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

Authors: Asma Jayari; Ahlem Jouini; Hager Boukhris; Safa Hamrouni; Chokri Damergi; Sami Ben Hadj Ahmed; Abderrazek Maaroufi
Journal: Journal of Food Quality
 (2021)

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## Abstract

The antibacterial effects of essential oils (EOs) extracted fromThymus capitatus and Thymus algeriensis were assessed and evaluated against four pathogenic bacteria (Escherichia coli (ATCC 25922), Listeria monocytogenes (ATCC 19118), Staphylococcus aureus (ATCC 25923), and Salmonella typhimurium (ATCC 1402)) and one spoilage bacterium (Pseudomonas aeruginosa (ATCC 27853)). Both investigated EOs presented significant antimicrobial activities against all tested bacteria with a greater antibacterial effect of T. capitatus EO. In fact, the results indicated that the minimum inhibitory concentrations (MICs) and the minimum bactericidal concentrations (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 0.012 and 0.025% and 0.05%, respectively. Furthermore, the inhibitory effects of both EOs were appraised against the spoilage bacterium P. aeruginosa, inoculated in minced beef meat, at two different loads (105 and 108 CFU) mixed with different concentrations of EOs (0.01, 0.05, 1, and 3%) and stored at 4°C for 15 days. The obtained data demonstrated that the antibacterial effect of tested EOs varies significantly in regard to the levels of meat contamination and the concentrations of EOs. In fact, in the presence of 0.01 and 0.05% of oils, a decrease in bacterial growth p<0.01 was observed; but, such an effect was more pronounced in the presence of higher concentrations of EOs (1 and 3%), regardless the level of meat contamination. Besides, at the low contamination level, both EOs exerted a rapid and a more pronounced antibacterial effect, as compared to the high contamination level. The results illustrated the efficacy of both EOs as preservatives in food against well-known pathogens of food-borne diseases and food spoilage, particularly in P. aeruginosa in beef meat. As regards sensory evaluation, the presence of T. capitatus EO proved to improve the sensory quality of minced beef meat.


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## Body

## 1. 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.The mechanisms responsible for meat spoilage are related to microbial growth, lipid oxidation, and enzymatic autolysis. The 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 [5–7]. The 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 likePseudomonas 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].Thyme oil is one of the top 10 EOs used as a natural preservative in food [17]. The genus Thymus L. is a member of the Lamiaceae family and contains about 215 species, particularly prevalent in the Mediterranean area [18]. Thyme species are aromatic plants widely used in Tunisia and they are well known for their antispasmodic, antimicrobial, expectorant, and antioxidant activities.Severalin vitro studies have reported the efficiency of plant EOs against food-borne pathogens, whilst few published papers have studied the antibacterial effect of Thymus EOs on pathogen growth, in meat.Thus, the purpose of the current work is to evaluate the antioxidant and the antimicrobial activities ofThymus capitatus (T. capitatus) and Thymus 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 105 CFU/g and 108 CFU/g at 4°C.


## 2. Materials and Methods

### 2.1. Extraction of the Essential Oils

The aerial parts ofT. capitatus and T. algeriensis were collected from Zaghouan region (north of Tunisia) in June 2018. The freshly cut plants were dried for two weeks, in the shade, at room temperature. They were grounded into powder, followed by hydrodistillation in a Clevenger-type apparatus for 3 hours. The EOs were extracted, dried over anhydrous sodium sulphate (Na2SO4), filtered, and then stored in the dark at 4°C.


### 2.2. Free Radical Scavenging Assay

The DPPH (2, 20-diphenyl-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. The mixture was shaken vigorously and incubated for 30 min, in the dark, at room temperature. The absorbance was then measured at 517 nm using a UV spectrophotometer, and the percentage of activity inhibition (I%) was calculated by the following formula: (I%) = [(A0 − At/A0) × 100], where A0 is the absorbance of the control sample (without EO) and At is the absorbance of the EO with DPPH at 30 min.The EO concentration providing anI% of 50 (IC50) is calculated from the regression equation prepared from the concentrations of the EO and the inhibition percentages. The experiment was carried out in triplicate.


