ANTIMICROBIAL COMPOSITION

Field

The present invention relates to materials useful for packaging that comprise an active antimicrobial agent. Such materials can be particularly useful in food packaging applications.

Introduction

In the packaged food industry, particularly in the packaging of fresh meat in plastic materials, the protection of meat from bacteria and bacterial growth is very important to meat processors, packagers, and retailers. In the fresh meat space, cost effectiveness and long-lasting antimicrobial efficacy are important considerations. Most commercial antimicrobial technologies in the fresh beef, pork and poultry markets are carcass applications that utilize an antimicrobial spray or bath to apply the antimicrobial agent to the carcass.

It is desirable to provide multiple hurdles for pathogens along the supply chain in the preparation and packaging of fresh meats. As such, significant research has gone into developing antimicrobial solutions that are delivered along or with the packaging. A number of techniques have been attempted including, for example: incorporating an antimicrobial agent directly into a packaging material (e.g., via compounding or blending); immobilizing an antimicrobial agent onto the packaging (e.g., via surface treatment, reactive bonding, etc.), incorporating the antimicrobial agent via functionalization of the polymer resin (e.g., a quat polymer), and delivering the antimicrobial agent via a coating applied to the packaging surface. Each of these techniques has serious limitations.

Incorporation of the antimicrobial agent into the packaging material is limited by the antimicrobial agent's temperature compatibility with extrusion temperatures (most organic antimicrobial agents are not compatible). Use of an antimicrobial agent (e.g., a temperature- stable metal-based particle such as silver) can result in the agent leaching out of the packaging material and onto the food. In addition, certain antimicrobial agents can induce changes to organoleptics (e.g., acids, essential oils, etc.) associated with the fresh meat. The functionalization of polymers to include functionalities with antimicrobial properties is typically not cost effective. Applying an antimicrobial agent to a packaging surface as a liquid is also not effective as such are typically low viscosity and aqueous- based, and therefore drip off the surface of the foodstuff and collect in pockets in the package. Finally, immobilization of an antimicrobial agent onto a packaging substrate inherently reduces the mobility of the antimicrobial agent and affects efficacy.

It would thus be desirable to have alternative approaches for providing antimicrobial agents in food packages and in particular, packages of fresh meat.

Summary

The present invention' s combination of a dry film layer comprising an active antimicrobial agent with a substrate to provide materials suitable for packaging as described herein advantageously addresses many of the limitations associated with prior attempts to provide antimicrobial agents in food packaging. For example, in various embodiments, the present invention provides materials suitable for packaging that comprise a dry film with an active antimicrobial agent which can be positioned on an inner surface of a package (e.g., the food side of a food package). The dry film comprises a water soluble carrier that provides appropriate food contact times (significantly increased over low viscosity, aqueous-based systems) due to the viscosity of the carrier after hydration. Once the carrier is hydrated, the active antimicrobial agent can remain fully mobile within the carrier' s matrix, allowing it to freely travel to infection sites, and thus improve efficacy over alternative approaches. Upon hydration, the carrier can have a viscosity that avoids dripping and/or substantial flow of the carrier (and thereby the active antimicrobial agent) off of the substrate. Further, the water solubility of the carrier can impact the rate of release of the active antimicrobial agent so as to prolong food contact time.

In one aspect, the present invention provides a material suitable for packaging that comprises (a) a substrate and (b) a dry film layer adjacent to the substrate, the dry film layer comprising (i) an active antimicrobial agent and (ii) a carrier, wherein the carrier is water soluble.

These and other embodiments are described in more detail in the Detailed

Description.

Brief Description of the Drawings

Figure 1 is a bar graph illustrating the results of Example 1.

Figure 2 is a bar graph illustrating the results of Example 2.

Figure 3 is a bar graph illustrating the results of Example 4.

Detailed Description

Unless specified otherwise herein, percentages are weight percentages (wt%) and temperatures are in °C. A "food surface" is an outer surface of any food product. Food products include, without limitation, meats, cheeses, fruits and vegetables. Meats are the flesh of animals intended for use as food. Animals include mammals (e.g., cows, pigs, sheep, buffalo, etc.), birds (e.g., chickens, turkeys, ducks, geese, etc.), fish and shellfish. Meats include fresh meats (e.g., animal carcasses, cut meat pieces, etc.), processed meats, and processed meat products, such as sausage, cured meats, meat spreads, deli meats, sliced meats, and ground meats. Such meats can include for example, fresh meats, processed meats, and processed meat products that are to be stored, transported, displayed, and/or sold under refrigerated conditions (e.g., at temperatures of 2 to 6° C). A "meat surface" is an outer surface of any meat product.

Following slaughter of an animal, meats are typically processed in a variety of ways prior to packaging for sale to consumers. In the case of some meat products (e.g., steaks, chicken breasts, etc.), the meats may only be cut and trimmed to smaller sizes. As another example, meat products, such as deli meat, may be cut, seasoned, cooked, and then sliced. Meats can be prepared in a wide variety of other ways known to those of skill in the art. Once prepared and/or processed for preparation to sale to consumers, the meats are packaged in a variety of ways.

At some point, prior to or after packaging, the meats are refrigerated or frozen. The packaged meats desirably remain refrigerated or frozen until purchase and/or use by the consumer. In the case of refrigerated meats, the meats and packages containing meats are typically held between 2° C and 6° C, often 4° C. This is often the situation for fresh meats to be sold in retail stores. In some situations, there may be variation in refrigeration temperatures along the supply chain (e.g., between a meat processor and a retail location) such that the storage temperature of the meat packages may be between 4° C and 12° C. At temperatures within that range (2° C and 12° C), the potential for bacterial growth on the meat surface remains, even after packaging. Furthermore, it is well known in the field that the bacterial makeup on the meat surface can be vastly different between 2° C and 12° C than at other temperatures. Furthermore, the bacterial makeup on the meat surface is generally also vastly different across the temperature range of 2° C to 12° C. Thus, some embodiments of the present invention are directed toward materials suitable for packaging that can be used with packaged meat products to prevent and/or inhibit the growth of bacteria on the meat surface at temperatures between 2° C and 12° C. Some embodiments of the present invention are directed toward materials suitable for packaging that can be used with packaged meat products to prevent and/or inhibit the growth of bacteria on the meat surface at a broad range of temperatures including temperatures less than 2° C and/or greater than 12° C.

In one aspect, the present invention provides a material suitable for packaging that comprises (a) a substrate and (b) a dry film layer adjacent to the substrate comprising (i) an active antimicrobial agent and (ii) a carrier, wherein the carrier is water soluble. The water solubility of the carrier is advantageous because it facilitates hydration of the dry film layer so as to release the active antimicrobial agent. Thus, in some embodiments, the carrier hydrates upon contact with water. Depending on the embodiment, the water can be a liquid within a package or moisture in the air within a package. For example, when materials of the present invention are incorporated into a food package according to some embodiments, hydration can be triggered by moisture on the surface of a food item upon contact with the dry film layer, or by moisture in the atmosphere of the package. Thus, in the context of a food package, the dry film layer can contact, adhere, cling to, wet, or be wetted by the surface of a food product (e.g., meat) in order to hydrate the carrier and facilitate transfer/migration of the active antimicrobial agent.

