Australian Patents Act 1990 - Regulation 3.2A ORIGINAL COMPLETE SPECIFICATION STANDARD PATENT Invention Title "Enhanced antimicrobial activity of plant essential oils" The following statement is a full description of this invention, including the best method of performing it known to me/us:- CANRPonbDCC\RBRuol2R7_l.DOC.19f/2012 ENHANCED ANTIMICROBIAL ACTIVITY OF PLANT ESSENTIAL OILS This application is a divisional application of Australian Application No. 2009201763 the specification and drawings of which as originally filed are incorporated 5 herein in their entirety by reference. CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. § I19(e) to provisional application Serial No. 61/053,216 filed May 14, 2008, herein incorporated by reference in its entirety. 10 FIELD OF THE INVENTION This invention relates to use of plant essential oils as antimicrobial compositions, and to enhancing their effectiveness by addition of antimicrobial enhancers and by use of combinations of essential oils. 15 BACKGROUND OF THE INVENTION It is known in the art that plant essential oils, that is oils derived from plants by distillation, expression or extraction may have antimicrobial activity when exposed to bacterial cells. Consumer acceptance of these essential oils is high because they usually 20 have the fragrance of the plant from which they were extracted. When used for animal use, the animals do not commonly shy away from them because again, they have the odor of plants from which they were derived, and such odors are not unfamiliar to many animals. Essential oils mixed with carriers have a lot of potential veterinary and human uses. For example in the veterinary world they may be used as teat dips, or disinfecting topicals 25 for skin ulcers, for shampoos, for topical gels and creams, for anti-fungals, and even to be taken internally for use in the GI tract. There is a continuing need for increasing the cellular uptake of plant essential oils in order to enhance their anti-bacterial effect. Some researchers have theorized that plant essential oils soften the walls of the bacteria then permeate them thus causing the enhanced 30 anti-bacterial effect. (see, Vaara, "Agents That Increase the Permeability of the Outer Membrane", Microbiological Reviews, September 1992, Vol. 56(3); and Johnson U.S. - 1 - C \NRPonbf\DCC\RBRWO8i827_L DOC-/9/2012 Patent No. 6,319,958 that teaches addition of at least one sesquiterpenoid to advance the antimicrobial effect of antimicrobial compounds. BRIEF SUMMARY OF THE INVENTION 5 In the ever increasing efforts to enhance antimicrobial effectiveness Applicant has now discovered that a combination of plant essential oils provides increased enhancement; and moreover the combination of oils may be used with other known enhancers to even further maximize effectiveness. 10 One or more embodiments of the present invention may provide plant essential oil derived antimicrobial compositions, that use a combination of essential oils, and in some cases a combination of enhancers also used with the oils, to achieve a maximized antimicrobial effect. One or more embodiments of this invention may advantageously allow for the 15 preparation a variety of different antimicrobial compositions based on the above discovery that are useful for veterinary or human use. The method or means of accomplishing at least the above embodiments and their advantages will become apparent from the detailed description of the invention which follows hereinafter. 20 Antimicrobial compositions based on a combination of plant essential oils are of enhanced antimicrobial effectiveness and are prepared by adding to at least two plant essential oils a small but antimicrobial enhancing effective amount of an enhancer selected from the group consisting of polyionic organic enhancers and polyionic inorganic enhancers. The composition comprises a mixture of plant essential oils wherein at least 25 one of the oils is oregano oil. Thus, the present invention provides antimicrobial compositions having an oil component based on plant essential oils, said anti-microbial being of enhanced anti microbial effectiveness, comprising: at least two plant essential oils providing enhanced anti-microbial effectiveness, 30 with one being oregano oil, as a major component of the oil component; and a small but antimicrobial enhancing effective amount of an enhancer selected from the group consisting of polyionic organic enhancers and polyionic inorganic enhancers. -2- C:WRPonbDCC\RBR\4081827l1. DOC.l9/2012 DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT This invention relates to antimicrobial compositions derived from plant essential oils, more importantly a combination of essential oils to enhance antimicrobial effectiveness. The essential oil component may contain from 40% to 95% by weight oils, 5 but is preferably from 50% to 90% by weight of a combination of essential oils. The most preferred essential oils being a I to 1 by weight mix of at least two different essential oils, with one of them being oregano oil. As a major component as herein defined means at least 40% by weight. Essential oils are volatile aromatic oils which may be synthetic or may be derived 10 from plants by distillation, expression or extraction, and which usually carry the odor or - 2a flavor of the plant from which they are obtained. In the combination compositions of this invention, antiseptic activity is provided by essential oils. Some of these essential oils also act as flavoring agents. Besides oregano oil and thymol, the essential oils of this invention may include but are not limited to menthol, methyl salicylate (wintergreen oil), eucalyptol, 5 carvacrol, camphor, anethole, carvone, eugenol, isoeugenol, limonene, osimen, n-decyl alcohol, citronel, a-salpineol, methyl acetate, citronellyl acetate, methyl eugenol, cineol, linalool, ethyl linalaol, safrola vanillin, spearmint oil, peppermint oil, lemon oil, orange oil, sage oil, rosemary oil, cinnamon oil, pimento oil, laurel oil, cedar leaf oil, and clove oil. In embodiments of the invention wherein organic phenolic compounds are obtained 10 from plant oil extracts, the oil is preferably extracted from a member of the Labiatae (also called Lamiaceae) or Verbenaceae family. Plants in the family Labiatae or Verbenaceae include hybrids of plants produced from individual plants in those two families. The common name for members of the Labiatae family, a large family of mostly annual or perennial herbs, is the "mint family." The mint family is classified in the division 15 Magnoliphyta, class Magnoliopsida, and order Lamiales. The Labiatae family includes about 200 genera, such as Salvia, Rosmarinus, Mentha, Ocimum, Thymus, Marrubium, Monarda, Trichostema, Teucrium, Hyptis, Physostegia, Lamium, Stachys, Scutellaria and Lycopus. Plants which are preferably used for extraction of organic phenolic compounds 20 include, but are not limited to, Ocimum spp., Saturea spp., Monarda spp, Origanum spp, Thymus spp., Meniha spp., Nepeta spp., Teucrium gnaphalodes, Teucrium polium, Teucrim divaricatum, Teucrim kotschyanum, Micromeria myrifolia, Calamintha nepeta, Rosmarinus officinalis, Myrtus communis, Acinos suaveolens, Dictamnus albus, Micromeriafruticosa, Cunila origanoides, Mosla Japonoica Maxymowitz, Pycnanthemum 25 nudum, Micromeria Juliana, Piper betel, Trachyspermum ammi, Lippia graveolens Escholcia splendens, and Cedrelopsis grevei, as well as others. In a preferred composition, the oil is extracted from Esholtia splendens, Cedrelopsis grevei, Lippia graveolens or a plant of the species Nepeta, including but not limited to Nepeta racemosa (catmint), Nepeta citriodora, Nepeta elliplica, Nepeta 30 hindostoma, Nepeta lanceolata, Nepeta leucophylla, Nepeta longiobracteata, Nepeta mussinii, Nepeta nepetella, Nepeta sibthorpii, Nepeta subsessilis and Nepeta tuberosa. 3 Most preferably, the oil is extracted from a hybrid plant produced from crossing Nepeta racemosa, Esholtia splendens, Cedrelopsis grevei, and Lippia graveolens. Plants of the Labiatae and Verbenacea families are found throughout the world and are relatively easy to cultivate. To cultivate the plants, seeds, preferably those of plants that 5 are expected to yield a high percentage (e.g., at least about 70 wt %, more preferably at least about 80 wt %), of organic phenolic compounds, are planted in fine loose soil, preferably in a sub-tropical climate. Hybrid seeds having a high percentage of organic phenolic compounds can be produced by known techniques. Crossing Nepela racemosa, Esholtia splendens, Cedrelopsis grevei, and Lippia graveolens produces one such hybrid 0 that is a preferred source of the organic phenolic compounds. The seeds are then cultivated using known agricultural techniques, such as watering, and artificial fertilizing. Most preferably, the plants are cultivated and grown without the use of any synthetic pesticides. Because the leaves contain a high amount of oil upon blossoming, it is preferred that the plants be harvested soon after the plants begin to blossom. Preferably, the