SYNERGISTIC CATIONIC SURFACTANTS RESULTING IN MICROBIAL

INHIBITION ON FOOD SURFACES AND HARD SURFACES

FIELD OF THE INVENTION

The presently disclosed subject matter relates to antimicrobial chemical compositions and materials, used as sprays, washes, films, coatings or dips, which are useful in the processing of food and other products. The presently disclosed subject matter also relates to processes for the applications of such compositions and

materials, and to the use of the compositions and materials in antimicrobial applications.

BACKGROUND

During processing, preparation, and packaging food products can encounter microorganisms that can negatively impact the shelf life and food safety that make the food unsuitable for consumption. The microorganisms can originate from the food itself, the food contact surfaces, cross contamination and/or the surrounding environment. To this end, the safety of food products has been a subject of increasing concern as a result of several well-publicized outbreaks of foodborne pathogens in fresh and ready- to-eat foods. In the United States, foodborne illness affects about 40 to 80 million people per year, causing 9,000 deaths and an estimated cost of 7 billion dollars. It is therefore critical for food products to be processed, handled, and packaged in the safest manner possible to help reduce microbial contamination.

The food industry has responded in various ways in an attempt to reduce microbial contamination. For example, aseptic packaging, pre-fill sterilization, and post- fill sterilization are commonly applied as possible microbial control methods. However, these methods often result in undesirable changes in food quality characteristics. In addition, fresh and minimally processed foods often cannot be preserved by such approaches and must rely on other methods. Modified atmosphere packaging is another common strategy used by the food industry to extend the shelf life of food products, particularly fresh produce and/or meat. In modified atmosphere packaging, the rate of food deterioration is reduced by modifying the initial concentrations of oxygen and carbon dioxide inside the package. However, the modified gas concentrations change over time. Also, the absence of oxygen can affect freshness and flavor perception as well as encourage the growth of harmful anaerobic microorganisms. The food industry has also attempted to incorporate antimicrobial agents directly into the food (e.g., preservatives such as BHT, sodium lactate or sodium citrate) as a means to control contamination. However, such additives can affect color, flavor, and/or smell of the food product. Accordingly, there is a need in the art for improved compositions, products and methods to control microbial contamination.

SUMMARY The presently disclosed subject matter is directed to a method of reducing microbes on a food surface. The method may include applying 0.02 wt% to 1 wt% of a first cationic surfactant and 0.02 wt% to 3 wt% of a second cationic surfactant to a food surface. The first cationic surfactant and the second cationic surfactant may form an antimicrobial composition. The first cationic surfactant may be a quaternary ammonium cation.

In some embodiments, the presently disclosed subject matter is directed to a second method for reducing the growth of microbes on a food surface. The method may include contacting the food surface with an effective amount of an antimicrobial composition. The antimicrobial composition may have two parts. The first part may be 0.02 wt% to 1 wt% of a first cationic surfactant and the second part may be 0.02 wt% to 3 wt% of a second cationic surfactant. The first cationic surfactant may be a quaternary ammonium cation. In some embodiments, the two parts may be applied to the food surface concurrently. In other embodiments, the two parts may be applied to the food surface sequentially. In some embodiments, the presently disclosed subject matter is directed to a method of reducing microbes on a hard surface. The method may include applying 0.02 wt% to 1 wt% of a first cationic surfactant and 0.02 wt% to 3 wt% of a second cationic surfactant to a hard surface. The first cationic surfactant and the second cationic surfactant may form an antimicrobial composition. The first cationic surfactant may have a quaternary ammonium cation.

In other embodiments, the method for reducing the growth of microbes on a hard surface may include contacting the hard surface with an effective amount of an antimicrobial composition. The antimicrobial composition may have two parts. The first part may have 0.02 wt% to 1 wt% of a first cationic surfactant. The second part may have 0.02 wt% to 3 wt% of a second cationic surfactant. The first cationic surfactant may have a quaternary ammonium cation. The two parts may be applied to a hard surface either concurrently or sequentially.

In further embodiments, the presently disclosed subject matter is directed to a method of reducing microbes on a surface. The method may include applying 1 % to 3% of a second cationic surfactant, 1 % to 3% of a lantibiotic, and 0.25% to 1 % of an aminopolycarboxylic acid to a surface. The second cationic surfactant, lantibiotic, and aminopolycarboxylic acid may form an antimicrobial composition.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a bar graph that illustrates the efficacy and synergy testing results of LAE/CPC blend on E. coli on skin-on chicken.

FIG. 2 is a bar graph that illustrates the efficacy testing results of LAE/CPC blend on E. coli on different food products. DETAILED DESCRIPTION I General Considerations

The presently disclosed subject matter is directed to a method of reducing microbes on a food surface and a hard surface. The method may include applying an antimicrobial composition including a first cationic surfactant and a second cationic surfactant. _ \l Definitions

While the following terms are believed to be well understood by one of ordinary skill in the art, the following definitions are set forth to facilitate explanation of the presently disclosed subject matter.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which the presently disclosed subject matter belongs.

Following long standing patent law convention, the terms "a", "an", and "the" refer to "one or more" when used in the subject application, including the claims. Thus, for example, reference to "a composition" includes a plurality of such compositions, and so forth.

Unless indicated otherwise, all numbers expressing quantities of components, compositions, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in the instant specification and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by the presently disclosed subject matter. As used herein, the term "about", when referring to a value or to an amount of mass, weight, time, volume, concentration, percentage, and the like can encompass variations of, and in some embodiments ±20%, in some embodiments ±10%, in some embodiments ±5%, in some embodiments ±1 %, in some embodiments ±0.5%, and in some embodiments ±0.1 %, from the specified amount, as such variations are appropriate in the disclosed composition, package and methods.

As used herein, the term "additive" refers to any substance, chemical, compound, composition or formulation that is added to an initial substance, chemical, composition compound or formulation in a smaller amount than the initial substance, chemical, compound, composition or formulation to provide additional properties or to change the properties of the initial substance, chemical, compound, composition or formulation.

As used herein, the term "preservative" refers to any chemical or compound that prevents degradation or breakdown of a compound, composition or formulation. A preservative also prevents bacteria from spoiling a compound, composition or formulation during storage or use.

As used herein, the term "buffer" refers to any chemical, compound, composition or solution that is used to control the pH of a composition, formulation, system, or solution. A "buffer system" refers to any composition or system where there are two or more components that are used to control the pH of a composition, formulation, system, or solution, such as an acid and a base. The components are any chemical, compound, composition, formulation or solution.

As used herein, the term "antimicrobial" refers to microbicidal activity or microbe growth inhibition in a microbe population. As used herein, the term "kill rate" refers to the number of microorganisms over time that the disclosed antimicrobial composition can effectively kill or inactivate.

