3. Results and discussion

Essential oil composition: EO analysis revealed the presence of 37 different compounds accounting for 99.86% of the tested oil. Those making up 0.1% or more of the EO are displayed in Table 3, the most abundant compounds being carvacrol (31.87%), β-cymene (also known as meta-cymene) (25.22%), linalool (17.86%), 1Rα-pinene (8.33%), and γ-Terpinene (6.04%).

It its worth noticing that although major thyme essential oil components found in this study agree with other reports [11, 12], thymol, a characteristic and major component of thyme essential oil [13], was not found to be present in the tested essential oil; however, its monoterpene precursor (γ-terpinene) [14] was identified. Despite this, the antimicrobial activity of the tested thyme essential oil did not seem to be affected. Ruiz-Navajas and her team [15] determined that the predominant compounds found in Thymus piperella essential oil were carvacrol (31.92%), pcymene (16.18%), γ-Terpinene (10.11%), and α-terpineol (7.29%); this essential oil proved to be effective, decreasing aerobic mesophilic and lactic acid bacterial counts when added to chitosan edible films that were applied to cooked cured ham as coatings.

It can be observed that values are equally effective against both strains. Similar results have been obtained in other studies where 0.1% (v/v) of thyme EO was enough to inhibit L. monocytogenes [11]; Hammer et al. [16] reported that more than 2.0% (v/v) of thyme EO was needed to inhibit the same S. typhimurium strain.

L. monocytogenes (Scott A) S. Typhimurium (ATCC 14028) 0.0740 0.0800 0.0927 0.1000

Looking for the Killer Combination: pH, Protein, and Thyme Essential Oil Interactions that…

Minimum inhibitory and minimum bactericidal concentrations of tested thyme essential oil\* against two of the

(w/w)

MIC % (v/v)

MBC % (w/w)

MBC % (v/v)

Strain MIC %

DOI: http://dx.doi.org/10.5772/intechopen.90099

Since other authors reported the need for an increased concentration of studied EOs for bacterial control when assessed in food matrixes instead of model media [17] such as in the case of ham [18] and barbecued chicken [19], the second set of tests were performed to determine EO concentrations suitable for the experimental design. Concentrations of 0.22, 0.25, 0.28, 0.30, and 1.00% (w/w) were tested with 10% ISP at pH 6.5 against Salmonella cocktails. It was observed (data not shown) that the proportion of survivor cells declined considerably with 0.22%(w/w) thyme EO, while no growth was observed at concentrations above 0.30% (w/w); therefore, 0.19, 0.22, and 0.25% (w/w) were set as EO concentration levels for the

In contrast with MICs derived from optimal growth conditions, in model media with increased protein content, an increased EO concentration was needed to inactivate bacteria. This is in accordance with several studies reporting that food components can interact with EO constituents impairing their function [3, 5]. The antimicrobial activity of Myagropsis myagroides ethanol extract was reduced in the presence of soybean oil, as a fat component, and >5% beef extract, as protein component but was not influenced by starch, as carbohydrate component [20]. Microbial inactivation: The complete experimental design and bacterial culture

The significant effect of the three studied factors was evaluated by analyzing the response using uncoded units. The results for the Box-Behnken experimental design on the log CFU/mL of Listeria and Salmonella cocktails allowed the construction of reduced quadratic models by means of stepwise backward elimination for those terms where p > 0.10 for the Listeria cocktail model and p > 0.05 for the Salmonella cocktail model. In the case of Listeria cocktail, the lack of fit was nonsignificant (p > 0.05), while for the Salmonella cocktail, the experimental data exhibited a good fit to the model with an R<sup>2</sup> of 0.914. The terms and uncoded coefficients included in

As can be seen from these results, the interaction between ISP and thyme EO concentration, as well as pH quadratic interaction, and the linear effects of ISP and thyme EO concentration have a significant effect on the log CFU/mL of Listeria cocktail. For Salmonella cocktail, all variables (pH, ISP, and EO concentration) and their interactions have a significant effect on the log CFU/mL except the quadratic

It is clear from Figure 1 that, for Listeria cocktail, as ISP concentration decreases

and thyme EO concentration increases, the number of bacteria (log CFU/mL) decreases, being these effects more effective at pH 6.0. For the Salmonella cocktail (Figure 2), a decrement in the number of bacteria (log CFU/mL) can be observed as thyme EO concentration increases and pH and ISP concentration decrease.

experimental design.

