ANTIBACTERIAL MODE OF ACTION OF THE ROSEMARY/EUCALYPTUS OIL COMBINATION ON THE MEMBRANE INTEGRITY OF SELECTED FOOD BORNE PATHOGENS

Pooja Patial 1,* , Dr. Savita Jandaik 2 and Rakesh Kumar 1 . 1. Research Scholar, Department of Microbiology, Shoolini University of Biotechnology and Management Sciences, Bhajhol, Solan (H.P). 2. Assistant Professor, Department of Plant Pathology University of Horticulture and Forestry, Nauni, Solan (H.P). ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History

Deterioration of food can be caused by various factors such as physical, chemical, enzymatic or microbiological. Due to increasing side effect of chemicals in food, consumers demand for some natural alternative to these chemical preservatives. One of these alternatives is use of plant essential oils. The aim of this study was to understand the effect of combination of two commercial essential oils (rosemary/eucalyptus oil) on the membrane of one gram positive and one gram negative bacteria at MIC concentration. The release of cell constituents at 260 nm and potassium ions from the bacterial cell were measured after they were treated with essential oil combination of rosemary /eucalyptus oil at their MIC concentrations. The result showed that there was damage in the cell covering of bacteria because of the treatment of combination of oils. The assays performed to study the effect of combination of oil on cell membrane of both selected bacteria exhibited that the combination at MIC concentration damaged the cell membrane of both bacteria. These results provide the useful information that the rosemary /eucalyptus oil combination might be used as efficient and safe natural antimicrobial agent in food industry.

Introduction:-
Essential oils are the aromatic liquids, result of secondary metabolism in plants (Guenther, 1948). Essential oils are natural antimicrobials, which are used in flavor and fragrance industries (Van de Braak and Leijten, 1999). In food industries the essential oils are used as natural preservatives, which are the demand of time. As the chemical preservatives have their own set of problems like carcinogenicity, teratogenicity, acute toxicity and the environmental problems they cause due to long degradation time taken by them (Smid and Gorris, 1999).
Essentials oils have antibacterial, antifungal, antioxidant and anticarcinogenic properties which help them to be used as food additives. Essential oils when used as antimicrobial in food are required in higher quantity, which can demonstrate negative effect in taste and odor of food (Yamazaki et al., 2004.). To tackle this problem the combinations of different oil can be used which solves two purposes, one being the increase in antimicrobial effect and second is the decrease of concentration required of the oils.

ISSN: 2320-5407
Int. J. Adv. Res. 5(1), 1407-1412 1408 Rosemary and Eucalyptus oils are good antimicrobials alone. There is no information regarding their antibacterial action on bacteria. Hence, in the present study the mode of action of this combination at MIC value for E. coli and Micrococcus yunnanensis will be studied on their cell walls by determining the release of cellular materials at 260 nm and potassium ion efflux.

Materials and Methods:-
Material:-Essential oil combination:-Rosemary/Eucalyptus oil combination. Bacterial strains:-Micrococcus yunnanensis, E. coli. Methods:-Preparation of inoculum:-The bacterial inoculum used for testing the activity was prepared in nutrient broth. Five ml of broth was suspended in test tubes and autoclaved. The medium was cooled and inoculated with a loopful of bacterial cultures from the nutrient agar slants under aseptic conditions and then incubated at 37±1 o C for 24 hours. In order to standardize the inoculums density for test, BaSo 4 turbidity standard equivalent to 0.5 McFarland (turbidity equals to 0.5= 1 to 2×10 8 CFU /ml) was used (Burt, 2004).

Determination of minimal inhibitory concentration (MIC):-Investigation of the MIC of Rosemary and
Eucalyptus essential oils was done in 96 well titre plate (Schelz et al., 2006). In each well Muller Hinton broth (90 μl) was added. The essential oils alone were taken from range100 μl to 3.25 μl along x axis. 10 μl of working inoculum suspension (8×10 8 CFU/ml) was added to the each well. The bacteria tested were 80 µl in which well. Ciprofloxacin was used as positive control and DMSO was used as negative control. The plate was incubated at 37 o C for 24 hours. The plates were then incubated for 24 hour at 37°C. After incubation, O.D was measured using ELISA reader at 595 nm. The lowest dilution showing no visible growth was considered as the MIC for that individual oil. The tests were performed in triplicate.

