The following bacterial cultures were procured from the Microbial type culture collection, in Chandigarh, India:

Bacterial cultures were kept on Luria Bertani agar and preserved at 4 °C in the refrigerator. The cultures were sub-cultured routinely once a month.

The essential oils (EOs) used were as follows:

EOs were of food grade, purchased from Sai Export India, Kannauj, India. The stock solution of EOs was prepared by using 10% Dimethyl sulphoxide (DMSO, HiMedia, India) and Tween-80 (0.005%, HiMedia, India), stored in McCartney vials, in the dark, below 25 °C.

For the preparation of the bacterial inoculum, two to three colonies of bacterial test organism were inoculated in Muller Hinton Broth (HiMedia, India) and incubated at 4 °C. The broth was incubated until the turbidity of the broth reached 0.5 McFarland (≈10⁶ cfu/ml). The test culture was spread uniformly on a Muller Hinton Agar (HiMedia, India) plate using a sterile spreader. The plates were dried for 15 minutes. Three sterile discs (HiMedia, 6mm) were placed on each plate at equal distances. With the help of a micropipette, 10 μl of a particular EO were dropped onto the sterile discs. 10% DMSO was used as control.

The plates were incubated at 37 °C for 24 h. After the incubation, the diameter of the zone of inhibition (in mm) was measured using an ‘Antibiotic Zone Scale’ (HiMedia) caliper. Three readings were noted, finally calculated as average ± standard error (SE). An inhibition zone of 14 mm or greater was considered as high antibacterial activity (Prabuseenivasan et al., 2006; Malik and Singh, 2010; Malik and Singh, 2015). A chloramphenicol disc (30 mcg, HiMedia) was taken as a positive control.

Dilution susceptibility testing was carried out for the determination of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC). In brief, Muller Hinton Broth (HiMedia, India) and a bacterial inoculum were added to the wells of the microtiter plate (Greiner Bio-one, Germany). Subsequently, EO was diluted (from 8 to 0.01562% v/v). EO was diluted in series in the wells of the microtiter plates, and they were incubated at 37 °C for 18 h. After incubation, 20 μl of resazurin dye (HiMedia) were added to every well. The microtiter plates were incubated in the dark at 37 °C for 2 h, and the color was noted. The appearance of pink/violet color or colorless liquid indicated the inhibition of bacteria in the medium. The lowest concentration showing blue color in the wells was considered to be MIC. For the determination of Minimum Bactericidal Concentration (MBC), subculturing was performed, and the lowest concentration, in which growth was absent, was noted as the MBC value of that particular oil (Malik and Singh, 2010; Malik and Singh, 2015; Malik et al., 2015). Similarly, the MIC and MBC of chemical preservatives [sodium nitrite, sodium benzoate, and potassium sorbate (HiMedia, India)] were also determined.

The EO and chemical preservatives showing the lowest MIC were selected for the evaluation of their combined antimicrobial effect. For the combination studies, the checkerboard method was adopted. In brief, dilutions (MIC, MIC/2, MIC/4 and MIC/8) of both EO and chemical preservatives were prepared following the same broth microdilution method which was also adopted to assess the MIC of these individual antimicrobial agents.

The analysis of the combination was obtained by calculating the Fractional Inhibitory Concentration (FIC) index in the following steps:

The interpretation of the FIC index is as follows:

A cucumber model media was prepared by taking two to three medium-sized fresh cucumbers which were washed with distilled water. The surface sterilization was done by soaking the cucumbers in 1.5% sodium hypochlorite for 20 min followed by 70% ethyl alcohol for 30 s and then allowed to air-dry inside a laminar airflow cabinet on sterile paper towels. The cucumbers were then peeled and chopped into fine pieces using a clean knife. Sterile de-ionized water (1:2; w/v) was added to the chopped cucumber and it was ground in a mixer for 2 to 3 minutes to make a smooth suspension. The suspension was filtered using muslin cloth; and the pH of the cucumber juice was adjusted to 5.5 using a pH meter (Eutech Instruments, India). 50 ml of the filtered juice were dispensed in a conical flask, then autoclaved at 121°C for 20 minutes. The Banana model was prepared by taking two to three ripe bananas, peeling, cleaning, and surface sterilizing them in the same manner as described above. The banana pulp was aseptically homogenized with sterile distilled water (1: 2, w/v), and the pH was adjusted to 4.7. 50 ml of the banana pulp were transferred to a sterilized 250 ml flask and autoclaved as described above. The gram flour (chickpea) soup model (10% w/v) was prepared by using the flour purchased from a local shop; the pH was adjusted to 5.6 using a pH meter. 50 ml of the soup were dispensed into a sterilized 250 ml flask and autoclaved. For preparing the meat broth model, mutton meat steaks were purchased from a local meat shop, external fat was removed and the meat was cut into pieces of uniform sizes (3 x 3 x 3 cm). The mutton pieces were boiled in distilled water (90 °C /20 min), the resulting broth was filtered using the Whatman filter paper no. 1 (HiMedia), and the filtrate was sterilized by autoclaving (121 °C/20 min) and stored in aliquots at -20 °C.

In all the food models, EO/chemical preservatives were added at different concentrations (MIC, 2MIC) and inoculated with 10 ³ cells of the test bacterial culture, and incubated at 37 °C for 24 hours. The bacterial counts were determined at regular intervals and expressed as log cfu/ml (Gutiérrez et al., 2009; Catherine et al., 2012).

All the experiments were carried out in triplicate. Using SPSS software package version 16.0, the results of the zone of inhibition were analyzed using analysis of variance (one-way ANOVA) followed by post hoc Tukey’s test. The significance was checked at three p-levels, 0.01, 0.1 and 0.5 levels. The results of inhibition studies obtained in model food experiments were compared with the control by t-test, at both 0.1 and 0.5 levels.

The screening of antimicrobial activity by disc diffusion methods showed the inhibition of bacteria in different magnitudes. All the EOs under study showed inhibition, although the highest inhibition was observed by C. winterianus EO against both A. hydrophila and L. monocytogenes. Both the bacteria Aeromonas hydrophila and Listeria monocytogenes were found to be sensitive to chloramphenicol (Table 1).

The MIC value (13 mg/ml) was found to be the lowest for EO against both Aeromonas hydrophila and Listeria monocytogenes. Among the chemical preservatives, sodium nitrite showed the lowest MIC as well as MBC (125 and 250 mg/ml) against both Aeromonas hydrophila and Listeria monocytogenes (Figure 1, sheet 4). For the chemical preservatives, the lowest MIC (125 mg/ml) and the lowest MBC (250 mg/ml) were determined for sodium nitrite against both Aeromonas hydrophila and Listeria monocytogenes (Figure 2).

The results of the combined studies (T. ammi EO and sodium nitrite) depicted the values of FICI to be 0.50 and 0.375 against A. hydrophila and L. monocytogenes, respectively. Both values for FICI were found to be ≤ 0.5, which indicated synergism between the two tested agents (Table 2).

Both T. ammi EO and sodium nitrite were effective in reducing the bacterial counts when added alone in the food-based broth media. However, a higher and more significant reduction in the bacterial counts was observed when T. ammi EO and sodium nitrite were added at twice the inhibitory concentrations (p < 0.01). A longer lag period was also observed in the presence of antimicrobial agents throughout the storage period as compared to the control (p < 0.01) (Figures 3 and 4).