\*Density = 0.9277 0.0008 g/mL.

Table 4.

studied strains.

responses are shown in Table 5.

interaction of ISP concentration.

97

the generated models are displayed in Table 6.

Minimum inhibitory and bactericidal concentrations: Table 4 exhibits the values of tested thyme essential oil MIC and MBC against L. monocytogenes (Scott A) or S. Typhimurium (ATCC 14028).


Table 3.

Main compounds found in tested thyme essential oil determined by gas chromatography-mass spectrometry.

Looking for the Killer Combination: pH, Protein, and Thyme Essential Oil Interactions that… DOI: http://dx.doi.org/10.5772/intechopen.90099


Table 4.

3. Results and discussion

as coatings.

Table 3.

96

α-pinene (8.33%), and γ-Terpinene (6.04%).

Technology, Science and Culture - A Global Vision, Volume II

or S. Typhimurium (ATCC 14028).

Essential oil composition: EO analysis revealed the presence of 37 different compounds accounting for 99.86% of the tested oil. Those making up 0.1% or more of the EO are displayed in Table 3, the most abundant compounds being carvacrol (31.87%), β-cymene (also known as meta-cymene) (25.22%), linalool (17.86%), 1R-

It its worth noticing that although major thyme essential oil components found in this study agree with other reports [11, 12], thymol, a characteristic and major component of thyme essential oil [13], was not found to be present in the tested essential oil; however, its monoterpene precursor (γ-terpinene) [14] was identified. Despite this, the antimicrobial activity of the tested thyme essential oil did not seem to be affected. Ruiz-Navajas and her team [15] determined that the predominant compounds found in Thymus piperella essential oil were carvacrol (31.92%), pcymene (16.18%), γ-Terpinene (10.11%), and α-terpineol (7.29%); this essential oil proved to be effective, decreasing aerobic mesophilic and lactic acid bacterial counts when added to chitosan edible films that were applied to cooked cured ham

Minimum inhibitory and bactericidal concentrations: Table 4 exhibits the values of tested thyme essential oil MIC and MBC against L. monocytogenes (Scott A)

Compound Percentage 1R-α-Pinene 8.33 Camphene 0.85 1–2-methylene-(1S)-beta-pinene 0.81 β-Pinene 0.86 β-Cymene 25.22 γ-Terpinene 6.04 Terpinolene 0.80 Linalool 17.86 Carvacrol 31.87 Caryophyllene 0.98 5-Isopropenyl-2-methyl-7-oxabicyclo [4.1.0] heptan-2-ol 0.11 Caryophyllene oxide 3.12 12-Oxabicyclo [9.1.0] dodeca-3,7-diene 0.13 Cembrene 0.14 2,4,7,9-tetramethyl-5-decyne-4,7-diol 0.10 1,2,5,5,8a-Pentamethyl1,2,3,5,6,7,8,8a-octahydronaphthalen-1-ol 0.50 α-Isomethyl ionone 0.57 Androst-4-en-11-ol-3, 7-dione, 9-thiocyanato 0.48 Retinoic acid 0.19 Abietic acid 0.33

Main compounds found in tested thyme essential oil determined by gas chromatography-mass spectrometry.

Minimum inhibitory and minimum bactericidal concentrations of tested thyme essential oil\* against two of the studied strains.

