Effect of thyme essential oil ( Thymus vulgaris ) on the growth of Salmonella Enteriditis and Salmonella paratyphi on surfaces of chilled raw bovine meat CURRENT STATUS: UNDER REVIEW

Essential oils (EOs) are mostly composed of terpenes and phenolic compounds that are stored in secretory tissues of aromatic plants. This study evaluated the effect of both commercial and experimental thyme oil (cEO and eEO, respectively) on ATCC strains of Salmonella enteritidis and Salmonella paratyphi present on surfaces of chilled raw beef. The composition of each EO was analyzed chemically by gas chromatography coupled with spectrometry (GC-MS). The antimicrobial activity was assessed through the agar diffusion method and the minimum inhibitory concentration by means of the macro dilution method in broth, thus proving that a higher concentration of both cEO and eEO is required to reduce the growth of S. enteritidis compared to S. paratyphi . Similar results were obtained when pieces of beef previously inoculated with 10 4 cfu / ml and kept at 0°C, 4°C and 10°C for 72 hours were treated with three concentrations of cEO and eEO. The in vivo approach showed that S. paratyphi is more sensitive to the action of EO than S. enteritidis and such sensitivity decreases when temperature increases. Overall, the cEO proved to be more effective than the eEO on the growth inhibition of the two species, showing in all cases a reduction greater than 10% for S. paratyphi at the concentration of 50% (v / v) and greater than 7% at the concentrations of 40 and 30% (v / v), exhibiting a significant variation at a p-value < 0.05. Regarding S. enteritidis , the values of reduction were 4.5% at 0°C and 4°C and 3.5% at 10°C for the cEO, and higher than 1% for the eEO. It is concluded that the application of thyme EO on surfaces of meat contributes to reduce the presence of Salmonella spp. on this type of foodstuff.


Introduction
Meat is one of the final products of the bovine agroindustry and, due to the lability of its components, is considered as a food with a high public health risk (de Bogota 2013), because of several factors that have a direct relation with the predominant decomposing biota present in meat, including the availability of nutrients, oxygen, temperature and time of storage, pH and rate of proliferation of microorganisms (Ray et al. 2010). Meat provides a variety of nutrients, including proteins of high biological value which are necessary for bones, teeth and muscles genesis, and also promotes an increase in the rate of hemoglobin, characteristics that make this food one of high nutritional level preparation of human disinfectants, mouthwashes and other antimicrobial agents used at home (Alzate et al. 2009;Rueda-Puente et al. 2018)In order to contribute to the implementation of effective and natural strategies to address the above challenges, the use of essential oils (EO) has assumed an important role, as the mixture of terpene components exhibits activity against common bacteria (Barrera et al. 2013) through mechanisms such as damage to membrane structure and function, inhibition of biosynthesis and function of nucleic acids, interference with essential metabolic processes, and disruption of normal cellular communication, among others (Gallegos-Flores et al. 2019;Radulovic et al. 2013). Based on the above context the objective of this study was to evaluate the effect of the essential oil obtained from thyme (Thymus vulgaris) on the growth of Salmonella enteritidis and Salmonella paratyphi present on surfaces of chilled raw beef.
Two sources of thyme essential oil were used: a commercial oil produced by the NOW Foods industry (Bloomingdale, IL, USA) and another experimental oil extracted from thyme plants purchased in the local market.
-Commercial oil. According to the manufacturer's website (https://www.nowfoods.com/essentialoils/white-thyme-oil), this product is 100% pure essential oil derived from flowers of two varieties of white thyme: the common thyme (Thymus vulgaris) and the "salsero" thyme (Thymus zygis), obtained through a steam distillation method.
-Experimental oil: The second type of essential oil was obtained from leaves and stems of vulgar thyme acquired at the local market, which was extracted by steam distillation method using a pilot plant with a capacity of 25 kg (University of Pamplona, Villa del Rosario).

Determination of the Chemical Composition of Essential Oils.
