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Antibacterial Effect of Combinations Essential Oils On Escherichia coliBrendan DettmerLoras College1450 Alta Vista StreetDubuque IA, 52001Antibacterial Effect of Combinations Essential Oils On Escherichia coliAbstractEssential oils have been apart of human culinary arts for a long time. Often times these oils are used in combination. The goal of this paper is to look at potential preservative and antimicrobial effects of these oils and when they are used in combinations. Thyme when used in combination with Tea Tree lead to an increase in inhibition against E. coli while Black Pepper and Tea Tree inhibited Clove and Thyme’s antimicrobial effects. The inhibition and synergistic effects may be due to different attack areas against bacterial cytoplasmic membranes. Future studies should look at combinations against a variety of microbes and fungus.IntroductionEssential oils are organic compounds that oxygen atoms and sometimes nitrogen and sulphur atoms. These oils are used in?perfumes, cosmetics, and?soaps?because of their volatile?aroma compounds (Bauer et al., 2001). Essential oils are also often used in food and drink for flavoring. In the past essential oils have been used to protect livestock and food from disease, pests and spoilage. (Dorman and Deans 2000). Spoilage in food is due to lipid deterioration, oxidation and spoilage by microorganisms. (Sacchetti et. Al. 2005). Essential oils, however, do not have significant nutrient content. In the past essential oils and their constituents have been found to possess antimicrobial properties, which is hypothesized why humans use them in their diet. (Lis-Balchin & Deans 1997; Billings and Sherman 1998). The mechanism for each essential oil’s antimicrobial properties is a method that is poorly understood. (Dorman and Deans 2000). Terpenoids can be a constituent of an essential oil. They are composed of isoprene groups and oxygen functional groups. (Dorman and Deans 2000). Previous studies have suggested that terpenoids attack the phospholipid bilayer of bacteria’s plasma membrane. The plasma membrane in bacteria is vital for energy conservation in bacteria, because they don’t have mitochondria. Terpenoids cause inhibition of electron transport, protein translocation, phosphorylation steps and other enzyme-dependent reactions in the plasma membrane of the bacteria. (Knobloch et al. 1986). Another constituent of an essential oil can be phenols. Phenol groups are composed of a benzene ring that has an alcohol group attached to it. Phenolic compounds have been shown to be highly active against microorganisms. They are also known to be either bactericidal or bacteriostatic agents. (Pelczar et al. 1988). The hydroxyl group in phenolic compounds has been found to be a major part of phenol’s antimicrobial properties. (Dorman and Deans 2000). Oregano, thyme, and clove essential oils have a lot of phenolic compounds in them. (Dorman and Deans 2000). Other groups have been found to increase antimicrobial activity. Acetates have been found to increase the activity of the parent chain. (Dorman and Deans 2000). Alcohols are known to possess bactericidal properties. Alcohols have the potential to work as protein denaturing agents, solvents or dehydrating agents against bacteria. (Dorman and Deans 2000).Aldehydes are also know to possess powerful antimicrobial properties. A proposed theory is that aldehydes high electronegativity has an antibacterial effect. (Moleyar & Narasimham 1986). Highly electronegative compounds may have inhibiting effect on electron transfer and may react with vital nitrogen components in bacteria such as proteins and nucleic acids. Therefore, these effects cause inhibition of growth of microorganisms. (Dorman and Deans 2000). Overall, previous studies have showed that essential oils with phenolic groups are the most effective against bacterial growth. (Dorman and Deans 2000). A potential factor other than essential oil structure that can effect effectiveness of bacterial inhibition of essential oils, is whether or not the bacteria is a gram negative or gram positive bacteria. Gram positive bacteria have a thick peptidoglycan layer covering their plasma membranes, while gram negative bacteria have an outer membrane covering a thin layer of peptidoglycan over their plasma membranes. The question of which bacteria is better at preventing essential oils from attacking their plasma membrane is one that scientists have many different