It can be observed that values are equally effective against both strains. Similar results have been obtained in other studies where 0.1% (v/v) of thyme EO was enough to inhibit L. monocytogenes [11]; Hammer et al. [16] reported that more than 2.0% (v/v) of thyme EO was needed to inhibit the same S. typhimurium strain.

Since other authors reported the need for an increased concentration of studied EOs for bacterial control when assessed in food matrixes instead of model media [17] such as in the case of ham [18] and barbecued chicken [19], the second set of tests were performed to determine EO concentrations suitable for the experimental design. Concentrations of 0.22, 0.25, 0.28, 0.30, and 1.00% (w/w) were tested with 10% ISP at pH 6.5 against Salmonella cocktails. It was observed (data not shown) that the proportion of survivor cells declined considerably with 0.22%(w/w) thyme EO, while no growth was observed at concentrations above 0.30% (w/w); therefore, 0.19, 0.22, and 0.25% (w/w) were set as EO concentration levels for the experimental design.

In contrast with MICs derived from optimal growth conditions, in model media with increased protein content, an increased EO concentration was needed to inactivate bacteria. This is in accordance with several studies reporting that food components can interact with EO constituents impairing their function [3, 5]. The antimicrobial activity of Myagropsis myagroides ethanol extract was reduced in the presence of soybean oil, as a fat component, and >5% beef extract, as protein component but was not influenced by starch, as carbohydrate component [20].

Microbial inactivation: The complete experimental design and bacterial culture responses are shown in Table 5.

The significant effect of the three studied factors was evaluated by analyzing the response using uncoded units. The results for the Box-Behnken experimental design on the log CFU/mL of Listeria and Salmonella cocktails allowed the construction of reduced quadratic models by means of stepwise backward elimination for those terms where p > 0.10 for the Listeria cocktail model and p > 0.05 for the Salmonella cocktail model. In the case of Listeria cocktail, the lack of fit was nonsignificant (p > 0.05), while for the Salmonella cocktail, the experimental data exhibited a good fit to the model with an R<sup>2</sup> of 0.914. The terms and uncoded coefficients included in the generated models are displayed in Table 6.

As can be seen from these results, the interaction between ISP and thyme EO concentration, as well as pH quadratic interaction, and the linear effects of ISP and thyme EO concentration have a significant effect on the log CFU/mL of Listeria cocktail. For Salmonella cocktail, all variables (pH, ISP, and EO concentration) and their interactions have a significant effect on the log CFU/mL except the quadratic interaction of ISP concentration.

It is clear from Figure 1 that, for Listeria cocktail, as ISP concentration decreases and thyme EO concentration increases, the number of bacteria (log CFU/mL) decreases, being these effects more effective at pH 6.0. For the Salmonella cocktail (Figure 2), a decrement in the number of bacteria (log CFU/mL) can be observed as thyme EO concentration increases and pH and ISP concentration decrease.


\*n = 6.

\*\*Initial count of cocktail =8.32 0.16 log CFU/mL (n = 24).

\*\*\*Initial count of cocktail = 8.61 0.05 log CFU/mL (n = 24).

#### Table 5.

Box-Behnken design with independent variables and obtained response values.


(w/w) of thyme EO was enough to inactivate the bacterial strains L. monocytogenes (Scott A) and S. Typhimurium (ATCC 14028). However, in model media conditions, 0.25% (w/w) of thyme EO still allows for the growth of more than 5 and 6 log

Contour plots for Listeria cocktail (log CFU/mL) as a function of (a) thyme essential oil and pH, (b) thyme

Looking for the Killer Combination: pH, Protein, and Thyme Essential Oil Interactions that…