As used herein, the term "LAE" refers to Na-Lauroyl-L-arginine ethyl ester monohydrochloride, ethyl lauroyl arginate, ethyl n-lauroyl -L-arginate hydrochloride salt ("LAE HCI"), ethyl n-lauroyl-L-arginate laurate complex ("LAE monolaurate"), N a - cocoyl arginine ethyl ester salt and N a -lauryl arginine iso-propyl ester salt.

As used herein, the term "CPC" refers to cetylpyridinium chloride, palmitylpyridinium chloride, C16-alkylpyridinium chloride, 1 -hexadecylpyridinium chloride, acetoquat CPC, aktivex, ammonyx CPC, Cecure®, ceepryn chloride, cepacol, ceprim, cepacol chloride, cetafilm, cetamium, dobendan, halset, ipanol, medilave, mercocet, merothol, pionin B, pristacin, pyrisept and asept.

The term "food product" or "food" refers to any food or beverage item that may be consumed by humans or mammals. Some non-limiting examples of a "food product" or "food" include the following: meat products, poultry products, processed meat and poultry products, raw meat and poultry products; fish products including cooked and raw fish, shrimp, and shellfish; produce including whole or cut fruits and vegetables and cooked or raw fruits and vegetables; eggs, and egg-based products and pre-made food items. The present invention is particularly useful for meat, poultry and produce products. Additionally, the present invention can be used on ready-to-eat products such as hot dogs, sausages, turkey, ham, pre-packaged meals and bacon.

The term "meat" refers to any myoglobin-containing or hemoglobin-containing tissue from an animal, such as beef, pork, veal, lamb, mutton, chicken or turkey; and game such as venison, quail, fish, seafood and duck. It may also be used with fruits & vegetables, seeds, nuts or combinations. It can also be used on raw or processed foods. The meat can be in a variety of forms including primal cuts, subprimal cuts, and/or retail cuts as well as ground, comminuted or mixed. The meat or meat product is preferably fresh, raw, uncooked meat, but also includes cooked meat, commingled meat, frozen, hard chilled or thawed. Meat also refers to any meat that has been subjected to other irradiative, biological, chemical and/or physical treatments. The suitability of any particular such treatment can be determined without undue experimentation in view of the present disclosure.

As used herein, the term "microbe" or "microorganism" refers to any organism capable of contaminating meat, food, or other products, thereby making such product unsuitable or unhealthy for human or animal consumption or contact. For example, in some embodiments, microbes can include bacteria, fungi, yeasts, algae, molds, mycoplasmids, protozoa, viruses and the like.

As used herein, the term "package" refers to packaging materials configured around a product being packaged. The phrase "packaged product," as used herein, refers to the combination of a product that is surrounded by a packaging material. All compositional percentages used herein are presented on a "by weight" basis, unless designated otherwise.

Although the majority of the above definitions are substantially as understood by those of skill in the art, one or more of the above definitions can be defined hereinabove in a manner differing from the meaning as ordinarily understood by those of skill in the art, due to the particular description herein of the presently disclosed subject matter.

III. The Disclosed Composition

The presently disclosed invention is directed to a method of reducing microbes on a food surface. The method may include a first cationic surfactant and a second cationic surfactant. The first and second cationic surfactant may be in the form of an antimicrobial composition. The first cationic surfactant may be a quaternary ammonium cation.

The first cationic surfactant and the second cationic surfactant may have a synergistic effect when combined. As set forth above, the disclosed antimicrobial composition may be a synergistic combination of a first cationic surfactant and a second cationic surfactant. The first cationic surfactant may be based on the cationic quaternary ammonium salts, such as the cetylpyridinium moiety. The second cationic surfactant may be based on cationic surfactants of N a -acyl acidic amino acid ester salts. As a result, the disclosed solution may have an enhanced antimicrobial effect, i.e., it is capable of destroying or inhibiting the growth of microorganisms, greater than either surfactant would be if used alone.

The first cationic surfactant may be at least one antimicrobial agent characterized as a quaternary ammonium compound. While any suitable quaternary ammonium derivative can be used, particularly useful quaternary ammonium compounds have been shown to have long alkyl chains. Examples are alkylpyridinium, tetra- alkylammonium and alkylalicyclic ammonium salts. The first cationic surfactant may be at least one of benzalkonium chloride, benzethonium chloride, methylbenzethonium chloride, cetalkonium chloride, cetylpyridinium bromide, cetylpyridinium chloride (CPC) and salts thereof. In some embodiments, the first cationic surfactant may be CPC. In other embodiments, the first cationic surfactant may be cetylpyridinium bromide. The above mentioned quaternary ammonium compounds are lethal to a wide variety of organisms except endospores, mycobacterium tuberculosis and non- enveloped viruses. CPC is frequently used in toothpastes, mouthwashes and ingestible products.

The second cationic surfactant may be derived from the condensation of fatty acids and esterified dibasic amino acids, according to the following formula (I), where: X is Br, CI or HS04, R1 : is an alkyl chain derived from a fatty acid, or hydroxy acid from 6 to 30 atoms of carbon bonded to the a-amino acid group through an amidic bond, R2 is a linear or branched alkyl chain from 1 to 18 carbon atoms or an aromatic moiety, R3: is Formula (II), where n can be from 0 to 4, and

While any suitable N a -acyl acidic amino acid ester salt derivatives can be used, particularly useful moieties include (but are not limited to): ethyl n-lauroyl -L-arginate hydrochloride salt ("LAE HCI"), ethyl n-lauroyl-L-arginate laurate complex ("LAE monolaurate"), N a -cocoyi arginine ethyl ester salt, N a -lauryl arginine iso-propyl ester salt. In one or more embodiments, X is chloride, bromide, or a counter ion derived from an organic or inorganic acid or a phenolic compound. Examples of acids that may be the source of the counter ion, X, include acetic acid, citric acid, lactic acid, fumaric acid, maleic acid, gluconic acid, propionic acid, sorbic acid, benzoic acid, carbonic acid, glutamic acid, lauric acid, oleic acid, linoleic acid, phosphoric acid, nitric acid, and sulfuric acid. Examples of phenolic compounds that may be the source of the counter ion, X, include butylated hydroxyanisole (BHA), butylated hydroxytoluene, tertiary butylhydroquinone, methylparaben, ethylparaben, propylparaben, and butylparaben. In some embodiments, the second cationic surfactant may be at least one of ethyl n- lauroyl -L-arginate hydrochloride salt, ethyl n-lauroyl-L-arginate laurate complex, N a - cocoyi arginine ethyl ester salt and N a -lauryl arginine iso-propyl ester salt. In other embodiments, the second cationic surfactant may be Na-Lauroyl-L-arginine ethyl ester monohydrochloride (LAE).

