2. Materials and methods

Essential oil and its composition: Thyme essential oil was obtained from the Laboratorios Hersol S.A. de C.V. (San Mateo Atenco, Estado de México, Mexico). It was selected based on both its reported antimicrobial activity and potential flavor compatibility with ready-to-eat meat products.

Tested EO was analyzed by gas chromatography–mass spectrometry (GC–MS) using a gas chromatograph (Agilent Technologies, 6850 N model, Santa Clara, CA) coupled to a mass selective detector with triple axis (Agilent Technologies, 5975C VL model, Santa Clara, CA) following the methodology from Ávila-Sosa et al. [6] using a fused silica HP-5MS (5% phenyl-95% polydimethylsiloxane) capillary column (30 m 0.250 mm; film thickness, 0.25 μm) and helium as the carrier gas (flow rate 1.1 mL/min). The injection volume was 1 μL of the EO samples prepared by dilution in ethanol (5100 v/v). Injector and detector temperatures were set at 250 and 280°C, respectively, and column oven temperature will be programmed from 60°C (4 min) to 240°C (10 min) at 4°C/min. Retention indices were calculated using a homologous series of n-alkanes C8 to C18 (Sigma, St. Louis, MO), and compounds were identified by comparing with retention indices from literature and the mass profile of the compounds available from the US National Institute of Standard Technology (NIST) library. The relative content of each component was calculated by % peak area. Thyme EO density was determined in triplicate by the relationship between mass and volume in accordance with the 962.3 AOAC method [7].

Microorganisms and cocktail preparation: Salmonella and Listeria strains (Table 1) were maintained in trypticase soy agar (Bixon BD, Cuautitlán Izcalli, Estado de México, Mexico) at 4°C and cultured in 10 mL trypticase soy broth at 35°C for 18 h prior to use.

Cocktails were prepared as equal amounts of each strain by mixing 1 mL of a fresh culture of each strain containing approximately 1 <sup>10</sup><sup>8</sup> CFU/mL in a sterile tube. Serial dilutions of each cocktail were made to obtain the desired inoculum concentration.

Minimum inhibitory (MIC) and bactericidal (MBC) concentrations: Minimum inhibitory concentration was determined by the agar dilution method [8, 9] tested in Petri dishes with trypticase soy agar for the strains L. monocytogenes (Scott A) and Salmonella Typhimurium (ATCC 14028). EO was added to the agar after sterilization at 45°C with Tween 80 (0.2%) as an emulsifier. For each strain, 50 μL of a fresh culture diluted to contain approximately 1x10<sup>4</sup> CFU/mL was spiral plated Looking for the Killer Combination: pH, Protein, and Thyme Essential Oil Interactions that… DOI: http://dx.doi.org/10.5772/intechopen.90099


#### Table 1.

achieve the desired effect. This, in turn, is an advantage since high concentrations of an EO can have a negative impact on the sensory acceptability of food products [4]. Although some studies have shown the efficacy of EOs as antimicrobials in meat products, others have reported very low antimicrobial activity and the need

In this context, there is a need to evaluate EOs directly in food products or in model systems that simulate food composition [4]. The use of food model media before food application is important since conditions can be controlled in order to determine the interactions between EOs and food components that could influence

In this regard, the main objective of this work was to, by means of a Box-Behnken design (BBD) of response surface methodology, evaluate the effects of pH, protein, and thyme essential oil concentration on the log of colony-forming units (CFU)/mL obtained after either a Salmonella or Listeria cocktail was subjected to

Essential oil and its composition: Thyme essential oil was obtained from the Laboratorios Hersol S.A. de C.V. (San Mateo Atenco, Estado de México, Mexico). It was selected based on both its reported antimicrobial activity and potential flavor