One advantage of some embodiments of the present invention is that prior to hydration, the dry film layer is not tacky and adheres to the substrate (e.g., a packaging film) such that the material (e.g., the substrate and the dry film) can be easily handled and rolled.

Another advantage of some embodiments of the present invention is the potential for "controlled release" of the active antimicrobial agent. For example, the components of the dry film layer can be selected so as to provide appropriate solubilization properties. For instance, a dry film layer where the polymer hydrates more slowly will result in slower release of the active antimicrobial agent, which can then be delivered over a longer period of time. As another example, the dry film layer can incorporate two active antimicrobial agents with one being highly soluble and one being less soluble. In such an example, the highly soluble active antimicrobial agent may migrate or move more quickly over a food product than the less soluble antimicrobial agent to provide a delayed release. Thus, the properties of the dry film layer can be tailored so as to extend shelf life in some

embodiments. Thus, two features of some embodiments of the present invention are improved handling of the materials to be used in a package, and the potential for controlled release of the active antimicrobial agent. A further advantage, in some embodiments, is that these features can be achieved independently of active antimicrobial agent used. For example, the active antimicrobial agent can be a variety of materials as discussed further below such as a bacteriophage, a bacteriocin, or other peptides, or a non-biological active, such as small organic molecules (e.g., lactic acid, lauric arginate, cetylpyridinium chloride, etc.), or metal- based actives (e.g., silver, zinc, copper-based systems), or others.

In some embodiments, the film layer forms a hydrogel upon contact with water. The term "hydrogel" is used herein in a manner consistent with the understanding of those of skill in the art. In general, a hydrogel refers to a nonfluid colloidal network or polymer network that is expanded throughout its whole volume primarily by water. When cut into two pieces, a hydrogel will not typically rejoin to form a single unit, whereas a non-gel, viscous liquid will over time lose shape and the two pieces will rejoin.

In some embodiments, upon hydration, the film layer can form a hydrogel at temperatures between 2° C and 12° C. As a hydrogel, the composition facilitates prolonged contact time for the active antimicrobial agent on the surface of the food. Furthermore, the active antimicrobial agent can remain fully mobile within the carrier's matrix, allowing it to freely travel to infection sites, and thus improve efficacy over alternative approaches.

A number of active antimicrobial agents can be used as discussed further below. In addition, a number of carriers can be used as discussed further below. In some

embodiments, materials can comprise a plurality of film layers comprising an active antimicrobial agent and a carrier, wherein the carrier is at least partially water soluble. In some such embodiments, an outermost film layer can be more water soluble than an inner film layer such that the active antimicrobial agent in the outermost film layer is delivered first upon hydration of the carrier in the outermost film layer. In general, the substrate can be any material that can be combined with the dry film layer and from which a package can be formed. The substrate is a polymeric film in some embodiments. In some embodiments, the substrate comprises a monolayer film while in other embodiments, the substrate comprises a multilayer film. In some embodiments where the substrate comprises a multilayer film, the film can comprise a surface layer adjacent to the antimicrobial film layer. The surface layer, in some embodiments, can be corona treated, plasma treated, or otherwise modified. Such surface treatment is believed, in some embodiments, to increase the surface energy of the surface layer and thus improve the wetting and/or adhesion between the substrate and the dry film layer. For example, surface treatment of the substrate can improve the wettability of the surface of the substrate by providing polar character or higher surface tension which, when an aqueous solution (e.g., an aqueous solution comprising an active antimicrobial agent and carrier to form a film layer) is applied, can result in more uniform coverage of the surface. In some embodiments, the adhesion between the substrate and the dry film layer can be enhanced in other ways. For example, in some embodiments, the surface layer can comprise a functional polymer such as, but not limited to the following: an ethylene vinyl acetate copolymer, an ethylene butyl acrylate, an ethylene ethyl acrylate, an ethylene methyl methacrylate, an ethylene methacrylic acid copolymer, a maleic anhydride grafted copolymer, an ethylene acrylic acid copolymer, an ethylene vinyl acetate carbon monoxide terpolymer, an ethylene methyl acrylate copolymer, an ionomer of any of the foregoing, or a combination thereof.

In some embodiments, the substrate and/or the film layer can comprise one or more additives such as antioxidants, surfactants, heat and/or light stabilizers (e.g., hindered phenols, ultraviolet light absorbers, etc.), buffers, plasticizers, other viscosity modifiers, pH adjusters, scavengers (e.g., odor, oxygen, moisture, etc.), slip aids, antiblocks, pigments, dyes, polymer processing aids, and/or other additives, as well combinations of different such additives. In some embodiments, the substrate comprises a molded article, a tray, an absorbent pad, or a sheet.

In some aspects, the present invention relates to a package comprising any of the materials suitable for packaging described herein. In packages incorporating materials of the present invention, the film layer incorporating the active antimicrobial agent is positioned on the inner surface of the substrate or sealant layer. In a further embodiment, the material incorporating the dry film layer may be sealed, preferably heat sealed to itself using techniques known to those of skill in the art. For example, in some embodiments, the material can be heat sealed to provide a peel strength of 3 pounds per inch or more. The package, in some further embodiments, comprises a food product, such as a meat product. In some embodiments, the dry film layer (or the hydrated film layer (e.g., hydrogel)) is in contact with the food product. While the dry film layer or hydrated film layer (e.g., hydrogel) is in contact with the food product in some embodiments, the substrate, the dry film layer, or the entirety of the hydrated film layer may not necessarily be in contact with the food product in some embodiments. For example, while the inner surface of the packaging material (or a portion of the inner surface of the packaging material) may not be in contact with the food product, the hydrated film layer (e.g., hydrogel) may still drip and spread across the surface of the food product over time.

Materials of the present invention can be adapted to prevent or inhibit the growth of a variety of bacteria including, for example:

(a) Escherichia coli including Shiga Toxin producing Escherichia coli (STEC) (also including strains of Verotoxin-producing Escherichia coli that have been linked with the severe complication hemolytic-uremic syndrome (HUS)); enterohemorrhagic E. coli (EHEC), shiga-like toxin-producing E. coli (STEC or SLTEC) (the specific seven serogroups of STEC include (0157:H7, 026, O103, 045, Ol l l, 0121 and 0145) of enterohemorrhagic E. coli that have been declared adulterants in non-intact raw beef by U.S. Department of Agriculture, hemolytic uremic syndrome-associated enterohemorrhagic E. coli (HUSEC) and

verocytotoxin- or verotoxin-producing E. coli (VTEC);

(b) other Escherichia coli strains that have been variously referred to by other virulence properties, such as enteroinvasive (EIEC), enteropathogenic (EPEC), enterotoxigenic (ETEC), enteroaggregative (EAEC or EAgEC);

(c) Salmonella species, including, but not limited to Salmonella enterica strains with the following subspecies based on serotyping:

