Synergistic antimicrobial interaction of plant essential oils and extracts against foodborne pathogens

Abstract Essential oils (EOs) and plant extracts have demonstrated inhibitory activity against a wide range of pathogenic bacteria. In this study, the chemical composition of manuka, kanuka, peppermint, thyme, lavender, and feijoa leaf and peel EOs and feijoa peel and leaf extracts were analyzed, and their antimicrobial activity against Escherichia coli, Salmonella enterica Typhimurium, Staphylococcus aureus, Bacillus cereus, and Listeria monocytogenes were determined. The results showed that the major compounds varied among different EOs and extracts, with menthol in peppermint EO, thymol and carvacrol in thyme EO, linalool in lavender EO, β‐caryophyllene in feijoa EO, and flavones in feijoa extract being the most prevalent. The study found that while EOs/extracts had antimicrobial activity alone, no individual EO/extract was highly effective against all tested species. Therefore, their combinations were tested to identify those that could broaden the spectrum of activity and act synergistically. The checkerboard method was applied to assess the possible synergism between the paired combinations of EOs/extract. The peppermint/thyme, peppermint/lavender, and peppermint/feijoa peel extract combinations exhibited a synergistic effect against E. coli and L. monocytogenes, with the peppermint/thyme and peppermint/feijoa peel extract combinations being the most effective against all five pathogens. Time‐to‐kill kinetics assays demonstrated that peppermint/thyme and peppermint/feijoa peel extract combinations achieved complete eradication of E. coli within 10–30 min and L. monocytogenes within 4–6 h. This study provides a promising approach to developing a natural alternative for food preservation using synergistic combinations of EOs/extracts, which could potentially reduce the required dosage and broaden their application in food products as natural preservatives.


