Essential Oils from Thymus capitatus and Thymus algeriensis as Antimicrobial Agents to Control Pathogenic and Spoilage Bacteria in Ground Meat

Institute Pasteur of Tunis, BP 74, 13 Place Pasteur, Belvédére, 1002 Tunis, Tunisia University of Tunis El Manar, 2092 Tunis, Tunisia Laboratory Analysis of Food Quality, National Institute of Agronomy of Tunisia, University of Carthage, Tunis, Tunisia Laboratory of Protein Engineering and Bioactive Molecules (LIP-MB), National Institute of Applied Sciences and Technology (INSAT), Centre Urbain Nord, University of Carthage, Tunis, Tunisia Medical Laboratory Sciences Department, College of Applied Medical Sciences, King Khalid University, Abha, Saudi Arabia


Introduction
Meat and meat products represent one of the most perishable foodstuffs [1] due to their complex composition which consists of proteins, saturated and unsaturated lipids, carbohydrates, vitamins, pigments, high water content, and moderate pH [2,3]. A large amount of spoiled meat has to be discarded engendering significant economic losses. According to the European Regulation (EC) No 178/ 2002 [4], spoiled meat is considered as unsafe, unsuitable food for human consumption and forbidden by the law. e mechanisms responsible for meat spoilage are related to microbial growth, lipid oxidation, and enzymatic autolysis. e breakdown of fats, proteins, and carbohydrates in meat leads to the formation of off-odors, off-flavors, and slime formation, rendering meat unacceptable for the consumer [5][6][7]. e presence of pathogenic bacteria such as E. coli, Salmonella, and S. aureus in minced meat and contact surface samples can cause serious health risks [8]. Microorganisms which are generally responsible for meat spoilage are Pseudomonas spp., Enterobacteriaceae, and Brochothrix thermosphacta [9]. Pseudomonas causes meat and meat product spoilage and develops repulsive characteristics as putrefaction of proteins and lipids with changes in pH continue [10,11].
Additionally, grinding has several detrimental effects on meat by increasing the surface area exposed to air and bacterial contaminations [12]. It increases losses of intracellular reductants as well as polyunsaturated fat, leading to deterioration of meat and the warmed-over flavors [13,14].
To extend the shelf life and decrease bacterial growth on meat, the common method used is refrigeration. However, lower temperatures might also modify the composition of the microbiota present on meat, such as psychrotrophic bacteria like Pseudomonas spp., which could grow at low temperature [1]. For this, it is an important challenge to find a solution to prevent bacterial contamination of meat.
Many strategies are being used to control the growth of pathogenic and spoilage bacteria and prolong the shelf life of meat. Since ancient times, plants and plant extracts have been used as flavoring agents in the food processing industry; they also exhibit some antibacterial, antifungal, and antioxidant properties [15]. Several aromatic plants, essentially rosemary, garlic, lavender, leek, olive leaf, onion, oregano, pepper, peppermint, sage, and Satureja montana, are being added to meat and meat products [16].
yme oil is one of the top 10 EOs used as a natural preservative in food [17]. e genus ymus L. is a member of the Lamiaceae family and contains about 215 species, particularly prevalent in the Mediterranean area [18]. yme species are aromatic plants widely used in Tunisia and they are well known for their antispasmodic, antimicrobial, expectorant, and antioxidant activities.
Several in vitro studies have reported the efficiency of plant EOs against food-borne pathogens, whilst few published papers have studied the antibacterial effect of ymus EOs on pathogen growth, in meat.
us, the purpose of the current work is to evaluate the antioxidant and the antimicrobial activities of ymus capitatus (T. capitatus) and ymus algeriensis (T. algeriensis) EOs against pathogenic bacteria and the impact of different concentrations of such EOs on the proliferation of spoilage bacterium P. aeruginosa at two contamination levels of 10 5 CFU/g and 10 8 CFU/g at 4°C.