hypothesis. Zaika (1998) had hypothesized that, gram positive bacteria would be more resistant to essential oils’ antimicrobial effects. Deans and Ritchie (1987) on the other hand predicted that the susceptibility of bacteria to plant volatile oils and the Gram reaction has little in?uence on growth inhibition. What Dorman and Deans found, however, was that with some essential oils the gram reaction had little influence on growth inhibition but with others gram negative bacteria were more resistant to the essential oils. (Dorman and Deans 2000). Given that essential oils have antimicrobial properties, the next question to look into is how do we use them? As food preservatives, volatile oils may have their greatest potential use. Spices that are used for flavoring are present in insuf?cient quantities for their antimicrobial properties to be signi?cant against microbes. (Dorman and Deans 2000). However, if the volatile oils if added to foods, would cause no loss of organoleptic properties, thus would retard microbial contamination, and therefore reduce the onset of spoilage. (Dorman and Deans 2000). Billings and Sherman (1998) wanted to test if; spices should kill or inhibit microorganisms that cause food spoilage, if spice use should be used heaviest in hot climates because food spoils the most rapidly in hotter climates, and whether spices with the most potent microbial activities should be favored in areas where food is quick to spoil. What they found was that people in hotter climates used more spices than people that lived in cooler climates. Hotter conditions were ideal for microbial growth. They also found that people that lived in hotter climates used more spices in combination with their recipes. (Billings and Sherman 1998). What Billings and Sherman’s (1998) findings suggest is that there may be a cooperative effect of different essential oils against bacteria. Dorman and Deans (2000) also found that different essential oils and their components have different effects on different bacteria. They also found that essential oils from thyme, oregano, clove, and nutmeg inhibited a wide range of bacteria. (Dorman and Deans 2000). Billings and Sherman found that garlic, oregano, and thyme inhabited the widest variety of spices. (1998). However, both these studies did not look at potential cooperative and synergistic effect that essential oils might have against gram negative and gram positive bacteria. Therefore the goal of this research is to look for possible synergistic effects that different essential oils used in combination will have against a variety of bacteria. The findings of this study might help better understand essential oils effect on different bacteria and how to better preserve our food. Materials and MethodsEssential Oil SamplesEssential oils from oregano, thyme, clove, tea tree, and black pepper were purchased. Vegetable oil was also purchased from Hy-vee to function as a control for the experiment. MicroorgansimEscherichia coli was the bacteria used for this experiment. E. coli is a foodborne pathogen found in animal feces. E. coli can cause hemor-rhagic colitis, hemolytic uremic syndrome (HUS), and thrombotic thrombocytopenic purpura (TTP) and is of great concern to the meat industry. (Kim et. al. 1995). E. coli was chosen because it is known to cause food poisoning in humans. (Kim et. al. 1995). The E. coli was obtained from Carolina Biological Supply Company. E. coli was used to test the effectiveness of essential oils, and their possible synergetic effects. Bacteria were grown up on Difco Nutrient Agar on 95 x 15 mm polystyrene plates. Anti-microbial TestAntimicribial tests of various essential oils were to be tested by paper disc diffusion using petri dishes that contain 0.5-0.8 cm of Difco nutrient agar. Bacterial colonies were spread as a thin layer using a sterile cotton swab from plates already containing the correct bacterial strain and then allowed to grow for 24 hours at room temperature to be used for other microbial tests within the experiment. Petri dishes were then to be spread with a lawn of the appropriate bacteria to be tested against each essential oil. For testing individual oils, each oil was mixed with vegetable oil and then 1 ?l of the combined vegetable oil/essential oil mixture is to be added to the paper disc which was 0.5 cm long and composed of Whitman Filter Paper. After that, the disc was added to the bacterial plate. This is to be repeated for each essential oil and each bacterium. Also 