| INTRODUC TI ON
Foodborne illness is a significant public health issue worldwide, with an estimated 600 million cases reported annually, resulting in 420,000 deaths globally each year (WHO, 2022).In recent years, foodborne diseases have been associated with the emergence of antibacterial-resistant pathogens, posing an added challenge and paving the way for humankind to enter into an anticipated postantibiotic era (Soulaimani et al., 2021).Resistant foodborne pathogens have been frequently detected in the food chain.They are most likely to enter the food chain during processing, packaging, and distribution, causing food quality degradation and financial loss to the food industry (El amrani et al., 2021).Previous studies have reported the presence of foodborne pathogens, like Escherichia coli, Salmonella spp., Listeria monocytogenes, Staphylococcus aureus, and Bacillus cereus directly in food or from the food processing environment (Ayari et al., 2020;Karagozlu et al., 2011).
In the food industry, eradication of foodborne pathogens to ensure food safety is achieved mainly by using chemical preservatives, for example, chlorine-based preservatives, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), and nitrates and nitrites (Santos et al., 2017).However, chemical preservatives have been associated with several side effects, such as nausea, headache, weakness, seizures, mental retardation, and anorexia (Bag & Chattopadhyay, 2015).
They are also known for their carcinogenic effects, posing a significant risk to human health (Olmez & Kretzschmar, 2009).Increased awareness of the side effects related to the use of chemical preservatives in food has resulted in an outcry of consumers to develop natural antimicrobial preservatives as an alternative.
Natural substances such as plant essential oils (EOs) and their extracts comprise an extensive reservoir of antimicrobial compounds, which could offer an alternative solution to combat the growth of foodborne pathogens (Soulaimani et al., 2021).Researchers have extensively studied the antimicrobial activity of individual EOs and their plant extracts against various pathogens (Ez zoubi et al., 2020;Qian et al., 2020;Raj et al., 2020;Singh et al., 2018;Yang et al., 2021).
However, a closer look at the literature reveals that a single EO or extract requires higher concentrations to accomplish similar effects in situ compared to those established in vitro (Angane et al., 2022;Bassole & Juliani, 2012;Burt, 2004).It is noted that there are disadvantages to this approach, such as negative organoleptic perception (alteration in food's taste and aroma) when higher concentrations of EOs/extracts are present in the food matrix (Khaleque et al., 2016).
Another disadvantage is that EOs/extracts have demonstrated strong antimicrobial potential mainly against gram-positive bacteria, but exert moderate-to-weak antimicrobial activity against gram-negative bacteria (Deng et al., 2020;Soulaimani et al., 2021).
A solution to these disadvantages is to combine EOs/extracts using a synergistic approach to inhibit foodborne pathogens (Milagres de Almeida et al., 2023).The advantage of using this approach is that combinations of EOs/extracts will reduce the required total concentration of EOs/extracts, thereby minimizing the undesirable aromas and flavors that they may individually impart to the food (Kim et al., 2021).It will also give a broad spectrum of antimicrobial activity against gram-positive and gram-negative bacteria and increase their efficacy in complex food matrices (Cava-Roda et al., 2021).
Recent research has reported the improved antimicrobial potential of various EO/extract combinations applied at lower doses.For example, Soulaimani et al. (2021) demonstrated synergistic and additive effects of combining lavender, thyme, and rosemary EO against gram-negative bacteria.Similarly, Chaichi et al. (2021) reported a synergistic and additive activity of the combination of thyme, cinnamon, and clove EO against E. coli, St. aureus, and Pseudomonas fluorescens, and Cava-Roda et al. (2021) observed synergistic effects of vanillin/clove EOs and vanillin/cinnamon bark EOs against E. coli O157:H7 and L. monocytogenes.Also, the combination of cinnamon and thyme EOs was identified to have a synergistic anti-Listeria effect in organic tomato juice (Kim et al., 2021).The abovementioned studies demonstrated two-to fourfold reductions in the EO concentration when used in combination compared to single treatments.
In light of these studies, it is conceivable that using combinations of EOs/extracts is an effective approach to lowering the concentrations required to have a bacteriostatic or bactericidal effect.Hence, we seek to identify effective combinations of EOs and plant extracts that could exhibit a broad spectrum of activity against foodborne pathogens, utilizing locally sourced materials from New Zealand where possible.We assessed their effectiveness against gram-negative and gram-positive organisms as an initial measure of their spectrum.For this purpose, we selected Manuka and Kanuka EOs derived from native New Zealand plants with documented antimicrobial properties (Chen et al., 2016;Fratini et al., 2019;Mathew et al., 2020).To represent less explored plant species in New Zealand with potential antimicrobial applications, we included feijoa peel and leaf EOs and extracts.Additionally, we incorporated peppermint, thyme, and lavender EOs based on the existing literature (Erland & Mahmoud, 2016;Hejna et al., 2021;Lages et al., 2021) and preliminary screening, suggesting their potential activity against either gram-negative (peppermint EO) or gram-positive (thyme and lavender EO) bacteria.This research aims to evaluate the chemical composition and antibacterial activity of commercially available manuka (Leptospermum scoparium), kanuka (Kunzea ericoides), peppermint (Mentha piperita), thyme (Thymus vulgaris), lavender (Lavandula intermedia) Eos, and laboratory-derived feijoa (Acca sellowiana) EOs (leaf and peel) and their ethanol extract (leaf and peel) against five foodborne pathogens (E. coli, S. enterica Typhimurium, St. aureus, B. cereus, and L. monocytogenes).Some existing studies in the broader literature have examined the antimicrobial potential of manuka (Mathew et al., 2020), kanuka (Essien et al., 2019), peppermint (Camele et al., 2021;Hejna et al., 2021), thyme (Lages et al., 2021), lavender (Adaszyńska-Skwirzyńska et al., 2021), feijoa EO and its extracts (Santos et al., 2021;Smeriglio et al., 2019) individually, but the effect of combining these EOs as well as feijoa extracts has not been established.Feijoa is a subtropical fruit widely cultivated in many parts of the world, including Australia and New Zealand (Bell et al., 2018).Despite studies on its antimicrobial (Phan et al., 2019), antioxidant (Peng et al., 2019b), anticancer (Bontempo et al., 2007), and anti-inflammatory activities (Peng et al., 2018), there has been little discussion on its potential synergistic applications as a natural preservative.
Our primary goal was to identify a synergistic or additive combinations that are active against gram-negative and gram-positive bacteria.In this context, we evaluated the susceptibility of E. coli and L. monocytogenes to paired combinations of EOs/extracts to detect synergistic, additive, indifferent/no interaction, or antagonistic effects in vitro.Synergistic combinations were also investigated for bactericidal effects against St. aureus, B. cereus, and S. enterica Typhimurium to ensure broad-spectrum activity using the proposed combinations, and time-to-kill kinetics was performed to validate the efficacy of EO/extract combinations.The outcomes of this study are expected to benefit the food industry by bringing a new class of natural plant-based preservatives with a broad spectrum of antimicrobial activities.

