In Vitro Antimicrobial Activities of Various Essential Oils Against Pathogenic and Spoilage Microorganisms

Background: Plant-derived Essential Oils (EOs) have shown remarkable antimicrobial activity against spoilage and pathogenic microorganisms isolated from food products. The objective of the current study was to determine in vitro antimicrobial effects of selected EOs against these microorganisms. Methods: Antimicrobial activity of EOs against food-borne and spoilage microorganisms was screened by disk diffusion assay; then, the Minimum Inhibitory Concentration (MIC) and Minimal Bactericidal Concentration (MBC) were determined. Statistical analysis was done using SPSS 23.0 software for Windows. Results: Oregano and thyme EOs showed the highest antimicrobial activity and the lowest MICs, while anise, fennel, garlic, and ginger showed a lower activity with significant differences ( p <0.05). It was demonstrated that Salmonella Typhimurium, Escherichia coli , Proteus mirabilis , and Yersinia enterocolitica were the most sensitive bacteria to all the EOs tested ( p <0.05). Among Gram-positive bacteria, Listeria innocua was demonstrated to be the most sensitive to most of the EOs ( p <0.05). Furthermore, Staphylococcus aureus and Listeria monocytogenes were shown to be more sensitive than Enterococcus spp. ( p <0.05). Yeasts were significantly ( p <0.05) more sensitive than bacteria and were inhibited by most of the EOs. Conclusion: The use of the analyzed EOs may be interesting to food processors because of their antimicrobial properties. However, it is necessary to test their use in food products and gauge their sensory implications.

effective against several microorganisms associated with several food products (Burt, 2004;Selim, 2010). EOs have been proposed as a viable alternative for application in food processing to avoid the use of traditional chemical additives (García-Diez et al., 2016;Ghabraie et al., 2016).
The differences in antimicrobial activity by different EOs are usually associated with their various chemical compositions that change according to seasons, geographical location of plants and/or the methodology used in EOs extraction (García-Diez et al., 2016;Kokkini et al., 1997). However, their antimicrobial activity may be attributed to their ability to penetrate through bacterial membranes and inhibit functional and lipophilic properties of the cell (Burt, 2004;Calo et al., 2015;Trombetta et al., 2005).
In recent years, consumers demand minimally processed foods. The negative perception of consumers about chemical food additives makes natural methods of preservation and natural preservatives receiving increased attention by the food industry (García-Diez et al., 2016). Non-phytotoxic oils are safe as food additives and declared as "Generally Recognized As Safe" (GRAS), which increased consumer acceptability (Jayasen and Jo, 2013). However, application of EOs is limited by taste and odor impacts, especially when used at high concentrations (Ghabraie et al., 2016). In fact, the organoleptically acceptable concentration depends on individual EO, the specific food systems, the method of application, and food product cooking methods. Indeed, it would be changed when other compounds are added to the food, too. Therefore, it is necessary to determine their lowest concentration with acceptable sensorial level in order to use them in food without any changes in smell and taste (Turgis et al., 2012).
The aim of the current study was to determine in vitro antimicrobial activity of various EOs against selected pathogenic and spoilage microorganisms, aiming for a future utilization in the manufacture of some food products.

Microorganisms and growth conditions
All strains used in this study (Table 1) were stored at -20 ºC in Tryptic Soy Broth (TSB; Pronadisa, Madrid, Spain) with 6 g/L of Yeast Extract (YE; Lab M, Bury, UK) containing 30% (v/v) glycerol (Sigma, Steinheim, Germany), and sub-cultured twice before use in assays. Each bacterial strain was grown on Tryptic Soy Agar (TSA, Pronadisa) with 6 g/L of YE (Lab M, Bury, UK) at 37 ºC for 24 h and yeasts in Yeast Malt Agar (YMA; Lab M, Bury, UK) at 25 ºC for 48 h.