-Gas Chromatography -Mass Spectrophotometry of thyme essential oil: A determination of the volatile composition of the two types of oils was carried out through gas chromatography with a selective mass detector (GC-MS), according to the CM-PTSE-04 procedure, version 01, 2014-04-04; based on the ISO 7609, 1985(E) (ISO 1985). The chromatographic analysis was performed on an AT 6890 Series Plus gas chromatograph (Agilent Technologies, Palo Alto, California, USA), coupled to a selective mass detector (Agilent Technologies, MSD 5975) operated in full scan mode. The columns used in the analysis were DB-5MS (J and W Scientific, Folsom, CA, USA) (5%-phenyl_poli (dimethylsiloxane), 60 m x 0.25 mm x 0.25 μm). Injection was performed in Split mode (30:1), Viny =2 μl.

EOs treatment approach.
Different treatments applied both in vitro as in vivo to determine the antibacterial activity of both EOs are represented in Table 1.  (4574 2007) for which a phase of pre-enrichment in a non-selective medium was performed, and along with a phase of selective enrichment, with the purpose of increasing the population of interest and inhibiting other microorganisms present. For this purpose, Rappaport broth was used, taking 1 ml of the preenrichment culture and sowing it in a tube containing 10 ml of broth, which was incubated at 42 °C for 24 hours. Afterwards, a differential isolation was performed by taking an inoculum and seeding it in Lysine Xylose Deoxycholate (XLD), on Hektoen agar (Merck). The presence of Salmonella was not found in these tests after an incubation at 37ºC for 24 hours.
-Salmonella enteritidis ATCC 17036 and Salmonella paratiphy ATCC 9150 strains obtained from University of Pamplona, Colombia. The strains were cultivated in nutritive broth and soy tripticase broth at 37ºC for 24 hours before use.
-Inoculum standardization: An inoculum was taken from the strains and seeded in a tube containing 0.5 and 99.5 ml of 0.18M barium chloride and 0.048M sulfuric acid, respectively. Such inoculum was incubated at 37°C for 2 to 4 hours and adjusted to 0.5 on the MacFarland scale to obtain 1.5 x 10 8 cells/ml.
-Determination of antibacterial activity by disk diffusion method: Antibacterial activity assessment was carried out according to Costa et al. (2013) with some modifications. Petri agar plates of Mueller-Hinton (BBL Becton Dickinson and Company) were inoculated by means of massive sowing with 0.1 ml of 10 6 ufc/ml of the standardized inoculum with Salmonella enteritidis and Salmonella paratiphy, each inoculum was allowed to dry for 5 minutes. Subsequently, sterile filter paper discs of 9 mm in diameter were permeated with 30 microliters of each essential oil at concentrations of 10%, 20%, 30%, 40% and 50% and then placed on the surface of the Mueller Hinton culture previously inoculated with each Salmonella strain. Plates were left for 15 minutes at 4°C to allow the pre-diffusion of the essential oils before incubation (Ramirez and Castaño 2009). Plates were then incubated at 37±0.5°C for 18 hours, as described by López (López-Oviedo et al. 2006). The standard method recommends that all final readings be taken at 18 hours, as it has been established that this is the point in which the interaction between microorganisms and the inhibitory effect of antibiotics is optimal. Ending the incubation, the inhibition halos were measured and the results were interpreted based on Celikel and Kavas (2008), -see Table 2a-. The assays were carried out in triplicates and discs permeated with absolute ethanol were used as negative control.

-Determination of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration
(MBC). MIC was identified through macro-dilution in broth method. Briefly, 0.1 ml of standardized inoculum was added to tubes containing 9.8 ml of sterile nutritive broth plus 0.1 ml of commercial (cEO) and experimental (eEO) essential oil of thyme according to the concentrations identified in the agar diffusion assay described in table 1. Tubes were incubated at 37°C for 18 hours. As control, one of the tubes did not contain EO. The experiments were developed in triplicates. After the identification of the MIC, a volume of 0.1 ml was taken from the tubes that did not show increased in turbidity and sowed in nutritive agar to corroborate the inhibition of growth, allowing to determine the MBC. The first tube in the series that when seeded did not exhibit colony development represented the MBC of the antimicrobial compounds under study.