Extraction of the Essential Oils.
e aerial parts of T. capitatus and T. algeriensis were collected from Zaghouan region (north of Tunisia) in June 2018. e freshly cut plants were dried for two weeks, in the shade, at room temperature. ey were grounded into powder, followed by hydrodistillation in a Clevenger-type apparatus for 3 hours. e EOs were extracted, dried over anhydrous sodium sulphate (Na 2 SO 4 ), filtered, and then stored in the dark at 4°C.

Free Radical Scavenging Assay.
e DPPH (2, 20diphenyl-1-picryl hydrazyl) radical scavenging capacity was measured according to the method described by Boulanouar et al. [19]. One ml of each concentration of the EO extract (200, 300, 400, and 500 μg/mL) was mixed with 250 μl of 0.2 mM methanolic DPPH solution. A negative control was prepared by mixing the same amounts of methanol and DPPH solution.
e mixture was shaken vigorously and incubated for 30 min, in the dark, at room temperature. e absorbance was then measured at 517 nm using a UV spectrophotometer, and the percentage of activity inhibition (I%) was calculated by the following formula:

Determination of MIC and MBC.
e minimum inhibitory concentrations (MICs) and the minimum bactericidal concentrations (MBCs) of T. capitatus and T. algeriensis were determined using the medium dilution method with minor modifications (NCCLS). e tested microorganisms were cultured at 37°C and diluted to approximately 10 6 CFU/ml, the negative control containing only the tested bacteria. e inoculated plates were inverted and incubated at 37°C for 24 h. e MIC was defined as the lowest concentration of EO at which no visible growth of bacteria is shown. e plates showing a concentration of EO greater than or equal to MIC were incubated at 37°C for further 24 h. e concentration at which no visible growth is noticed was defined as the MBC.
e experiment was carried out in triplicate.

Inhibitory Effect of EO against Pseudomonas aeruginosa Inoculated in Minced Beef Meat.
e procedure reported by Careaga et al. [21] was followed with some slight modifications to study the inhibitory effect of EOs.

Preparation of the Meat Model.
Four kilos and 500 g of fresh beefsteaks were obtained from a local meat supermarket. Meat samples were collected and transported for analysis in an insulated cooler. Each piece of meat was plunged in boiling water for 5 min to reduce the number of microorganisms attached to the beef muscle surface, which was eliminated with a sterile knife under aseptic conditions.

Treatment of Minced Beef Meat.
To evaluate the antimicrobial activity of T. capitatus EO against bacteria in meat samples, pieces of meat were minced in a sterile grinder with 19 cm in diameter, and portions of 22 ± 0.1 g were put into a high-density polyethylene bag.
Decimal dilutions were prepared from a fresh culture of 24 h. For each dilution, the optical density at 620 nm and the CFU were determined by subculturing on agar. e data obtained were used to make a calibration. us, the initial inocula (10 5 CFU P. aeruginosa and 10 8 CFU P. aeruginosa) were obtained based on a spectrophotometer reading.
Halves of the meat samples were inoculated with 10 5 CFU P. aeruginosa/g of beef and the remaining halves with 10 8 CFU P. aeruginosa/g of beef. en, the samples were treated with different concentrations (0.01, 0.05, 1, and 3%) of T. capitatus or T. algeriensis EO, dissolved in 10% DMSO and homogenized in a stomacher for 5 min. For the control sample, the EO extract was replaced by DMSO. Finally, all bags containing the meat samples were stored at 4°C and examined every three days, during 15 days of storage [20].
e experiment was carried out in triplicate.

Bacterial
Enumeration. P. aeruginosa count was done by adding 9 ml of BHI broth to 1 g meat sample placed in a polyethylene bag. Bacterial strain enumeration was determined by the plate colony count technique. For this, a series of dilutions was performed with physiological saline solution, and 100 µL of each sample dilution was spread onto the surface of Pseudomonas agar F "King's Medium B" plates, followed by incubation at 37°C for 24 hours. e obtained results were expressed as log 10 CFU/g of meat.

