Assessing the potential and safety of Myrtus communis flower essential oils as efficient natural preservatives against Listeria monocytogenes growth in minced beef under refrigeration.

Abstract In this research, the chemical composition and biological properties of Tunisian Myrtus communis (McEO) flowers were investigated. The antibacterial effect of McEO toward some bacteria was assessed, alone and in combination with nisin. The major components of McEO were α‐pinene, 1,8‐cineol, limonene, and linalool. McEO exhibited cytotoxicity toward HepG2 and MCF‐7 cell lines. The microbiological data showed that Gram‐positive bacteria were more susceptible to McEO. McEO had a bactericidal effect against L. monocytogenes. McEO is able to prevent lipid oxidation, microbial development at noncytotoxic concentrations, when used alone or in combination with nisin. It can improve sensory attributes within acceptable limits and improve the conservation of shelf life of minced beef meat during the 4°C storage period. The most potent preservative effect was obtained with the mixture: 0.8% McEO with 500 IU/g of nisin. This combination may be a good alternative for the development of natural preservatives.


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DHIFI et al. a high risk for listeriosis. Food such as soft cheese, cold-smoked fish, and meat (especially deli meat, sausages, cooked, cured, and/ or fermented meat products) are considered high-risk food causing listeriosis. The use of chemical additives is therefore essential to prolong the shelf life and to prevent the growth by L. monocytogenes in refrigerated meat products (Mir, Masoodi, & Raja, 2017). Given the growing safety concerns associated with the use of synthetic additives and preservatives, natural products such as plant essential oils (EOs) and bacteriocins (e.g., nisin) gained popularity as an alternative for food preservation including that of meat (Alizadeh Behbahani, Tabatabaei Yazdi, Shahidi, Mortazavi, & Mohebbi, 2017;Ben Hsouna, Ben Halima, Smaoui, & Hamdi, 2017;Smaoui et al., 2016). Due to their antioxidant and antibacterial activities, bacteriocins and some EOs can act as natural preservatives (Smaoui et al., 2016).

Nisin is a bacteriocin secreted by Lactococcus lactis or
Streptococcus uberis strains and is generally considered a safe added ingredient (Castro et al., 2017). While most bacteriocin peptides exhibit a narrow spectrum of activity, nisin is commonly used as meat preservative thanks to its large activity field against Gram-positive bacteria including L. monocytogenes (Alizadeh Behbahani et al., 2017;Hansen & Sandine, 1994).
Myrtus communis L. commonly known as myrtle belongs to the Myrtaceae family. Myrtle has been traditionally used in folk medicine for its anantiseptic, disinfectant, anti-inflammatory, and hypoglycemic properties (Jabri et al., 2016). Various parts of myrtle have been used in the preparation of cosmetics and as additives to alleviate the flavor of some food (Aleksic & Knezevic, 2014). Most studies available on myrtle focus only on the activity of its leaves and berries EOs (Bajalan & Pirbalouti, 2014), and not on its flower EOs. The antimicrobial and natural preservative potential of EOs have been investigated alone (Bellili et al., 2018;Ozcan, Sagdic, & Ozcan, 2003) and in combination with nisin (Gao et al., 2014;Solomakos, Govaris, Koidis, & Botsoglou, 2008). Nonetheless, the effect of EOS ex-

| Plant material
Myrtle flowers were gathered during the month of June 2016 from the region of Elkef in Tunisia (located at 35.23°N, and 11.11°E).

The flower identification was conducted by Professor Ferjani Ben
Abduallah, botanist in the Faculty of Sciences of Sfax-Tunisia.

| Essential oil extraction
Hydrodistillation in a Clevenger for 3 hr of 1 kg of air-dried myrtle flowers allowed the extraction of its essential oils. Dichloromethane (3 × 50 ml) and anhydrous sodium sulfate were used to extract and then dry the aqueous phase, respectively. Following filtration, a rotary evaporator was employed to eliminate the solvent by distillation under reduced pressure. The extracted oil was then refrigerated (4°C) in the dark (Ben Hsouna & Hamdi, 2012). The EO yields were determined based on the dry weight of plant material used as follows:

| Gas chromatography-mass spectrometry (GC-MS)
The chemical composition of McEO was analyzed by GC-MS (Agilent 6890N; Agilent Technologies), equipped with a capillary HP-5MS column (60 m length, 0.25 mm diameter, 0.25 mm film thickness), and coupled with a mass selective detector (Agilent MSD5973 model; Agilent Technologies). The ionization voltage was 70 eV. The carrier gas was Helium (1.2 ml/min flow rate). The oven temperature was programmed for 1 min at 100°C, increased from 100 to 280°C at a rate of 5°C/min, and then set at 280°C for 25 min. The temperatures of the injector and detector were 250 and 310°C, respectively. The injection (µl) was conducted manually in the split mode (1:50 split ratio).