| Sample collection and storage
Feijoa fruit and leaf samples were handpicked from regional orchards located in the Ngatea region of the North Island of New Zealand.The fruit was collected during the peak season from February to March 2021, and leaves were collected during the pruning season from May to June 2021.The feijoa fruits and leaves were transported to the laboratory, where they were thoroughly washed under running tap water to remove excess dust, debris, and mud from the surface.The feijoa fruit was peeled manually, and the leaves were air-dried, then packed in ziplock bags and stored at −20°C until analysis.Manuka and kanuka EOs were purchased from New Zealand Manuka Bioactives Ltd., Opotiki, New Zealand and Pure Nature, Auckland, New Zealand, respectively.Peppermint, thyme, and lavender EOs were provided by Pure Ingredients, Glendene, New Zealand.

| Extraction of feijoa peel and leaf EO
The feijoa peel was cut into small pieces, and air-dried feijoa leaves were ground to a fine powder using a Waring blender (Vitamix, Cleveland, OH).The peels and the ground leaves were subjected to hydrodistillation for 8 h, using Clevenger-type apparatus, as described by Peng et al.'s (2019a) study.The EO was then transferred to an airtight, amber-colored glass vial, flushed with nitrogen gas, and stored at −80°C until analysis.

| Preparation of feijoa peel and leaf ethanol extract
Ethanol extraction was conducted according to Peng et al. (2018).
The peel and leaf samples were transferred to −80°C storage for at least 8 h and then freeze-dried for 72 h (Labconco freeze dryer, USA).The freeze-dried peel and leaf samples were stored at −80°C in an airtight container before extraction.For the fruit peels, 20 g of the freeze-dried powder was mixed with 1 L of 50% (v/v) ethanol and then extracted for 45 min using a magnetic hot plate stirrer set at 60°C and 600 rpm.For the feijoa leaves, 20 g of freeze-dried powder was mixed with 1 L of 30% (v/v) ethanol and then extracted for 10 min using a magnetic hot plate stirrer set at 50°C and 600 rpm.

| Chemical characterization
Gas chromatography-mass spectrometry (GC-MS) was used to determine the chemical composition of EO, as described by Peng et al. (2019a) with some modifications.Briefly, 1 μL of feijoa peel and leaf EO was dissolved in 1 mL hexane and 10 μL of internal standard (2-methyl-3-heptanone, 312 μg/mL) was added.The GC-MS (Agilent 7890A) used was coupled to a DB-5MS column (30 m × 250 μm × 0.25 μm).Helium was used as the carrier gas at a 1.5 mL/min flow rate.The sample was injected using splitless mode at a temperature of 250°C.The oven temperature was maintained at 35°C for 0 min, followed by an increase at 8°C/min to 105°C, then 3°C/min to 150°C, and the final ramp was set to increase at a rate of 25°C/min to 280°C.The ion source temperature was set at 200°C, with electrons generating at 70 eV with a scanning range from 41 to 500 m/z.The volatile compounds in EO were identified by using the NIST Mass Spectral Library.Liquid chromatography-mass spectrometry (LC-MS) was used to determine the chemical composition of feijoa extracts, as described by Peng et al. (2019b) without any modifications.

