Four essential oils viz., Cinnamon bark oil, Cinnamon leaf oil, Clove oil and Garlic oil were obtained from M/s Kancor essential oils, Ernakulam, Kerala. All these essential oils were stored in amber colored bottles at 4°C for further use.

All the four essential oils were characterized by Gas Chromatography- Mass Spectrometry system (789°A,

Agilent Technologies Inc., Santa Rosa, CA, USA) to know the chemical constituents present in them. This system is equipped with a HP-5 MS capillary column (30 m x 0.25 mm, 0.25 mm, Agilent Technologies Inc., Santa Rosa, CA, USA) and injected volume of each sample was 1 uL. Helium gas was used as carrier gas which has flow rate of 1 mL/min. The temperature of the injection port was 250°C and the temperatures programmed in the oven were: 50°C for 2 min, then increased to 180°C at a rate of 5°C/min, followed by an increase to 270°C at a rate of 20°C/min and maintenance at 270°C for 5 min. The mass spectra used -EI ion source temperature of 230°C, ionization energy of 70eV and a mass scan range of 40–500 amu. The essential oil components were tentatively identified by comparing their mass spectra with the standards present in NIST library.

Four L. monocytogenes isolates obtained from milk, soil, beef, along with positive control (MTCC 1143) were used to determine antibacterial efficacy of essential oils. All these isolates were inoculated in Buffered Listeria Enrichment Broth (BLEB) and incubated at 37°C for 24 hrs. After incubation, the inoculum was centrifuged at 8000 rpm for 10 minutes and the supernatant was discarded. Then the sediment was washed twice with normal saline and centrifuged at 8000 rpm for 10 min. Supernatant was discarded and the cells were mixed with normal saline to get 0.5 McFarland standard turbidity which indicates 1.5x10⁸ cells/mL.

Essential oils were diluted a day prior to use with 1% of Tween 80 in requirement of concentrations of 2%, 1%, 0.5% and 0.25%. The diluted essential oils were stored at 4°C for further use.

The antibacterial activity of essential oils was tested by agar well diffusion method (Gupta et al., 2008). The standard L. monocytogenes isolate was inoculated in nutrient broth and incubated at 37°C for 24 hrs. The inoculum was adjusted to 10⁶ CFU/ml with McFarland standards. A 100 μl of test bacterial culture (10⁶ CFU/ml) was spreaded on dried Mueller Hinton agar plates and left for 10 minutes. Holes were punched on the agar plates with 6 mm cork borer and 50 ul each of four essential oils were poured in to different wells after sealing wells bottom with 1% agar. Sterile Tween 80 was kept as negative control. These plates were kept aside for one hour for proper diffusion of essential oils and incubated at 37°C for 24 hrs. The plates were prepared in duplicate. After incubation, the zones of inhibition were measured in mm and the sensitivity of each oil was compared with standards was given by Ponce et al., 2003 and Moreira et al., 2005: < 8 mm-Non sensitive; 9 to 14mm-Sensitive; 15 to 19mm- Very sensitive; >20mm-Extremely sensitive.

The Minimum Inhibitory Concentration (MIC) of Cinnamon bark oil, Cinnamon leaf oil, Clove bud oil, Garlic oil was also determined by agar well diffusion method (Gupta et al., 2008). The MIC was defined as the lowest concentration of essential oils at which no visible growth ofbacteria was observed within 24 hrs of incubation at 37°C (Thongson et al., 2004). Various concentrations of essential oils were prepared by two-fold dilution series to get concentrations of 2%, 1%, 0.5% and 0.25%. A 50 ul of various concentrations of essential oils were poured in to wells which were punched on Mueller Hinton agar plates spreaded with 10⁶CFU/ml inoculum and incubated at 37°C for 24 hrs. The tests were done for twice. After incubation, zones of inhibitions were measured and the lowest concentration at which zone of inhibition was clear can be considered as MIC value of that essential oil.