Examples of commercially available LAE moieties include Aminat and Mirenat®- N (available from Vedeqsa, Inc., New York, New York, United States of America), Ethyl Lauroyl Arginate HCI by Jetide Health, Wuhan, China and CytoGuard LA® (available from A&B Ingredients, Fairfield, New Jersey, United States of America). In some embodiments, complexes may be formed with LAE, such as LAE palmitate, LAE stearate, LAE lactate, LAE citrate, LAE oleate, LAE benzoate, LAE acetate, LAE hydrogen sulfate, LAE phosphonate, and the like. Advantageously, the LAE moieties disclosed above have been approved in the

United States and Europe for food applications. To this end, the LAE moieties are nontoxic, non-allergenic and have been determined to be harmless to human and/or animal health. Additionally, the LAE moieties are effective against a broad range of microorganisms without destroying or damaging meat or produce tissues. It has been shown that LAE moieties also do not impart any off-tastes, odors, or changes in color. In addition, LAE moieties are stable at a wide range of temperatures, lighting, and environmental conditions.

While not intended to be bound by any theory, the antimicrobial activity of LAE and CPC is believed to be due to the physicochemical properties, namely the ability to insert into bacterial membranes. LAE and CPC exhibit cationic surfactant properties. Cationic surfactants are known to disrupt the integrity of cell membranes in a broad spectrum of bacteria, yeasts, and molds.

The first cationic surfactant and second cationic surfactant may form an antimicrobial composition. In some embodiments, the antimicrobial composition may have 0.02 wt% to 1 wt% of a first cationic surfactant. In other embodiments, the antimicrobial composition may have 0.02 wt% to 0.8 wt% of a first cationic surfactant, 0.1 wt% to 0.8 wt% of a first cationic surfactant, 0.2 wt% to 0.8 wt% of a first cationic surfactant or any range between any of these values. In further embodiments, the antimicrobial composition may have 0.02 wt% of a first cationic surfactant, 0.04 wt% of a first cationic surfactant, 0.05 wt% of a first cationic surfactant, 0.06 wt% of a first cationic surfactant, 0.08 wt% of a first cationic surfactant, 0.1 wt% of a first cationic surfactant, 0.15 wt% of a first cationic surfactant, 0.175 wt% of a first cationic surfactant, 0.2 wt% of a first cationic surfactant, 0.25 wt% of a first cationic surfactant, 0.3 wt% of a first cationic surfactant, 0.32 wt% of a first cationic surfactant, 0.35 wt% of a first cationic surfactant, 0.4 wt% of a first cationic surfactant, 0.45 wt% of a first cationic surfactant, 0.5 wt% of a first cationic surfactant, 0.55 wt% of a first cationic surfactant, 0.6 wt% of a first cationic surfactant, 0.65 wt% of a first cationic surfactant, 0.7 wt% of a first cationic surfactant, 0.75 wt% of a first cationic surfactant, 0.8 wt% of a first cationic surfactant, 0.85 wt% of a first cationic surfactant, 0.9 wt% of a first cationic surfactant, 0.95 wt% of a first cationic surfactant, 1 wt% of a first cationic surfactant or any range between any of these values. In some embodiments, the antimicrobial composition may have 0.32 wt% of a first cationic surfactant.

In some embodiments, the antimicrobial composition may have 0.02 wt% to 3 wt% of a second cationic surfactant. In other embodiments, the antimicrobial composition may have 0.1 wt% to 2.5 wt% of a second cationic surfactant. In further embodiments, the antimicrobial composition may have 0.25 wt% to 2 wt% of a second cationic surfactant. In further embodiments, the antimicrobial composition may have 0.02 wt% of a second cationic surfactant, 0.05 wt% of a second cationic surfactant, 0.1 wt% of a second cationic surfactant, 0.2 wt% of a second cationic surfactant, 0.25 wt% of a second cationic surfactant, 0.3 wt% of a second cationic surfactant, 0.35 wt% of a second cationic surfactant, 0.4 wt% of a second cationic surfactant, 0.45 wt% of a second cationic surfactant, 0.5 wt% of a second cationic surfactant, 0.55 wt% of a second cationic surfactant, 0.6 wt% of a second cationic surfactant, 0.65 wt% of a second cationic surfactant, 0.7 wt% of a second cationic surfactant, 0.75 wt% of a second cationic surfactant, 0.8 wt% of a second cationic surfactant, 0.9 wt% of a second cationic surfactant, 1.0 wt% of a second cationic surfactant, 1 .25 wt% of a second cationic surfactant, 1.5 wt% of a second cationic surfactant, 1 .75 wt% of a second cationic surfactant, 2 wt% of a second cationic surfactant, 2.25 wt% of a second cationic surfactant, 2.5 wt% of a second cationic surfactant, 2.75 wt% of a second cationic surfactant, 3 wt% of a second cationic surfactant or any range between any of these values. In some embodiments, the antimicrobial composition may have 2 wt% of a second cationic surfactant.

In some embodiments, the antimicrobial composition may have 0.2 wt% of a first cationic surfactant and 0.5 wt% of a second cationic surfactant. The antimicrobial composition may have 0.4 wt% of a first cationic surfactant and 1 wt% of a second cationic surfactant. The antimicrobial composition may have 0.6 wt% of a first cationic surfactant and 0.5 wt% of a second cationic surfactant. The antimicrobial composition may have 0.8 wt% of a first cationic surfactant and 1 wt% of a second cationic surfactant. The antimicrobial composition may have 0.6 wt% of a first cationic surfactant and 1 .5 wt% of a second cationic surfactant. The antimicrobial composition may have 0.2 wt% of a first cationic surfactant and 1 .5 wt% of a second cationic surfactant. The antimicrobial composition may have 0.4 wt% of a first cationic surfactant and 2 wt% of a second cationic surfactant. The antimicrobial composition may have 0.8 wt% of a first cationic surfactant and 2 wt% of a second cationic surfactant. The antimicrobial composition may have 0.32 wt% of a first cationic surfactant and 2 wt% of a second cationic surfactant.

The antimicrobial composition may also contain additives as are commonly known in the art. In some embodiments, the additives may be at least one acidulant, antioxidant, buffer, carrier, chelating agent, coupling agent, dye, excipient, ionic and non-ionic emulsifier, essential oil, fatty compound, film-forming agent, flavoring aide, defoaming agent, fragrance, gelling agent, jellying hydrophilic agent, opacifier, oxidizer, potentiator, preservative, solvent, surface active agent, surfactant and thickening agent. The fatty compounds may be a mineral oil, an animal oil, a vegetable oil, a synthetic oil, a silicone oil, an alcohol, a fatty acid, a wax and any combination thereof. The additive may be at least one preservative. The at least one preservative will be generally recognized as safe (GRAS) or food law approved. In some embodiments, the preservative may be a carbamate, a quaternary ammonium compound, an alkyl amine, an isothiazoline and combinations thereof. The isothiazoline may be benzylisothiazolinone, 5-chloroisothiazolinone, methylisothiazolinone and combinations thereof. In other embodiments, the preservative may be 1 ,2-benzisothiazolin-3-one sodium salt and 3-iodo-2-propynyl butyl carbamate.