Enteritidis, Kentucky, Typhimurium, Typhimurium Covariant V, Heidelberg, Hadar , Newport , Georgia, Agona, Grampian, Senftenberg , Alachua, Infantis, Reading, Schwarzengrund, Mbandaka, Montevideo, Berta and Thompson;

(d) Pseudomonas species (including P. fragi, P. lundensis, P. fluorescens);

(e) Campylobacter species (including Campylobacter jejuni);

(f) Clostridium perfringens; Clostridium botulinum;

(g) Listeria species (including Listeria monocytogenes);

(h) Shigella spp. (including serotypes A, B, C and D);

(i) Staphylococcus species including Staphylococcus aureus (including Methicillin resistant Staphylococcus and including species causing Staphylococcal enteritis;

(j) Streptococcus species;

(k) Vibrio species including Vibrios cholera (including serotypes 01 and non-01, Vibrio parahaemolyticus, and Vibrio vulnificus);

(1) Yersinia species including Yersinia enterocolitica and Yersinia pseudotuberculosis;

(m) Acinetobacter species (including Ac. johnsoni); (n) Moraxella species;

(o) Psychrobacter species (including Psychr. immobilis);

(p) Shewanella species (including Shewanella putrefaciens);

(q) Enterobacter species;

(r) Serratia species;

(s) Lactobacillus species; and/or

(t) Brochothrix thermosphacta.

A variety of active antimicrobial agents can be used in various embodiments to inhibit or prevent the growth of bacteria. One important factor in selecting an active antimicrobial agent is the type(s) of bacteria to be targeted.

In some embodiments, the active antimicrobial agent comprises one or more quaternary ammonium salts. Quaternary ammonium salts suitable for use in embodiments of the present invention include, for example, those effective in inhibiting growth of bacteria, including spoilage bacteria. Preferably, the quaternary ammonium salt has at least one aromatic substituent (e.g., pyridinium or benzyl). Preferably, the quaternary ammonium salt has at least one C8-C25 alkyl group, preferably C ₁₀ -C2 ₀ . An example of a quaternary ammonium salt that can be used in some embodiments is cetyl pyridinium chloride. Another example of a quaternary ammonium salt that can be used in some embodiments is dimethyloctadecyl[3-(trimethoxysilyl)propyl]ammonium chloride, which is a silyl quat. Preferred quaternary ammonium salts include those effective against preventing or inhibiting the growth of one or more of the bacteria listed above. One factor in selecting a particular quaternary ammonium salt for use in some embodiments of the present invention is the bacteria to be targeted.

In embodiments utilizing quaternary ammonium salts as an antimicrobial agent, it is applied to the food surface with a carrier (as described in more detail below). As set forth herein, the carrier can be in the form of a dry film but upon hydration, becomes an aqueous medium such as a hydrogel. The concentration of quaternary ammonium salt in the dry film layer can be selected based on its activity, the target viscosity upon hydration of the dry film layer, the amount and/or surface area of the food product, and other factors in accordance with the teachings herein.

In some embodiments, the active antimicrobial agent comprises one or more amino acid derivatives. Amino acid derivatives suitable for use in embodiments of the present invention include, for example, those effective in inhibiting growth of bacteria, including spoilage bacteria. One exemplary amino acid derivative that can be used in some embodiments is ethyl-N ^a -lauroyl-L-arginate (CAS number 60372-77-2, as the HC1 salt) (also known as lauric arginate). Preferred amino acid derivatives include those effective against preventing or inhibiting the growth of one or more of the bacteria listed above. One factor in selecting a particular amino acid derivative for use in some embodiments of the present invention is the bacteria to be targeted.

In embodiments utilizing lauric arginate as an antimicrobial agent, it is applied to the food surface with a carrier (as described in more detail below) initially as part of a dry film layer, but upon hydration, becomes an aqueous medium such as a hydrogel. The concentration of lauric arginate in the dry film layer can be selected based on its activity, the target viscosity upon hydration of the dry film layer, the amount and/or surface area of the food product, and other factors in accordance with the teachings herein.

In some embodiments, the active antimicrobial agent comprises one or more organic acids. An "organic acid" is a compound containing carbon and hydrogen atoms and having a pK _a (measured at room temperature) from 2 to 6, preferably from 2.5 to 5.5, preferably from 3 to 5. For example, organic acids that can be used in some embodiments include carboxylic acids. Organic acids suitable for use in embodiments of the present invention include, for example, those effective in inhibiting growth of bacteria, including spoilage bacteria. The organic acids may be partially or even completely in their ionized form, i.e., as their salts. Preferably, organic acids do not contain nitrogen atoms. More than one organic acid may be used in combination. Preferably, the organic acid has from two to ten carbon atoms, preferably from two to eight, preferably from three to six. Examples of organic acids that can be used in some embodiments include lactic acid, benzoic acid, sorbic acid, citric acid, acetic acid, propionic acid and octanoic acid. In some embodiments, the organic acid comprises lactic acid. Preferred organic acids include those effective against preventing or inhibiting the growth of one or more of the bacteria listed above. One factor in selecting a particular organic acid for use in some embodiments of the present invention is the bacteria to be targeted. In embodiments utilizing organic acids as an antimicrobial agent, it is applied to the food surface with a carrier (as described in more detail below) initially as a dry film layer, but upon hydration, as an aqueous medium such as a hydrogel. The concentration of organic acids in the dry film layer can be selected based on its activity, the target viscosity upon hydration of the dry film layer, the amount and/or surface area of the food product, and other factors in accordance with the teachings herein.

In some embodiments, the active antimicrobial agent comprises one or more peptides. Peptides suitable for use in embodiments of the present invention include, for example, those effective in inhibiting growth of bacteria, including spoilage bacteria.

Examples of peptides that can be used in some embodiments include, for example, nisin, epsilon-polylysine, bacteriocins and colicins; preferably nisin and epsilon-polylysine.

Preferred peptides include those effective against preventing or inhibiting the growth of one or more of the bacteria listed above. One factor in selecting a particular peptide for use in some embodiments of the present invention is the bacteria to be targeted. In embodiments utilizing peptides as an antimicrobial agent, it is applied to the food surface with a carrier (as described in more detail below) initially as a dry film layer, but upon hydration, as an aqueous medium such as a hydrogel. The concentration of peptide in the dry film layer can be selected based on its activity, the target viscosity upon hydration of the dry film layer, the amount and/or surface area of the food product, and other factors in accordance with the teachings herein.