### 2.3. Microorganisms and Growth Conditions

The bacteria used in the present study were obtained from the culture collections of ATCC and Institute Pasteur of Tunis. The strains ofL. monocytogenes (ATCC 19118) were cultivated in PALCAM Listeria agar (Biokar Diagnostics), S. aureus (ATCC 25923) in Baird-Parker (Biokar Diagnostics), E. coli (ATCC 25922) in Mac Conkey Sorbitol (Biolife), S. typhimurium (ATCC 1402) in Hektoen (Biolife), and P. aeruginosa (ATCC 27853) in Pseudomonas agar F (King’s Medium B) (Biolife), at 37°C. Working cultures were prepared by adding a loopful of each test bacterium to 5 ml of Luria–Bertani Medium (LB) (Oxoid Ltd., UK) and then incubated at 37°C for 18 h [20].


### 2.4. Determination of MIC and MBC

The minimum inhibitory concentrations (MICs) and the minimum bactericidal concentrations (MBCs) ofT. capitatus and T. algeriensis were determined using the medium dilution method with minor modifications (NCCLS). The tested microorganisms were cultured at 37°C and diluted to approximately 106 CFU/ml, the negative control containing only the tested bacteria.The inoculated plates were inverted and incubated at 37°C for 24 h. The MIC was defined as the lowest concentration of EO at which no visible growth of bacteria is shown. The plates showing a concentration of EO greater than or equal to MIC were incubated at 37°C for further 24 h. The concentration at which no visible growth is noticed was defined as the MBC. The experiment was carried out in triplicate.


### 2.5. Inhibitory Effect of EO againstPseudomonas aeruginosa Inoculated in Minced Beef Meat

The procedure reported by Careaga et al. [21] was followed with some slight modifications to study the inhibitory effect of EOs.


### 2.6. 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.


### 2.7. Treatment of Minced Beef Meat

To evaluate the antimicrobial activity ofT. 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. The data obtained were used to make a calibration. Thus, the initial inocula (105 CFU P. aeruginosa and 108 CFU P. aeruginosa) were obtained based on a spectrophotometer reading.Halves of the meat samples were inoculated with 105 CFU P. aeruginosa/g of beef and the remaining halves with 108 CFU P. aeruginosa/g of beef. Then, 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]. The experiment was carried out in triplicate.


### 2.8. 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. The obtained results were expressed as log10 CFU/g of meat.


### 2.9. Sensory Analysis

The sensory test was carried out at the Laboratory of Epidemiology and Veterinary Microbiology, Institute Pasteur of Tunis, Tunisia. The 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. The samples should be of uniform size. These were placed in aluminum trays covered with aluminum foil identified and put in a conventional oven. The 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. The 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. The attributes were ranged from the lowest intensity of each trait to the highest. They 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].


### 2.10. Statistical Analysis

For each test, the results were presented as mean ± SD of three independent samples. The inhibitory concentration 50% (IC50 values) for antioxidant activities was calculated by a nonlinear regression analysis using GraphPad Prism, version 5.0. The in situ antibacterial activity was also performed using GraphPad Prism, version 5.0. The 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. The 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). The residual mean square error was used as the error term. Means were separated using Duncan’s test with a significance level ofp<0.05 (SAS, 9.1).


## 2.1. Extraction of the Essential Oils

The aerial parts ofT. capitatus and T. algeriensis were collected from Zaghouan region (north of Tunisia) in June 2018. The freshly cut plants were dried for two weeks, in the shade, at room temperature. They were grounded into powder, followed by hydrodistillation in a Clevenger-type apparatus for 3 hours. The EOs were extracted, dried over anhydrous sodium sulphate (Na2SO4), filtered, and then stored in the dark at 4°C.


