WO2017158607A1

WO2017158607A1 - Antimicrobial compositions and use thereof

Info

Publication number: WO2017158607A1
Application number: PCT/IL2017/050336
Authority: WO
Inventors: Boaz Mizrahi
Original assignee: Technion Research & Development Foundation Ltd.
Priority date: 2016-03-17
Filing date: 2017-03-16
Publication date: 2017-09-21

Abstract

Compositions comprising crosslinked polymers comprising one or more volatile oils and uses same for e.g., food preservation is disclosed.

Description

ANTIMICROBIAL COMPOSITIONS AND USE THEREOF

FIELD OF THE INVENTION

The present invention, in some embodiments thereof, relates to polymeric compositions comprising one or more volatile oils, processes of preparing same, and uses thereof in, for example, reducing or preventing growth of microorganisms in packages.

BACKGROUND OF THE INVENTION

Food retailers and wholesalers want packing that prolongs the shelf life of the foods they market; consumers are demanding fresh, convenient and safe products. The spoilage of fresh foods each year all over the world incurs great economic losses and often threatens consumers' health. These concerns have led to a search for better systems for maintaining food quality as well as for more effective safety regulations.

Over the past decade, there has been an immense effort to develop anti-microbial packaging systems that can potentially improve food safety throughout the supply chain. Active packaging has been one of the most promising approaches to protect fresh food and increase its shelf life. These packages interact with the product or the headspace between the package and the food matrix to restrict the growth of microorganisms and slow other deterioration processes. As a result, food safety and quality is improved while shelf life is extended. Nevertheless, active food packages have several drawbacks including consumers' refusal to purchase food with synthetic additives, the useless addition of antibiotic agents, which may lead to increased development of anti-microbial-resistant mutants and taste bad.

Volatile oils such as essential oils (EOs) are natural substances, recognized as

GRAS (Generally Recognized as Safe), derived from plants that possess antimicrobial activity against a wide range of microorganisms, including bacteria, yeast and molds (Friedman, M., Journal of Agricultural and Food Chemistry 2014, 62 (31), 7652-7670). These compounds are commonly used as safe, effective, and natural remedies for diseases in traditional medicine.

US 2005/0220375 discloses packages with active agents, such that at least one active agent is associated with at least one of the body panels and includes a freshness- extension agent to inhibit spoilage of perishable product disposed in the package, an odor management agent to reduce, mask, or neutralize odors from garbage or waste disposed in the package. SUMMARY OF THE INVENTION

According to one aspect, the present invention provides a composition-of-matter comprising a polymer having incorporated within one or more volatile oils.

According to some embodiments, the one or more volatile oils are characterized by vapor pressure of at least about 0.001 mmHg at about 4-7 °C. In some embodiments, the one or more volatile oils are selected from the group consisting of oregano, carvacrol and thymol, lemon grass, or isomers, derivatives and mixtures thereof. In some embodiments, the one or more volatile oils have an antimicrobial effect.

According to some embodiments, said polymer has surface area of at least 0.1 to 4 m²/g. According to some embodiments, said polymer has surface area of 0.4 to 2 m²/g.

According to some embodiments, said polymer has a pore volume of 0.001 to 0.01 cm³/g.

According to some embodiments, at least 1% of said oil is non-covalently incorporated within said polymer. According to some embodiments, at least 1% of said oil does not undergo oxidization within said polymer for a pre-determined time.

According to some embodiments, wherein said polymer is an amphiphilic polymer.

According to some embodiments, wherein said polymer is a hydrophobic polymer.

According to some embodiments, wherein said polymer is a cross-linked polymer. According to some embodiments, the polymer is selected from the group consisting of polyacrylic acid, carbohydrate, polyallyl methacrylates, cellulose, starch, maltodextrin, and mixtures, copolymers or derivatives thereof. In some embodiments, the cellulose is selected from the group consisting of: microcrystalline cellulose and croscarmellose. According to some embodiments, the composition further comprises one or more adhesive materials. According to some embodiments, the composition further comprises one or more adhesive materials in an amount sufficient to adhere the polymer to a substrate.