In some embodiments, the active antimicrobial agent comprises a metal-based antimicrobial agent. Metal-based antimicrobial agents suitable for use in embodiments of the present invention include, for example, those effective in inhibiting growth of bacteria, including spoilage bacteria. Examples of such metal-based antimicrobial agents include in some embodiments silver-based antimicrobial agents, zinc-based antimicrobial agents, and copper-based antimicrobial agents. Such metal-based antimicrobial agents can be in any form known in the art to be suitable for food applications including, for example, metal salts, metal oxides, nanoparticles, metals supported onto inorganic materials such as zeolites and clays, and combinations thereof. Preferred metal-based antimicrobial agents include those effective against preventing or inhibiting the growth of one or more of the bacteria listed above. One factor in selecting a particular metal-based antimicrobial agent for use in some embodiments of the present invention is the bacteria to be targeted. In embodiments utilizing metal-based antimicrobial agents, it is applied to the food surface with a carrier (as described in more detail below) initially as a dry film layer, but upon hydration, as an aqueous medium such as a hydrogel. The concentration of metal-based antimicrobial agent in the dry film layer can be selected based on its activity, the target viscosity upon hydration of the dry film layer, the amount and/or surface area of the food product, and other factors in accordance with the teachings herein.

In some embodiments, the active antimicrobial agent comprises a bacteriophage. Bacteriophages suitable for use in some embodiments of the present invention include, for example, those effective in inhibiting growth of bacteria, including spoilage bacteria.

Preferred bacteriophages include those effective against the bacteria previously listed. One factor in selecting a particular bacteriophage for use in some embodiments of the present invention is the bacteria to be targeted. In embodiments utilizing bacteriophages as an antimicrobial agent, it is applied to the food surface with a carrier (as described in more detail below) initially as a dry film layer, but upon hydration, as an aqueous medium such as a hydrogel. In one preferred embodiment, the aqueous medium is buffered, preferably to a pH from 4 to 9, preferably from 5 to 8.5, preferably from 6 to 8. The concentration of bacteriophages in the dry film layer can be selected based on its activity, the target viscosity upon hydration of the dry film layer, the amount and/or surface area of the food product, and other factors in accordance with the teachings herein.

In some embodiments, antimicrobial compositions of the present invention do not include a bacteriophage, or a combination of bacteriophages as the only active antimicrobial agent. For example, embodiments of the present invention are targeted at the prevention or inhibition of bacterial growth at lower temperatures (e.g., refrigeration temperatures of 2° C to 12° C). Bacteriophages are understood to be less effective at lower temperatures due to lower microbial activity which is required for bacteriophage propagation, such that their inclusion may be less desirable in some embodiments of the present invention. For example, a higher concentration of bacteriophage may be needed to obtain antibacterial activity equivalent to the activity at higher temperatures where the bacteria are active. Another limitation of bacteriophages is that a cocktail of many individual bacteriophages would be needed to target the broad range of bacteria that could found in meat packaging, for example, as bacteriophages are specific to individual strains of bacteria. Thus, in some embodiments, to the extent that bacteriophages are used, one or more bacteriophages can be used in combination with another active antimicrobial agent disclosed herein that is not a bacteriophage. In some embodiments, an antimicrobial composition does not include any bacteriophages.

In addition to the above active antimicrobial agents, other active antimicrobial agents that are effective in preventing or inhibiting the growth of bacteria can also be used in some embodiments of the present invention. Examples of such active antimicrobial agents can include, for example, naturally-derived antimicrobial agents including essential oils, such as for instance Myristica fragrans, Origanum vulgare, Pelargonium graveolens, Piper nigrum, Syzygium aromaticum, Thymus vulgaris; essential oil extracts and other naturally-derived active ingredients, such as for instance borneol, δ-3-carene, carvacrol, carvacrol methyl ester, cis/trans -citra , eugenol, geraniol, thymol, a-terpineol, terpinen-4-ol, (+)-linalool, (-)-thujone, geranyl acetate, nerol, menthone, -pinene, R(+)-limonene, a- pinene, a-terpinene, citronellal, p-cymene, cinnamaldehyde and bornyl acetate; inorganic salts, such as for instance sodium chloride, acidified sodium chlorite, calcium hypochlorite, sodium metasilicate and trisodium phosphate; and organic salts such as sodium acetate.

In some embodiments, combinations of active antimicrobial agents can be provided in some embodiments of materials of the present invention. For example, certain active antimicrobial agents may be more effective against certain varieties of bacteria, such that a combination of antimicrobial agents can provide better efficacy against a wider range of bacteria. In some embodiments, a dry film layer can comprise any two or more of the active antimicrobial agents disclosed herein. For example, a dry film layer, in some embodiments, can comprise a bacteriophage and at least one other active antimicrobial agent disclosed herein. As another example, a dry film layer may comprise a highly soluble active antimicrobial agent and a less soluble active antimicrobial agent such that upon hydration of the film layer, the highly soluble active antimicrobial agent may reach a food surface more quickly than the less soluble antimicrobial agent, resulting in a controlled release of the antimicrobial agents. Persons skilled in the art can determine various combinations of active antimicrobial agents, relative amounts, and concentrations based on the teachings herein.

In accordance with the present invention, the active antimicrobial agent is provided in a carrier as part of a dry film layer. In some embodiments, the active antimicrobial agent is generally uniformly dispersed within the dry film layer. The dry film layer in combination with a substrate (discussed further herein) form a material suitable for packaging. The carrier is preferably water soluble. The water solubility of the carrier facilitates hydration of the dry film layer so as to release the active antimicrobial agent. Thus, in some embodiments, the carrier hydrates upon contact with water (e.g., a liquid within a package, moisture in the air within a package, etc.). The carrier can be selected so as to have a water solubility that provides a desirable viscosity upon hydration so as to facilitate contact between the active antimicrobial agent and a food product, for example. For example, a dry film layer where the carrier hydrates more slowly can result in a slower release of the active antimicrobial agent, which can then be delivered over a longer period of time. In other words, the properties of the dry film layer can be tailored so as to extend shelf life in some embodiments.

The carrier can comprise components that result in the dry film layer becoming a hydrogel upon hydration, in some embodiments. The viscosity of the hydrated film layer, particularly as a hydrogel, can enable prolonged contact time between the active antimicrobial agent and the food or meat surface. Further, the viscosity of the hydrated film layer, particularly as a hydrogel, can facilitate the ability of the active antimicrobial agent to remain fully mobile with the matrix of the carrier, allowing the agent to freely travel to infection sites.

As embodiments of the present invention are directed to materials suitable for the packaging of fresh meats that are to be processed, shipped, and/or stored at refrigerated temperatures (2° C to 12° C), the hydrated film layer can be a hydrogel within that temperature range for the reasons set forth herein.

In some embodiments, the carrier can comprise components that result in the dry film layer becoming a viscous liquid upon hydration, wherein the liquid has a viscosity of at least 50 centipoise at temperatures between 2° C and 12° C. At such viscosities, the viscous liquid facilitates prolonged contact time for the active antimicrobial agent on the surface of a food item. Furthermore, the active antimicrobial agent can remain fully mobile within the carrier's matrix, allowing it to freely travel to infection sites, and thus improve efficacy over alternative approaches. The upper limit of viscosity can vary depending on the particular active antimicrobial agent and carrier. In general, for a particular composition, the viscosity will be too high if there is a significant impact on mobility and/or efficacy of the active antimicrobial agent used. In general, the viscosity will be too low if significant dripping is observed such that the liquid does not maintain adequate contact with the food item. Thus, in considering a target viscosity range for a particular hydrated film layer, important factors will include, for example, the particular antimicrobial agent(s) used (including combinations of antimicrobial agents) , the amount of antimicrobial agent used, the content and composition of the carrier, the surface area of the food item, and other factors. By way of example, in some embodiments, the hydrated film layer can have a viscosity of up to 200,000 centipoise at temperatures between 2° C and 12° C. As used herein, and unless otherwise stated, all references to viscosity values refer to viscosities measured using an ARES RFS3 rheometer with a cup and bob fixture containing 17 mL of solution at a temperature of 4° C and at a shear rate of 10s ^"1 .