## 2.2. Free Radical Scavenging Assay

The DPPH (2, 20-diphenyl-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. The mixture was shaken vigorously and incubated for 30 min, in the dark, at room temperature. The absorbance was then measured at 517 nm using a UV spectrophotometer, and the percentage of activity inhibition (I%) was calculated by the following formula: (I%) = [(A0 − At/A0) × 100], where A0 is the absorbance of the control sample (without EO) and At is the absorbance of the EO with DPPH at 30 min.The EO concentration providing anI% of 50 (IC50) is calculated from the regression equation prepared from the concentrations of the EO and the inhibition percentages. The experiment was carried out in triplicate.


## 2.3. Microorganisms and Growth Conditions

The bacteria used in the present study were obtained from the culture collections of ATCC and Institute Pasteur of Tunis. The strains ofL. monocytogenes (ATCC 19118) were cultivated in PALCAM Listeria agar (Biokar Diagnostics), S. aureus (ATCC 25923) in Baird-Parker (Biokar Diagnostics), E. coli (ATCC 25922) in Mac Conkey Sorbitol (Biolife), S. typhimurium (ATCC 1402) in Hektoen (Biolife), and P. aeruginosa (ATCC 27853) in Pseudomonas agar F (King’s Medium B) (Biolife), at 37°C. Working cultures were prepared by adding a loopful of each test bacterium to 5 ml of Luria–Bertani Medium (LB) (Oxoid Ltd., UK) and then incubated at 37°C for 18 h [20].


## 2.4. Determination of MIC and MBC

The minimum inhibitory concentrations (MICs) and the minimum bactericidal concentrations (MBCs) ofT. capitatus and T. algeriensis were determined using the medium dilution method with minor modifications (NCCLS). The tested microorganisms were cultured at 37°C and diluted to approximately 106 CFU/ml, the negative control containing only the tested bacteria.The inoculated plates were inverted and incubated at 37°C for 24 h. The MIC was defined as the lowest concentration of EO at which no visible growth of bacteria is shown. The plates showing a concentration of EO greater than or equal to MIC were incubated at 37°C for further 24 h. The concentration at which no visible growth is noticed was defined as the MBC. The experiment was carried out in triplicate.


## 2.5. Inhibitory Effect of EO againstPseudomonas aeruginosa Inoculated in Minced Beef Meat

The procedure reported by Careaga et al. [21] was followed with some slight modifications to study the inhibitory effect of EOs.


## 2.6. 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.


## 2.7. Treatment of Minced Beef Meat

To evaluate the antimicrobial activity ofT. 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. The data obtained were used to make a calibration. Thus, the initial inocula (105 CFU P. aeruginosa and 108 CFU P. aeruginosa) were obtained based on a spectrophotometer reading.Halves of the meat samples were inoculated with 105 CFU P. aeruginosa/g of beef and the remaining halves with 108 CFU P. aeruginosa/g of beef. Then, 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]. The experiment was carried out in triplicate.


## 2.8. 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. The obtained results were expressed as log10 CFU/g of meat.


## 2.9. Sensory Analysis

The sensory test was carried out at the Laboratory of Epidemiology and Veterinary Microbiology, Institute Pasteur of Tunis, Tunisia. The 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. The samples should be of uniform size. These were placed in aluminum trays covered with aluminum foil identified and put in a conventional oven. The 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. The 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. The attributes were ranged from the lowest intensity of each trait to the highest. They 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].


## 2.10. Statistical Analysis

For each test, the results were presented as mean ± SD of three independent samples. The inhibitory concentration 50% (IC50 values) for antioxidant activities was calculated by a nonlinear regression analysis using GraphPad Prism, version 5.0. The in situ antibacterial activity was also performed using GraphPad Prism, version 5.0. The 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. The 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). The residual mean square error was used as the error term. Means were separated using Duncan’s test with a significance level ofp<0.05 (SAS, 9.1).