According to some embodiments, the composition-of-matter is in a form of a patch or a tablet. In some embodiments, the patch or the tablet are in the form of a single layer. In some embodiments, the patch or the tablet are in the form of a double layer. According to some embodiments, the polymer and the oil are compressed, so as to form the patch or tablet disclosed herein. According to some embodiments, the single layer or double layer comprises at least one layer comprising one or more adhesive materials.

According to some embodiments, said composition comprises an antimicrobial effective amount of one or more volatile oils. According to some embodiments, said antimicrobial effective amount of one or more volatile oils is at least 1 % by total weight of said composition. According to some embodiments, a weight ratio of the oil and the polymer is in a range of between 1 to 100. According to some embodiments, a weight ratio of the oil and the polymer is in a range of between 1 to 60. According to some embodiments, a weight ratio of the oil and the polymer is in a range of between 5 to 60.

According to some embodiments, the composition-of-matter further comprises one or more agents selected from a flavoring agent, a binder, a lubricant or a combination thereof.

According to some embodiments, the composition-of-matter is for use preventing microbial growth. According to some embodiments, the composition-of-matter is identified for use for a preservative activity.

According to another aspect, the present invention provides an article comprising any one of the compositions disclosed herein. According to some embodiments, the article is selected from the group consisting of a food package, and medical device package.

According to another aspect, the present invention provides a method of inhibiting or reducing a formation of load of a microorganism and/or a formation of a biofilm within an article, the method comprising incorporating any one of the compositions disclosed herein.

According to some embodiments, the microorganism is selected from bacteria, molds and fungi. According to another aspect, the present invention provides a method for the preparation of an antimicrobial composition, the method comprising: (i) providing a polymer having a surface area of 0.1 to 4 m²/g; (ii) providing an antimicrobial effective amount of one or more volatile oils; and (iii) saturating said polymer with the antimicrobial effective amount of one or more volatile oils.

According to some embodiments, the method further comprises a step of pressing the saturated polymer under a pressure of at least 1 ton.

Other features and advantages of the present invention will become apparent from the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced.

In the drawings:

FIG. 1 presents a schematic illustration of a fabrication process of a tablet or a patch according to some embodiments of the invention;

FIGs. 2A-B present images demonstrating the texture of the patch and the dimensions thereof (FIG. 2A) and patches being adhered to a food package (FIG. 2B).

FIGs. 3A-B are bar graphs showing the absorption percentage of carvacrol oil (FIG. 3A) or lemon grass oil (FIG. 3B) in various tested SAPs.

FIGs. 4A-B present the relative fumes concentration curves over time at 25°C (FIG. 4A) and 4°C (FIG. 4B).

FIGs. 5A-C are bar graphs showing the bacterial cell counts upon inserting the patches within packages of various sandwiches (FIG. 5A, 5B, 5C) (vis-a-vis a control bar of a package without patches).

FIG. 6 is a bar graph showing the coliform miloda cell counts of Tilapia fish packages upon inserting patches with various oils after 10 days at 7 °C (vis-a-vis a control bar of a package without patches). DETAILED DESCRIPTION OF THE INVENTION

The present invention, in some embodiments thereof, relates to a polymer saturated with one or more volatile oils, processes of preparing same, and uses thereof for, e.g., reducing or preventing growth of microorganisms, and/or preserving fresh food.

Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways.

In one embodiment, the present invention provides compositions comprising absorbent polymer (also referred to herein as "polymer structure") absorbed with one or more anti-microbial volatile oils. The invention further provides non-covalent incorporation of the oil into the polymer structure. Advantageously, the polymer shows prolonged and efficient bacteriocidal, as well as fungicidal activity.

Compositions-of-matter:

According to an aspect of some embodiments of the present invention there is provided a composition-of-matter comprising one or more volatile oils absorbed within an absorbent polymeric structure.

The term "polymer" refers to a molecule (or a material composed of such molecules) of high relative molecular mass, the structure of which essentially comprises the multiple repetition of units derived, actually or conceptually, from molecules of low relative molecular mass, also termed: monomeric units. In some embodiments, the substrate comprises a combination (e.g., blend) of one or more polymers.