| Cell culture conditions
The human liver (HepG2) and breast (MCF-7) cancer cell lines were used in the cytotoxicity screens. These cell lines were grown in Roswell Park Memorial Institute (RPMI) 1640 medium (Thermo Fisher Scientific) supplemented with 10% (v/v) fetal calf serum (FCS) and 2 mM L-Glutamine in tissue culture flasks (Nunc, Thermo Fisher Scientific) and passaged twice a week. Cell lines were preserved at 37°C ± 5% CO 2 .
About 0.25 ml of DPPH radical solution (0.2 mM) was added to the reaction mixture. The percentage of free radicals inhibition in percentages was calculated as follows: where A corresponds to the absorbance at 517 nm.

| β-Carotene bleaching assay
The β-carotene bleaching method defined by Solomakos et al. (2008) was used to determine the antioxidant activity of McEO. Tests were run in triplicate. All the tests detailed in the below sections ("Agar well diffusion method" and "Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)") were run in triplicates.

Agar well diffusion method
About 100 μl of inoculum was evenly spread on the surface of Mueller-Hinton agar plates. Sterile Pasteur pipette was used to punch 6-mm wells into the agar plates after they had aseptically Plates were then incubated at 37°C for 24 hr. About 50 μl of each of gentamicin (10 µg) and 1:9 DMSO/water (1:9) solution (50 μl) served as positive and negative controls, respectively. The test zone diameter of growth inhibition around the punched well was measured.

Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)
The broth microdilution method was employed to determine the

| Sample preparation
Raw minced beef meat was purchased from a supermarket in Sfax (Tunisia) and transported to the laboratory under refrigeration conditions within less than 30 min. Each meat sample was divided into

| Thiobarbituric acid-reactive substances value (TBARS)
The distillation method was employed to measure TBARS and evaluate the oxidation of lipids in minced beef meat samples (Eymard et al., 2005). Results were given in mg of malonaldehyde (MDA) equivalents per kg of the sample (mg/kg) using the molar extinction coefficient of the MDA-2-thiobarbituric acid (TBA) at 532 nm (1.56 × 10 5 M −1 cm −1 ).

| Microbiological analysis
About 25 g of each meat sample was dissolved in 225 ml of sterile peptone water (0.1 g/100 ml) and homogenized in a stomacher for 90 s at room temperature. A serial 10-fold dilution series was prepared in peptone water (0.1 g/100 ml

| Sensory evaluation
Eighteen experienced panelists were chosen from the staff members of the University of Sfax to assess the color, appearance, odor, and overall acceptability of the minced meat samples. The assessment was performed using a nine-point scale, where nine correspond to "like extremely" and one corresponds to "dislike extremely." Values of or above five (which corresponds to "neither like nor dislike") were regarded to as acceptable (Smaoui et al., 2016).

| Statistical analysis
The SPSS 19 statistical software (SPSS Ltd.) was exploited to evaluate significant differences between the treated meat samples using the one-way analysis of variance (ANOVA) and Turkey's post hoc test. Bacterial counts data were transformed into logarithms of the CFU per g of ground beef. Corresponding means, standard errors, and variances were analyzed. Differences between the mean values of the different treatments were assessed by the least significant difference test. A probability level of p < .05 was adopted to test the statistical significance of all the experimental data.

| Cytotoxicity assay
McEO exhibited a significant concentration-dependent cytotoxicity against HepG2 and MCF-7 human cancer cell lines with IC 50 values of 131.3 and 204.33 µg/ml, respectively (Figure 1). This is the first report on the cytotoxic activity of McEO against human cancer cell lines; nonetheless, the cytotoxic effect of essential oils extracted from different medicinal plants has been investigated (Innocenti et al., 2010).
The essential oil major components such as limonene, terpinen-4-ol, and β-Caryophyllene have been reported to exhibit an antitumor activity against different cell lines (Lu et al., 2014;Zhang, Scialis, Feng, & Leach, 2013). The cytotoxicity of essential oils could be attributed to various mechanisms including the disruption of the mevalonate pathway (Talib & Mahasneh, 2010), inducing of apoptosis (Kumar, D'Souza, Gaonkar, Rai, & Salimath, 2008) and the alteration of cell membranes, by either increasing its permeability and/or reducing the activity of its enzymes (Rezende, 2012;Zhang et al., 2013). While the cytotoxicity of McEO could be attributed to its major components (Lu et al., 2014;Zhang et al., 2013), minor components could contribute to that activity, individually or in synergy with major components.

| Antioxidant activities
The result of DPPH test ( Figure 2)
Since α-terpineol (3.86%) and β-caryophyllene oxide (1.49%) were minor constituents of McEO, we assume that they act synergistically with major components to give the observed antimicrobial activity.
Further studies are required to identify the McEO components responsible for its antimicrobial activity.