## 3. Results and Discussion

### 3.1. Antioxidant Activity: Free Radical Scavenging Assay

The EOs of herbs possess antioxidant properties that improve the shelf life of food. Thus, 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 IC50 value of 213.53 µg/ml than that of T. algeriensis showing an IC50 value of 861.12 µg/ml, butylated hydroxytoluene (BHT) presenting an IC50 value of 30 ± 0.01 µg/ml (Table 1). These results agreed with those of Amarti et al. [26], who showed that T. capitatus EOs possess strong antioxidant activities with IC50 equal to 69.04 µg/ml. However, the used T. algeriensis EO demonstrated weaker antioxidant effect with IC50 equal to 745 µg/ml.Table 1
Antioxidant and antimicrobial activities ofThymus capitatus and Thymus algeriensis essential oils.
Thymus capitatus essential oilsThymus algeriensis essential oilsMain essential oil compounds∗Carvacrol (88.98%), thymol (0.51%),p-cymene (1.14%), and α-terpinene (0.40%)Linalool (17.62%), camphor (13.82%), terpinen-4-ol (6.80%),α-terpineol (6.41%), and α-terpenyl acetate (6.27%)Antioxidant activities IC50 (µg/ml)213.53861.12Antimicrobial activities (%)MICMBCMICMBCE. coli0.006Ba0.012Ba0.025Aa0.05AaS. aureus0.006Aa0.012Ba0.020Ba0.05AaL. monocytogenes0.012Bb0.025Bb0.025Aa0.05AaP. aeruginosa0.012Bb0.025Bb0.025Aa0.05AaS. typhimurium0.006Aa0.012Ba0.025Ba0.05Aa∗The detailed results were reported by El Abed et al. and Ahmed et al. [24, 25]. IC50: 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.The antiradical activity ofT. 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 [27–29]. Based on these results, T. capitatus can be used as a natural antioxidant in food or for pharmaceutical applications.


### 3.2.In Vitro Antibacterial Effect of Thyme Essential Oil

According to the results of MIC and MBC, illustrated in Table1, both investigated EOs’ activities presented an antimicrobial activity against all tested bacteria with a greater antibacterial effect of T. capitatus EO. As illustrated in a previous study of El Abed et al. [24], the EOs of T. capitatus, harvested from Zaghouan region, presented 19 compounds with the presence of several bioactive compounds, including carvacrol (88.98%), thymol (0.51%), p-cymene (1.14%), and α-terpinene (0.40%). As reported by Ben Hadj Ahmed et al. [25], the EOs of T. algeriensis from Zaghouan region presented 39 compounds, with linalool as a major compound (17.62%), followed by camphor (13.82%), terpinen-4-ol (6.80%), α-terpineol (6.41%), and α-terpinyl acetate (6.27%).In addition, the results indicated that the MICs and MBCs ofT. 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. These 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 thatT. 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. These results are in accordance with previous studies revealing that the weakest activity of T. pectinatus EO is observed against P. aeruginosa [31]. The 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. These findings are in agreement with previous works carried out with several Thymus species [33].The 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. This 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%) ofT. 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 ofP. aeruginosa is significantly p<0.0001 more sensitive to EO of T. capitatus than that of T. algeriensis. These results are consistent with the previous report [39].