plants 5 are harvested within 24 hours after blossoming, more preferably within 12 hours after blossoming. Most preferably, harvesting is undertaken early in the morning or late in the evening hours (after blossoming begins) when the leaves are not exposed to the sun. Because the majority of the oil is found in the leaves and blossoms of the plant, it is preferred that only the leaves and blossoms be utilized in the extraction process. Use of 0 other parts of the plant may increase impurities and decrease yield, but may be utilized. Thymol, also known by the chemical formula 5-methyl-2-(1-methylethyl) phenol, is obtained from the essential oil of Thymus vulgaris Labiatae and Monarda punctata Labiatae. Thymol is a white crystalline powder with an aromatic odor and taste and is soluble in organic solvents but only slightly soluble in deionized water. Thymol along with 25 oregano oil are preferred for at least one of the combination of essential oils Menthol is isolated principally from the oil of Mentha arvensis. In its commercial form, menthol is available as L-menthol crystals obtained from a process involving cooling of the oil. Fractional distillation of peppermint oil which usually contains from about 40% to about 65% menthol represent another important source of menthol. Synthetic sources of 30 L-menthol are also available. 4 Eucalyptol, another essential oil with antiseptic properties, is derived from the eucalyptus tree. Having a camphoraceous odor and cooling taste, this essential oil is often combined with other essential oils such as menthol in confection formulations to impart medicinal effect. Combinations of menthol and eucalyptol are widely used. Particularly 5 preferred uses of the menthol-eucalyptol combination include, according to the present invention, dentifrices such as toothpastes or dental gels. Methyl salicylate is the main ingredient in many essential oils, constituting about 99% of oil of wintergreen (Gaultheria procumbens) and sweet birch (Betula lenta). Methyl salicylate, which has a distinctive refreshing aroma, is used widely in mouthwashes, 0 chewing gums and other oral and pharmaceutical preparations. In the most preferred compositions of the present invention it is preferred that at least one of the essential oils have as their active ingredient a combination of thymol and carvacrol. The most preferred is oregano oil. One very satisfactory oil blend is 47.5% by weight oregano oil, 23.75% by weight 5 cinnamon bark oil, and 23.75% by weight clove oil and 5% capsicum oil resin. Other oil blends may also be used such as: 46% by weight oregano, 22% cinnamon bark, 22% clove, 5% nerolidol and 5% capsicum. A third blend formulation is 30% oregano, 30% cinnamon bark, 30% clove, 5% nerolidol and 5% capsicum. 10 A fourth blend formulation is 36.20% oregano, 18% cinnamon bark, 17% clove, 4% nerolidol, .8% oleoresin capsicum, 4% cranberry, 6.60% geranium, 6.67% patchouli, and 6.67% tea tree. A fifth blend formulation is 33% regular oregano, 33.34% clove, and 33.34 cinnamon. 25 A sixth blend formulation is 95% rosemary oil and 5% nerolidol. The essential oil can be mixed in a variety of physical formats, with one preferred one being so called beads. Bead format is from 0.5% to 50% of a combination of oils or pure oil added to a mixture of alginate, shellac and seaweed carriers to provide a carrier bead with the oil. This allows for a convenient and easy subsequent processing. The 30 process is known and can be accomplished by a variety of manufacturers. 5 Products based on essential oils, such as those containing organic phenolic compounds, tend to be absorbed at a level greater than 90% in the small intestines. Therefore, most of the activity of such products tends to be localized in the stomach and/or small intestine. However, there are many microbial infections that occupy portions of the 5 gastrointestinal tract beyond the small intestine. Therefore, it may be desirable to extend the activity of the combination essential oil based product into the large intestine. Microencapsulation is one method that can help extend the-activity of the antimicrobial composition throughout the entire gastro-intestinal tract (GIT). Microencapsulation is a micro-packaging technique which involves the coating of small 0 particles of solids, liquid droplets, or dispersion of solids, within liquids. Microencapsulated antimicrobial compound may be used to treat infections located in the end of the small intestines (e.g., jejunum and/or ileum) and beginning of the large