory properties, regulatory limits, and industrial feasibility were not assessed in this study. Future research should therefore evaluate these aspects against spoilage microorganisms and further investigate the effectiveness of these combinations across a wider range of L. monocytogenes strains, other foodborne microorganisms, and different food matrices and processing conditions. Future studies should also incorporate higher numbers of biological replicates as part of more detailed validation efforts. Integration with complementary preservation strategies may also help optimize efficacy while maintaining product quality.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

N.A-D. and S.D. contributed to writing the original draft of the manuscript and performed the results analysis. S.M. contributed to the study design, methodology, data interpretation, and manuscript review and editing. L.K. supervised the study design, methodology, data interpretation, contributed to writing the original draft, review and editing of the manuscript. All authors read and approved the final manuscript.

Funding

This research was supported by Qatar University Collaborative grant QUCG-CHS-25/26–677 and Postdoc Grant 1438. Open Access funding provided by the Qatar National Library.

Data availability

All relevant data are included within the manuscript and its supplementary materials.

Declarations

Competing interests

The authors declare no competing interests.