| Bacterial cultures
The EOs and extracts were tested against 2 g-negative bacteria, E.

| Determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)
were further diluted at a ratio of 1:1 with Mueller-Hinton broth (MHB; Difco™, Becton, Dickinson & Co.) + 1% Tween 80 (Sigma-Aldrich).The final concentration of each working stock solution was 200 mg/mL of EO/extract (20%) and 30% of ethanol.In a 96well plate, 100 μL of homogenized EO/extract solution was added to the first row, and a twofold serial dilution was carried out with MHB.A 50-μL of bacterial suspension prepared in MHB adjusted to approximately 1 × 10 6 CFU/mL was then dispensed into each well.The last row was treated as a positive control.The plates were incubated at 37°C for 24 h in a shaking incubator operating at 200 rotations per minute (rpm) in a sealed box humidified with moistened tissue paper.MIC was determined as the lowest concentration at which no visible growth occurred.MBC was determined by plating 10 μL from each well on the Mueller-Hinton agar (MHA; Difco™) plate, and the plate was incubated at 37°C for 24 h.MBC was determined as the lowest concentration at which no bacterial colonies were observed, indicating a >1000fold reduction in the number of viable cells remaining after the test period (Appendix S1; European Committee for Antimicrobial Susceptibility Testing [EUCAST], 2000).The EOs and extracts tested contributed to the turbidity of the medium; hence, MIC was not recorded.

| Synergistic assay
To determine antimicrobial synergy between EO and extracts, the stock solutions were prepared at the maximum concentration of 4× MBC in ethanol.Two compounds were tested per assay and labeled Compound A and Compound B. The stock solution containing Compound A was added to the first row of Plate 1, and the stock solution containing Compound B was added to all wells in the last column of Plate 2. Twofold dilutions were carried out in MHB, and the last row and column were maintained as a positive control.Furthermore, 25 μL was withdrawn from the corresponding wells of both plates and mixed gently in the matched well of the third plate (Appendix S2).The bacterial suspensions adjusted to a concentration of 10 6 CFU/mL was added to all wells of Plate 3. Plates were incubated at 37°C for 24 h.The drop plate was performed on the MHA plate to assess bactericidal concentrations and combinations.
The fractional bactericidal concentration (FBC) index was calculated using the following formula: where A and B are the MBC of each compound in combination, and MBC A and MBC B are the MBC of individual compounds.Interactions between EO/extracts were interpreted as follows: synergistic (<0.5), additive (>0.5-1), indifferent/no interaction (>1-4), and antagonistic (>4) (Bag & Chattopadhyay, 2015).An isobologram was then plotted (Appendix S2) to depict the hypothetical scenario of synergistic, additive, and antagonistic interactions (EUCAST, 2000).

| Time-to-kill kinetic
A time-to-kill assay was conducted to assess the effectiveness of synergistic combinations of EOs and extracts with the lowest FBC index as per the method described by Lim et al. (2023).Bovine serum albumin (BSA) was added to the samples to mimic the protein burden in the food matrix.Samples were prepared at FBC, ½ FBC, and ¼ FBC concentrations in MHB, and a bacterial suspension was added to achieve a concentration of 1 × 10 7 CFU/mL.A positive control consisting of a bacterial suspension in MHB without EO/extract was also included.The control and samples were incubated at 37°C with shaking at 200 rpm, and aliquots were taken at 10 min, 30 min, and 1, 2, 4, 6, and 8 h.Serial 10-fold dilutions were performed in the letheen broth to deactivate the EO/extract, and then appropriate dilutions were plated on MHA.After incubating the plates at 37°C for 24 h, the number of surviving bacteria was enumerated.