intestines (e.g., ascending colon and transverse colon). The microencapsulation prevents release of the active ingredients in the stomach or in the beginning of the small intestines (e.g., 5 duodenum). If the antimicrobial compound is not microencapsulated, the acidic environment of the stomach will tend to break the association between the antimicrobial compound and most carriers in the pharmaceutical composition (such as dextrose, starch, etc.) and thereby activate the antimicrobial compound in the stomach. For example, a microencapsulated form of the antimicrobial compositions may be 0 used to treat Cryptosporidia spp. infections and/or chronic enteritis in humans; Cryptosporidia infections in animals, Lawsonia intracellularis and Treponema hyodesynteriae infections in pigs, and others. One example of a microencapsulation process includes encapsulating the antimicrobial composition in a multi walled capsule such that the layers of the wall 25 dissolve as the capsule travels through the gastrointestinal tract. Thus, the components that make up each layer of the capsule wall are chosen based on the conditions in the specific region of the gastrointestinal tract in which they are desired to dissolve. For example, the pH along the gastrointestinal tract (GIT) varies: in the stomach, the pH is between 2 and 5; in the duodenum, 4 and 6; jejunum, 4 and 6; ileum, 6.5 and 7.5; caecum 5.5 and 6.5; colon, 30 6.5 and 7; and rectum, 6.5 and 7. Therefore, the components of the wall layers may differ depending on what type of an ailment is to be treated, or its location, and whether the final 6 C:\NRPonbiKDCC\RBRWOBI 27 _LDOC.I/9/2012 formulation is meant to treat humans or animals. Each layer of the wall may also contain the composition of the invention so that upon dissolution of that wall layer, it can be released to effectuate treatment of the ailment. Suitable coating matrices include fatty acids, waxes, sugars, and shellac. 5 Encapsulation techniques are known. An example of one encapsulation technique (called fluidized bed coating) is provided below. In a fluidized bed, a suspension of solid particles is transformed into a fluid-like state by an upward gas flow through the system. Because of the intensive heat and mass transfer, fluidized bed reactors are widely used, e.g. in chemical industry for solid-catalyzed gas-phase reactions. To maximize the yield of 10 such reactors, liquid reactants can locally be injected into the fluidized bed. The injected liquid reactants penetrate the fluidized bed and evaporate. For design purposes and the achievement of optimal operating conditions, the spatial distribution of the concentration of components and temperature has to be predicted. Fluidized bed coating can be used to encapsulate the antimicrobial compound in a 15 coating material which includes ethyl cellulose and plant oil. First, the antimicrobial compound described is combined in the fluid bed mixer with the ingredients to form a powder, such as the ingredients shown in the table below. While the above disclosure has been emphasizing blends of at least two oils with at least one being oregano, multi-oil blends with at least one being oregano also work. 20 The especially preferred three-oil blend formulas include the following: -7- C:\NRPorDCC\R BRI27_1.DOC.1/9/l012 1" Oil Blend Formula: Base Formula* 33.34% Regular Oregano Oil 33.34% Rosemary Oil 11.11% Licorice Powder 11.11% Cinnamon Bark 11.11% 100% 2 "d Oil Blend Formula: Base Formula* 33.34% Regular Oregano Oil 33.34% Rosemary Oil 11.11% Chamomile Oil 11.11% Peppermint Oil (high menthol content) 11.11% 100% 3 rd Oil Blend Formula: Base Formula* 33.34% Regular Oregano Oil 33.34% Cinnamon Bark Oil 11.11% Rosemary Oil 11.11% Peppermint Oil (high menthol content) 11.11% 100% * Base Formula as used here includes 33% Geranium oil, 33.34% Patchouli and 33.34% Tea Tree oil. 5 Scours Finished Product Example Two scours treating products were made, each containing combinations of essential oils, as listed below in the finished product. Product A contained sodium polyphosphate inorganic enhancer. Product B contained three enhancers Nerolidol, PEI, and sodium polyphosphate. A B Ingredient Percentage Ingredient Percentage Purified Water 44.60% Purified Water 39.60% Activated Charcoal 10.00% Activated Charcoal 10.00% Regular Oregano Oil 7.50% Regular Oregano Oil 7.20% Cinnamon Bark Oil 3.25% Cinnamon Bark Oil 7.20% Redistilled Clove Leaf Oil 2.75% Redistilled Clove Leaf Oil 3.60% Nerolidol 0.50% Nerolidol 1.00% Capsicum Oleoresin 0.75% Capsicum