Sensory
Analysis. e sensory test was carried out at the Laboratory of Epidemiology and Veterinary Microbiology, Institute Pasteur of Tunis, Tunisia. e day before the event tasting. meat samples were thawed in a refrigerator at 4°C. Minced beef meat samples were cooked with no added salt and divided into samples of 10 g. e samples should be of uniform size. ese were placed in aluminum trays covered with aluminum foil identified and put in a conventional oven.
e beef meat samples were warmed before the evaluation, covered with aluminum foil. and presented to the panelists. Twelve trained panelists, comprised student and employees of the Laboratory of Epidemiology and Veterinary Microbiology, Institute Pasteur of Tunis, were served five meat samples: 1, control; 2, treated with 1% T. capitatus; 3, treated with 3% T. capitatus; 4, treated with 1% T. algeriensis; 5, treated with 3% T. algeriensis, with water and an unsalted snack in between to remove the remaining flavor. Coffee was also served to neutralize their noses between samples. e panel evaluated each sample in triplicate. Judges were requested to evaluate the cooked beef meat (offered in a randomized order) with a 3-digit code. Each attribute was scored on a scale of 10 cm for each characteristic: taste, color, tenderness, flavor, juiciness, and odor. e attributes were ranged from the lowest intensity of each trait to the highest. ey measured overall acceptability in beef meat samples using the 9-point hedonic scale (1: dislike extremely, 2: dislike very much, 3: dislike moderately, 4: dislike slightly, 5: neither like nor dislike, 6: like slightly, 7: like moderately, 8: like very much, and 9: like extremely) [22].

Statistical Analysis.
For each test, the results were presented as mean ± SD of three independent samples. e inhibitory concentration 50% (IC 50 values) for antioxidant activities was calculated by a nonlinear regression analysis using GraphPad Prism, version 5.0. e in situ antibacterial activity was also performed using GraphPad Prism, version 5.0. e results were analyzed by two-way analysis of variance (ANOVA) to evaluate different antimicrobial treatment effects during the time of storage of 0, 3, 6, 9, 12, and 15 days. e statistical data analysis processed by ANOVA was calculated at a significance level of p < 0.05 using Bonferroni's multiple comparison tests.
CMI, CMB, and sensory data were analyzed by one-way ANOVA with the general linear model procedure of SAS (9.1). e residual mean square error was used as the error term. Means were separated using Duncan's test with a significance level of p < 0.05 (SAS, 9.1).