Disk Diffusion Assay (DDA)
Each inoculum was prepared by resuspending isolated colonies of each strain, previously cultured on TSA+YE or YMA, in sterile Ringer solution (Lab M, Bury, UK) in order to obtain turbidity equivalent to 0.5 in McFarland scale (bioMérieux, Marcy-l'Etoile, France).
The antimicrobial impact of EOs was evaluated by the DDA as indicated by Zaika (1987), with some modifications. Briefly, plates prepared with Mueller-Hinton Agar (MHA; Biokar, Beauvais, France), or YMA for yeasts were dried and 100 μl of standardized inoculum were uniformly spread. After that, sterilized filter paper disks (Whatman No. 5, 6 mm diameter) were applied to the surface of the seeded agar plates and 5 μl of each sterilized EO (0.22 m syringe filter) was applied to each disk. The plates were kept at 4 ºC for 2 h to allow dispersion and incubated for 18 to 24 h at 37 ºC for all bacteria; also, the yeasts were incubated for 48 h at 25 ºC. The antimicrobial activity was visually evaluated as inhibition zone surrounding the disk and the disk diameter was measured in mm. Inhibition was only considered if the halos were greater than 10 mm, according to García-Díez et al. (2016). The DDA assay was carried out in triplicate.

Determination of Minimum Inhibitory Concentration (MIC) and Minimal Bactericidal Concentration (MBC)
The MIC and MBC were studied only for all EOs that resulted in inhibition halos greater than 10 mm. The assay was based on the procedures described in CLSI (2012) using 96-wells microtiter plates. The dilutions of Downloaded from jfqhc.ssu.ac.ir at 16:18 IRST on Tuesday October 16th 2018 the EOs were provided according to the inhibitory profile with the DDA (halos greater than 10 mm). EOs dilutions were prepared directly in the Mueller-Hinton Broth (MHB, Biokar, France) to achieve in the well each of the followings concentrations: 50, 25, 12.5, 6.25, 3.125, 1.56, 0.78, 0.39, 0.195, 0.0975, 0.0488, 0.0244, 0.0129, and 0.0060%. In each well, it was mixed 80 µl of MHB, 100 µl of each EO dilution, and 20 µl of standard suspension of each target microorganism (prepared in MHB to achieve a final cell density in each well of ca. 5 log colony forming unit/ml). Un-inoculated negative controls were included. The plates were covered, incubated for 24 h and then checked for visible growth (turbidity) in each well. The MIC was determined as the lowest concentration of EO which prevented growth. To determine the MBC, 10 μl of each well, in which no microbial growth was seen, was spread into MHA and incubated for 24 h, as stated by García-Díez et al. (2016).

Statistical analysis
The comparison of the antimicrobial activity of EOs against each microorganism was carried out by one-way Analysis of Variance (ANOVA). The Tukey-Kramer test was used to determine the significant differences (p<0.05) among group means. Statistical analysis was done using SPSS 23.0 software for Windows.

Results
The data of the antimicrobial activity assessed by DDA showed that in general the antimicrobial activities of the different tested EOs were varied and dependent on the type of oil and type of microorganism (data not shown). In general, oregano from "Casa das Essências": C.E. and oregano from "Ventós" Chemical: V. had the EOs that showed significantly (p<0.05) higher antimicrobial activity than the others. On the other hand, EOs of lemon, ginger, and anise were the ones that showed meaningfully (p<0.05) lower antimicrobial activity. It was demonstrated that S. Typhimurium, Escherichia coli, Proteus mirabilis, and Yersinia enterocolitica were the most sensitive bacteria to all the tested EOs (p<0.05). Among Gram-positive bacteria, L. innocua was demonstrated to be the most sensitive to most of the EOs (p<0.05). Furthermore, Staphylococcus aureus and also L. monocytogenes were shown to be more sensitive than Enterococcus spp. (p<0.05). Yeasts were significantly (p<0.05) more sensitive than bacteria and were inhibited by most of the EOs.
Results of MIC and MBC of the tested EOs are presented in Tables 2 to 6. Through the results obtained in the DDA, all EOs with halos lower than 10 mm were excluded and not tested for determination of MICs. Values of MIC and MBC were, on average, higher for Gram-negative microorganisms than for Gram-positives.
EOs of bay, cloves, oregano (C.E.), oregano (V.), and thyme presented MICs between 0.0488% and 1.56% for Gram-negative bacteria (Tables 2 and 3). However, the EO that demonstrated the lowest inhibitory concentration against all the analyzed microorganisms was the oregano (V.) (0.0975-0.0488%). Hence, the most sensitive microorganisms were S. Typhimurium, E. coli, as well as Y. enterocolitica.
Regarding Gram-positive bacteria, there was a higher number of EOs demonstrating low inhibitory concentrations. For the non-spore forming bacteria, EOs of bay, cloves, coriander, cumin, marjoram, oregano (C.E.), oregano (V:), rosemary, and thyme presented MICs between 0.0244 and 3.125% (Tables 4 to 6). Strains of St. aureus, L. monocytogenes, and L. innocua were the most sensitive. Enterococcus spp. were the most resistant.
Regarding yeasts, these were extremely sensitive, demonstrating low MICs for most of the EOs, with the exception of anise, basil, fennel, juniper berries, nutmeg, and parsley that showed higher MICs (between 3.125% and 100%; data not shown).