### 3.3. Antibacterial Efficacy ofT. capitatus Essential Oil against Pseudomonas aeruginosa in Minced Beef Meat

To study the antibacterial effect ofT. capitatus EO, depending on the inoculum concentrations, two different loads of P. aeruginosa (105 CFU/g and 108 CFU/g) were used before treating minced beef meat with EOs. As shown in Figure 1, an increase in the Pseudomonas count was detected, from the first day of incubation, when a high inoculum (108 CFU/g) was used. P. aeruginosa count then increased by 2.41 log10 CFU/g and reached 10.42 log10 CFU/g, fifteen days later. In contrast and according to the results shown in Figure 2, lower inoculum (105 CFU/g) induced an exponential increase of P. aeruginosa growth by 3.04 log10, reaching 8.05 log10 CFU/g at the end of the incubation period. Thus, 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].Figure 1
Time-related survival, at 4°C, of the high meat contamination level (108 CFU/g) of P. aeruginosa, following treatment with increasing concentrations of T. capitatus EO. The 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: Thymus capitatus.Figure 2
Time-related survival, at 4°C, of the low meat contamination level (105 CFU/g) of P. aeruginosa, following treatment with increasing concentrations of T. capitatus EO. The 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: Thymus capitatus.In this study, we have tested the antimicrobial effect of bothT. 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. This 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 (105 CFU/g), in the presence of low concentrations of T. capitatus EOs (0.01% and 0.05%), showed approximately 5.73 and 5.44 log10 CFU/g of P. aeruginosa, respectively, as compared to 8.05 log10 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 (108 CFU/g) presented the same trend of growth with a reduction of 2 log10 CFU/g. In fact, Pseudomonas growth titers achieved 8.82 and 8.57 log10 CFU/g in the presence of 0.01% and 0.05% of T. capitatus EO, respectively, as compared to 10.42 log10 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 log10 CFU/g, reaching 3.88 log10 CFU/g after 15 days of storage at 4°C.When beef meat was inoculated with a high concentration ofP. aeruginosa, in the presence of 1% of T. capitatus EO, a reduction in the bacterial load, from 7.03 to 3.39 log10 CFU/g, was obtained. Treatment with 3% of T. capitatus EO showed a greater antibacterial effect than in the presence of 1, 0.05, and 0.01%. Moreover, a 3% concentration of T. capitatus EO induced a bacteriostatic effect, leading to a very significant p<0.0001 reduction of bacterial growth, from 6.59 and 6.44 log10 CFU/g to 1.46 and 3.98 log10 CFU/g, for low and high inoculum loads, respectively, after a 15-day storage period.


### 3.4. Antibacterial Efficacy ofT. algeriensis Essential Oil against Pseudomonas aeruginosa in Minced Beef Meat

As shown in Figures3 and 4, meat samples contaminated with two different concentration levels of P. aeruginosa and then treated with EOs of T. algeriensis, at 0.01 and 0.05%, displayed a bacterial growth significantly lower than that of untreated meat samples p<0.0001, by the end of the storage period. Based on the data shown in Figure 4, applying an initial inoculum of 105 CFU/g and 0.01 and 0.05% of T. algeriensis EO induced a reduction of 2.22 and 2.46 log10 CFU/g in bacterial growth, respectively. As shown in Figure 3, practically, the same evolution was observed; using the higher inoculum of 108 CFU/g, a reduction of 1.56 and 1.68 log10 CFU/g, respectively, was obtained after 15 days of storage.Figure 3
Time-related survival, at 4°C, of the high meat contamination level (108 CFU/g) of P. aeruginosa, following treatment with increasing concentrations of T. algeriensis EO. The 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: Thymus algeriensis.Figure 4
Time-related survival, at 4°C, of the low meat contamination level (105 CFU/g) of P. aeruginosa, following treatment with increasing concentrations of T. algeriensis EO. The 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: Thymus algeriensis.On the contrary, a concentration of 1% ofT. algeriensis EO allowed a high bacteriostatic effect, leading to a significant decrease in bacterial titers p<0.0001 of 3.67 log10 CFU/g for the low contamination level as compared to 2.86 log10 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 log10 CFU/g for low initial inoculum and 3.76 log10 CFU/for high initial inoculum.Therefore, it is important to note that both used EOs are effective and able to inhibit the growth ofP. aeruginosa. Hence, increasing the concentrations of EOs to the treated samples allowed a gradual decrease in bacteria counts. This 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.The antibacterial effect ofT. 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 ofT. 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 (105 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. These 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 (105 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 109 CFU/ml and 6-fold slower at 108 CFU/ml than at 106 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 105 CFU/g of P. aeruginosa and weak and delayed antibacterial activities at a high concentration of initial inoculum of 108 CFU/g. This 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.