The components of the carrier at the target temperature range and the water/moisture content adjacent to the dry film layer (e.g., on the interior of a food package) are the key factors affecting the hydration of the dry film layer and whether it becomes a hydrogel or a viscous liquid upon hydration. The carrier preferably comprises a rheology modifier that can both form a dry film layer and also form a hydrogel (or a viscous liquid) upon hydration so as to distribute the active antimicrobial agent over a food item. For example, the dry film layer can be formed by mixing the rheology modifier, water, and the active antimicrobial agent, and then drying the mixture, as described in more detail below. When the dry film layer is contacted with a certain amount of moisture, the film layer hydrates to form a hydrogel or a viscous liquid.

Examples of rheology modifiers that can be included in various embodiments of the present invention to form a hydrogel include cellulose ether polymers, gelatin, pectin, xantham gum, guar gum, and other rheology modifiers that others skilled in the art can identify based on the teachings herein. The particular type of rheology modifier and amount can be selected so as to have the ability to form a dry film layer and also combine with water (hydrate) to form a hydrogel or viscous liquid (as discussed above) at temperatures between 2° C and 12° C.

One particularly desirable rheology modifier for use in some embodiments of the present invention is a cellulose ether polymer. Examples of cellulose ether polymers that can be used in some embodiments of the present invention include methylcellulose polymers, ethyl cellulose polymers, hydroxypropyl methylcellulose polymers, and combinations thereof. Such cellulose ether polymers are commercially available from The Dow Chemical Company under the names METHOCEL™ and ETHOCEL™. The amount of cellulose ether polymer, including potential combinations of cellulose ether polymers that can be used in embodiments of the present invention is an amount adequate to form a dry film layer comprising an active antimicrobial agent, and to later form a hydrogel upon hydration. In some embodiments, a combination of cellulose ether polymers can be selected so as to facilitate a controlled release of the active antimicrobial agent; in other words, the rheology modifier(s) can be selected so as to provide a desired breakdown rate of the dry film layer upon hydration which will impact the rate at which the active

antimicrobial agent is delivered to a food surface in a package environment, for example.

In the present invention, a specific hydroxypropyl methylcellulose polymer that exists as a hydrogel when in solution at 37° C is as follows. The hydroxypropyl methylcellulose has anhydroglucose units joined by 1-4 linkages. Each anhydroglucose unit contains hydroxyl groups at the 2, 3, and 6 positions. Partial or complete substitution of these hydroxyls creates cellulose derivatives. For example, treatment of cellulosic fibers with caustic solution, followed by a methylating agent, yields cellulose ethers substituted with one or more methoxy groups. If not further substituted with other alkyls, this cellulose derivative is known as methylcellulose.

An essential feature of the specific methylcellulose used in some embodiments of the present invention is the position of the methyl groups. The composition for delivery of the invention comprises a methylcellulose wherein hydroxy groups of anhydroglucose units are substituted with methyl groups such that s23/s26 is 0.36 or less, preferably 0.33 or less, more preferably 0.30 or less, most preferably 0.27 or less or 0.26 or less, and particularly 0.24 or less or 0.22 or less. Preferably s23/s26 is 0.08 or more, 0.10 or more, 0.12 or more, 0.14 or more, or 0.16 or more.

In the ratio s23/s26, s23 is the molar fraction of anhydroglucose units wherein only the two hydroxy groups in the 2- and 3 -positions of the anhydroglucose unit are substituted with methyl groups and s26 is the molar fraction of anhydroglucose units wherein only the two hydroxy groups in the 2- and 6-positions of the anhydroglucose unit are substituted with methyl groups. For determining the s23, the term "the molar fraction of

anhydroglucose units wherein only the two hydroxy groups in the 2- and 3-positions of the anhydroglucose unit are substituted with methyl groups" means that the two hydroxy groups in the 2- and 3-positions are substituted with methyl groups and the 6-positions are unsubstituted hydroxy groups. For determining the s26, the term "the molar fraction of anhydroglucose units wherein only the two hydroxy groups in the 2- and 6-positions of the anhydroglucose unit are substituted with methyl groups" means that the two hydroxy groups in the 2- and 6-positions are substituted with methyl groups and the 3-positions are unsubstituted hydroxy groups. Formula I below illustrates the numbering of the hydroxy groups in anhydroglucose

(Formula I) In one embodiment, hydroxy groups of anhydroglucose units are substituted with methyl groups such that the s23/s26 of the methylcellulose is 0.27 or less, preferably 0.26 or less, more preferably 0.24 or less or even 0.22 or less. In this embodiment, s23/s26 of the methylcellulose preferably is 0.08 or more, 0.10 or more, 0.12 or more, 0.14 or more, 0.16 or more, or 0.18 or more. A general procedure of making methylcelluloses of this embodiment is described in International Patent Applications WO 2013/059064, pages 11 - 12 and WO 2013/059065, pages 11 - 12.

In another embodiment, hydroxy groups of anhydroglucose units are substituted with methyl groups such that the s23/s26 of the methylcellulose is more than 0.27 and up to 0.36, preferably more than 0.27 and up to 0.33, and most preferably more than 0.27 and up to 0.30. Methylcelluloses wherein hydroxy groups of anhydroglucose units are substituted with methyl groups such that s23/s26 is about 0.29 are commercially available under the trade name METHOCEL™ SG or SGA (The Dow Chemical Company). They gel at a relatively low temperature, at 38 °C to 44 °C at a concentration of 2 wt. % in water. US Patent No. 6,235,893 teaches the preparation of methylcelluloses of which 1.5 wt. % solutions in water exhibit onset gelation temperatures of 31 - 54 °C, most of them exhibiting gelation temperatures of 35 - 45 °C.

The methylcellulose preferably has a DS(methyl) of from 1.55 to 2.25, more preferably from 1.65 to 2.20, and most preferably from 1.70 to 2.10. The degree of the methyl substitution, DS(methyl), also designated as DS(methoxyl), of a methylcellulose is the average number of OH groups substituted with methyl groups per anhydroglucose unit.

The determination of the % methoxyl in methylcellulose is carried out according to the United States Pharmacopeia (USP 34). The values obtained are % methoxyl. These are subsequently converted into degree of substitution (DS) for methyl substituents. Residual amounts of salt have been taken into account in the conversion.

