Peppermint oil was procured from Kanta Enterprises Pvt. Ltd, Noida, UP, India. As per the certificate of analysis provided with the product, it has refractive index in range of 1.449-1.467 and has specific gravity in range of 0.890¬0.905. The oil contained 39.50% of menthol as its main constituent. All the reagents and chemicals used in the study were of analytical grade.

Ten pure freeze dried cultures were procured from Institute of Microbial Technology (IMTECH), Chandigarh, India viz. Bacillus cereus (MTCC 1272), Escherichia coli (MTCC 723), Enterococcus faecalis (MTCC 890), Klebsiella pneumoniae (MTCC 109), Listeria monocytogenes (MTCC 1143), Proteus mirabilis (MTCC 425), Pseudomonas aeruginosa (MTCC 741), Salmonella enterica serovar Typhi (MTCC 733), Shigella flexneri(MTCC 1457), and Staphylococcus aureus (MTCC 96). These cultures were revived and stock cultures were prepared and are being maintained at -80°C in the department by regular passaging.

Antimicrobial potential of Peppermint essential oil (PEO) was tested on ten entero-pathogenic bacterial cultures (Bacillus cereus, Escherichia coli, Enterococcus faecalis, Klebsiella pneumonia, Listeria monocytogenes, Proteus mirabilis, Pseudomonas aeruginosa, Salmonella enterica, Shigella flexneri and Staphylococcus aureus), which were cultured into fresh broth media and brought into log phase of growth by incubating at 37^oC for 24 hours before being used. The antibacterial activity was assessed using Disc diffusion assay (Bauer et al, 1966). Mueller-Hinton agar (HiMedia Laboratories) plates were inoculated with one of the selected bacterial species using a sterile swab dipped into a bacterial suspension containing 10⁷ CFU/ ml. Then a sterile paper disk (5 mm ID, Whatmann #1) was placed onto the centre of the agar plate and 5 µl of EO was placed onto the centre of the disk. Plates were then incubated at 37 °C for 24 h. Following incubation, growth inhibition zones were observed. Diameters of these zones were measured and tabulated.

It was estimated by method followed by Kumar et al.(2017) with some modifications. Uniform concentrations of log phase bacterial cultures were prepared by adjusting their absorbance at 600 nm. Concentrations of Peppermint essential oil were adjusted to 0.125%, 0.25%, 0.1%, 0.3%, 0.5%, 1.0%, 2.0% and 3.0% with help of DMSO. In the Microtiter plates, 100 pl of each culture were added to 30 pl of oil dilution and 170 pl of nutrient broth. The plates were kept for incubation at 37 ^oC for 24 hours. Absorbance of samples was measured at 600 nm to observe growth inhibition. The growth inhibition was also confirmed by streaking the samples on nutrient agar plates and observing for bacterial growth after 24 hours incubation at 37 ^oC.

The antioxidant activity of the peppermint essential oil was measured as free radical scavenging activity. The ability of essential oil to donate hydrogen atoms or electrons was evaluated from bleaching of coloured methanoloic solution of 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity. The possibility of free radical scavenging activity is higher if number of hydroxyl groups are more.

Radical scavenging potential of Peppermint Oil was assessed using a methanolic solution of the “stable” free radical, DPPH. The method of (Blois, 1958) was used in studying the effect of various oil concentrations on DPPH radicals with some modifications. A solution of DPPH (0.15 mmol/L) in methanol was prepared. Oil concentrations (0.5, 1.0, 1.5, 2.0, and 2.5%) were prepared in methanol and 200 pl of each dilution was mixed with 50 pl of DPPH solution in a 96 well microtitre plates. The mixture was allowed to stand at room temperature in dark for 30 min. The decrease in absorbance at 517 nm was measured. Butylated hydroxyanisole (BHA) was used as positive control. The radical scavenging activity was measured as a decrease in absorbance of DPPH.

% Radical Inhibition = {(Control OD - Sample OD)/ Control OD} x 100

ABTS cation decolorization assay was conducted on various concentrations of Peppermint oil (0.5, 1.0, 1.5, 2.0, 2.5%) made in methanol (Re et al, 1999) with slight modifications. ABTS radical cation was freshly prepared by mixing 14mM ABTS with equal volume of 4.95 mM potassium persulphate and kept for 24 hours at room temperature. The ABTS radical cation was used for the assay after dilution with Phosphate Buffer Saline (PBS) appropriately. To 50 pl of various concentrations of PEO, 150 pl of ABTS solution was added. After 1 min incubation at room temperature, absorbance was measured at 732 nm. Methanol was used as blank solution and ABTS solution without essential oil served as control. The cation scavenging activity was measured same as with DPPH.

% Radical Inhibition = {(Control OD - Sample OD)/ Control OD} x 100

Biofilm inhibition was achieved by aliquoting 10 pL of a standardized (~10⁶ CFU/ml) L. monocytogenes and P.mirabilis culture into each 96-well microtitre plate followed by the addition of 30 pL of PEO (1250 ppm), respectively (Galvao et al, 2012). Following incubation at 37 ^oC for 24 h, the plates were rinsed three times with phosphate-buffered saline (PBS, pH 7.2) to remove loosely attached cells. The plates were air-dried and then the wells were stained with 250 pL of 0.1% crystal violet and incubated at room temperature for 30 min. After incubation, the plates were washed and then left to dry. Finally, 250 pL of 33% glacial acetic acid was added to solubilize the dye and the OD₅₇₀ was recorded using microplate reader. The per cent biofilm inhibition was estimated following the formula:

The positive control was the amount of biofilm formed with a pure culture of L. monocytogenes and P.mirabilis.