### 3.5. Sensory Analysis

The sensory evaluation results are reported in Figures5 and 6 and Table 2. Panelists ranked the samples treated with 3% of T. capitatus EO as superior p<0.05 to the other samples and to the control for smell. Notably, taste score showed that samples treated with 3% of T. capitatus EO were significantly higher p<0.05 compared to the samples treated with 1% of T. capitatus EO and the samples treated with 3 and 1% of T. algeriensis EO. Moreover, the samples treated with 1% of T. capitatus EO and the samples treated with 3 and 1% of T. algeriensis EO were scored significantly higher p<0.05 compared with the control. For the flavor, the treated samples with 3% of T. capitatus EO were ranked as superior p<0.05 to the treated samples with 1% of T. capitatus EO followed by treated samples with 1% of T. algeriensis EO and treated samples with 3% of T. algeriensis EO. For the tenderness attribute, samples treated with T. capitatus EO and T. algeriensis EO showed that the application of EO made 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. The 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. These 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.Figure 5
Sensory assay of meat samples treated withT. capitatus EO.Figure 6
Sensory assay of meat samples treated withT. algeriensis EO.Table 2
Mean (standard deviation) of sensory attributes of minced beef meat samples.
Sensory attributeControlTC1TC3TA1TA3Odor2.44c6.55ab7.77a6.22ab5.22bFlavor3.11c6ab7.66a5.33b4.77bcTaste2.66c6.44ab7.66a5.66b5.11bJuiciness4.22a4.11a5.44a3.88a4aTenderness5.11a3.55abc4.44ab2.66c3.33bcColor3a4.22a4.33a4a4aOverall acceptability4.55b7a6.55a5b4.66bMeans in the same row followed by the same letter are not significantly differentp>0.05. Acceptance was evaluated using a 9-point scale, where 1 = extremely dislike and 9 = extremely like.


## 3.1. Antioxidant Activity: Free Radical Scavenging Assay

The EOs of herbs possess antioxidant properties that improve the shelf life of food. Thus, 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 IC50 value of 213.53 µg/ml than that of T. algeriensis showing an IC50 value of 861.12 µg/ml, butylated hydroxytoluene (BHT) presenting an IC50 value of 30 ± 0.01 µg/ml (Table 1). These results agreed with those of Amarti et al. [26], who showed that T. capitatus EOs possess strong antioxidant activities with IC50 equal to 69.04 µg/ml. However, the used T. algeriensis EO demonstrated weaker antioxidant effect with IC50 equal to 745 µg/ml.Table 1
Antioxidant and antimicrobial activities ofThymus capitatus and Thymus algeriensis essential oils.
Thymus capitatus essential oilsThymus algeriensis essential oilsMain essential oil compounds∗Carvacrol (88.98%), thymol (0.51%),p-cymene (1.14%), and α-terpinene (0.40%)Linalool (17.62%), camphor (13.82%), terpinen-4-ol (6.80%),α-terpineol (6.41%), and α-terpenyl acetate (6.27%)Antioxidant activities IC50 (µg/ml)213.53861.12Antimicrobial activities (%)MICMBCMICMBCE. coli0.006Ba0.012Ba0.025Aa0.05AaS. aureus0.006Aa0.012Ba0.020Ba0.05AaL. monocytogenes0.012Bb0.025Bb0.025Aa0.05AaP. aeruginosa0.012Bb0.025Bb0.025Aa0.05AaS. typhimurium0.006Aa0.012Ba0.025Ba0.05Aa∗The detailed results were reported by El Abed et al. and Ahmed et al. [24, 25]. IC50: 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.The antiradical activity ofT. 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 [27–29]. Based on these results, T. capitatus can be used as a natural antioxidant in food or for pharmaceutical applications.