2.5. Effects of EO and refrigeration temperature on the growth of Salmonella in fresh meat.
-Meat preparation. First quality beef was cut into 1cm 2 pieces that were previously disinfected with 540 ppm Citrosan (Brand and city) for 2 mins. Later, the pieces were washed with sterile distilled water and the presence of Salmonella was evaluated in a fraction of 25 grams by the method described on the NTC 4574 (4574 2007). In addition, mesophilic aerobes, molds and yeasts were analyzed to rule out microbial contamination.
Ten grams of meat were taken and placed in a bottle with 90 ml of buffered water. Dilutions were made up to 10 -3 , and those of 10 -2 and 10 -3 were inoculated by pour plating seeded in deep plate using in plate counting agar and Saboraud agar. The remaining cuts were wrapped in sterile plastic wrapping foil and frozen until the results of Salmonella spp. were obtained.
-Microorganisms and development conditions. Salmonella enteritidis and Salmonella paratyphi strains were kept in Soy Tripticase Broth (STB) (Acumedia, UK) at 4°C. The inoculum was obtained by cell culture in nutritive broth for 24 hours at 37°C. The cell suspension was diluted with peptone water (Acumedia, UK) to provide an initial cell count close to 1.5x10 8 ufc/ml (McFarland 0.5). 24 hours prior to each experiment 0.1 ml of culture was transferred to 9.9 ml of nutrient broth.
-Inoculation of Salmonella spp. in beef. We applied a modified procedure to inoculate the meat (Dussault et al. 2014). After an initial period of incubation for 24 hours in STB, bacteria were resuspended in fresh medium and incubated for another 24 hours prior to the experiment, seeking to achieve working cultures of approximately 10 9 cfu/ml. On the day of meat inoculation, each culture was diluted 10 4 times in peptone water in order to prepare a 10 4 to 10 5 cfu/ml inoculum solution and thus obtain a final concentration of 10 3 to 10 4 cfu/g in the meat pieces.
-Effect of essential oils. Once the meat was inoculated, each piece was individually sprayed with five different concentrations of cEO and eEO as described in table 1, plus controls. They were arranged in sterile plastic boxes and incubated at three temperatures: 0°C, 4°C and 10°C, for 24, 48 and 72 hours. In addition, controls used consisted of pieces of meat sprayed with ethanol, essential oils, and positive controls were pieces of meat with the bacteria without any treatment. The whole experiment was carried out in triplicate with three biological repetitions.
-Microbiological analysis. After incubation at different temperatures (0, 4 and 10ºC), a piece of meat was placed in peptone water (9ml), the microbial population diluted and then inoculated by pour plating in Tripticase Soy Agar (TSA) medium. Subsequently, the number of colonies present in the agar was counted and compared with the initial colony count to determine the percentage reduction at each time interval in each treatment used.

Data analysis
The effect of the essential oils was assessed by determining the growth/death rate for 72 hours, using a non-linear regression analysis with DMFit software, as well as determining the percentage reduction by comparing the final microbial load to the initial one.

-Statistical Analysis. For the statistical process an experimental design was applied in a Randomized
Complete Block Design (RCBD) with three replicas for each treatment studied, thus covering all the possible combinations for each factor. The statistical evaluation was carried out by means of ANOVA/MANOVA variance analysis, and the Tukey-HSD multiple range test was used to compare means and identified statistically significant differences (p < 0.05) applying the Statgraphics Centurion XV.II software. In the case of outliers, a Kruskal-Wallis Test was used.
Final reduction percentages of Salmonella enteritidis and paratyphi were determined by comparing the mean of the counts for each time interval (24 h, 48 h and 72 h) versus the initial count (0 h). With these data, the mean speed of death was determined by means of non-linear regression analysis for each treatment using DMFit software.