Antioxidant Activity: Free Radical Scavenging Assay.
e EOs of herbs possess antioxidant properties that improve the shelf life of food.
us, incorporation of EOs directly into food helps preserving it from oxidation phenomena [23]. In this context, it was shown that the antioxidant activity of EO of T. capitatus exhibits higher antiradical activity with an IC 50 value of 213.53 µg/ml than that of T. algeriensis showing an IC 50 value of 861.12 µg/ml, butylated hydroxytoluene (BHT) presenting an IC 50 value of 30 ± 0.01 µg/ml (Table 1). ese results agreed with those of Amarti et al. [26], who showed that T. capitatus EOs possess strong antioxidant activities with IC 50 equal to 69.04 µg/ml. However, the used T. algeriensis EO demonstrated weaker antioxidant effect with IC 50 equal to 745 µg/ml. e antiradical activity of T. capitatus EO could be attributed to its high content of carvacrol (88.89%). On the contrary, T. algeriensis EO presented a weaker activity because of its poor content in phenolic compounds. In fact, a highly positive link between phenolic compounds and antioxidant activity was provided in this study, which is in agreement with other reported findings [27][28][29]. Based on these results, T. capitatus can be used as a natural antioxidant in food or for pharmaceutical applications.
In addition, the results indicated that the MICs and MBCs of T. capitatus EO are in the range of 0.006-0.012% and 0.012-0.025%, respectively, while those of T. algeriensis EO ranged between 0.020 and 0.025% and 0.05%, respectively. ese results are similar to those presented by Amarti et al. [30], which reported that T. capitatus EO from Morocco, mainly composed of carvacrol (70.92%), inhibits the growth of E. coli and S. aureus at a concentration of 1/2000 (v/v). A previous study, carried out in Tunisia by Aouadhi et al. [27], showed that the T. capitatus plant from Bizerte, containing thymol (81.49%), exhibits significantly higher antibacterial activity than T. capitatus from Sousse that contains thymol (69.95%), with MIC values ranging between 0.025 and 0.8%. Our results indicated that the used T. capitatus EO, collected from Zaghouan, exhibits the strongest antibacterial effect due to its high content of carvacrol (88.98%) [24,31,32].
Findings from the present study indicated that T. capitatus EO has a very significant (p < 0.0001) antibacterial action against E. coli, S. typhimurium, and S. aureus, whereas L. monocytogenes and P. aeruginosa seem to be the least sensitive. ese results are in accordance with previous studies revealing that the weakest activity of T. pectinatus EO is observed against P. aeruginosa [31]. e EO extracted from the T. algeriensis plant exhibited a moderate antimicrobial effect against most tested bacteria, without any significant difference (P > 0.05) between them. Our results showed that both EOs did not have selective antibacterial activity on the basis of the cell wall differences of bacterial microorganisms. ese findings are in agreement with previous works carried out with several ymus species [33].
e mechanism of action of EOs and phenolic compounds on microorganisms has not been elucidated; it is generally proved that these not only attack the cytoplasmic membrane, thus destroying its permeability and releasing intracellular constituents, but also could cause membrane dysfunction with respect to electron transport, nutrient absorption, nucleic acid synthesis, and ATPase activity. is could be the result of the alteration of various enzymatic systems, including those involved in the production of energy and the synthesis of structural components [34].
Besides, our results showed that the antibacterial properties could be attributed to the high percentages of linalool (17.62%) and camphor (13.82%) of T. algeriensis EO [25,35]. In this regard, the research of Liu et al. [36] showed a good antibacterial activity of linalool against P. aeruginosa with MIC and MBC values in the range of 431 and 832 μg/ml, respectively. Likewise, the study carried out by Rezzoug et al. [37] showed that the EO of T. algeriensis grown in the Atlas Algerian Sahara and composed of linalool (1.2%) inhibits P. aeruginosa growth with a MIC value of 512 µg/ml, while that of Moroccan T. algeriensis, composed of camphor (27,7%), showed a weak antibacterial effect against E. coli and S. aureus, with a MIC value of 1/100 [38]. It is worth noting that the chemical compositions of the EOs of T. algeriensis from Algeria and Morocco are completely distinct from that of Zaghouan, used in the present work.
Our study revealed that the antimicrobial activity of P. aeruginosa is significantly (p < 0.0001) more sensitive to EO of T. capitatus than that of T. algeriensis. ese results are consistent with the previous report [39].