The chemical constituents present in Cinnamon bark oil, cinnamon leaf oil, clove bud oil and garlic oil were represented in Fig. 1 to 4. The present study identified cinnamaldehyde (74.76%) as the major chemical constituent in cinnamon bark oil and this is similar to findings of Mareim et al., 2020 and Jham et al., 2005 who reported 74.56% and 75% of cinnamaldehyde as major component in their studies. Sun et al., 2016 obtained a little high prevalence of cinnamaldehyde of 79.39% while Zhang et al. (2016) reported a very high percentage of cinnamaldehyde (92.4%) in their studies respectively. Shan et al., 2007 in their study also identified 60–80% of cinnamaldehyde from the barks of Cinnamon zeylanicum which is almost similar to the present study. In contrary to this, other researchers identified 45.13% and 12.5% of cinnamaldehyde respectively (El-Baroty et al., 2010; Bayoub et al., 2010).

Eugenol (78.413%) is the main component found in cinnamon leaf oil in the present study which is similar to the findings of Bayoub et al, 2010 (76.91%). Singh et al. (2007) also reported eugenol (85%) as predominant component in cinnamon leaf oil which is slightly higher than the present study.

The main components identified in clove bud are Eugenol (77.004%) and Caryophyllene (10.688%) were similar to the Mith et al. (2014) who found same compounds with little high percentages - Eugenol (84.75%) and beta caryophyllene (4.60%). The studies of Cava et al. (2007) also determined cinnamaldehyde (67%) and eugenol (4%) as the important components in cinnamon bark oil while 4% cinnamaldehyde and 77% eugenol in cinnamon leaf oil; 86% eugenol and <0.1% cinnamaldehyde in clove essential oils respectively.

On characterization of garlic oil in the present study, it was found that diallyl trisulphide (37.144%), diallyl disulphide (28.478%) and diallyl sulphide (5.741%) were the main constituents. Mareim et al. (2020) recorded presence of diallyl trisulphide and diallyl disulphide in garlic oil with different percentages of 25.13 and 22.74. Razavi Rohani et al. (2011) also identified 27.33% of diallyl trisulphide, 24.67% of diallyl disulphide, 19.11% of methyl allyl trisulphide, 8.59% propenyldithiopropanoate, 2.18% dimethyl trisulfide, 2.13% diallyltetrasulfide, 1.49% of 3-vinyl-[4H]-1,2 dithin (3-VDT) and 1.25% 2-vinyl-[4H]-1,2 dithin (2-VDT). Similarly, El-Sayed et al, 2017 and Zhang et al., 2016, have also found similar compounds in garlic oil but with different concentrations.

These variations in the composition of essential oils from the present study to other studies may due to species, subspecies and variety of plants selected for preparation of essential oils, various agroclimatic conditions viz., climatic, seasonal and geographic of the regions, harvesting seasons (Mith et al., 2014), maturity stage and adaptive metabolism of plants, distillation conditions and the plant part analyzed (El-Baroty et al., 2010). The antimicrobial activity these essential oils also depends on the concentration of the phenolics compounds present in them, where at low concentrations, these phenolics compounds affects enzyme activity related to energy production and at high concentrations, causes protein precipitation (Prindle and Wright, 1997).

All the four essential oils viz., cinnamon bark, cinnamon leaf, clove bud and garlic oils have shown antibacterial activity against L. monocytogenes. Among the four essential oils, cinnamon bark oil has shown with widest zone of inhibition (27±0 mm), followed by cinnamon leaf oil (17±0 mm), clove bud oil (14±0 mm) and garlic oil (12±0 mm).Similar to the present study, Ghabraie et al., 2015 in their agar diffusion assay studies, found that cinnamon bark oil and clove bud oil has shown highest inhibition diameters of approximately 27.8±0.6 mm and 14.8±0.3 mm among 32 essential oils used against L. monocytogenes. Babu et al., 2011 also observed that cinnamon oil (25±0.06) has shown more zone of inhibition followed by clove (21±0.08) and garlic (17±0.18) oils in their studies. Hoque et al., 2008 also found 33 mm and 14 mm inhibition zones by testing the antimicrobial activity of cinnamon and clove essential oils against L. monocytogenes. In contrary to the present study, Mareim et al. (2020) recorded high zone of inhibition by garlic oil (31±1.7 mm) than cinnamon oil (12.3±0.5) against L. monocytogenes. While, Smith and Palmer et al. (1998) have found 6.8 mm, 9.2 mm, 8.4 mm and 4 mm zones of inhibition showed by cinnamon bark, cinnamon leaf, clove and garlic oils against L. monocytogenes respectively. Bayoub et al. (2010) observed more zone of inhibition for clove oil (25±10 mm) than cinnamon oil (22±0 mm) against L. monocytogenes in their preliminary screening of essential oils. Similarly, in a study conducted by Shan et al., 2007, clove bud oil (13.7 mm) showed more zone of inhibition than cinnamon oil (8.9 mm) against L. monocytogenes by agar well diffusion method.