3. Results And Discussion 3.1. Physicochemical Characterization of Thyme Essential Oils. Table 2b shows some biological features of both EOs. The physical composition of cEO and eOE was similar in terms of density, refractive index, odor and color. As for the percentage of yield obtained, it could only be determined for the eEO, exhibiting 0.15%, value that falls within the nominal range of essential oils content for herbs and spices. In this regard, Tajkarimi report that herbs and spices contain essential oils (EOs) in a range of 0.05-0.1% (Tajkarimi et al. 2010). Table 3  Sensitive + Between 15 and 19 mm very sensitive ++ Greater than 20 mm Extremely sensitive +++ (Celikel and Kavas 2008) b) Table 2 a) Classification of the individual sensitivity to essential oils by the diameter of the inhibition zones b) Physical and organoleptic characteristics of each thyme EO. From this evaluation, 19 components were found in cEO, all of which were identified (100%), while 33 components were found in the eEO, of which 32 were identified (97%). The relative abundance of the cEO was represented by two major components (80%): thymol (45.3%) and p-cymene (30.3%), and a smaller proportion by γ-terpinene (8.4%). Regarding the eEO, its relative composition varied substantially as the p-cymene is presented as the major component (43.8%), following by trans-βcaryophyllene (6.2%), thymol (5.7%), α-pinene and limonene (4.2% each). The compositions described are similar to other essential oils obtained from the same plant species with variation in their abundance percentages. It is worth mentioning that in the eOE the quantities of thymol were low and the presence of carvacrol was not found, unlike what was seen in the cEO. In this sense, some authors have found a relationship between thymol production and the presence or absence of carvacrol (D'Auria et al. 2005), which seems to be in accordance with the observations made in this study. However, the results obtained are similar to those found in another study, in which four main components were identified in hydroponically grown thyme essential oil in two nutrient solutions and three planting densities (Guerrero-Lagunes et al. 2011). These were thymol, p-cymene, γ-terpinene and carvacrol, which presented an average abundance of 23.3%, 7.4%, 4.9% and 1.1%, respectively.
These are comparable to those found by Omidbaigi and Arjmandi, who identified thymol as the major component of the essential oil on this species (Omidbaigi and Arjmandi 2001). However, the composition of thymol can range from 10-64% according to Lens (Lens-Lisbonne et al. 1987).
The lower composition variability identified in the cEO may be indicative of higher purity, as referred to by the supplier both on the commercial label and on the manufacturer's website (https://www.nowfoods.com/essential-oils/white-thyme-oil) (100% purity). It should also be borne in mind that this is an essential oil obtained from two varieties of white thyme in its floral state, the common thyme (Thymus vulgaris) and the "salsero" thyme (Thymus zygis), by means of a steam distillation method under standardized conditions on an industrial scale; whereas the eEO was obtained from dried and fresh leaves and stems of vulgar thyme acquired in a local market, not knowing the phenological state of the plant or growing conditions, which may provide a lower purity, less abundance of active components and greater variety of components, as reflected on the table above.   Figure 2 shows the spectrum corresponding to γ-terpinene obtained in the cEO sample, in which the molecular ion limit was 136 (figure 2a), and the peak base was 93 (figure 2b) for the same component, but obtained from the Wiley 275.L database; data indicative of the presence of this molecule. In this way, the compounds of each peak obtained from the chromatographic analysis from both EOs were identified, according to its mass spectrum and compared with the most similar spectrum of the reference database.
Although the biosynthesis and accumulation of thyme essential oil are genetically controlled, its chemical composition is also affected by a number of geographical, geobotanical and environmental factors such as temperature, light, climate, altitude, soil type and rainfall (Baranauskienė et al. 2003;Curioni et al. 2002;Figueiredo et al. 2008;Tajkarimi et al. 2010). Within the compendium of factors associated with differences in oil composition it has been proved that agronomic conditions such as fertilization, sowing density and cultivation method influence the quantity and quality of essential oil (Boskovic et al. 2015;Guerrero-Lagunes et al. 2011;Naghdi Badi et al. 2004). Other elements such as time and mode of collection, plant part, material handling and the obtaining process can affect composition (Albado Plaus et al. 2001;Bandoni et al. 2009), making it almost impossible to obtain two identical essential oils (Bandoni et al. 2009).