The antimicrobial composition may have at least one solvent. The at least one solvent may be acetic acid, acetone, benzyl alcohol, diethylene glycol, ethanol, glycerin, propylene glycol, water and any combination thereof. In some embodiments, the at least one solvent may be water and propylene glycol. The water may be boiled water, deionized water, distilled water, filtered water, hard water, mineral water, purified water, reverse osmosis treated water, soft water, spring water, tap water or any combination thereof. In other embodiments, the antimicrobial composition may not have a solvent.

In some embodiments, the antimicrobial composition may have at least 80 wt% solvent. In other embodiments, the antimicrobial composition may have 80 wt% solvent, 85 wt% solvent, 90 wt% solvent, 91 wt% solvent, 91 .5 wt% solvent, 92 wt% solvent, 92.5 wt% solvent, 93 wt% solvent, 93.5 wt% solvent, 94 wt% solvent, 94.5 wt% solvent, 95 wt% solvent, 95.5 wt% solvent, 96 wt% solvent, 96.5 wt% solvent, 97 wt% solvent, 97.1 wt% solvent, 97.2 wt% solvent, 97.3 wt% solvent, 97.4 wt% solvent, 97.5 wt% solvent, 97.6 wt% solvent, 97.7 wt% solvent, 97.8 wt% solvent, 97.9 wt% solvent, 98 wt% solvent, 98.1 wt% solvent, 98.2 wt% solvent, 98.3 wt% solvent, 98.4 wt% solvent, 98.5 wt% solvent, 98.6 wt% solvent, 98.7 wt% solvent, 98.8 wt% solvent, 98.9 wt% solvent, 99 wt% solvent, 99.1 wt% solvent, 99.2 wt% solvent, 99.3 wt% solvent, 99.4 wt% solvent, 99.5 wt% solvent, 99.6 wt% solvent or any range between any of these values. In further embodiments, the antimicrobial composition may have 97.2 wt% solvent. In yet further embodiments, the antimicrobial composition may have 97.48 wt% solvent.

The antimicrobial composition may have deionized water, CPC and LAE. In some embodiments, the antimicrobial composition may have deionized water, CPC, LAE and propylene glycol. In other embodiments the antimicrobial composition may have 97.2 wt% deionized water, 0.32 wt% CPC, 2 wt% LAE and 0.48 wt% propylene glycol.

IV. Methods of Making the Disclosed Solutions

The presently disclosed composition may be formulated using any of a wide variety of conventional techniques well-known in the art. The disclosed antimicrobial composition may include one or more active ingredients. In some embodiments, the first cationic surfactant and the second cationic surfactant may be dissolved in water. The water may be boiled water, deionized water, distilled water, filtered water, hard water, mineral water, purified water, reverse osmosis treated water, soft water, spring water, tap water or any combination thereof. In some embodiments, the water may be deionized water. In other embodiments, the water may be distilled water. In other embodiments, the first cationic surfactant and the second cationic surfactant may not be dissolved in water.

Additives may be added to the composition. In some embodiments, the additives may include at least one acidulant, antioxidant, buffer, carrier, chelating agent, coupling agent, dye, excipient, ionic and non-ionic emulsifier, essential oil, fatty compound, film- forming agent, filter, flavoring aide, foaming agent, fragrance, gelling agent, jellying hydrophilic agent, opacifier, oxidizer, potentiator, preservative, solvent, surface active agent, surfactant and thickening agent. The additional ingredients may be selected from food grade dyes, GRAS dyes, colorants, pH stabilizers and buffers, non-ionic surfactants, fragrance, fragrance enhancers, viscosity modifiers, light stabilizers, emulsifiers, colloids, edible polymeric materials and combinations thereof.

The first cationic surfactant and second cationic surfactant are combined to result in a synergistic blend. The synergistic blend may be an antimicrobial composition that offers enhanced antimicrobial properties. The first cationic surfactant and second cationic surfactant may be combined in any order. In some embodiments, the additives and/or additional ingredients may be added at any time during the combination of the two surfactants. The first cationic surfactant may be formulated as a coating, dust, emulsion, liquid, powder or any combination thereof. The second cationic surfactant may be formulated as a coating, dust, emulsion, liquid, powder or any combination thereof.

The antimicrobial composition may be formulated by any known formulation to one of skill in the art. In some embodiments, the antimicrobial composition may be formulated as a coating, dust, emulsion, liquid, powder or any combination thereof. \A Methods of Using the Disclosed Composition

The previously disclosed first cationic surfactant, second cationic surfactant, and antimicrobial composition may be used to reduce microbes on a hard surface. Non- limiting examples of a hard surface may include non-porous materials, such as, metals, like stainless steel and aluminum, glass, rigid plastics, porcelain, and tile. The previously disclosed first cationic surfactant, second cationic surfactant, and antimicrobial composition may be used to reduce microbes on a food surface. The combination of the first cationic surfactant and the second cationic surfactant provides a synergistic effect. The combination enhances the antimicrobial efficacy of the first cationic surfactant and the second cationic surfactant without any adverse impact on food and/or product appearance or organoleptic issues.

The presently disclosed subject matter is directed to a method of reducing microbes on a hard surface. The presently disclosed subject matter is directed to a method of reducing microbes on a food surface. The disclosed method may be used in the production, handling and packaging of food and/or food products. The method may include applying a first cationic surfactant and a second cationic surfactant to a food surface. In some embodiments, the method may include applying a first cationic surfactant and a second cationic surfactant to process water that contacts a food surface. The first cationic surfactant and the second cationic surfactant may form an antimicrobial composition. In some embodiments, the first cationic surfactant may be applied to a hard surface separately from the second cationic surfactant. In some embodiments, the first cationic surfactant may be applied to a food surface separately from the second cationic surfactant. In other embodiments, the first cationic surfactant and the second cationic surfactant may be applied to a food surface at the same time. In some embodiments, the first cationic surfactant may be applied at a diluted concentration. The first cationic surfactant may be diluted with at least one solvent to a 0.02 wt% to 1 wt% solution. In other embodiments, the first cationic surfactant may be diluted with at least one solvent to a 0.02 wt% to 0.8 wt% solution, 0.1 wt% to 0.8 wt% solution, 0.2 wt% to 0.8 wt% solution or any range between any of these values. In further embodiments, the first cationic surfactant may be diluted with at least one solvent to a 0.02 wt% solution, 0.04 wt% solution 0.05 wt% solution, 0.06 wt% solution, 0.08 wt% solution, 0.1 wt% solution, 0.15 wt% solution, 0.175 wt% solution, 0.2 wt% solution, 0.25 wt% solution, 0.3 wt% solution, 0.32 wt% solution, 0.35 wt% solution, 0.4 wt% solution, 0.45 wt% solution, 0.5 wt% solution, 0.55 wt% solution, 0.6 wt% solution, 0.65 wt% solution, 0.7 wt% solution, 0.75 wt% solution, 0.8 wt% solution, 0.85 wt% solution, 0.9 wt% solution, 0.95 wt% solution, 1 wt% solution or any range between any of these values. In some embodiments, the first cationic surfactant may be diluted with at least one solvent to a 0.32 wt% solution. In other embodiments, the first cationic surfactant may be diluted with at least one solvent to a 0.32 wt% solution. In some embodiments, the second cationic surfactant may be applied at a diluted concentration. The second cationic surfactant may be diluted with at least one solvent to a 0.02 wt% to 3 wt% solution. In other embodiments, the second cationic surfactant may be diluted with at least one solvent to a 0.1 wt% to 2.5 wt% solution. In further embodiments, the second cationic surfactant may be diluted with at least one solvent to a 0.25 wt% to 2 wt% solution. In further embodiments, the second cationic surfactant may be diluted with at least one solvent to a 0.02 wt% solution, 0.05 wt% solution, 0.1 wt% solution, 0.2 wt% solution, 0.25 wt% solution, 0.3 wt% solution, 0.35 wt% solution, 0.4 wt% solution, 0.45 wt% solution, 0.5 wt% solution, 0.55 wt% solution, 0.6 wt% solution, 0.65 wt% solution, 0.7 wt% solution, 0.75 wt% solution, 0.8 wt% solution, 0.9 wt% solution, 1 .0 wt% solution, 1 .25 wt% solution, 1.5 wt% solution, 1 .75 wt% solution, 2 wt% solution, 2.25 wt% solution, 2.5 wt% solution, 2.75 wt% solution, 3 wt% solution or any range between any of these values. In some embodiments, the second cationic surfactant may be diluted with at least one solvent to a 2 wt% solution.