## 3.2.In Vitro Antibacterial Effect of Thyme Essential Oil

According to the results of MIC and MBC, illustrated in Table1, both investigated EOs’ activities presented an antimicrobial activity against all tested bacteria with a greater antibacterial effect of T. capitatus EO. As illustrated in a previous study of El Abed et al. [24], the EOs of T. capitatus, harvested from Zaghouan region, presented 19 compounds with the presence of several bioactive compounds, including carvacrol (88.98%), thymol (0.51%), p-cymene (1.14%), and α-terpinene (0.40%). As reported by Ben Hadj Ahmed et al. [25], the EOs of T. algeriensis from Zaghouan region presented 39 compounds, with linalool as a major compound (17.62%), followed by camphor (13.82%), terpinen-4-ol (6.80%), α-terpineol (6.41%), and α-terpinyl acetate (6.27%).In addition, the results indicated that the MICs and MBCs ofT. 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. These 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 thatT. 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. These results are in accordance with previous studies revealing that the weakest activity of T. pectinatus EO is observed against P. aeruginosa [31]. The 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. These findings are in agreement with previous works carried out with several Thymus species [33].The 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. This 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%) ofT. 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 ofP. aeruginosa is significantly p<0.0001 more sensitive to EO of T. capitatus than that of T. algeriensis. These results are consistent with the previous report [39].


## 3.3. Antibacterial Efficacy ofT. capitatus Essential Oil against Pseudomonas aeruginosa in Minced Beef Meat

To study the antibacterial effect ofT. capitatus EO, depending on the inoculum concentrations, two different loads of P. aeruginosa (105 CFU/g and 108 CFU/g) were used before treating minced beef meat with EOs. As shown in Figure 1, an increase in the Pseudomonas count was detected, from the first day of incubation, when a high inoculum (108 CFU/g) was used. P. aeruginosa count then increased by 2.41 log10 CFU/g and reached 10.42 log10 CFU/g, fifteen days later. In contrast and according to the results shown in Figure 2, lower inoculum (105 CFU/g) induced an exponential increase of P. aeruginosa growth by 3.04 log10, reaching 8.05 log10 CFU/g at the end of the incubation period. Thus, 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].Figure 1
Time-related survival, at 4°C, of the high meat contamination level (108 CFU/g) of P. aeruginosa, following treatment with increasing concentrations of T. capitatus EO. The 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: Thymus capitatus.Figure 2
Time-related survival, at 4°C, of the low meat contamination level (105 CFU/g) of P. aeruginosa, following treatment with increasing concentrations of T. capitatus EO. The 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: Thymus capitatus.In this study, we have tested the antimicrobial effect of bothT. 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. This 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 (105 CFU/g), in the presence of low concentrations of T. capitatus EOs (0.01% and 0.05%), showed approximately 5.73 and 5.44 log10 CFU/g of P. aeruginosa, respectively, as compared to 8.05 log10 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 (108 CFU/g) presented the same trend of growth with a reduction of 2 log10 CFU/g. In fact, Pseudomonas growth titers achieved 8.82 and 8.57 log10 CFU/g in the presence of 0.01% and 0.05% of T. capitatus EO, respectively, as compared to 10.42 log10 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 log10 CFU/g, reaching 3.88 log10 CFU/g after 15 days of storage at 4°C.When beef meat was inoculated with a high concentration ofP. aeruginosa, in the presence of 1% of T. capitatus EO, a reduction in the bacterial load, from 7.03 to 3.39 log10 CFU/g, was obtained. Treatment with 3% of T. capitatus EO showed a greater antibacterial effect than in the presence of 1, 0.05, and 0.01%. Moreover, a 3% concentration of T. capitatus EO induced a bacteriostatic effect, leading to a very significant p<0.0001 reduction of bacterial growth, from 6.59 and 6.44 log10 CFU/g to 1.46 and 3.98 log10 CFU/g, for low and high inoculum loads, respectively, after a 15-day storage period.