The viscosity of the methylcellulose is generally at least 2.4 mPa»s, preferably at least 3 mPa»s, and most preferably at least 10 mPa»s, when measured as a 2 wt. % aqueous solution at 5 °C at a shear rate of 10 s ^"1 . The viscosity of the methylcellulose is preferably up to 10,000 mPa»s, more preferably up to 5000 mPa»s, and most preferably up to 2000 mPa»s, when measured as indicated above.

Cellulose ether polymers other than, or in addition, to methylcellulose can be used as carrier in some embodiments. In some embodiments, an ethylcellulose can be used instead of methylcellulose or in combination with methylcellulose. Examples of ethylcellulose that can be used in some embodiments are commercially available from The Dow Chemical Company under the ETHOCEL™ product name.

Carriers used in embodiments of the present invention can include other rheology modifiers. Such rheology modifiers can be provided in addition to cellulose ether polymers in some embodiments. In other embodiments, a cellulose ether polymer may not be present with such rheology modifiers. Such other rheology modifiers can be used as the carrier of the active antimicrobial agent in dry film layer and upon hydration, in the hydrogel or other viscous liquid. In general, the amount of rheology modifier can be determined using techniques known to those of skill in the art so as to prepare a composition that can form a dry film layer comprising an active antimicrobial agent, and to later form a hydrogel or other viscous liquid upon hydration. In some embodiments, the rheology modifier can comprise gelatin. In general, any gelatin that is approved for use in food applications can be used. Preferably, the gelatin exists as a gel at temperatures between 2° C and 12° C. Non-limiting examples of gelatins that can be used in some embodiments of the present invention include gelatin

commercially available from Sigma- Aldrich Co. The amount of gelatin that can be used in embodiments of the present invention is an amount adequate for the antimicrobial composition to form a dry film layer comprising an active antimicrobial agent, and to later form a hydrogel or other viscous liquid upon hydration.

Examples of other rheology modifiers that can be used in some embodiments of the present invention include pectin, xantham gum, guar gum, and others that persons of skill in the art can identify based on the teachings herein. The amount of such rheology modifiers can be selected so as to prepare a composition comprising an active antimicrobial agent that can form a dry film layer and that upon hydration, can be a hydrogel (or other viscous liquid) at temperatures between 2° C and 12° C.

As indicated above, the carrier can also comprise a plurality of rheology modifiers

(e.g., combinations of those described herein). The particular rheology modifiers and relative amounts can be selected so as to prepare a dry film layer comprising an active antimicrobial agent and upon hydration, a hydrogel (or other viscous liquid) at temperatures between 2° C and 12° C, and so as to avoid potential compatibility issues that might impact the performance of the dry film layer/hydrogel or viscous liquid and the safety of the product.

In addition to rheology modifier(s), the carrier, in some embodiments, can comprise other ingredients. Such ingredients can include, for example, antioxidants, surfactants, stabilizers, buffers, scavengers (e.g., odor, oxygen, moisture, etc.), slip aids, antiblocks, polymer processing aid, as well as others that persons of skill in the art can identify based on the disclosure herein. In some embodiments, solvents, such as glycol solvents (e.g., propylene glycol, or glycerol), can be included. Preferably, when solvents are included they are present in an amount no greater than 10 wt%, preferably no greater than 7 wt%, preferably no greater than 4 wt%, preferably no greater than 3 wt%, preferably no greater than 2 wt%.

The dry film layer can generally be prepared by mixing an aqueous solution comprising the active antimicrobial agent(s) with an aqueous solution comprising the rheology modifier. A thin film of the antimicrobial agent/rheology modifier solution can be cast on a substrate and then allowed to dry to form the dry film layer. A thin film layer comprising the active antimicrobial agent/rheology modifier can also be formed on the substrate using other techniques including, for example, casting (e.g., tape casting, slot die casting), spraying, coating (e.g., gravure or flexographic coating) or extruding (e.g., extrusion coating), or using techniques known to those of skill in the art based on the teachings herein. Other details regarding formation of the dry film layer on the substrate are discussed herein.

In some embodiments, materials suitable for packaging of the present invention can comprise a plurality of dry film layers with each additional dry film layer comprising an active antimicrobial agent and a carrier. The carrier in each dry film layer can hydrate upon contact with water as discussed above, in some embodiments. In some embodiments, each dry film layer can form a hydrogel or viscous liquid upon contact with water as discussed above. It should be understood that the different dry film layers may not hydrate and/or form a hydrogel/viscous liquid at the same time. For example, a dry film layer in contact with a food item or a wet environment is expected to hydrate and/or form a

hydrogel/viscous liquid before a more interior layer, though depending on the amount of moisture and/or the thickness of the layers, the time difference may not be substantial.

In general, the active antimicrobial agent and carrier can be any of those disclosed herein in connection with dry film layers for use in embodiments of the present invention. Each dry film layer in some such embodiments can comprise substantially the same composition. That is, in some embodiments, each dry film layer can comprise the same active antimicrobial agent and the same carrier in generally the same relative amounts. However, in other embodiments, different film layers may comprise different active antimicrobial agents, different concentrations of active antimicrobial agents, and/or different carriers.

In some such embodiments, each dry film layer can have a thickness of up to 0.7 mil, or up to 1.0 mil. In some embodiments, each dry film layer can have a thickness of 0.1 mil or greater, of 0.2 mil or greater, or of 0.3 mil or greater.

The dry film layers can be formed sequentially on a substrate in some embodiments. For example, a first dry film layer can be formed on a substrate as described herein. Once the first dry film layer is formed, a second dry film layer can be formed on the first dry film layer in the same manner. Likewise, a third dry film layer can be formed on the second dry film layer, and so on.

In materials suitable for packaging of the present invention, a dry film layer is adjacent to a substrate. In general, the substrate can be any material suitable for use in forming a food package. In typical embodiments of the present invention, the substrate will be a polymeric film. In general, any polymeric film known to those of skill in the art to be suitable for food packaging can be used. In various embodiments, the substrate can be a monolayer film or a multilayer film. In general, any monolayer or multilayer film known to those of skill in the art to be suitable for food packaging can be used. Non- limiting examples of polymers used in such films or film layers include polyethylene,

polypropylene, polyethylene terephthalate, polyamide, ethylene vinyl alcohol, and others.

A substrate for use in a material suitable for packaging of the present invention will typically have opposing facial surfaces. The dry film layer will be formed on one of those surfaces according to some embodiments of the present invention. For example, a substrate may be a sealant film or an inner layer or sealant layer of a multilayer film. In some embodiments, to facilitate adhesion, the surface of the substrate may be treated prior to application of the dry film. For example, the surface of the substrate can be corona treated to increase the surface energy of the substrate and to facilitate adhesion with the film layer comprising the active antimicrobial agent. The surface of the substrate can be corona treated or similarly modified using techniques known to those of skill in the art. In some embodiments, the dry film can also act as a sealant. In some embodiments, the dry film does not interfere with the sealing of the substrate (e.g., through a sealant layer in the substrate) to form a package. In some embodiments, the dry film may not fully cover the surface of the substrate such that a sealant layer in the substrate can permit sealing of the multilayer film to form a package.