In some embodiments, the first cationic surfactant and the second cationic surfactant may form an antimicrobial composition before application to a hard surface. In other embodiments, the first cationic surfactant may be in a first container and the second cationic surfactant may be in a second container before the step of applying to a hard surface. In further embodiments, the first cationic surfactant may be in a first container and the second cationic surfactant may be in a second container, and both are combined to form an antimicrobial composition in a third container before the step of applying to a hard surface. In yet further embodiments, the first cationic surfactant and the second cationic surfactant form an antimicrobial composition at the same time as the step of applying to a hard surface.

In some embodiments, the first cationic surfactant and the second cationic surfactant may form an antimicrobial composition before application to a food surface. In other embodiments, the first cationic surfactant may be in a first container and the second cationic surfactant may be in a second container before the step of applying to a food surface. In further embodiments, the first cationic surfactant may be in a first container and the second cationic surfactant may be in a second container, and both are combined to form an antimicrobial composition in a third container before the step of applying to a food surface. In yet further embodiments, the first cationic surfactant and the second cationic surfactant form an antimicrobial composition at the same time as the step of applying to a food surface.

The containers may be made from metal, glass, paper, cardboard, plastic and combinations thereof. In some embodiments, the containers may include polyethylene terephthalate (PET), polyethylene naphthalate (PEN) or polycarbonate (PC), boxes, metal cans, glasses, vessels, refillable cans, barrels or vessels. The containers may be of any shape. For example, the containers may be in the shape of a circle, a diamond, an oval, a square, a rectangle, a pentagon, a hexagon, a heptagon, an octagon or combinations thereof. The device to dispense the first cationic surfactant, second cationic surfactant and/or antimicrobial composition may include a dispensing nozzle. In some embodiments, the dispensing nozzle may be connected to the containers. In other embodiments, the dispensing nozzle may not be connected to the containers. The first cationic surfactant, second cationic surfactant and/or antimicrobial composition may be pumped from the containers and dispensed from the nozzle.

In some embodiments, the first cationic surfactant and the second cationic surfactant may form an antimicrobial composition before applying 0.02 wt% to 1 wt% of a first cationic surfactant and 0.02 wt% to 3 wt% of a second cationic surfactant to a food surface. In other embodiments, the first cationic surfactant may be in a first container and the second cationic surfactant may be in a second container before the step of applying 0.02 wt% to 1 wt% of a first cationic surfactant and 0.02 wt% to 3 wt% of a second cationic surfactant to a food surface. In further embodiments, the first cationic surfactant may be in a first container and the second cationic surfactant may be in a second container, and both are combined to form an antimicrobial composition in a third container before the step of applying 0.02 wt% to 1 wt% of a first cationic surfactant and 0.02 wt% to 3 wt% of a second cationic surfactant to a food surface. In yet further embodiments, the first cationic surfactant and the second cationic surfactant form an antimicrobial composition at the same time as the step of applying 0.02 wt% to 1 wt% of a first cationic surfactant and 0.02 wt% to 3 wt% of a second cationic surfactant to a food surface.

In some embodiments, the step of applying a first cationic surfactant and a second cationic surfactant to a food surface may include covering greater than 50% of the food surface with the composition. In some embodiments, the step of applying a first cationic surfactant and a second cationic surfactant to a food surface may include covering greater than 55% of the food surface with the composition, 60% of the food surface with the composition, 65% of the food surface with the composition, 70% of the food surface with the composition, 75% of the food surface with the composition, 80% of the food surface with the composition, 85% of the food surface with the composition, 90% of the food surface with the composition, 95% of the food surface with the composition, 96% of the food surface with the composition, 97% of the food surface with the composition, 98% of the food surface with the composition, 99% of the food surface with the composition, 100% of the food surface with the composition or any range between any of these values. In other embodiments, the step of applying a first cationic surfactant and a second cationic surfactant to a food surface may include covering greater than 95% of the food surface with the composition.

In some embodiments, the step of applying 0.02 wt% to 1 wt% of a first cationic surfactant and 0.02 wt% to 3 wt% of a second cationic surfactant to a food surface may include covering greater than 85% of the food surface with the composition. The step of applying 0.02 wt% to 1 wt% of a first cationic surfactant and 0.02 wt% to 3 wt% of a second cationic surfactant to a food surface may include covering greater than 90% of the food surface with the composition. The step of applying 0.02 wt% to 1 wt% of a first cationic surfactant and 0.02 wt% to 3 wt% of a second cationic surfactant to a food surface may include covering greater than 95% of the food surface with the

composition. In other embodiments, the step of applying 0.02 wt% to 1 wt% of a first cationic surfactant and 0.02 wt% to 3 wt% of a second cationic surfactant to a food surface may include covering greater than 99% of the food surface with the

composition.