| Microbiological characteristics
In treated samples, the mesophilic (APC), psychrotrophic (PTC) bacterial, and Enterobacteriaceae counts were lower than in controls (p < .05; Table 3  PTC count of T2, T3, T4, and T5 was below the detectable levels of plate counts (log 6.7 CFU/g) after 21 days at 4°C (Table 3). Moreover, the treatment of samples with the combination McEO-nisin (T5: McEO at 0.8% v/w with nisin at 500 AU/g) was most effective in delaying the bacterial growth rate in meat. Similarly to Smaoui et al. (2016), the APC and PTC were lower in McEO-treated meat than in controls. This observation may be attributed to the antioxidant activity of phenolic compounds present in essential oils (Joukar, Hosseini, Moosavi-Nasab, Mesbahi, & Behzadnia, 2017;Ozogul et al., 2017). In fact, essential oils that exhibit an antimicrobial activity against foodborne pathogens are characterized with a high percentage of phenolic compounds (Joukar et al., 2017;Ozogul et al., 2017).
Following the addition of 0.4% (T1) and 0.8% (T2) McEO, a reduction in growth of Enterobacteriaceae was noted (in comparison with the control C; Table 3). Similarly, it has been shown that the addition of essential oils to meat was effective in reducing the Enterobacteriaceae count (Smaoui et al., 2016). Nonetheless, the combination of McEO and nisin is much more effective. The combination of McEO and nisin at 500 AU/g (T5) kept the level of Enterobacteriaceae below the detection limit which is 2 log10 CFU/g until the end of storage at 4°C (Table 3). The Enterobacteriaceae count was reduced to 1.66 log10 CFU/g after McEO addition at 0.8% combined with nisin at 500 AU/g (T5; Table 3). This is in accordance with the work of Smaoui et al. (2016) who reported that the combination of Mentha piperita essential oils and bacteriocin (named BacTN635) can be considered as an effective antimicrobial on minced beef meat during refrigerated storage (Smaoui et al., 2016).

| Kill-time analysis: effect of McEO (alone and in combination with nisin)
The

| Physicochemical characteristics
We noted alterations in the pH values of raw minced beef meat samples (C, T 1-5 ) during storage period at 4°C (Table 4). The initial pH of minced beef meat samples (C, T 1-5 ) was of 5.60. The pH value of the control sample (C) increased the most among all the samples (increase in pH from 5.60 ± 0.26 to 6.92 ± 0.24, with a difference of about 1.37 in pH between day 21 and day 0 of storage). The pH increase in treated minced beef samples (T 1-5 ) was lower than that of the control sample (C). The lowest pH values increase over the period of storage (difference in recorded pH values between day 21 and day 0) was recorded for T 5 (about 0.51 in pH difference, which was < T 4 <T 3 < T 2 < T 1 < C

TA B L E 5 Effect of
McEO and their combination with nisin on color, odor, and overall acceptability of raw minced meat beef stored at 4°C and day 0) over the storage period (day 0-21; Table 4). The threshold for the acceptability of TBARS is 2 (Campo et al., 2006). The TBARS recorded for T 5 was <2, indicative of the protective antioxidant effect of the combination used in T 5 . The rapid rate of increase in lipid oxidation in the control sample (C) can be attributed to the absence of antioxidants (Ben Hsouna et al., 2017). The antioxidant activity of McEO (alone or in combination with nisin) slowed down the free radical propagation process and the oxidation rate (TBARS < 2.3 in T 1-5 ; Table 4). Table 5 summarized the findings relative to the sensory analysis of minced beef meat treated with essential oil tested individually or in mixture. Sensory evaluation showed a decrease in all groups evaluated sensory attributes (color, odor, and appearance and overall acceptability) during the storage. These results revealed a best sustainability for the group treated with the combination

| Sensory scores
McEO at 0.8% v/w + nisin at 500 IU/g (T 5 ) followed by T 4 (McEO at 0.4% v/w with nisin at 500 AU/g). For the samples treated with McEO and McEO/nisin combinations, color scores above five corresponding to the rejection limit of raw minced beef meat were not reached until the 14th day of storage. The overall acceptability of treated minced beef (T 1-5 ) was maintained for 14 days (p < .05).
The untreated control sample (C) was unacceptable from day 7 (p < .05). The decrease in overall acceptability scores during storage (C, T 1-5 ) might be due to a decline in scores of other sensory attributes or characteristics such as color, odor, and appearance (Smaoui et al., 2016).

| CON CLUS ION
In

ACK N OWLED G M ENTS
This study was supported by a grant from Ministry of Higher Education and Scientific Research of Tunisia.

CO N FLI C T O F I NTE R E S T
All authors declare that there is no conflict of interest.

E TH I C A L S TATEM ENT
There was no human or animal testing in this study.