The minimum inhibitory concentration (MIC) of the four essential oils viz., cinnamon bark oil, cinnamon leaf oil, clove bud oil and garlic oil were given in Table 1.

Among four essential oils, Cinnamon bark oil alone was found to be sensitive against L. monocytogenes at tested concentrations (0.25%, 0.5%, 1% and 2%) with MIC of 1%. These findings are similar to the studies of Gupta et al. (2008) who recorded 1.25% MIC of cinnamon bark oil. In contrary, Mith et al. (2014) observed antilisteria activity of cinnamon oil against L.monocytogenes with a MIC of 0.125 Lil/ml; Bayoub et al.(2010) had MIC of 0.4 mg/ml for cinnamon oil against L. monocytogenes.

Though the cinnamon leaf oil, clove bud and garlic oils have shown antibacterial activity, but they didn't shown any zone of inhibitions at required concentrations (2%, 1%, 0.5% and 0.25%) while determining MIC. In contrast to this, Wafaa et al. (2019) investigations showed the MIC of cinnamon leaf and bark oils against L. Monocytogenes was 0.5% (v/v) and also reported that these oils can be used as potential antimicrobial agents to keep fresh produce safe from Listeria for human consumption.

In contrary to the clove bud oil findings in the present study, Menon and Garg (2001), Gupta et al. (2008), Barbosa et al. (2009) and Bayoub et al. (2010) have found the minimum inhibitory action of clove oil at a concentration of 1%, 5%, 0.09% and 0.25 mg/mL against L. monocytogenes. Hoque et al. (2008) reported MIC values for cinnamon and clove essential oils were 1.25 mg/ml and 2.5 mg/ml respectively against L. monocytogenes.

Cava et al. (2007) also observed antimicrobial effect of cinnamon bark, cinnamon leaf and clove oils against L. monocytogenes in pasteurized milk stored at 7°C and 35°C. They also observed strongest antimicrobial activity by cinnamon bark oil while cinnamon leaf and clove oils required higher concentrations for complete inhibition of growth of L. monocytogenes in pasteurized milk. They detected MIC value of 500 ppm for cinnamon bark oil and 3000 ppm of each for cinnamon leaf and clove oils respectively.

Similar to the present study, Indu et al. (2006) studies also found that garlic oil didn't show any inhibitory effect against growth of L. monocytogenes by agar well method. Kim et al. (2004) also found that garlic oil and its constituents have weak antibacterial activity with MIC value of 300 Hg/mL and Razavi et al. (2011) had found 100 ng/mL MIC values against L. monocytogenes. Kumar and Berwal (1998) had found 85% inhibition of L. monocytogenes at 10% garlic concentration and also determined MIC value at 8.8% respectively. Allicin or diallylthiosulphinic acid or diallyl disulphide was responsible for antimicrobial activity of garlic oil (Indu et al., 2006). Till now, only few works has been carried out on antibacterial activity of garlic oil due to rapid decomposition of the allicin, the major antibacterial compound in garlic, during the preparation of garlic essential oil (Razavi Rohani et al., 2011). The results may vary from one researcher to others due to selection of bacterial strains, volume of inoculum, incubation time and temperatures used in the experiments (Mith et al., 2014).

Our present study was mainly aimed to know the concentration of essential oils at which antibacterial activity was shown without change in the organoleptic characteristics of foods. In the present study, cinnamon bark oil only has shown activity against L. monocytogenes at 2% and 1% respectively while the remaining three essential oils didn't show any activity at required concentrations. The results obtained from the present study highlights the use of cinnamon bark oil in food industries as food preservative in foods against L. monocytogenes.