The method of reducing microbes on the food surface may include disinfecting a hard surface. The method of reducing microbes on the food surface may include disinfecting the food surface. The method of reducing microbes on the food surface may not include disinfecting the food surface. In some embodiments, the food surface may be a meat surface. In other embodiments, the food surface may be a beef surface, fish surface, fruit surface, lamb surface, pork surface, poultry surface, seafood surface, vegetable surface and combinations thereof. The step of applying may include coating, dipping, misting, rinsing, showering, spraying, foaming or washing. In some embodiments, the step of applying may include coating, dipping, misting, rinsing, showering, spraying, foaming or washing 0.02 wt% to 1 wt% of a first cationic surfactant and 0.02 wt% to 3 wt% of a second cationic surfactant to a food surface. In some embodiments, a second method may be used for reducing the growth of microbes on a hard surface. The method may include contacting the hard surface with an effective amount of an antimicrobial composition. In some embodiments, a second method may be used for reducing the growth of microbes on a food surface. The method may include contacting the food surface with an effective amount of an antimicrobial composition. The antimicrobial composition may have two parts. The first part may be a first cationic surfactant. The second part may be second cationic surfactant. In some embodiments, the first part may be 0.02 wt% to 1 wt% of a first cationic surfactant and the second part may be 0.02 wt% to 3 wt% of a second cationic surfactant. In some embodiments, the two parts may be applied to the food surface either concurrently. In other embodiments, the two parts may be applied to the food surface sequentially.

The second method may have the two parts applied to the food surface with a sufficient contact time to maximize wetting of the food surface. Sufficient contact time requires at least 2 seconds. In some embodiments, sufficient contact time may be at least 2 seconds, 3 seconds, 4 seconds, 5 seconds, 6 seconds, 7 seconds, 8 seconds, 9 seconds, 10 seconds, 15 seconds, 20 seconds, 25 seconds, 30 seconds or any range between any of these values. In some embodiments, the antimicrobial composition may contact the food surface before the food surface is loaded into a packaging material. In other embodiments, the antimicrobial composition may contact the food surface after the food surface is loaded into a packaging material. In further embodiments, the antimicrobial composition may contact the food surface at the same time the food surface is loaded into a packaging material.

In some embodiments, the antimicrobial composition may contact process water that contacts the food surface. The antimicrobial composition may help keep the microbes under control in the process water, allowing the process water to be re-used in the same applications or other applications for more than 1 production day. In other embodiments, the antimicrobial composition may contact the process water and then the process water with the antimicrobial composition contacts the food surface.

In some embodiments, the antimicrobial composition may contact a food processing surface before sanitation that comes into contact with the food surface. In other embodiments, the antimicrobial composition may contact a food processing surface after sanitation that comes into contact with the food surface. The antimicrobial composition may help keep the microbes under control on the food processing surface, allowing the food processing surface to be hygienic for food production. In other embodiments, the antimicrobial composition may contact the food processing surface and then the food processing surface with the antimicrobial composition contacts the food surface. In other embodiments, the first and the second cationic surfactants may be first applied to a food processing surface and then the step of applying the first and second cationic surfactants to a food surface is performed by the transference of the first and the second cationic surfactant from the food processing surface to the food surface after the food surface comes into contact with the food processing surface. The food processing surface may be a hard surface.

In some embodiments, at least one of the first part and/or the second part may be incorporated into a packaging material. The incorporating into a packaging material may include extrusion, co-extrusion, surface coating, or dusting of at least one of the first part and the second part into a packaging material. The first and/or the second part may be contained on the surface of a plastic packaging material with a spray application onto the surface of the plastic packaging material. The surface with the spray application would then make direct contact with the food surface. In some embodiments, the first part may be applied to the food surface and the second part may be contained within or on the surface of a plastic packaging material and direct contact may be made with the food surface and the plastic packaging material to contact the food surface with an effective amount of the antimicrobial composition. In other embodiments, the first part may be contained within or on the surface of a plastic packaging material and the second part may be applied to the food surface and direct contact may be made with the food surface and the plastic packaging material to contact the food surface with an effective amount of the antimicrobial composition. In further embodiments, the first part and the second part may be each contained within or on the surface of a plastic packaging material and direct contact may be made with the food surface and the plastic packaging material before packaging of the food surface.

In some embodiments, the kill rate of the antimicrobial composition may be 90% to 99.99%. The kill rate of the antimicrobial composition may be 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%. 99.9%, 99.95%, 99.99% or any range between any of these values. In other embodiments, the log kill rate of the disclosed solution exhibits a log E. coli kill rate of at least about 1 log CFU/g.

In addition, the disclosed invention may also be directed to a method of reducing microbes on a hard surface, e.g. food processing surface. The method of reducing microbes may be on a food surface. The method may include applying a second cationic surfactant, a lantibiotic, and an aminopolycarboxylic acid. The second cationic surfactant, lantibiotic, and aminopolycarboxylic acid may be in the form of an antimicrobial composition. The second cationic surfactant may be any compounds previously mentioned above. The second cationic surfactant may be Na-Lauroyl-L- arginine ethyl ester monohydrochloride (LAE). The lantibiotic may be Nisin, Nisin A, Nisin Z, Nisin Q, Nisin F, Nisin U, or combinations thereof. The lantibiotic may be Nisin Z. The aminopolycarboxylic acid may be ethylenediaminetetraacetic acid (EDTA) or a salt of EDTA. The salt of EDTA may be disodium EDTA, calcium disodium EDTA. The aminopolycarboxylic acid may be ethylenediaminetetraacetic acid (EDTA). The antimicrobial composition may have 1 % to 3% of a second cationic surfactant, 1 % to 3% of a lantibiotic, and 0.25% to 1 % of an aminopolycarboxylic acid. The antimicrobial composition may have 1 %, 1 .25%, 1 .5%, 1 .75%, 2%, 2.25%, 2.5%, 2.75%, 3%, or any range between these values of a second cationic surfactant. The antimicrobial composition may have 2% of a second cationic surfactant. The antimicrobial composition may have 0.25%, 0.3%, 0.35%, 0.4%, 0.41 %, 0.42%, 0.43%, 0.44%, 0.45%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1 %, or any range between these values of a lantibiotic. The antimicrobial composition may have 0.41 % of a lantibiotic. The antimicrobial composition may have 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1 %, 1 .1 %, 1 .2%, 1 .3%, 1 .4%, 1 .5%, 1 .6%, 1.7%, 1 .8%, 1.9%, 2%, or any range between these values of an aminocarboxylic acid. The antimicrobial composition may have 1 % of an aminocarboxylic acid. In some embodiments, the antimicrobial composition may have 1 % to 3% LAE, 0.25% to 1 % Nisin Z, and 0.25% to 2% EDTA. In other embodiment, the antimicrobial composition may have 2% LAE, 0.41 % Nisin Z, and 1 % EDTA.

The method of reducing microbes on a surface may include applying 1 % to 3% of a second cationic surfactant, 1 % to 3% of a lantibiotic, and 0.25% to 1 % of an aminopolycarboxylic acid. In some embodiments, the method of reducing microbes on a surface may include applying 2% LAE, 0.41 % Nisin Z, and 1 % EDTA to a surface. The surface may be a food surface. The surface may be a hard surface.