L. monocytogenes and P.mirabilis biofilms, with or without treatment with PEO, were grown on pre-sterilized glass cover slips (Musthafa et al, 2010). An aliquot of 80 pL/ well (~10⁶ CFU/mL) of overnight grown test pathogens were added to 6 well tissue culture plate containing sterile glass cover slips, BHI medium (1680 pL/well) and PEO (240 pL/well). The bacterial suspensions were used as controls. Biofilms were stained with crystal violet and the stained biofilms were visualized under light microscope at 40X magnification (Olympus Microscope, USA).

Data was analyzed statistically on ‘SPSS-16.0’ (SPSS Inc., Chicago, II USA) software package as per standard methods (Snedecor and Cochran, 1994). Whole set of experiment was repeated three times and mean values were reported along with standard error. The statistical significance was estimated at 5% level (p<0.05) and evaluated with Duncan’s Multiple Range Test (DMRT).

Antimicrobial activity was performed by disc diffusion method against ten foodborne pathogens (Fig. 1). Clear demarcated zone appeared which is an indicative of inhibition of various microorganisms by the peppermint oil used in our study.

Inhibition zone size of PEO ranged between 12.33 to 34.66 mm and maximum inhibition zone diameter was observed for Proteus mirabilis (34.66±1.20) followed by Staphylococcus aureus (26.66±1.76) whereas minimum inhibition zone diameters were observed for Klebsiella pneumonia (12.33±0.88) followed by Salmonella enterica(14.33±1.20) (Fig. 2). The MIC (ppm) of the oil against the above mentioned organisms is presented in Fig. 3. MIC (ppm) of peppermint essential oil against the tested organisms was observed in the range of 5000-20000 ppm. A significant correlation was also observed between MIC and ZOI values. It was observed that the peppermint essential oil was showing effective results against both Gram-positive as well as Gram-negative microorganisms.

However, the Gram-negative organisms were found slightly more susceptible than Gram-positive organisms. Rusenova et al. (2009) reported in-vitro antimicrobial activity of twelve selected essential oils including peppermint essential oil against Gram-positive and Gram¬negative microorganisms and found that the peppermint essential oil was effective against all the tested organisms. Mimica- Dukic et al. (2003) conducted a study on the antibacterial activity of essential oils of three Menthaspecies against a range of five Gram-positive strains and eight Gram-negative strains of bacteria. The results revealed that generally essential oils were more effective against Gram-positive bacteria than Gram-negative strains, but the test strains of multiresistant Gram-negative bacteria also exhibited a remarkable susceptibility to the examined essential oils. In the present study, though a strong positive correlation was observed between zone inhibition diameters and MIC values, however, a little discrepancy of obtained values as compared with previously conducted studies might be due to variation in origin and type of peppermint plant, method of extraction employed, final composition of peppermint essential oil, methodology adopted for evaluation of ZOI and MIC etc. (Kanth et al., 2018).

Results for antioxidant activity viz. DPPH and ABTS results for PEO are tabulated in Table 1 respectively. DPPH radical-scavenging action is due to their hydrogen- donating ability which is directly proportional to the total number of hydroxyl groups.

Therefore, more the number of hydroxyl groups, the greater is the hydrogen-donating ability and higher is the DPPH radical-scavenging activity (Blois, 1958). DPPH radical scavenging activity of oil was determined by using method of Kumar et al. (2017) with slight modification. A solution of DPPH (0.15 mmol/L) in methanol was used along with five different concentrations of oil (5000, 10000, 15000, 20000, 25000 ppm) and it was observed that there was an increasing trend of radical scavenging activity with increase in the concentration of peppermint essential oil. The different concentrations of essential oil to be tested were selected on the basis of MIC values as depicted in Fig. 3. ABTS decolourization is an excellent tool for the evaluation of antioxidant activity of hydrogen donating antioxidants. It was observed that there was an increasing trend of radical scavenging activity with increase in the concentration of peppermint essential oil. The presence of active principle i.e. menthol might be responsible for the higher radical scavenging activity of peppermint oil. The findings indicate that essential oil under investigation has potential antioxidant and free radical scavenging activity. Along with proven health benefits, these attributes can be helpful in controlling oxidative rancidity in high fat food products such as meat, thereby extending its storage stability.

In our study, we had explored the effect of peppermint essential oil on formation of biofilms by pathogens. The obtained results demonstrated that peppermint oil inhibited biofilm formation by L. monocytogenes and P. mirabilis strains with 45.80 and 73.01%, respectively (Fig. 4). In addition, the obtained results showed a correlation with anti-microbial activity. Light microscopic analysis revealed that the control slides showed a well-developed biofilm growth of test bacterial pathogens, whereas, pathogens treated with the peppermint oil (5000 ppm) showed poor and loose biofilm architecture that established its promising ability to destroy the biofilm structures. Similarly, Bazargani et al. (2016) reported that peppermint essential oil showed highest activity in the inhibition of biofilm growth against S.aureus (Gram positive) and E.coli (Gram negative) bacteria.