When the substrate is a multilayer film, in some embodiments, the multilayer film can comprise an inner layer or sealant layer comprising a functional polymer. As part of an inner layer of the substrate, one or more functional polymers, in some embodiments, can facilitate adhesion between the substrate and the dry film layer comprising the active antimicrobial agent. Examples of film layers that can be used in some embodiments include ethylene vinyl acetate copolymers, ethylene butyl acrylates, ethylene ethyl acrylates, ethylene methyl methacrylates, ethylene methacrylic acid copolymers, maleic anhydride grafted copolymers, ethylene acrylic acid copolymers, ethylene vinyl acetate carbon monoxide terpolymers, ethylene methyl acrylate copolymers, ionomers of any of the foregoing, and combinations of any of the foregoing. Some of the foregoing functional polymers are commercially available from The Dow Chemical Company under the names PRIMACOR™ and AMPLIFY™, as well as from other sources. Such functional polymers can be incorporated into a surface layer of a multilayer film using techniques known to those of skill in the art. In some embodiments, the substrate comprises a nonwoven material or a molded tray. In some embodiments, the substrate comprises an absorbent pad such as those used in food package applications. For example, such absorbent pads can be designed to absorb water, food juices, grease, fat, or other liquids within a package. The dry film layer can be provided on a surface of such pads. In some embodiments, substrates such as molded trays or absorbent pads, for example, can comprise a plurality of microcapillaries within the tray or pad.

Some embodiments of the present invention relate to packages comprising a material of the present invention having a substrate and a dry film layer comprising an active antimicrobial agent and a water soluble carrier. The type and size of package can depend on the substrate that is used, the expected contents of the package, and other factors. Embodiments of the present invention are particularly well-suited for food packages, like barrier shrink bags and thermoformed bags, for example. In some embodiments, the material of the present invention having a substrate and a dry film layer can be sealed to form the package. In some embodiments, the package is hermetically sealed. Persons of skill in the art will recognize that materials of the present invention can be incorporated into any number of types, shapes, and sizes of food packages depending, for example, on the substrate, the food product to be provided in the package, and other factors. In a food package, the dry film layer comprising the active antimicrobial agent can be on an inner surface of the package. By being positioned on the inner surface of the package, moisture in the package or on the surface of the food product can hydrate the film layer and release the active antimicrobial agent as discussed further herein. Some embodiments of the present invention are directed to such food packages wherein the package comprises a meat product. In such embodiments, the film layer can be in contact with or near the meat product such that upon hydration of the film layer, the active antimicrobial agent is released onto the surface of the meat product. In some embodiments, rather than being incorporated into the food package itself, materials of the present invention can be provided as an insert in a food package (e.g., a pad in the bottom of a fresh or frozen chicken package).

Some embodiments of the invention will now be described in detail in the following Examples.

Examples

Preparation of Samples

The below Examples utilize the following active antimicrobial agents as specified in the particular Examples: cetylpyridinium chloride ("CPC") and lauric arginate ester ("LAE"). The below Examples utilize the following rheology modifiers in the carriers for the active antimicrobial agents as specified in the particular Examples: METHOCEL™ F4M, METHOCEL™ F15, METHOCEL™ E6, and a gelatin. METHOCEL™ F4M, METHOCEL™ F15, and METHOCEL™ E6 are cellulose ether polymers (methylcellulose) commercially available from The Dow Chemical Company. The gelatin is a gelatin derived from porcine skin having a bloom of 175 commercially available from Sigma- Aldrich Co. as catalog number G2625.

Aqueous solutions of the specified active antimicrobial agent are prepared by dissolving the active antimicrobial agent in deionized water and stirring to fully dissolve the agent. Aqueous solutions comprising an active antimicrobial agent and cellulose ether polymers are prepared by mixing a concentrated solution of the cellulose ether with the aqueous solution of active antimicrobial agent at the appropriate ratio to achieve the desired concentration.

In some of the examples, the rheology modifier is a cellulose ether polymer (commercially available from The Dow Chemical Company as METHOCEL™ F4M, METHOCEL™ F4M, or METHOCEL™ E6 as specified in the examples). Stock solutions of methylcellulose are prepared by dispersing the specified METHOCEL™ solid polymer in hot water (at a temperature of at least 80° C) while stirring to fully mix the polymer in the water. Stirring is then continued while the solution was cooled to 4° C. The solution is then stored overnight at 4° C to complete the polymer hydration. The antimicrobial

compositions are then prepared by mixing the methylcellulose stock solution with the specified active antimicrobial agents and water to achieve the desired concentrations. Dry film layers comprising an active antimicrobial agent in a cellulose ether carrier are prepared by casting a thin film layer of the above-described antimicrobial agent/cellulose ether solution on a corona treated, seven-layer polyethylene film substrate using a #5 wire wound draw down rod to produce an approximately 0.5 mil layer. The samples are allowed to dry at ambient conditions for at least 12 hours prior to use. In certain Examples, multiple layers of the antimicrobial agent/cellulose ether solution are cast in order to achieve a thicker film. The target thickness of each layer (as cast) in such Examples is 0.5 mil; the thickness of the dry film layer is less due to water evaporation during drying. When multiple layers are cast, each layer is allowed to fully dry before the next layer was added.

Gelatin gel samples are prepared by mixing the specified gelatin with hot water (-80° C) to solubilize the gelatin. The solution is cooled while stirring, and the active antimicrobial agent is added once the solution reaches room temperature (~20°C). Once thoroughly mixed, the solution is cooled overnight at 4°C to solidify the gelatin into a hydrogel for certain of the Examples. In other examples, dry film layers comprising an active antimicrobial agent in a gelatin carrier are prepared by casting thin films of the above-described antimicrobial agent/gelatin solution on a corona treated polyethylene film substrate after mixing the components and cooling to room temperature; a dry film layer is formed when water in the antimicrobial agent/gelatin carrier mixture evaporates.

Efficacy Testing

Efficacy of the formulations is tested by applying the treatment to the surface of chicken samples inoculated with bacteria, refrigerating the samples to allow for

antimicrobial activity, and quantification of the remaining bacteria. Chicken breast purchased from a local grocer is cut into 1.5 inch cubes. The chicken samples are rinsed with water but are not otherwise pretreated or decontaminated. A chicken sample is inoculated with bacteria by dispensing 0.5 mL of a liquid bacteria culture on the top face of the cube, allowing the excess to drip from the surface, and rotating the sample several times to ensure complete contact with the pooled bacteria culture. For the materials utilizing a dry film layer comprising the active antimicrobial agent, a piece of the film material is placed on the top face of the chicken sample and the combination is placed in a sealed plastic bag (having a zipper-style closure) and placed in a 4°C refrigerator for the overnight trial (approximately 18 hours). In each sample bag, an untreated control is also included that is covered with a piece of corona treated polyethylene with no active antimicrobial agent/carrier. Additional control samples of aqueous antimicrobial solutions are included in some of the studies as discussed below, and are prepared by dispensing 0.5 mL of the aqueous solution comprising an active antimicrobial agent on the surface of the chicken with the concentration selected to match the dose of active antimicrobial agent applied per surface area in samples treated with the material having the film layer. Each test condition is repeated in triplicate.