Oleoresin 0.50% -8- C:WRPortbl\DCC\RBRO81827_l-DOC-I/9/2012 Cranberry Extract 0.25% Cranberry Extract 0.50% Dextrose 5.00% Dextrose 5.00% Agro-Pect 5.00% Agro-Pect 5.00% Arabic Gum 2.50% Arabic Gum 2.50% Glycine 2.50% Glycine 2.50% Psylium Seed Hulls 2.50% Psylium Seed Hulls 2.50% Betaine Hydrochloride 2.00% Betaine Hydrochloride 2.00% Seaweed Meal---Nori 2.00% Seaweed Meal---Nori 2.00% Flakes Flakes Citric Acid 1.50% Citric Acid 1.50% Sodium Chloride (Extra 1.50% Sodium Chloride (Extra 1.50% Fine) Salt Fine) Salt Potassium Chloride 1.50% Potassium Chloride 1.50% (Muriate of Potash) (Muriate of Potash) Magnesium Chloride 1.00% Magnesium Chloride 1.00% Citricidal 1.00% Citricidal 1.00% Ascorbic Acid 1.00% Ascorbic Acid 1.00% Zinc Oxide 1.00% Zinc Oxide 1.00% Magnesium Oxide 0.30% Magnesium Oxide 0.30% Chlorophyll 0.10% Chlorophyll 0.10% Each of products A and B when mixed appeared physically as a gel delivery format. In several separate trials over 1000 baby calves were treated in the following manner. The gel is orally fed to the stricken calf at 30cc/calf as a 1-3 time dose depending 5 upon the severity of diarrhea. In nearly all cases after treatment the scours clearly up within days. As used in the appended claims, with respect to the oil blend concentrate, major component refers to at least 40% by weight of the amount of combined essential oil component; minors include other additives. 10 The polymeric polyionic organic enhancer can be the preferred polyethyleneimine (PEI) or can be others such as paramethoxyphenyl ethylmethylamine. The amount can be 0.1mM to 50mM, similar to the same amount of the sesquiterpenoids (herein before described). The polyionic inorganic enhancers preferably polyphosphate enhancers and can 15 include sodium tripolyphosphate, sodium hexametaphosphate, at similar levels. Other carriers may include minors used for a variety of purposes in various topicals, pills, gelatins, etc. and can include small amounts of Apple Powder, Citrus Pectins, Arabic Gum, Ascorbic Acid, Beeswax, Betaine Hydrochloride, Biotin, Calcium Carbonate (Thermocal), -9- C RPortbDCC\RBR408 1827_ .DOC-U1912012 Canola Oil, Cetyl Alcohol, Choline Chloride, Citric Acid, Cobalt Carbonate, Copper Sulfate, Corn Starch, Dextrose, Dry Sweet Orange Flavoring, Flaxseed Oil, Folic Acid, Glycine, Lanolin, Lavendar Oil, Lemon Powder, Lipase DS, Maltodextrin, Manganese Sulfate, Magnesium Chloride, Magnesium Oxide, Malic Acid, Niacin, Olive Oil, 5 Pantothenic Acid, Potassium Chloride, Potassium Sulfate, Polysorbate, Propylene Glycol, Purple Pigment, Pyridoxine HCL, Riboflavin, Seaweed Meal, Probiotics/Bacteria, Selenium, Silicon 50S, Silicon Dioxide, Sodium Acetate, Sodium Chloride, Sodium Citrate, Sodium Propinate, Sodium Silica Aluminate - MS, Spearmint Oil, SST (Activated Charcaol), Steryl Alcohol, Thiamine, Vaseline, Vitamin A, Vitamin B12 600mg, Vitamin 10 D3 500, Vitamin E, Vitamin K, Water, and Zinc Sulfate. Applicants have also discovered that further anti-microbial property enhancement is achieved if from .01% by weight to 10% by weight, preferably .05% by weight to 5.0% by weight of the concentrate is an organic acid anti-microbial selected from the group of acetic, citric and fumaric. 15 Testing of the combination of oils at Iowa State University during the year 2006/2007 revealed that the combination of oils was more effective from an antimicrobial standpoint than the single oils alone. Preferred was the oil blend previously described as the first oil blend and the second oil blend. These oils in combination with various carriers may be used to make a variety of veterinarian and human use compositions, as previously 20 stated and those include pills, gelatin capsules, skin topicals, gels, creams, liquid rub-ons, powders and shampoos and G.I. tract medicines. To make a final finished product the concentrate containing the plant essential oil blend is added to the remaining ingredients of the finished product to provide from 0.05% to about 25% by weight of the overall finished product, preferably from 0.5% to 10% by 25 weight of the finished product, and most preferably from 0.5% to 5% by weight of the finished product. - 10 - Example - Combinations having enhanced anti-microbial effectiveness Materials Essential Oils: Cinnamon Oil Lot No. 49086 Redistilled Oregano Oil Lot No. 49096 Van Beek Natural Science Orange City, IA Staphylococcus aureus ATTC No. 6538 Pseudomonas aeruginosa ATTC No. 15442 Salmonella choleraesuis ATTC No. 10708 Escherichia coli ATTC No. 11229 American