Antibacterial Efficacy of T. capitatus Essential Oil against Pseudomonas aeruginosa in Minced Beef Meat.
To study the antibacterial effect of T. capitatus EO, depending on the inoculum concentrations, two different loads of P. aeruginosa (10 5 CFU/g and 10 8 CFU/g) were used before treating minced beef meat with EOs. As shown in Figure 1,  [24,25]. IC 50 : concentration of essential oil required for 50% of inhibition. MIC: minimal inhibitory concentration. MBC: minimal bactericidal concentration. A and B: different letters in the same row indicate significant differences (p < 0.05). a and b: different letters in the same column indicate significant differences (p < 0.05).
an increase in the Pseudomonas count was detected, from the first day of incubation, when a high inoculum (10 8 CFU/g) was used. P. aeruginosa count then increased by 2.41 log 10 CFU/g and reached 10.42 log 10 CFU/g, fifteen days later. In contrast and according to the results shown in Figure 2, lower inoculum (10 5 CFU/g) induced an exponential increase of P. aeruginosa growth by 3.04 log 10 , reaching 8.05 log 10 CFU/g at the end of the incubation period.
us, it can be assumed that, at high inoculum concentration, bacteria growth is limited due to a deficiency in nutrients, as described by Mytle et al. [40].
In this study, we have tested the antimicrobial effect of both T. capitatus and T. algeriensis EOs against one of meat food-borne pathogens (P. aeruginosa), inoculated in minced beef meat, at different concentrations (0.01, 0.05, 1, and 3%). As demonstrated in Figure 1 and Table 1, the concentrations employed for the in situ tests are higher than those used for the MIC and MBC tests. is could be due to intrinsic and extrinsic factors (proteins, fat, temperature, and oxygen limitation) which may influence the behavior of bacteria in food ecosystems and their interactions with the antimicrobial agents [21]. In fact, high protein and fat contents in meat are known to solubilize phenolic compounds, decreasing their sensitivity to the antimicrobial action. It is worth noting that the antimicrobial effects of the spices are lower in food systems than in microbiological media [41]. In addition, some studies have reported that many plant extracts and EOs are used to decrease food pathogens in meat products [42].
On the contrary, meat samples inoculated with a low bacterial contamination level (10 5 CFU/g), in the presence of low concentrations of T. capitatus EOs (0.01% and 0.05%), showed approximately 5.73 and 5.44 log 10 CFU/g of P. aeruginosa, respectively, as compared to 8.05 log 10 CFU/g shown for the control untreated samples, at the end of the storage period (15 days). Moreover, meat samples inoculated with a high level of bacterial contamination (10 8 CFU/g) presented the same trend of growth with a reduction of 2 log 10 CFU/g. In fact, Pseudomonas growth titers achieved 8.82 and 8.57 log 10 CFU/g in the presence of 0.01% and 0.05% of T. capitatus EO, respectively, as compared to 10.42 log 10 CFU/g of the control untreated sample. Treatment based on low bacterial inoculum in the presence of a concentration of 1% of T. capitatus EO was able to significantly decrease P. aeruginosa growth by 4.17 log 10 CFU/g, reaching 3.88 log 10 CFU/g after 15 days of storage at 4°C.

Antibacterial Efficacy of T. algeriensis Essential Oil against Pseudomonas aeruginosa in Minced Beef Meat. As shown in
On the contrary, a concentration of 1% of T. algeriensis EO allowed a high bacteriostatic effect, leading to a significant decrease in bacterial titers (p < 0.0001) of 3.67 log 10 CFU/g for the low contamination level as compared to 2.86 log 10 CFU/g for the high contamination level, by the end of the storage days. An identical trend was observed after the addition of 3% of T. algeriensis EO, leading to a significant reduction in bacterial titers (p < 0.0001) of 4.75 log 10 CFU/g for low initial inoculum and 3.76 log 10 CFU/for high initial inoculum. erefore, it is important to note that both used EOs are effective and able to inhibit the growth of P. aeruginosa. Hence, increasing the concentrations of EOs to the treated samples allowed a gradual decrease in bacteria counts. is is in accordance with the findings of Emiroglu et al. [43], which revealed that Pseudomonas spp. was reduced in the ground beef patties when coated with thyme and oregano EOs. In contrast, the report of Ouattara et al. [44] did not show any significant effect of thyme oil on the growth of meat spoilage microorganisms such as Pseudomonas fluorescens. e antibacterial effect of T. capitatus EO reported in the present study was significantly stronger than that of T. algeriensis EO, for both meat contamination levels.
We have thus examined the impact of the initial inoculum on the antibacterial effects of T. capitatus and T. algeriensis EOs on the growth of P. aeruginosa. It was shown that both T. capitatus and T. algeriensis EOs induce a rapid antibacterial activity against a low inoculum (10 5 CFU/ g) of P. aeruginosa. In fact, our results demonstrated that the antibacterial effect of both EOs appears to be significantly weak when used at low concentrations but becomes more pronounced at higher EO concentrations, even in the presence of high inoculum. ese findings are in accord with those reported by Udekwu et al. [45] who stated that bacteria may appear susceptible to bioactive molecules when the inoculum is of a standard level (10 5 CFU/ml) but resistant if the inoculum is increased. Accordingly, Bulitta et al. [46] proved that killing P. aeruginosa is 23-fold slower at a concentration of 10 9 CFU/ml and 6-fold slower at 10 8 CFU/ ml than at 10 6 CFU/ml. Besides, our study did not show any immediate lethal (bactericidal) effect against the Pseudomonas population when T. capitatus and T. algeriensis EO are applied. In addition, T. capitatus EO did exhibit more pronounced antimicrobial activity than T. algeriensis EO.
Both EOs showed rapid antibacterial activities against a low initial inoculum of 10 5 CFU/g of P. aeruginosa and weak and delayed antibacterial activities at a high concentration of initial inoculum of 10 8 CFU/g. is may be explained by the fact that the EO activities depend on the type, the composition, and the concentration of used EO, as well as the dose of targeted microorganisms present in meat.