The combination of LAE, Nisin Z, and EDTA acts as an antimicrobial composition and may be used as a direct food contact intervention to minimize pathogens on meat and potentially to extend shelf life. This intervention may be extended to poultry, red meat, fish, ready-to-eat meat, seafood, fruits, and vegetables. In some embodiments, the antimicrobial composition may be used as a hard surface sanitizer. The antimicrobial composition may be used on hard, non-porous food processing surfaces, e.g. stainless steel. In some embodiments, the antimicrobial composition may be used on hard surfaces as mentioned above, previously. Examples of hard surfaces include, but are not limited to, chairs, tables, countertops, food processing surfaces, and floors. The above mentioned antimicrobial compositions have improved material compatibility than current antimicrobials, since they are less corrosive. All of the ingredients are also GRAS or have appropriate food contact allowances. The antimicrobial composition is not highly acidic and it is not an oxidizer, making it less likely to change the appearance of a food surface. There are numerous advantages to using the above mentioned antimicrobial compositions in food processing. There is the potential to reduce the number of applications of antimicrobial treatments in a food processor. It may be possible to reduce or eliminate the use of peroxyacetic acid (PAA) and all rinses, which would result in less chemicals used, reduced cost, less toxic work environment, less wastewater treatment, less corrosion, and increased equipment life.

These applications are for illustrative purposes only and are not intended as a limitation on the scope of the presently disclosed subject matter. EXAMPLES

The following Examples provide illustrative embodiments of the presently disclosed subject matter. In light of the present disclosure and the general level of skill in the art, those of ordinary skill can appreciate that the following Examples are intended to be exemplary only and that numerous changes, modifications, and alterations can be employed without departing from the scope of the presently disclosed subject matter.

EXAMPLE 1

Antimicrobial Test Solution

The following is an example of an antimicrobial composition (Sample 1 ) used in the method of the present invention. Sample 1 was prepared at room temperature according to the formulation in Table 1 below. The composition was physically and chemically stable. Table 1 Antimicrobial Composition

Sample 1

Raw Material Quantity (Wt. %) Water (deionized) 97.2 Cetyl Pyridinium Chloride (CPC) 0.32 Ethyl Lauroyl Arginate (LAE) 2.00 Propylene Glycol 0.48

EXAMPLE 2

Demonstration of Synergistic Effect

The following example determined the efficacy and synergistic effect of Sample 1 at reducing Escherichia coli (E. coli) or Salmonella Typhimurium on skin-on poultry. For this example a solution of 2% LAE, 0.32% CPC, and a Sample 1 were prepared. LAE and CPC can optionally be dissolved in propylene glycol or any other food grade solvent to provide a concentrated formulation for industrial use. Skin-on chicken was placed in an inoculation bath for 15 minutes. The inoculated chicken was allowed to dry for 20 minutes on sterile racks. Then five randomly selected chicken drumsticks were selected for enumeration of initial microbial levels as control chicken samples. The chicken drumsticks were diluted 1 : 10 in buffered peptone water and gently massaged for 1 min. Then the solution was serially diluted onto plates using neutralizing solution to 10 ^"2 , 10 ^"3 , 10 ^"4 , 10 ^"5 , 10 ^"6 . The plates were incubated for 48 hours at 37°C. Five Inoculated chicken samples were dipped in either a solution of 2% LAE, 0.32% CPC, or Sample 1 for 30 seconds at room temperature. The dip treated inoculated chicken samples were sampled in the same method discussed above.

The treatment of the skin-on chicken with 2% LAE and 0.32% CPC separately resulted in a >1 log reduction of E. coli. The treatment of the skin-on chicken with Sample 1 resulted in a >5 log reduction of E. coli. The blend of LAE and CPC (Sample 1 ) has a combined antimicrobial effect greater than the sum of its separate effects (See FIG. 1). Therefore, the presence of each component is necessary to achieve the observed antimicrobial efficacy of the combination as a whole.

EXAMPLE 3

Solution Treatment Study of Antimicrobial Material on Fresh Chicken

The following example compares the efficacy of other commercial antimicrobials and antimicrobial combinations at reducing E. coli on skin-on chicken. The log reduction or achievable inhibition of E. coli on skin-on chicken was measured for each chemical combination. For this example, the previously described test method in Example 2 was used. Skin-on chicken was placed in an inoculation bath for 15 minutes. The inoculated chicken was allowed to dry for 20 minutes on sterile racks. Then five randomly selected chicken drumsticks were selected for enumeration of initial microbial levels as control chicken samples. The chicken drumsticks were dilution 1 : 10 in buffered peptone water and gently massaged for 1 min. Then the solution was serially diluted onto plates using neutralizing solution to 10 ^"2 , 10 ^"3 , 10 ^"4 , 10 ^"5 , 10 ^"6 . The plates were incubated for 48 hours at 37°C. Five Inoculated chicken samples were dipped in each sample listed in Table 2 and Table 3 for 30 seconds at room temperature. The commercial antimicrobials, antimicrobial combinations, and efficacy results are shown in Table 2 and Table 3 below. Table 2

Commercial Antimicrobials

Sample Active Antimicrobials Log Reduction StDv

(ppm or wt. % when specified)

Sample 2 2% LAE 1 .18 0.945

Sample 3 1 % Lactic Acid 1 .56 0.429

Sample 4 0.32% CPC 1 .05 0.521

Sample 5 600ppm PAA 0.692 0.541

Sample 6 600ppm peroxymonosulfate 0.541 0.395

Table 3 Antimicrobial Combinations

Sample Antimicrobial Combinations Log StDv

(ppm or wt. % when specified) Reduction

Sample 7 200ppm LAE + 80ppm Tween20 1 .01 0.259

Sample 8 2% LAE + 600 ppm OSA 1 .04 0.400

Sample 9 0.32% CPC + 220ppm PAA (pH=3.57) 1 .37 0.140

Sample 10 0.32% CPC + 220ppm PAA (NaOH added to 0.900 0.194

reach pH=7.70)

Sample 1 2% LAE + 0.32% CPC >5 N/A

It can be seen in Table 2 and Table 3 that a blend of LAE and CPC (Sample 1 ) achieves a greater inhibition of E. coli on skin-on chicken compared to other commercial antimicrobials and antimicrobials used in combination, demonstrating the unexpected properties of the antimicrobial composition.

EXAMPLE 4

Use in Different Applications

The following example demonstrates the efficacy of Sample 1 on E. coli on different food products including poultry (skin-on chicken), fresh red meats (pork and beef), ready-to-eat meats, fruits and vegetables (citrus, potatoes, lettuce). LAE and CPC can optionally be dissolved in propylene glycol or any other food grade solvent to provide a concentrated formulation for industrial use. The log reduction or achievable inhibition of E. coli on each food product was measured. For this example, the previously described test method in Example 2 was used. FIG. 2 demonstrates the inhibition achieved on each food product using Sample 1 .