Type Culture Collection Manassas, VA Neutralizing Media: Dey-Engley with 0.07% Lecithin, 0.5% Tween 80, and 1.5% Glycine Dey-Engley Neutralizing Medium (DE) Lot No. 1165828, Exp. 4/14 Becton Dickinson Sparks, MD Lecithin Lot No. C19WO22, Ass. Exp. 3/14 Alfa Aesar Ward Hill, MA Tween 80 Lot No. 031712, Ass. Exp. 2/14 Fisher Scientific Fair Lawn, NJ - 11 - Glycine Lot No. A019730001, Ass. Exp. 2/14 Acros Organics NJ Mineral oil Lot No. 107725, Ass. Exp. 8/14 Lot No. 110685, Ass. Exp. 9/14 Fisher Scientific Cysteine Trypticase Soy Agar (CTA) Lot No. 995396, Exp. 1/15 Remel Lenexa, KS Nutrient Broth (NB) Lot No. 1005358, Exp. 1/15 Becton Dickinson Nutrient Agar (NA) Lot No. 1059433, Exp. 7/15 Becton Dickinson Serological Pipettes, 1.0 and 10.0 ml VWR Scientific Products Sugarland, TX pH 211 pH Meter Hanna Instruments Petri Plates, 15x 100 mm, Plastic, Disposable BVA Scientific San Antonio, TX Test Tubes, Stainless Steel Closures, and Support Racks; 18x 150mm, 25x 150mm Water Bath Capable of 35±1"C (thermostatically controlled) Incubator Capable of 35+2*C Thermometers Tissue Homogenizer, 40 ml, Ten-Broeck Steam Sterilizer, Model STM-E. Market Forge. - 12 - Miscellaneous Laboratory Glassware Laboratory Timer Magnetic Stir Bars and Stir Plate Ohaus B300D Top-Loading Balance - 13- Procedure 1. Preparation of Bacterial Cultures The bacterial stocks were obtained fresh from the American Type Culture Collection within six months from use in this test. S. aureus, S. choleraesuis, and Escherichia coli stocks were maintained on nutrient agar slants at 3+2"C after monthly transfers to fresh nutrient agar slants grown for 48+8 hours at 35+2"C. P. aeruginosa stock was maintained in cysteine trypticase soy agar (CTA) stabs with monthly transfers grown for 48+8 hours. From the bacterial stock cultures of S. aureus, S. choleraesuis, Escherichia coli, and P. aeruginosa, one loopful of bacteria was added to 10 ml of nutrient broth and incubated at 35+2*C for 24+4 hours. Consecutive transfers were made by transferring 0.1 ml into 10 ml of nutrient broth and incubated at 35+2*C for 24+4 hours. 2. Preparation of Essential Oils The Essential oils tested in this study were one lot of Cinnamon oil and one lot of Re distilled Oregano Oil. Oils were tested alone and in combination to test for synergism of the oils. In Combination 1 the essential oils are mixed so that each oil dilutes the other and the final concentrations are the same as the individual essential oils tested. In Combination 2 the essential oils are mixed so that each oil dilutes the other and the final concentrations are half the individual essential oils tested. Several preliminary studies were done to determine the appropriate dilution of each essential oil for a moderate rate of kill that would provide the opportunity to test for synergism when the oils were combined. This dilution was different for each microbe. The oils were diluted into mineral oil, which has no antimicrobial action. For S. aureus, Cinnamon oil was tested at 10X and 20X while Oregano oil was tested at lOX, 20X, 40X, 60X, and 80X. It was found that 20X Cinnamon oil and 80X Oregano oil diluted with mineral oil provided optimal results to test for synergism when combined. The oils were combined in two different ways to test for synergism: Combination I) 1 part 10X Cinnamon oil + part 40X Oregano oil and Combination 2) 1 part 20X Cinnamon oil + 1 part 80X Oregano oil. For P. aeruginosa, Cinnamon oil was tested at lOX, 20X, 30X, 40X, 50X, 60X, and 80X while Oregano Oil was tested at 10X, 20X, 30X, 40X. It was found that 40X Cinnamon oil and 30X Oregano oil diluted with mineral oil provided optimal results to test for synergism when combined. The oils were combined in two different ways to test for synergism: Combination 1) 1 part 20X Cinnamon oil + I part 15X Oregano oil and Combination 2) 1 part 40X Cinnamon oil + 1 part 40X Oregano oil. For E. coli, Cinnamon oil was tested at 40X while Oregano oil was tested at 40X, 80X and 1OX. It was found that 40X Cinnamon oil and 100X Oregano oil diluted with mineral oil provided optimal results to test for synergism when combined. The oils were combined in two different ways to test for synergism: Combination I) 1 part 20X - 14 - Cinnamon oil + 1 part 50X Oregano oil and Combination 2) 1 part 40X Cinnamon oil + I part lOOX Oregano oil. For S. choleraesuis, Cinnamon oil was tested at 40X, 60X and 80X while Oregano oil was tested at 40X, 80X, 120X and 160X. It was found that 60X Cinnamon oil and 160X Oregano oil diluted with mineral