## 3.4. Antibacterial Efficacy ofT. algeriensis Essential Oil against Pseudomonas aeruginosa in Minced Beef Meat

As shown in Figures3 and 4, meat samples contaminated with two different concentration levels of P. aeruginosa and then treated with EOs of T. algeriensis, at 0.01 and 0.05%, displayed a bacterial growth significantly lower than that of untreated meat samples p<0.0001, by the end of the storage period. Based on the data shown in Figure 4, applying an initial inoculum of 105 CFU/g and 0.01 and 0.05% of T. algeriensis EO induced a reduction of 2.22 and 2.46 log10 CFU/g in bacterial growth, respectively. As shown in Figure 3, practically, the same evolution was observed; using the higher inoculum of 108 CFU/g, a reduction of 1.56 and 1.68 log10 CFU/g, respectively, was obtained after 15 days of storage.Figure 3
Time-related survival, at 4°C, of the high meat contamination level (108 CFU/g) of P. aeruginosa, following treatment with increasing concentrations of T. algeriensis EO. The 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: Thymus algeriensis.Figure 4
Time-related survival, at 4°C, of the low meat contamination level (105 CFU/g) of P. aeruginosa, following treatment with increasing concentrations of T. algeriensis EO. The 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: Thymus algeriensis.On the contrary, a concentration of 1% ofT. algeriensis EO allowed a high bacteriostatic effect, leading to a significant decrease in bacterial titers p<0.0001 of 3.67 log10 CFU/g for the low contamination level as compared to 2.86 log10 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 log10 CFU/g for low initial inoculum and 3.76 log10 CFU/for high initial inoculum.Therefore, it is important to note that both used EOs are effective and able to inhibit the growth ofP. aeruginosa. Hence, increasing the concentrations of EOs to the treated samples allowed a gradual decrease in bacteria counts. This 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.The antibacterial effect ofT. 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 ofT. 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 (105 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. These 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 (105 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 109 CFU/ml and 6-fold slower at 108 CFU/ml than at 106 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 105 CFU/g of P. aeruginosa and weak and delayed antibacterial activities at a high concentration of initial inoculum of 108 CFU/g. This 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.


## 3.5. Sensory Analysis

The sensory evaluation results are reported in Figures5 and 6 and Table 2. Panelists ranked the samples treated with 3% of T. capitatus EO as superior p<0.05 to the other samples and to the control for smell. Notably, taste score showed that samples treated with 3% of T. capitatus EO were significantly higher p<0.05 compared to the samples treated with 1% of T. capitatus EO and the samples treated with 3 and 1% of T. algeriensis EO. Moreover, the samples treated with 1% of T. capitatus EO and the samples treated with 3 and 1% of T. algeriensis EO were scored significantly higher p<0.05 compared with the control. For the flavor, the treated samples with 3% of T. capitatus EO were ranked as superior p<0.05 to the treated samples with 1% of T. capitatus EO followed by treated samples with 1% of T. algeriensis EO and treated samples with 3% of T. algeriensis EO. For the tenderness attribute, samples treated with T. capitatus EO and T. algeriensis EO showed that the application of EO made 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. The 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. These 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.Figure 5
Sensory assay of meat samples treated withT. capitatus EO.Figure 6
Sensory assay of meat samples treated withT. algeriensis EO.Table 2
Mean (standard deviation) of sensory attributes of minced beef meat samples.
Sensory attributeControlTC1TC3TA1TA3Odor2.44c6.55ab7.77a6.22ab5.22bFlavor3.11c6ab7.66a5.33b4.77bcTaste2.66c6.44ab7.66a5.66b5.11bJuiciness4.22a4.11a5.44a3.88a4aTenderness5.11a3.55abc4.44ab2.66c3.33bcColor3a4.22a4.33a4a4aOverall acceptability4.55b7a6.55a5b4.66bMeans in the same row followed by the same letter are not significantly differentp>0.05. Acceptance was evaluated using a 9-point scale, where 1 = extremely dislike and 9 = extremely like.


## 4. Conclusion

This study showed an interesting antioxidant effect using the DPPH assay and an interesting antimicrobial profile shown by MIC and MBC ofT. capitatus EO. The 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. Therefore, T. capitatus EO could be used as a safe and a natural biopreservative for the improvement of microbiological and sensory quality of beef meat.



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*Source: 5599374-2021-05-24.xml*