1 ?l of vegetable oil is to be tested as a control group. For the combination of each essential oil, the oils of equal volume are to be mixed together thoroughly. After that 1 ?l of the resulting mixture is to be added to the paper disc and added to the bacterium. This is to be done for each essential oil combination and each bacterium. Plates are then to be left at room temperature for 48 hours. Then the diameter of the inhibition is to be measured and averaged. Measurements for each inhibition were done by taking the diameter of the zone of inhibition and subtracting 0.5 cm to account for the disc. Each test is to be repeated at least ten times. Vegetable oil is to be used as a control while the other oils are to be tested for possible effects and synergistic against the bacterium. Data is to be analyzed by a t-test and graphs using Microsoft Excel. ResultsAs seen in Figure One individual oils had varying effects against E. coli. Black Pepper and Vegetable Oil were not statistically different from one another. All other oils were significantly different from Vegetable Oil (p value < 0.05). In regards to combinations all combinations were significantly different compared to the control of vegetable oil as seen in Figure 2. In most cases however there was no significant difference when the individual oil was compared to the combination. However there was a couple exceptions.Black Pepper actually statistically decreased the inhibition of Thyme and Clove as seen in Figure 3. Tea Tree when combined with Clove also decreased inhibition (Fig. 4). Thyme and Oregano lead to an increase of inhibition however (p value < 0.01) as seen in Figure 5.DiscussionFrom analyzing individual oils it is clear that Thyme, Clove, and Oregano were the most powerful oils that were tested. Thyme was the best at inhibition (Figure 1). It is of note that each of these oils contains a phenol group. (Sacchetti et al 2005). Phenolic compounds have in the past shown to have strong antimicrobial effects. (Deans and Ritchie 1987). Therefore the large difference between Thyme, Clove, and Oregano when compared to the rest of the oils is to be expected. The mechanism for how the phenols attack bacterial cells has been identified. Phenolic compounds act by disrupting lipid-containing bacterial membranes resulting in leakage. With higher concentrations the phenols can even precipitate cytoplasmic cell proteins. (Edris 2007). However, Thyme, Clove, and Oregano each had a varying effect on E. coli despite all of them containing phenols. This may be due to concentration of phenolic compounds in the oils. Thyme has the highest concentration of phenolic compounds. (Dorman and Deans 2000).Tea Tree had mild effects while Black Pepper did not perform better than the vegetable oil control. Tea Tree and Black Pepper essential oils both contain aldehyde and ketone functional groups. These in the past have shown to perform mild effects against gram negative bacterium. (Dorman and Deans, 2000; Siqueira + Lobes 1999). However, Tea Tree oil has been shown to have very potent antifungal activity. (Siqueira + Lobes 1999). When looking at combinations, a majority of the combinations were not better or worse at inhibition than just the “stronger” oil by itself. However, there were four exceptions. The first two involved Black Pepper. Black Pepper inhibited the effects of Thyme and Clove. On a similar hand Tea Tree inhibited Clove. Thyme when used in combination with Oregano however led to an increase in inhibition. One possible explanation for why these effects happened could be due to the mechanics of the antimicrobial effects. In the case of Black Pepper and Clove, aldehydes and ketones have both been predicted to attack the cytoplasmic membrane. (Dorman and Deans 2000). One possibility is that they inhibit Thyme from attacking the bacteria cytoplasmic membrane. On the other hand in regards to Thyme and Oregano it is possible that their phenol groups are attacking different locations of the cytoplasmic membrane leading to an increased effect. Previous studies have found that Thymol (phenol used in thyme) does have synergistic effects with other phenolic compounds such as eugenol + carvacrol. (Bassole + Juliani 2012). Carvacrol is the main phenol present in Oregano. (Dorman and Deans 2000). Also previous studies have shown that a lot of combinations show no synergistic or additive effects. (Bassole + Juliani 2012). In the case of antagonism, it is known to happen with phenols. Aldehydes have