oil provided optimal results to test for synergism when combined. The oils were combined in two different ways to test for synergism: Combination 1) 1 part 30X Cinnamon oil + I part 80X Oregano oil and Combination 2) 1 part 60X Cinnamon oil + I part 160X Oregano oil. 3. Test Procedure Nine (9.0) ml of test Essential oil or Essential oil mixture were placed into a sterile 25x150 mm capped test tube, and brought to temperature in a 35-1"C water bath. One (1.0) ml of bacterial suspension was added and the tube was agitated on a vortex mixer. After exposure times of 1.0, 3.0 and 5.0 minutes at 35+1*C, 1.0 ml of the oil/culture solution was removed and placed into 19 ml of neutralizing recovery medium. Serial ten fold dilutions were made as 1.0 ml into 9 ml portions of recovery medium. One-half (0.5) ml portions of the dilution tubes were transferred onto the surface of nutrient agar for bacterial cultures in petri plates. Petri plates were incubated for >48 hours at 35±2*C. Colonies were counted and multiplied by appropriate dilution factors to determine the number of surviving colony forming units (CFU) in the reaction tube at the various exposure times. 4. Determine the Number of CFU of Bacteria Originally in the Reaction Tube Serial ten-fold dilutions of bacterial cultures, as used in the study, were made as 1.0 ml into 9 ml of nutrient broth. One half (0.5) ml portions of various dilution tubes were transferred to the surface of nutrient agar in petri plates. Tubes and plates were incubated at 35±2*C for >48 hours. The colonies were counted and multiplied by the appropriate dilution factors to determine the number of CPU of bacteria originally in the reaction tube. 5. Validation of Neutralization and Viability One (1.0) ml of test-strength Essential oil or Essential oil mixture was added to a tube of 19 ml neutralizing recovery medium. One ten-fold dilution was made as 1.0 ml into 9 ml neutralizing recovery medium. Each tube was spiked with approximately 103 CFU of bacteria held in 1.0 ml of broth. As a comparative viability control, a 19 and 9 ml tube of neutralizing recovery medium containing no oil were spiked with approximately 103 CFU of bacteria. Tubes were held for 10 minutes, and then 0.5 ml portions of each were transferred to the surface of nutrient agar in petri plates and incubated with the test. Similar numbers on all plates per bacteria validated neutralization of the test oils. - 15 - Results S. aureus There were 4.00x 102 surviving CFU of S. aureus in the reaction tube after 5.0 minutes of exposure to 20X Cinnamon oil diluted with mineral oil. There were 7.20x 10 3 surviving CFU of S. aureus in the reaction tube after 5.0 minutes of exposure to 80X Oregano oil diluted with mineral oil. There were 2.40x 103 surviving CFU of S. aureus in the reaction tube after 1.0 minutes of exposure and zero (total kill) surviving CFU after 3.0 minutes of exposure to 1 part lOX Cinnamon oil: 1 part 40X Oregano oil (Combination 1) resulting in 20X Cinnamon oil and 80X Oregano oil. There were an average of 2.16x108 CFU of S. aureus originally in the reaction tube. Neutralization by the recovery method was validated for 20X Cinnamon oil diluted with mineral oil, 80X Oregano oil diluted with mineral oil and 1 part lOX Cinnamon oil: I part 40X Oregano oil. S.aureus was tested a second time to look at a different combination of the Essential oil mixture: 1 part 20X Cinnamon oil: 1 part 80X Oregano (Combination 2) resulting in 40X Cinnamon oil and 160X Oregano oil. There were 9.60x 103 surviving CFU of S. aureus in the reaction tube after 5.0 minutes of exposure to 20X Cinnamon oil diluted with mineral oil. There were 9.32x 105 surviving CFU of S. aureus in the reaction tube after 5.0 minutes of exposure to 80X Oregano oil diluted with mineral oil. There were 4.24xl05 surviving CFU of S. aureus in the reaction tube after 5.0 minutes of exposure to 1 part 20X Cinnamon oil: 1 part 80X Oregano oil. There were an average of 1.38x10 8 CFU of S. aureus originally in the reaction tube. Neutralization by the recovery method was validated for 20X Cinnamon oil diluted with mineral oil, 80X Oregano oil diluted with mineral oil and 1 part 20X Cinnamon oil: I part 80X Oregano oil. P. aerufinosa There were 2.04x10 6 surviving CFU of P. aeruginosa in the reaction tube after 5.0 minutes of exposure to 40X Cinnamon oil diluted with mineral oil. - 16- There were 1.88x10 6 surviving CFU of P. aeruginosa in the reaction tube after 5.0 minutes of exposure to 30X Oregano oil diluted with mineral