Determination of Fractional Inhibitory Concentration Index (FICI):-Fractional inhibitory concentration index
was determined by checkerboard titration method but with some modifications (CLSI, 2005). Along the rows Rosemary oil concentration was reduced to half in each subsequent well, whereas Eucalyptus oil concentration was fixed at 50 µl and in another row eucalyptus oil concentration was reduced and rosemary oil concentration was fixed at 50 µl. The bacteria tested were 80 µl in which well. Ciprofloxacin was used as positive control and DMSO was used as negative control. The plates were then incubated for 24 hour at 37°C. After incubation, O.D was measured at 595 nm. The combination which showed maximum inhibition at lower concentration was taken as MIC. Where, EOA= rosemary oil and EOB= eucalyptus oil. The results were interpreted according to FIC indices as follows: FICI ≤0.5: Synergy; 0.5 < FICI ≤ 4: Additive; and FICI > 4: Antagonistic (Leclercq et al., 1991). All the experiments were repeated thrice.

Mode of action:-
The effect of combination of essential oils at its MIC concentration was studied on cell membrane of both selected bacteria by following assays: Potassium leakage assay:-The potassium leakage from cells was studied by following the methodology of Cox et al. (2000). 2 ml Suspensions of selected bacteria were exposed to essential oils combination (MIC) in sterile peptone water (0.1 g/ 100mL) for 0, 30, 60, and 120 minutes at 37 o C. After predetermined interval the samples were collected and extracellular potassium concentration was measured by photometric method. Controls were devoid of Essential oils combination. Results were calculated as amount of extracellular potassium (mmol/L) in the growth media at each interval of time.
Leakage of 260 nm and 280 nm cellular materials:-This assay was performed according to the methodology of Carson et al. (2002). To 2 ml of bacterial culture in sterile peptone water (0.1 g/ ml) essential oil(s) combination at their MIC concentration was added. The mixture was incubated at 37 o C for 0, 30, 60 and 120 minutes. Cells for measurement were collected after 0, 30, 60, and 120 minutes, cells were then centrifuged at 3500 rpm for 15 1409 minutes. The supernatant were collected and absorbance was measured at 260 nm UV/Vis Spectrophotometer. Controls were kept under same conditions, except that they were not treated with essential oils combination. The experiment was performed in triplicates and mean values were plotted against time.

Determination of Minimum Inhibitory concentration (MIC) and Fractional inhibitory concentration (FIC) of essential oils alone and in combination:-
The combination effect of oils on E. coli and Micrococcus yunnanensis (taken as models for further studies) was studied by Checkerboard method.
In tables (i) and (ii) the results of combination of rosemary oil and eucalyptus oil are summarized for E. coli and Micrococcus yunnanensis respectively. In case of E. coli when 6.25 µL of rosemary oil was mixed with 50 µL of eucalyptus oil, 0.125±0.01 MIC was obtained. Whereas for Micrococcus yunnanensis 0.098±0.01 MIC was obtained when 12.5 µL rosemary oil was mixed with 50 µL eucalyptus oil.  In tables (iii) and (iv) the results of combination of eucalyptus oil with rosemary oil are shown. For E. coli the best MIC value of 0.166±0.02 was depicted by eucalyptus oil (6.25 µL) with rosemary oil (50 µL). 0.144±0.01values were obtained in case of Micrococcus yunnanensis when mixed in ratio of 1:1 with rosemary oil. Eucalyptus oil combination with rosemary oil exhibited better result as compared to eucalyptus oil alone.   Mechanism of action:-The effect of rosemary and eucalyptus oil combination at their MIC concentration on cell membrane of E. coli and Micrococcus yunnanensis was studied using two assays: Assay of Potassium ions efflux:-The mode of antibacterial action of combination of oils (rosemary and eucalyptus) at MIC against two tested food borne pathogen was further confirmed using the assay for the release of K + ions from treated cells of E. coli and Micrococcus yunnanensis (figure i (A, B). Treatment of bacterial cells with oil combination at the MIC concentration induced a major efflux of intracellular K + ions. The leakage of potassium ions in case of both the bacteria increased with increase in time interval (figure i (A, B). However no K + ions efflux was observed in control cells of the tested bacteria during the study.

Leakage of Cellular Metabolites:-
The results of the release of cellular material at the absorbance value of 260nm from E. coli and Micrococcus yunnanensis cells exposed to concentration (rosemary + eucalyptus) of oils at the MIC concentrations indicated that the higher exposure time led to higher cell leakage of nucleic acids (figure ii(A, B). However, no changes in the absorbance of untreated cells (control) of tested bacteria were seen. At 30 and 60 minutes of treatment, significant increase in the absorbance of the bacterial cells treated with oil combination was demonstrated. The leakage of the material absorbing at 260nm from the bacterial cells treated with combination of rosemary and eucalyptus at MIC, confirms the damage of cell membrane.

Discussion:-
Essential oils are natural antimicrobials produced by many aromatic plants and exhibit a great potential to be used in food industry, but there use is restricted due to strong smell and alteration of taste food. To overcome such problem combination of oils can be good option.
In this study commercially available rosemary and eucalyptus essential oils were briefly studied, obtained from local market of Solan, Himachal Pradesh for their potential to be used as food preservative. Rosemary and eucalyptus oil