Discussion
Through the analysis of the results obtained for DDA and MIC, it was possible to verify that Gram-positive bacteria are more sensitive than Gram-negative ones, which is in accordance with previous reports (Hyldgaard et al., 2012;Nazzaro et al., 2013). The cell wall of Grampositive bacteria allows hydrophobic molecules to readily penetrate into cells and act on both cell wall and cytoplasm; but Gram-negative bacteria have an outer membrane that contains lipopolysaccharides that could act as a barrier against macromolecules and hydrophobic compounds, making them more resistant to these same compounds (Nikaido, 1994(Nikaido, , 2003 Regarding the used EOs, it was possible to state that oregano and thyme were the EOs with the greatest inhibitory capacity for all the bacteria tested in DDA. The MIC and MBC values that were observed in this study were similar to those previously reported in the literature (García-Diez et al., 2016;Sokovic et al., 2010). Oregano (V.) was the EO that presented the lowest MIC, with values between 0.195 and 0.0244%. The lowest MIC (0.0244%) was observed against L. innocua, nevertheless a MIC of 0.0975% was observed for the majority of the microorganisms under study. According to these concentrations, this EO could be considered with great inhibitory potential. These results are in agreement with previous studies, which also reported a high antimicrobial activity of oregano and thyme (Dobre et al., 2011;Semeniuc et al., 2017). Although two different oregano oils were used in our study, oregano (V.) has a greater inhibitory capacity, which may be due to their different origins. According to Kokkini et al. (1997), the type of extraction of EOs and the different seasons of the year could produce different amounts of compounds related to each EO. This antimicrobial activity is probably due to its main components, including carvacrol for oregano and thymol for thyme. Thymol as well as carvacrol are hydrophobic compounds, which induce structural and functional damages to cytoplasmic membrane (Sikkema et al., 1995). Hyldgaard et al. (2012) described that thymol could be involved in the rupture of the inner and outer membrane and the interaction with membrane proteins and intracellular targets, while carvacrol owes its mechanism of action to its ability to position into the membrane, which increases their permeability.       Silva et al. (2013), it was also demonstrated that among the evaluated EOs, the greatest effectiveness was achieved when thyme and oregano were used, which showed activity against all the tested bacterial strains. Similarly, Gutierrez et al. (2008) showed that B. cereus, E. coli, L. monocytogenes, and Ps. aeruginosa were sensitive to the oregano EO. Burt (2004) also reviewed that EOs had antimicrobial effect against some microorganisms in different food products such as boiled rice, carrots, soft cheese, and fish. Regarding yeasts, the results obtained in the present study are in agreement with other studies showing that oregano EO exhibited a broad spectrum of activity against Candida spp. (Khosravi et al., 2011) and Saccharomyces cerevisiae was the most sensitive microorganism to all EOs tested (Çoskun et al., 2016).

Conclusion
The current study demonstrated that tested EOs had in vitro antimicrobial effect against food-borne pathogens such as Salmonella spp., L. monocytogenes, St. aureus, and E. coli and also against some spoilage bacteria. EOs of oregano and thyme showed the greatest inhibitory effect against the different microorganisms. These differences could be associated to several factors such as chemical composition of the EOs or to the specific sensitivity of the target microorganism among others.
Taking into account the large number of oils and the number of investigated microorganisms, this study is of great importance to their potential users, namely the food industry, since it covers several food-borne pathogenic microorganisms. However, it is necessary to test their use in each food matrix and gauge their sensory implications.

Author contributions
H.A., M.C., and P.T. designed the study and wrote the manuscript; M.C. conducted the experimental work; H.A. and M.C. analyzed the data. All authors revised and approved the final manuscript.