Tested EO was analyzed by gas chromatography–mass spectrometry (GC–MS) using a gas chromatograph (Agilent Technologies, 6850 N model, Santa Clara, CA) coupled to a mass selective detector with triple axis (Agilent Technologies, 5975C VL model, Santa Clara, CA) following the methodology from Ávila-Sosa et al. [6] using a fused silica HP-5MS (5% phenyl-95% polydimethylsiloxane) capillary column (30 m 0.250 mm; film thickness, 0.25 μm) and helium as the carrier gas (flow rate 1.1 mL/min). The injection volume was 1 μL of the EO samples prepared by dilution in ethanol (5100 v/v). Injector and detector temperatures were set at 250 and 280°C, respectively, and column oven temperature will be programmed from 60°C (4 min) to 240°C (10 min) at 4°C/min. Retention indices were calculated using a homologous series of n-alkanes C8 to C18 (Sigma, St. Louis, MO), and compounds were identified by comparing with retention indices from literature and the mass profile of the compounds available from the US National Institute of Standard Technology (NIST) library. The relative content of each component was calculated by % peak area. Thyme EO density was determined in triplicate by the relationship between

mass and volume in accordance with the 962.3 AOAC method [7].

Microorganisms and cocktail preparation: Salmonella and Listeria strains (Table 1) were maintained in trypticase soy agar (Bixon BD, Cuautitlán Izcalli, Estado de México, Mexico) at 4°C and cultured in 10 mL trypticase soy broth at

Cocktails were prepared as equal amounts of each strain by mixing 1 mL of a fresh culture of each strain containing approximately 1 <sup>10</sup><sup>8</sup> CFU/mL in a sterile tube. Serial dilutions of each cocktail were made to obtain the desired inoculum

Minimum inhibitory (MIC) and bactericidal (MBC) concentrations: Minimum inhibitory concentration was determined by the agar dilution method [8, 9] tested in Petri dishes with trypticase soy agar for the strains L. monocytogenes (Scott A) and Salmonella Typhimurium (ATCC 14028). EO was added to the agar after sterilization at 45°C with Tween 80 (0.2%) as an emulsifier. For each strain, 50 μL of a fresh culture diluted to contain approximately 1x10<sup>4</sup> CFU/mL was spiral plated

for high concentrations of the EO to accomplish the desired effect [3].

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

their antimicrobial activity.

2. Materials and methods

35°C for 18 h prior to use.

concentration.

94

the selected factors in model culture media.

compatibility with ready-to-eat meat products.

Studied bacterial strains and providers.

(Autoplate 4000, Spiral Biotech, Norwood, MA) for each concentration of thyme EO tested, 0.065–0.093% (w/w).

MIC was considered as the lowest concentration of EO at which no bacterial growth was observed after 24 h of incubation at 35°C and MBC as the lowest concentration, which resulted in no visible growth on the plate and no growth on trypticase soy broth after streaking the plate surface and further incubated under optimal conditions [10].

Plate preparation and inoculation: The media for plates were prepared by mixing isolated soy protein (ISP) adjusted to the desired concentration, trypticase soy broth as the nutrient provider, bacteriological agar as solidifying media, NaCl to adjust water activity to 0.98, and distilled water. The mixture was heated until boiling under continuous agitation and sterilized by autoclaving for 5 min at 17 lb./ in2 . After the media cooled down to 45°C, pH was adjusted with citric acid (10%), and EO to the desired tested concentration was added during continuous agitation with a magnetic stirrer. The media was poured in Petri dishes and left to cool for at least 2 h prior to inoculation. Plates at room temperature were inoculated with 30 μL (in three drops of 10 μL) of the cocktail dilution which, by previous determination, allowed the count of 20–70 CFU in each drop.

Experimental design: A Box-Behnken design (BBD) of response surface methodology was applied using Minitab 18.1. (Minitab, Inc., State College, PA) to study the effect of medium pH, isolated soy protein (ISP) concentration (% w/w), and EO concentration (% w/w), as well as their interactions on the growth of Salmonella or Listeria cocktails measured as fraction of surviving microorganisms when compared to CFU/mL found in each cocktail.

Each factor was investigated at three different levels (1, 0, +1) shown in Table 2. The experimental design included 15 sets of test conditions, including 3 replicated center points as randomized experiments to avoid bias.


Table 2.

Studied independent variables and their levels for tested box-Behnken design.