Sensory Analysis.
e sensory evaluation results are reported in Figures 5 and 6 Figure 3: Time-related survival, at 4°C, of the high meat contamination level (10 8 CFU/g) of P. aeruginosa, following treatment with increasing concentrations of T. algeriensis EO. e results represent the means of three replicate experiments, and error bars represent the standard error of the mean. Statistical significance differences: * p < 0.05 (significant), * * p < 0.01 (very significant), and * * * p < 0.001 and * * * * p < 0.0001 (extremely significant). CFU: colony-forming unit; TC: ymus algeriensis. meat less tender. In terms of juiciness and color, there were no significant differences among samples treated with T. capitatus EO and samples treated with T. algeriensis EO and the control. e overall acceptability indicated that samples treated with T. capitatus EO were more acceptable (p < 0.05) than samples treated with T. algeriensis EO and the control. Generally, the present findings asserted that the application of T. capitatus EO has an important place in the improvement of the characteristic odor, taste, and flavor of minced beef meat. ese results are in agreement with those obtained by Shalaby et al. [47], which reported that using olive leaf extracts as a natural preservative on minced beef improves the sensory attributes.

Conclusion
is study showed an interesting antioxidant effect using the DPPH assay and an interesting antimicrobial profile shown by MIC and MBC of T. capitatus EO. e results of "in situ" antibacterial activity confirmed those obtained by "in vitro" tests. It was also shown that T. capitatus EO is more effective than T. algeriensis EO, inhibiting Pseudomonas growth in inoculated minced beef meat at high concentrations (1% and 3%). In addition, at the low level of contamination, both EOs exerted a rapid and a more pronounced antibacterial effect, as compared to the high level of contamination. However, based on the sensory data, minced beef meat treated with T. capitatus EO was most acceptable to the panelists. e results represent the means of three replicate experiments, and error bars represent the standard error of the mean. Statistical significance differences: * p < 0.05 (significant), * * p < 0.01 (very significant), and * * * p < 0.001 and * * * * p < 0.0001 (extremely significant). CFU: colony-forming unit; TC: ymus algeriensis.   Means in the same row followed by the same letter are not significantly different (p > 0.05). Acceptance was evaluated using a 9-point scale, where 1 � extremely dislike and 9 � extremely like. erefore, T. capitatus EO could be used as a safe and a natural biopreservative for the improvement of microbiological and sensory quality of beef meat.

Data Availability
All the data used to support the findings of this study are approved and included within this article.

Conflicts of Interest
e authors declare that there are no conflicts of interest regarding the publication of this paper.