DOI: http://dx.doi.org/10.5772/intechopen.90099

The interaction of food components and EO constituents can impair their function. These results reflect the protective effect of the protein on bacteria, which could be interacting with the EO and in that way reducing its effectiveness [21]. Veldhuizen and his team [22] incubated carvacrol with bovine serum albumin (BSA) for 15 min and then filtered the solution through a 10 kDa pore-size filter which could easily be traversed by carvacrol (molecular mass of 150 Da) but not BSA (molecular mass of 66 kDa). By measuring the amount of carvacrol recovered and compared to appropriate controls, these authors concluded that the reduction in the amount of carvacrol recovered was the result of the binding of carvacrol to BSA. This, in turn, explained the reduced antilisterial activity observed in the presence of egg yolk and BSA. The binding of carvacrol to the protein reduced the

The microstructure of the food matrix must be considered as another possible reason for the diminished antimicrobial activity of EOs when tested in real foods in contrast to culture broths [23]. In our case, the effect of higher protein concentration on the reduced antimicrobial activity of thyme EO could also be explained by the physical nature of the media. In solid form, a higher protein concentration could limit its diffusion and in turn decrease tested EO effectiveness [21, 23]. Skandamis et al. [23] studied S. typhimurium in the presence or absence of oregano EO at two

CFU/mL of Listeria and Salmonella cocktails, respectively.

essential oil and isolated soy protein, or (c) pH and isolated soy protein.

Figure 1.

99

effective concentration of carvacrol in solution.

\*Coefficients are presented in uncoded units. A, B, and C represent isolated soy protein concentration, pH, and essential oil concentration, respectively.

#### Table 6.

Coefficients of the reduced quadratic models for Listeria and Salmonella cocktails.

It can also be observed that the effect of thyme EO concentration becomes more relevant as ISP concentration increases. This becomes evident for both bacterial cocktails when considering the MIC and MBC values obtained from previous tests in trypticase soy agar; under those conditions a concentration of only 0.0927%

Looking for the Killer Combination: pH, Protein, and Thyme Essential Oil Interactions that… DOI: http://dx.doi.org/10.5772/intechopen.90099

#### Figure 1.

Contour plots for Listeria cocktail (log CFU/mL) as a function of (a) thyme essential oil and pH, (b) thyme essential oil and isolated soy protein, or (c) pH and isolated soy protein.

(w/w) of thyme EO was enough to inactivate the bacterial strains L. monocytogenes (Scott A) and S. Typhimurium (ATCC 14028). However, in model media conditions, 0.25% (w/w) of thyme EO still allows for the growth of more than 5 and 6 log CFU/mL of Listeria and Salmonella cocktails, respectively.

The interaction of food components and EO constituents can impair their function. These results reflect the protective effect of the protein on bacteria, which could be interacting with the EO and in that way reducing its effectiveness [21]. Veldhuizen and his team [22] incubated carvacrol with bovine serum albumin (BSA) for 15 min and then filtered the solution through a 10 kDa pore-size filter which could easily be traversed by carvacrol (molecular mass of 150 Da) but not BSA (molecular mass of 66 kDa). By measuring the amount of carvacrol recovered and compared to appropriate controls, these authors concluded that the reduction in the amount of carvacrol recovered was the result of the binding of carvacrol to BSA. This, in turn, explained the reduced antilisterial activity observed in the presence of egg yolk and BSA. The binding of carvacrol to the protein reduced the effective concentration of carvacrol in solution.

The microstructure of the food matrix must be considered as another possible reason for the diminished antimicrobial activity of EOs when tested in real foods in contrast to culture broths [23]. In our case, the effect of higher protein concentration on the reduced antimicrobial activity of thyme EO could also be explained by the physical nature of the media. In solid form, a higher protein concentration could limit its diffusion and in turn decrease tested EO effectiveness [21, 23]. Skandamis et al. [23] studied S. typhimurium in the presence or absence of oregano EO at two

It can also be observed that the effect of thyme EO concentration becomes more relevant as ISP concentration increases. This becomes evident for both bacterial cocktails when considering the MIC and MBC values obtained from previous tests in trypticase soy agar; under those conditions a concentration of only 0.0927%

Coefficients of the reduced quadratic models for Listeria and Salmonella cocktails.