The treatment of pork, skin-on chicken, potatoes and lettuce with Sample 1 resulted in a >5 log reduction of E. coli. The treatment of citrus, ready-to-eat meat, and beef with Sample 1 resulted in a >4 log reduction of E coli.

EXAMPLE 5

Concentration Testing

The following example determined the efficacy of an antimicrobial combination of LAE and CPC dissolved in propylene glycol and D. I water at different concentrations and ratios at reducing E coli on skin-on poultry. LAE and CPC can optionally be dissolved in any other food grade solvent to provide a concentrated formulation for industrial use. For this example solutions of ethyl lauroyl arginate and cetyl pyridinium chloride at varying concentrations were prepared. The log reduction or achievable inhibition of E coli on skin-on chicken was measured for each chemical combination. For this example, the previously described test method in Example 2 was used. The antimicrobial concentrations and efficacy results are shown in Table 4 below. Table 4 Inhibition Results on Skin-on Poultry at Varying Concentrations of CPC and LAE

Sample LAE (wt. %) CPC (wt. %) Log StDv

Reduction

Sample 1 2.00 0.32 >5 N/A

Sample 1 1 2.00 0.16 4.88 0.170

Sample 12 1 .50 0.08 4.35 0.280

Sample 13 1 .00 0.32 4.98 0.000

Sample 14 0.20 0.16 >5 0.090

Sample 15 0.20 0.08 >5 0.090

Sample 16 0.10 0.16 4.60 0.1 10

Sample 17 2.00 0.00 1 .18 0.945

Sample 18 0.00 0.32 1 .05 0.521

Sample 19 0.00 0.16 1 .56 0.150

Sample 1 and Samples 1 1 -16 resulted in a >4 log reduction of E. coli. Table 4 demonstrates that concentrations as low as 0.08% CPC and 0.1 % LAE can achieve a >4 log reduction of E. coli, as long as the appropriate LAE/CPC combination is applied. Sample 17-18 containing less than two components resulted in ~1 log reduction of E. coli, demonstrating that the presence of both LAE and CPC is necessary to achieve the observed antimicrobial efficacy of the combination as a whole.

EXAMPLE 6

Comparison of Invention with Additional Additives

The following example compares the efficacy of an antimicrobial combination of

LAE and CPC dissolved in propylene glycol and D. I water with the addition of the following products: fatty acids and chelating agents at reducing E. coli on skin-on poultry. LAE and CPC can optionally be dissolved in any other food grade solvent to provide a concentrated formulation for industrial use. For this example solutions of ethyl lauroyl arginate, cetyl pyridinium chloride, and either a fatty acid or chelating agent were prepared. The log reduction or achievable inhibition of E. coli on skin-on chicken was measured for each chemical combination. For this example, the previously described test method in Example 2 was used. The antimicrobial combinations and efficacy results are shown in Table 5 below. Table 5

Inhibition Results of LAE and CPC Blend Combined with Other Additives

Sample LAE (wt. CPC (wt. %) Additive Log StDv

%) Reduction

Sample 20 0.25 0.04 EDTA 0.5% 1.36 0.05

Sample 21 0.25 0.04 Decanoic Acid 1 .1 1 0.1 1

43ppm

Sample 22 0.25 0.04 N/A 0.80 0.08 It can be seen in Table 5 that a blend of LAE and CPC with the addition of either a fatty acid or chelating agent achieves a greater inhibition of E. coli on skin-on chicken compared to a low level of LAE and CPC blend alone.

EXAMPLE 7

Temperature Testing

The following example determined the efficacy of an antimicrobial combination of LAE and CPC dissolved in propylene glycol and D. I water at different temperatures at reducing E. coli on skin-on poultry. LAE and CPC can optionally be dissolved in any other food grade solvent to provide a concentrated formulation for industrial use. For this example a blend of 2% LAE and 0.32% CPC was prepared. For this example, the previously described test method in Example 2 was used, however, it was done at 22°C and 8°C.

Table 6

Inhibition Results of LAE and CPC Blend at Varying Temperatures

Sample LAE (wt. %) CPC (wt. %) Temperature Log Reduction StDv

Sample 23 2.00 0.32 22°C >5 N/A

Sample 24 2.00 0.32 8°C >5 0.17

It is demonstrated in Table 6 that a blend of LAE and CPC at varying temperatures can achieve >5 log reduction of E. coli on skin-on chicken. EXAMPLE 8

Antimicrobial composition of LAE, Nisin Z, and EDTA

The following is another example of an antimicrobial composition (Sample 2) used in the method of the present invention. Sample 2 was prepared at room temperature according to the formulation in Table 7 below. The composition was physically and chemically stable.

Table 7

Composition of Sample 2

Sample 2

Raw Material Quantity (Wt. %) Water (deionized) 96.59 LAE 2 Nisin Z 0.41 EDTA 1

Sample 3 was prepared with only Nisin Z and EDTA at 0.41 % and 1 %, respectively. Sample 4 was prepared with only LAE at 2% LAE. Both Sample 3 and 4 remaining weight percent was deionized water.

A culture of E.coli (ATCC 8739) was plated using tryptic soy agar (TSA) and grown overnight. The next day, 1 L of tryptic soy broth (TSB) was inoculated with E. coli and grown for 5 hours. A 50 gram product was aseptically placed in a sterile bin, and 1 L of the inoculated TSB was added to the bin. Each condition was done in replicates of 5 and a control was also added to the bin. All samples were left in the bin for 10 minutes. All products were aseptically removed and placed on a sterile rack in a biosafety cabinet for 20 minutes to dry. Randomly selected controls were placed individually in a sterile whirl-pak bag and weighed. Randomly selected treated products for each testing condition were placed individually in individual sterile baskets and weighed. Each basket was placed in a treatment solution of either Sample 2, Sample 3 (Nisin Z + EDTA), or Sample 4 (LAE) for 30 seconds. The baskets were then removed and placed on a sterile rack in a biosafety cabinet for 5 minutes to dry. A 1 : 10 dilution of each product tested with buffered peptone water was put into a bag and gently massaged for 1 minute. Samples were serially diluted using neutralized peptone water to 10 ^~1 , 10 ^"3 , 10 ^"5 . 100 microliters of each dilution was plated for a final dilution of 10 ^"2 , 10 ^"4 , 10 ^"6 . All plates were incubated for 48 hours at 37°C.

The results of Sample 4 (LAE) showed that LAE alone only has a 1 .18 log reduction. The results of Sample 3 (Nisin Z + EDTA) showed that this formulation only has a 1 .56 log reduction. But the combination of all 3 ingredients (Sample 2), resulted in >5 log reduction of E. coli. These results show that the combination of all 3 ingredients (LAE, Nisin Z, and EDTA) have a synergistic effect resulting in unexpected results of >5 log reduction of E. coli. These results equate to more than a 99.999% reduction in organisms as compared to a 1 log reduction which is a 90% reduction.