oil. There were 1.20x10 4 surviving CFU of P. aeruginosa in the reaction tube after 1.0 minutes of exposure and zero (total kill) surviving CFU after 3.0 minutes of exposure to 1 part 20X Cinnamon oil: 1 part 15X Oregano oil (Combination 1) resulting in 40X Cinnamon oil and 30X Oregano oil. There were an average of 1.42x109 CFU of P. aeruginosa originally in the reaction tube. Neutralization by the recovery method was validated for 40X Cinnamon oil diluted with mineral oil, 30X Oregano oil diluted with mineral oil and 1 part 20X Cinnamon oil: I part 15X Oregano oil. P. aeruginosa was tested a second time, this time with 40X concentrations of the individual Essential oils because they were similar to the 40X Cinnamon and 30X Oregano oil and would be simpler to look at the different Essential oil combination mixture: 1 part 40X Cinnamon oil: I part 40X Oregano (Combination 2) resulting in 80X Cinnamon oil and 8OX Oregano oil. There were 5.04x10 5 surviving CFU of P. aeruginosa in the reaction tube after 5.0 minutes of exposure to 40X Cinnamon oil diluted with mineral oil. There were 4.72x10 6 surviving CFU of P. aeruginosa in the reaction tube after 5.0 minutes of exposure to 40X Oregano oil diluted with mineral oil. There were 4.04x 105 surviving CFU of P. aeruginosa in the reaction tube after 5.0 minutes of exposure to 1 part 40X Cinnamon oil: 1 part 40X Oregano oil. There were an average of 6.80xl0 CFU of P. aeruginosa originally in the reaction tube. Neutralization by the recovery method was validated for 40X Cinnamon oil diluted with mineral oil, 40X Oregano oil diluted with mineral oil and I part 40X Cinnamon oil: I part 40X Oregano oil. E. coli There were 4.24x 106 surviving CFU of E. coli in the reaction tube after 5.0 minutes of exposure to 40X Cinnamon oil diluted with mineral oil. There were 1.38x106 surviving CFU of E. coli in the reaction tube after 5.0 minutes of exposure to 100X Oregano oil diluted with mineral oil. There were 1.04x10 5 surviving CFU of E. coli in the reaction tube after 1.0 minutes and zero (total kill) surviving CFU after 3.0 minutes of exposure to 1 part 20X Cinnamon oil: 1 part 50X Oregano oil (Combination 1) resulting in 40X Cinnamon oil and IOOX Oregano oil. - 17 - There were 1.20x107 surviving CFU of E. coli in the reaction tube after 5.0 minutes of exposure to I part 40X Cinnamon oil: 1 part 100X Oregano oil (Combination 2) resulting in 80X Cinnamon oil and 200X Oregano oil. There were an average of 3.64x10 8 CFU of E. coli originally in the reaction tube. Neutralization by the recovery method was validated for 40X Cinnamon oil diluted with mineral oil, 100X Oregano oil diluted with mineral oil, I part 40X Cinnamon oil: 1 part 100X Oregano oil and 1 part 20X Cinnamon oil: 1 part 50X Oregano oil. S. choleraesuis There were 3.36x108 surviving CFU of S. choleraesuis in the reaction tube after 5.0 minutes of exposure to 60X Cinnamon oil diluted with mineral oil. There were 3.16x10 7 surviving CFU of S. choleraesuis in the reaction tube after 5.0 minutes of exposure to 160X Oregano oil diluted with mineral oil. There were 1.88x 104 surviving CFU of S. choleraesuis in the reaction tube after 5.0 minutes of exposure to I part 30X Cinnamon oil: I part 80X Oregano oil (Combination 1) resulting in 60X Cinnamon oil and 160X Oregano oil. There were 1.44x10 7 surviving CFU of S. choleraesuis in the reaction tube after 5.0 minutes of exposure to I part 60X Cinnamon oil: I part 160X Oregano oil (Combination 2) resulting in 120X Cinnamon oil and 320X Oregano oil. There were an average of 1.38x10 8 CFU of S. choleraesuis originally in the reaction tube. Neutralization by the recovery method was validated for 60X Cinnamon oil diluted with mineral oil, 160X Oregano oil diluted with mineral oil, 1 part 60X Cinnamon oil: 1 part 160X Oregano oil and I part 30X Cinnamon oil: I part 80X Oregano oil. Conclusions For S. aureus, P. aeruginosa, E. coli, and S. choleraesuis, Combination I concentrations of Cinnamon oil and Oregano oil demonstrated a synergistic effect, killing the bacteria faster than the individual oils alone. Combination 2 concentrations demonstrated a smaller synergistic effect, killing these four bacteria at approximately the same rate as the individual oils, but with half the amount of each oil. - 18- C:NRPonbl\DCC\RBR\408827 1 DOC.1/9/2012 Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or 5 steps. The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general 10 knowledge in the field of endeavour to which this specification relates. - 19 -