Other studies found similar results, since these works also evaluated the essential oil of thyme as well as oregano (Boskovic et al. 2015;Muñoz López de Bustamante 2002), finding thymol with an abundance of 50.48%, followed by p-cymene with 24.79%, linalool with 4.69%, and y-terpinene with 4.14%, concentrations very similar to our observations. The presence of p-cymene, γ-terpinene, carvacrol and thymol in the two EO samples tested are an indicator that these four components are biologically and functionally related and supports the hypothesis that thymol is formed via p-cymene from y-terpinene in T. vulgaris (Burt 2004;Kokkini et al. 1997).s In the cEO sample, thymol (2-isopropyl-5-methylphenol) was the major component with 45.3%, whereas in the eEO it barely reached 5.7% (Table 2). One of the main characteristics and properties of thymol as a phenolic compound is its bactericidal, pesticidal and fungicidal potential. Thymol belongs to terpenes group of terpenes and can often be miscalled with the carvacol isomer, which can also be found in thyme essential oil and is structurally very similar to carvacrol, having the hydroxyl group in a different location of the phenolic ring (Lambert et al. 2001).
p-cymene (1-methyl-4-(1-methylethyl)benzene) found in the cEO represents the second constituent with a 30.3%, while in the eEO it was the main component with 43.8% (Table 2). This naturally occurring aromatic organic compound is classified as an alkylbenzene monoterpene obtained from aromatic plants (thyme, cumin). The biological precursor of carvacrol, p-cymene, is hydrophobic and causes cytoplasmic membrane swelling more largely than carvacrol (Ultee et al. 2002). p-cymene is not an effective antibacterial molecule when used alone (Dorman and Deans 2000;Juliano et al. 2000;Juven et al. 1994;Ultee et al. 2000), but in combination with carvacrol it is has been reported to have synergism against bacteria such as Bacillus cereus in vitro and in rice samples (Ultee et al. 2002). It is hypothesized that such synergy occurs when p-cymene is incorporated into the lipid bilayer which most likely facilitates the transport of carvacrol across the cytoplasmic membrane (Ultee et al. 2002).

Agar diffusion tests.
Results obtained from agar diffusion tests for Salmonella enteritidis and Salmonella paratyphi are shown in Table 4. Data are presented based on the sensitivity taking into account the inhibition halos as follows: Extremely sensitive (+++) for diameters above 20 mm, very sensitive (++) for diameters between 15 and 19 mm, sensitive (+) for diameters between 9 and 14 mm and non-sensitive (-) for diameters below 8 mm (Ponce et al. 2003).
According to the sensitivity obtained (Table 4), S. paratyphi showed to be more sensitive to thyme essential oil than S. enteritidis, as 50% of the treatments tested yielded inhibition halos with areas greater than 9 mm, whereas for S. enteritidis just treatment five (T5) yielded a halo greater than 9 mm. This could indicate that thyme essential oil can be considered as a possible natural additive for food preservation. The refractoriness of S. enteritidis does not represent a barrier to this potential application, it only endorses the claim that biological safety of food is based on one or more  Data obtained from these tests regarding the type of essential oil clearly showed that the cEO exhibits stronger effects, an observation that could be explained by its chemical composition as it has higher amounts of thymol and p-cymene, which act as antimicrobials. On the other hand, this oil was obtained from two varieties of thyme plants in floral state since it has been reported that EOs obtained from herbs and spices collected during or immediately after the flowering process have stronger antimicrobial activity (Burt 2004). It has been observed that the Thymus zygis' EO exhibits greater antibacterial power than its counterpart of T. vulgaris when tested against Salmonella enteritidis and Escherichia coli O157:H7 (Rota et al. 2008), hence the mixture of chemotypes in the cEO (T.zygis -T.vulgaris), as well as the higher presence of phenolic compounds, alcohols and thymol compared to the eEO suggests a synergistic action between such components and their relative amounts (Burt 2004).

Minimum Inhibitory Concentration and Minimum Bactericidal Concentration.