| Statistical analysis
All measurements were performed in triplicate, and median values were plotted for the antimicrobial assays.Analysis of variance (ANOVA) via Sigmaplot (version 13.5, Systat Software Inc.) was used to analyze the experimental data statistically.Tukey's honestly significant difference test was used to compare the means obtained by time-to-kill kinetics at a preset significance level of p < .05.
This has been previously reported by Kong (2019) who observed that the chemical composition of the feijoa leaf EO was comparable to that of the peel EO.Nevertheless, flavones were the primary phenolic compound detected in both feijoa leaf and peel extracts, followed by procyanidin B1, epicatechin, quercitrin 3-d-galactoside, procyanidin B2, epicatechin gallate (ECG), and ellagic acid (Table 2).

| Antimicrobial activity of EOs and extracts
This assay aimed to assess the antimicrobial properties of manuka, kanuka, peppermint, thyme, lavender, feijoa EOs, and feijoa extracts against five foodborne pathogens.We observed that the EOs demonstrated varying degrees of antimicrobial activity against the species tested, ranging from moderate (5% or 5 mg/mL) to strong (<5% or 5 mg/mL), with a few cases exhibiting only very weak activity (>5% or 5 mg/mL might restrict the dispersion of hydrophobic molecules through the cell wall of the bacteria (Angane et al., 2022).
In contrast, in gram-positive bacteria, the complex outer membrane is absent.Instead, they have a thick peptidoglycan layer comprising the lipophilic tail of the lipoteichoic acid, which might ease the entry of hydrophobic molecules such as EOs into the cell wall (Chouhan et al., 2017).Some authors have speculated that high levels of β-triketones in the manuka EO could be responsible for its inhibitory activity.Although Chen et al. (2016) found that kanuka and manuka EOs were equally effective at inhibiting E.
coli and St. aureus, this study found that kanuka EO exhibited poor inhibitory effects on all the tested pathogens.The reason for this rather contradictory result could be the larger molecular size of EO, which may limit the compound passing through the bacterial cell membrane ( Van de Vel et al., 2019).Another possible reason may be due to the effect of different varieties and sources of kanuka trees (Mathew et al., 2020).
Furthermore, the findings in this study agree with those of previous studies examining the effect of feijoa peel EO on St. aureus and E. coli (Smeriglio et al., 2019).They reported a MIC of 2.67 mg/ mL, MBC of 5.35 mg/mL against St. aureus, and no activity against E.
Peppermint, thyme, lavender EOs, and the feijoa extracts, however, followed a different trend, and their effect was random against gram-positive and gram-negative bacteria.Peppermint EO displayed a potent antibacterial effect against gram-negative E. coli at a concentration of 0.6% (6.25 mg/mL), exhibiting a strong activity.The primary contributors to the antibacterial activity of these EOs/extracts are expected to be menthol in peppermint EO (Bassole et al., 2010), thymol and carvacrol in thyme EO (Chaichi et al., 2021), linalool in lavender EO (Garzoli et al., 2020), and flavone in feijoa peel extract (Peng et al., 2019b).These results align with the study by Trombetta et al. (2005), where menthol, the major component of peppermint EO, was found to be more effective against E. coli than other terpene compounds tested.The effectiveness of thyme EO can be attributed to the hydrophobic nature of the thymol and carvacrol molecules (Chaichi et al., 2021), which can disrupt the outer membrane of gram-negative bacteria.This disruption results in the release of lipopolysaccharides and an increase in the permeability of the cytoplasmic membrane (Bassole et al., 2010).Furthermore, Motohashi et al. (2000) and Phan et al. (2019) reported the potent antibacterial activity of feijoa peel extract against E. coli, P. aeruginosa, St. epidermidis, and St. aureus.However, in their study, the agar disk diffusion method was used to evaluate the efficacy of the peel extracts.The agar disk diffusion method is a quick typing tool to determine the sensitivity of bacteria but cannot differentiate between bacteriostatic and bactericidal effects (Angane et al., 2022).This is the first publication to report the antibacterial activity of EOs of feijoa and ethanol extract of feijoa using the MBC assays.