Run Independent variables Log CFU/mL\*

Technology, Science and Culture - A Global Vision, Volume II

\*n = 6.

Table 5.

Table 6.

98

\*\*Initial count of cocktail =8.32 0.16 log CFU/mL (n = 24). \*\*\*Initial count of cocktail = 8.61 0.05 log CFU/mL (n = 24).

essential oil concentration, respectively.

Box-Behnken design with independent variables and obtained response values.

Term Listeria cocktail Salmonella cocktail

Constant 45.9 <0.001 57.03 <0.001 A 1.654 0.001 2.742 <0.001 B 18.79 0.371 5.58 <0.001 C 60.5 0.002 461.8 <0.001 <sup>B</sup><sup>2</sup> 1.582 0.016 1.148 <sup>&</sup>lt;0.001 C2 — — 455.8 <0.001 AB — — 0.2404 <0.001 AC 6.31 0.072 7.16 <0.001 BC — — 26.31 <0.001 \*Coefficients are presented in uncoded units. A, B, and C represent isolated soy protein concentration, pH, and

Coefficient\* P-value Coefficient\* P-value

 1 1 0 6.64 0.03 6.66 0.40 2 1 1 0 6.61 0.19 7.40 0.12 1 1 0 6.55 0.06 6.75 0.07 1 1 0 7.53 0.06 8.22 0.12 1 0 1 5.98 2.93 8.56 0.07 6 10 1 7.66 0.15 8.46 0.11 1 0 1 5.78 0.06 6.80 0.24 1 0 1 6.33 0.29 7.99 0.24 9 0 1 1 7.21 0.27 8.63 0.11 0 1 1 7.33 0.15 8.10 0.06 0 1 1 6.66 0.05 6.42 0.24 0 1 1 6.51 0.13 7.47 0.09 0 0 0 6.74 0.15 7.43 0.16 0 0 0 6.45 0.21 7.64 0.16 0 0 0 6.45 0.27 7.52 0.17

ABC Listeria cocktail\*\* Salmonella cocktail\*\*\*

Thyme EO

Acknowledgements

DOI: http://dx.doi.org/10.5772/intechopen.90099

numbers 2409 and 3555).

Author details

101

Puebla, Cholula, Puebla, Mexico

provided the original work is properly cited.

The author Lastra-Vargas gratefully acknowledges Universidad de las Américas

Looking for the Killer Combination: pH, Protein, and Thyme Essential Oil Interactions that…

Puebla (UDLAP) and the National Council for Science and Technology (CONACyT) of Mexico financial support for her PhD studies. This work was supported by CONACyT (grant number CB-2016-2101-283636) and UDLAP (grant

Leonor Lastra Vargas\*, Aurelio Lopez-Malo\* and Enrique Palou\*

\*Address all correspondence to: leonor.lastravs@udlap.mx, aurelio.lopezm@udlap.mx and enrique.palou@udlap.mx

Department of Chemical and Food Engineering, Universidad de las Américas

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

Figure 2.

Contour plots for Salmonella cocktail (log CFU/mL) as a function of (a) thyme essential oil and pH, (b) thyme essential oil and isolated soy protein, or (c) pH and isolated soy protein.

pH values comparing its growth in liquid and solid media; they found that while there was no significant effect of the physical state of the media on the growth of colonies in the absence of the EO, the type of media did have an influence on the inhibitory efficacy of oregano EO. Identical treatments with 0.03% oregano EO showed increased effectiveness in liquid culture than in gelatin gel, attributed to a better dispersion of tested EO in broth which could increase the interaction between cells and EO antimicrobial components.

For the Listeria cocktail, only a 2.54 log reduction could be expected at pH 6.0 with the lowest ISP (10% w/w) and highest thyme EO (0.25% w/w) tested concentrations, while for the Salmonella cocktail, the highest reduction (2.19 log) was achieved at an ISP concentration of 11.5% w/w with a pH 5.5 and 0.25% w/w of thyme EO.