Based on the results obtained from the above tests, MIC of both essential oils on bacterial proliferation was determined (Table 5 and 6). Data obtained related to MIC of both EOs on S.
enteritidis allowed inferring the absence of turbidity in culture broths in concentrations higher than 0.5%, yielding a MIC of 5.55 mg/ml as indicated in the yellow column (Table 5). Likewise, in relation to eEO, absence of turbidity was found at higher concentrations compared to its commercial counterpart (0.7%), so the MIC was placed at a concentration of 7.41 mg/ml. Regarding Salmonella paratyphi test, MIC data of both EOs is depicted in Table 6. In this case, results obtained from the disc diffusion test were confirmed, as S. paratyphi inhibition halos were higher compared for those of S. enteritidis, indicating a higher sensitivity of the first species. MIC for both oils against S.paratyphi was lower than that observed for S. enteritidis at 4.625 mg/ml (cEO) and 5.557 mg/ml (eEO), respectively (Table 6).
Regarding the Minimum Bactericidal Concentration it can be inferred from the results obtained that the bactericidal effect of commercial thyme oil on S. enteritidis and S.paratyphi strains was evidenced at a concentration of 5.55 mg/ml and 4.625 mg/ml, respectively, while the experimental thyme oil was able to kill the microorganisms at concentrations corresponding to 7.41 mg/ml and 5.55 mg/ml, respectively, as depicted in Tables 7 and 8, respectively These findings are similar to those reported by another study, where it was found that S. Enteritidis was more resistant than S. typhimurium to the action of thymol and carvacrol obtained from oregano's EO (Boskovic et al. 2015). Likewise, another approach also found comparable observations regarding the increased sensitivity of S. typhimurium compared to S. Enteritidis to the EOs of various thyme chemotypes (Rota et al. 2008).
The antimicrobial mechanism of carvacrol and thymol, which are the two main components of our EOs, is based on their ability to disintegrate the external membrane of Gram-negative bacteria due to loss of proton gradient and hence reduction in ATP synthesis (Tajkarimi et al. 2010).    Table 8 Assays In all cases it was observed that T5 treatment (50% cEO), equivalent to a concentration of 462.5 mg/ml, achieved the highest growth inhibition of S. paratyphi. This reduction was constant for 72 hours, reaching 12.68%, 11.45% and 10.88% at temperatures of 10 °C, 4 °C and 0 °C, respectively ( Fig. 3). On the other hand, treatments T3 and T4 (equivalent to 30% and 40% of cEO) significantly reduced the growth of S. paratyphi at all temperatures during the first 24 hours, and then stabilize the population for the following 48 hours, achieving a diminution of observational units from approximately 7-8% in all cases (Fig. 3), without finding statistically significant differences between these treatments. From this analysis, it could be considered that cEO at a concentration of 50% behaves as a medium potency bactericidal agent when applied on a meat matrix, while at concentrations between 30% and 40% the bactericidal effect is maintained during the first 24 hours and then reduced to a bacteriostatic agent.
The effect of the eEO on the growth of S. paratyphi was lower in all cases compared to its counterpart. Only treatment T10 (50%) managed to reduce proliferation at temperatures of 0 °C and 4 °C to 72 hours in a range of 7-8%, whereas T8 and T9 reduced growth by around 5.5% regardless of temperature. eEO can be considered as a medium to low-power bactericidal agent that maintains its bactericidal character for 72 hours (Fig. 3). The above results are congruent with the death rate values obtained for each treatment shown in Table 9. Table 9 Death rate of S. paratyphi for the most efficient treatments evaluated on chilled beef slice.
Although the properties of spice and herb essential oils and their components have long been studied, the mechanism of action of antimicrobial EO components is not known in detail (Burt 2004;Lambert et al. 2001). Given the large number of chemical components present in EOs, it is likely that the antibacterial activity is not consequence of a single mechanism, but they may rather act on several cellular targets (Carson et al. 2002;Skandamis et al. 2001). However, more specific studies evaluating the effect of temperature on the diffusion capacity are needed to clarify these assumptions.