been known to show antagonistic effects against some phenols. (Bassole + Juliani 2012). The reason for antagonistic effects has been credited to interactions between non-oxygenated and oxygenated monoterpene hydrocarbons. This would explain the complications of phenolic compounds when interacting with non-oxygenated monoterpenes. It is clear from the data though that some essential oil combinations are better than others in disrupting E. coli growth. Thyme and Oregano when used in combination was stronger than Thyme and Black Pepper used together. So while this data can remotely support the initial hypothesis, that some combinations are better at inhibiting microbial growth; a lot more needs to be done. One is the fact that this study only looked at E. coli. It is certainly possible that some combinations of oils are better against different microbes than other combinations. Previous studies have noted that some oils are potent against certain microbes and no effect on others. (Dorman + Deans 2000; Bassole + Juliani 2012). For example Tea Tree has shown to have high resistance to fungal activity. (Siqueira + Lobes 1999). In addition samples of more oils would help broaden the understanding of how certain combinations function. Also the methodology of the experiment was a bit crude. A better way of obtaining measurements would be desirable for future experiments. Furthermore, there were some contamination issues with the study. Some groups throughout the experiment were contaminated and were removed from the sample size. Finally, the essential oils used in the experiment were very concentrated. The concentration of the oils would not be pleasing to taste on regular food. Thus it would be interesting to see if the oils were still as potent in more dilute solutions comparable to what is used in cooking. Acknowledgments Dr. Fred Schnee, for helping me through the process of this research throughout a very complicated time…and for putting up my frequent problems.Dr. Aditi Sinha for being an awesome advisor throughout my time at Loras.Loras College Science Staff for all the information that has been provided too me.Loras College Staff for a great experience that will last me for all my years to come.Cherie and Karl Dettmer for providing support even when I didn’t want it.Ellen Reiss for providing me with the drive to continue on in school and in life’s challenges.ReferencesBauer, K. G., Garbe, D., & Surburg, H. (2001). Common fragrance and flavor materials preparation, properties and uses (4th, completely rev. ed.). Weinheim: Wiley-VCH.Bassolé, Ima?l, and Rodolfo Juliani. "Essential Oils in Combination and Their Antimicrobial Properties."?Molecules?17.4 (2012): 3989-4006.?Molecules. Web. <, J., & Sherman, P. (1998). Antimicrobial functions of spices: why some like it hot. The Quarterly Review of Biology, 73(1), 3-49.Deans, S., & Ritchie, G. (1987). Antibacterial properties of plant essential oils. International Journal of Food Microbiology, 5, 165-180.Dorman, H., & Deans, S. (2000). Antimicrobial agents from plants: antibacterial activity of plant volatile oils. Journal of Applied Microbiology, 88, 308-316.Kim, J., Marshall, M., & We, C. (1995). Antibacterial activity of some essential oil components against five foodborne pathogens. Journal of Agriculture and Food Chemistry, 43, 2839-2845.Knobloch, K., Pauli, A., Iberl, N., Weigand, N., & Weis, H. (1989). Antibacterial and antifungal properties of essential oil components. Journal of Essential Oil Research, 1, 119-128.Lis-Balchin, M., & Deans, S. (1997). Bioactivity of selected plant essential oils against Listeria monocytogenes. Journal of Applied Microbiology, 82, 759-762.Moleyar, V., & Narasimham, P. (1986). Antifungal activity of some essential oil component. Food Microbiology, 3, 331-336.Mukamolva, G. Kormer, S. Kell, D. & Kaperlyants, A. (1999). Stimulation of the multiplication of Micrococcus luteus by an autocrine growth factor. Archives of Microbiology, 172, 9-14Pelczar, M., Chan, E., & Krieg, N. (1988). Microbiology. New York: McGraw-Hill.Sacchetti, G., Muzzoli, M., Scaglianti, M., Manfredini, S., Radice, M., & Bruni, R. (2005). Comparative evaluation of 11 essential oils of different origin as functional antioxidants, antiradicals and antimicrobials in foods.. Food Chemistry, 91(4), 621-632.Zaika, L. (1988). Spices and herbs: their antimicrobial activity and its determination. Journal of Food Nutrition, 9, 97-118. ................
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