| Synergistic effects of EO and extracts on selected bacteria
The use of synergism can be an effective strategy for reducing the bactericidal concentration required for reducing foodborne pathogens.The primary objective of this study was to develop a broad-spectrum approach for targeting these pathogens while also improving the antibacterial interactions between EOs and extracts and minimizing any associated adverse sensory attributes.Various binary combinations were evaluated using the checkerboard method to achieve these objectives, and the fractional bactericidal concentration (FBC) index was determined.Given that meat products are commonly contaminated with gram-negative pathogens, manuka and kanuka EO were excluded from the synergistic study, as they require higher concentrations (10% or 10 mg/mL) to exhibit antibacterial activity against gram-negative bacteria.Peppermint EO was the most potent against gram-negative bacteria from our findings, so it was chosen for testing in combination with thyme, lavender, feijoa peel and feijoa leaf EO, feijoa peel extract, and feijoa leaf extract.Furthermore, we focused our synergistic study on two model organisms: E. coli to represent gram-negative bacteria and L. monocytogenes to represent gram-positive bacteria.Note: All experiments were performed in triplicate and median values were recorded.The EOs and extracts contributed to the turbidity of medium, hence MIC was not recorded.

| Synergistic effect of selected EOs and extracts
The combined effect of peppermint EO with other EOs and extracts was determined using the checkerboard method, and the FBC index was calculated using Equation 1.The FBC index results for E. coli are presented in Table 4 and Figure 4), while feijoa peel EO, leaf EO, and feijoa leaf extracts showed an additive effect (Figure 2c,d,f and Table 4).
However, the combination of peppermint EO and feijoa leaf EO showed indifferent/no interaction action against L. monocytogenes (Figure 2d and Table 4).
We found that the combination of peppermint/thyme demonstrated the highest potency with the lowest FBC index.One potential explanation for this observation is that thymol and carvacrol, which are major components of thyme and can disrupt the outer membrane of bacteria, may enhance the penetration of other EO molecules into the bacterial cell (Kim et al., 2021).The combination of peppermint/feijoa peel extract showed the second highest efficacy, followed by the combination of peppermint/lavender.It is worth noting that while peppermint, thyme, lavender, and feijoa peel extract individually displayed inhibitory effects at higher concentrations, the best synergistic effects were observed when coli, with thymol, carvacrol, and menthol identified as the primary components responsible for this effect (El amrani et al., 2021).
Similarly, Gallucci et al. (2009) found synergistic effects of menthol and thymol, in peppermint and thyme EO, respectively, against B.
cereus, but an additive effect against E. coli.In another study (Bassole et al., 2010), the combination of menthol and carvacrol in peppermint and thyme EO, respectively, had an indifferent/no interactive effect against L. monocytogenes, but a synergistic effect against E.

these
EOs and extracts were combined.The MBC value of each individual EO or extract was reduced significantly when used in combination with other EOs, indicating a combined antimicrobial effect.Peppermint EO exhibited a reduction in concentration by thirty times for E. coli and eight times for L. monocytogenes, while thyme EO showed a reduction of four times for E. coli and eight times for L. monocytogenes.In addition, peppermint EO combined with the lavender EO, and feijoa peel extract exhibited a reduction of four times in the inhibitory value for both E. coli and L. monocytogenes.These synergistic combinations were also tested against B. cereus, St. aureus, and S. enterica Typhimurium, and we found the combinations were effective at inhibiting all three pathogens.This result has further strengthened our confidence in our hypothesis that synergism effectively reduces the inhibitory concentration of the tested EOs and extracts.This study supports previous research indicating that Origanum compactum and Mentha piperita have a synergistic effect against E.
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