On the other hand, the antimicrobial effect of both oils on S. Enteritidis revealed that the highest decrease in growth was reached with treatment T5 (50% cEO), however, and unlike what was observed for S. Paratyphi, T3 and T4 treatments also reduced the population of S. Enteritidis to concentrations similar to T5 at all temperatures, without statistically significant differences (data not shown). The final reduction values were similar at temperatures of 0 °C and 4 °C (between 4% and 5%), but higher than those observed at 10 °C (< 3.5%) (data not shown). In all cases, the reduction of S. Enteritidis was evidenced during the first 24 hours, remaining stable for the next 48 hours. This suggests that treatment of S. Enteritidis with cEO appears to act as a bactericidal agent only in the first 24 hours of application and then reduced to a bacteriostatic agent.
All eEO treatments had minimal effect on S. Enteritidis, as the maximum reduction achieved (T10 at 10 °C) does not exceed 2.3% compared to the initial inoculated population. In this case, it could be indicated that this EO has sparse or no effect on S. enteritidis when applied as a food preservative.
The above results were reflected in the S. enteritidis mortality rate values obtained for each treatment, which are shown in Table 10. These data are comparable to those reached with this oil for S. paratyphi, since the highest death rate was achieved with the T5 treatment at 0 °C, a value that was 1.6 times lower than the highest death rate observed in S. paratyphi under the same approach (0.0092 log10 ufc/g*h vs 0.0145 log10 ufc/g*h, respectively). Table 10 Death rate of S. Enteriditis for the most efficient treatments evaluated on chilled beef slice.
According to the above, based on the results obtained from our in vitro and in vivo tests and in accordance to previous work (Tajkarimi et al. 2010), the two essential oils of thyme (commercial and experimental) can be used as agents that control or prevent the growth of pathogenic microorganisms, in particular Salmonella spp. rather than as preservatives (those that prevent or avoid processes of food alteration). The effectiveness of the essential oil of various spices, including thyme, depends on various factors such as pH, storage temperature, amount of oxygen, oil concentration and active components and the EOs applied directly to foods is decreased compared to in vitro activity tests, due to the influence of various factors that have not been tested under the same conditions (Stoicov et al. 2009;Tajkarimi et al. 2010). In addition to the above factors, it should be noted a high fat content in meat products can reduce the action of essential oils (Burt 2004).
All the above data are clear evidence that S. enteritidis is more resistant to the effect of EO than S.
paratyphi, and even with the low activity shown by the eEO, the application of these essential oils can prevent or at least control the growth of these food-borne pathogens.

Conclusions
As a result of the present research, the chemical composition of each essential oil was evaluated, finding 19 components in cEO and 39 in eEO, with thymol (45.3%) and p-cimene (30.3%) being the major components in cEO, and to a lesser extent y-terpineneh (8.4%), while the eEO exhibited pcimene (43.8%) as the main molecule and in lower concentrations trans-β-caryophyllus (6.2%) and thymol (5.7%).
We were also able to determine that there is antibacterial activity by the two oils evaluated in the Salmonella enteritidis and Salmonella paratyphi strains, observing a greater sensitivity of the former to the action of the oils. It was also found that commercial oil was more effective compared to oil extracted on a laboratory scale, due in part to its higher purity, type and concentration of chemical components, the flowering state of the plant at the time of collection, and perhaps due to the extraction method used. From this study, we were able to extract the evidence related to the greater antibacterial activity in vivo for commercial oil, which was obtained at a concentration of 465 mg/ml, reducing the growth of Salmonella paratyphi by more than 10% and that of Salmonella enteritidis by 3.35%.
It was also observed that thyme EO behaves in vitro as an antibacterial agent that decreases the presence of Salmonella enteritidis and Salmonella paratyphi, while in vivo testing in raw chilled meat shows it as a bactericidal-bacteriostatic agent, as it decreases the initial load of Salmonella spp. but does not inhibit its growth over time. Finally, it is noted that Salmonella enteritidis is more resistant to the effect of thyme essential oil than Salmonella paratyphi, but that even with the lower activity exhibited by eEO, the use of these essential oils could prevent or at least control the growth of these food-borne pathogens and thus increase the shelf life of foods, especially raw meats.   S paratyphi growth kinetics during 72 hours in chilled beef slices at different temperatures.

Abbreviations
Only the most effective treatments are shown.