Antimicrobial efficacy is evaluated by determining the amount of bacteria on the treated surface of the chicken sample relative to the untreated control. The polyethylene film is removed from the chicken surface and the top portion of the chicken that is in contact with the film is sliced from the remainder of the untreated chicken. The chicken is then vortexed in 10 mL of sterile Peptone Buffered Water (PBW) cell culture media. PBW was purchased from Sigma Aldrich (catalog number 08105). The slurry is then diluted lOx in PBW media resulting in a uniform solution of the bacteria extract. The bacteria concentration in this solution is quantified by plating serial dilutions on selective plate media. Eosin Methylene Blue (EMB) agar purchased from Sigma Aldrich (catalog number 70186) or MacConkey agar purchased from Sigma Aldrich (catalog number M7408) are used as the agar plates for quantifying bacteria. The agar plates streaked with the bacteria dilutions are incubated overnight at 37 °C for E.Coli samples or 30°C for Pseudomonas Fluorescens samples, with no control of humidity. Following overnight incubation the number of bacteria colonies on each plate are counted to determine the number of Colony Forming Units (CFUs) in each sample. Colony counts are converted to bacteria concentration in the extract solution by factoring in the dilutions of the samples prior to spreading the extract on the agar plate (See Turano, A. Pirali, F. "Quantification Methods in Microbiology," Laboratory Diagnosis of Infection Disease, 1968, pp. 8-13). Any counts of less than 1 log bacteria are recorded as 1 log to account for limitations in obtaining accurate concentrations below this level.

Example 1

In this example, the chicken cube samples are inoculated with Pseudomonas Fluorescens culture at a concentration of about 10 ² CFU/mL. Films and chicken samples are prepared as described above. The films each comprise a dry film layer having the specified active antimicrobial agent in a carrier comprising METHOCEL™ F4M. Three sets of samples are prepared for analysis after 1, 2, and 3 days (each also in triplicate for analysis of reproducibility). Samples are analyzed at the desired time point as described above. An initial data point is collected by processing a set of three samples immediately after inoculation, with no treatment. The results are reported as the average and standard deviation of the three replicates for each sample. The results are shown in Table 1 and Figure 1. Table 1 - Example 1 Efficacy Results

This Example shows that a dry film layer comprising an active antimicrobial agent in an ethyl cellulose polymer carrier can provide a reduction in bacteria, or a slowed growth rate of the bacteria, over time. For the samples using CPC as the active antimicrobial agent, the sample with a low level of CPC (0.005mg/cm ² ) does not show a significant effect when compared to the control (no treatment), but the higher dose of 5 mg/cm ² does show an initial reduction in bacteria followed by a slower growth rate than the control. The sample containing 5 mg/cm ² LAE exhibits no detectable level of bacteria at 1, 2 or 3 days. The sample containing the higher level of LAE (20 mg/cm ² ) exhibits a decrease in bacteria over the time analyzed, but less than the lower dose. Visually, it was observed that the dry film layer does not completely dissolve as rapidly with the higher dose of active antimicrobial agent, such that the delayed solubilization of the active antimicrobial agent may have contributed to the slower bacteria reduction rate. This could be beneficial in applications where a controlled release is required. Example 2

In this Example, the chicken cube samples are inoculated with Pseudomonas Fluorescens culture at a concentration of aboutlO ² CFU/mL. Films and chicken samples are prepared as described above. This Example also includes an active antimicrobial agent (CPC) provided in an aqueous solution. The films each comprise a dry film layer having the specified active antimicrobial agent in a carrier comprising METHOCEL™ F4M. Two sets of samples are prepared for analysis after 1 and 3 days (each also in triplicate for analysis of reproducibility). Samples are analyzed at the desired time point as described above. An initial data point is collected by processing a set of three samples immediately after inoculation, with no treatment. The results are reported as the average and standard deviation of the three replicates for each sample as shown in Table 2 and Figure 2.

Table 2 - Example 2 Efficacy Results

This example demonstrates that when compared to the control with no treatment or the aqueous solution with CPC, the dry film layers comprising an active antimicrobial agent in an ethyl cellulose polymer carrier each result in a decreased rate of bacteria growth.

Example 3

In this Example, the chicken cube samples are inoculated with E.coli culture at a concentration of about 10 ⁴ CFU/mL. Films and chicken samples are prepared as described above. The chicken samples are treated with a variety of formulation approaches (aqueous, gel, or dry film layer) with either gelatin or cellulose ether polymer (two METHOCEL™ products are used in this Example), at a range of CPC doses. Film layers containing METHOCEL™ F4M are cast from solutions containing 1 % polymer; film layers containing METHOCEL™ F15 are cast from solutions containing 10% polymer; film layers containing gelatin are cast from solutions containing 1 % polymer. Gels are formed from gelatin containing 1% gelatin. The results are reported as the average and standard deviation of the three replicates for each sample as shown in Table 3.

Table 3 - Example 3 Efficacy Results

This Example evaluates the form of the treatment (aqueous solution vs. gel vs. dry film) and the level of active antimicrobial agent. The greatest reduction in bacteria is observed for the dry film samples containing 3 mg/cm ² of CPC, the gelatin gel containing 1.5 mg/cm ² CPC, and the viscous METHOCEL™ F15 polymer solution with a dose of 1.5 mg/cm ² CPC. The Example shows that there are multiple approaches to delivering the active antimicrobial agent.

Example 4

In this Example, the chicken cube samples are inoculated with E.coli culture at a concentration of about 10 ⁴ CFU/mL. Films and chicken samples are prepared as described above. Each of the films contains 0.54 mg/cm ² of METHOCEL™ F4M cellulose ether polymer. This Example also includes an active antimicrobial agent (CPC) provided in aqueous solutions. The results are reported as the average and standard deviation of the three replicates for each sample as shown in Table 4 and Figure 3.

Table 4 - Example 4 Efficacy Results

This example demonstrates the increased efficacy of the dry polymer film when compared to the aqueous application of the active antimicrobial agent.

Example 5 In this Example, the chicken cube samples are inoculated with Pseudomonas Fluorescens and E. coli culture at a concentration of aboutlO ³ CFU/mL. Films and chicken samples are prepared as described above. This example includes film samples containing varying amounts of vinegar powder as the antimicrobial active in METHOCEL™ E6 polymer. The films are prepared by casting aqueous solution containing 7.5%

METHOCEL™ E6 and dissolved vinegar powder. Each layer of film is cast to 0.5 mils thickness of aqueous solution. Films are prepared by casting 1, 2, or 3 layers of solutions, allowing each layer to dry before applying the next. The final concentration of vinegar powder in the dry films was 4, 8, or 12 mg/cm ² . The results are reported as the average and standard deviation of the three replicates for each sample as shown below in Table 5.

Table 5