In some embodiments, the polymeric structure counts on superabsorbent polymers (SAPs). As used herein, SAPs are of a porous structure. In some embodiments, the porous structure is characterized by a low density, which gives the polymer the ability to absorb and release their volatile content in a controlled pattern. In some embodiments, these cross- linked polymers are also characterized with their capacity and ability to absorb large amounts of solvent and stay solid and stable.

By "large amounts" it is meant to refer to a capability to absorb and "trap" liquids

(e.g., the volatile oils of the invention) up to e.g., ten times of the polymer weight. In some embodiments, the term "liquid" as used herein may refer to hydrophilic solutions. In some embodiments, the term "liquid" as used herein may refer to hydrophilic and hydrophobic solutions. In some embodiments, at least 20%, 30%, 50%, 60%, 70%, 80%, 90% or 100% of the polymer is absorbed with one or more volatile and antimicrobial oils.

In some exemplary embodiments, the polymer or absorbent polymeric structure is characterized by internal porous structure and/or large BET (Brunauer, Emmett and Teller) surface area. In one embodiment, the absorbent polymeric structure has a BET surface area of 0.1 to 5 m²/g. In another embodiment, the absorbent polymeric structure has a BET surface area of 0.4 to 2 m²/g.

In another embodiment, the absorbent polymeric structure has a BET surface area of at least 0.1 m²/g. In another embodiment, the absorbent polymeric structure has a BET surface area of at least 0.2 m²/g. In another embodiment, the absorbent polymeric structure has a BET surface area of at least 0.3 m²/g. In another embodiment, the absorbent polymeric structure has a BET surface area of at least 0.4 m²/g. In another embodiment, the absorbent polymeric structure has a BET surface area of at least 0.5 m²/g.

In another embodiment, the absorbent polymeric structure has a BET surface area of at most 5 m²/g. In another embodiment, the absorbent polymeric structure has a BET surface area of at most 4 m²/g. In another embodiment, the absorbent polymeric structure has a BET surface area of at most 3 m²/g. In another embodiment, the absorbent polymeric structure has a BET surface area of at most 2 m²/g.

For purposes of this invention, the BET surface area measurement can be made as known to one skilled in the art. A variety of commercially available devices are known and useful for measuring BET surface area including a Micromeritics TRISTAE. 3000 device and a Quantachrome Monosorb tester. Samples are suitably outgassed prior to making the measurements, with suitable conditions such as 200°C at atmospheric pressure. An average of multiple data points can be used to determine the BET value.

One skilled in the art is capable of providing a polymeric structure with a defined surface area and/or porosity so as to absorb higher volumes of volatile oils.

In some embodiments, the absorbent polymeric structure is characterized by a pore volume of 0.001 to 0.01cm³/g. In some embodiments, the absorbent polymeric structure is characterized by a pore volume of at least 0.001 cm³/g. In some embodiments, the absorbent polymeric structure is characterized by a pore volume of at most 0.01cm³/g.

Herein, the term "porosity" refers to a pore volume of a substance (e.g., a "spongelike" material) which consists of voids. In another embodiment, porosity is measured according to voids within the surface area divided to the entire surface area (BET) (porous and non-porous).

In some embodiments, the porous structure of the disclosed polymers allows absorbing oil efficiently on the polymer surface. Without being bound by any particular theory, this surprising discovery can be explained in view of the disclosed polymer structure and its porosity.

In some embodiments, the absorbent polymeric structure comprises an amphiphilic polymer. An amphiphilic compound is one that has both a hydrophobic and hydrophilic segment. Thus, it comprises both a polar water soluble group (hydrophilic segment) and a nonpolar water insoluble group (hydrophobic segment). In some embodiments, the amphiphilic polymer comprises a hydrophobic domain and a hydrophilic domain. In some embodiments, the amphiphilic polymers comprise a higher ratio of hydrophobic to hydrophilic domains, thereby providing a relatively more oil soluble structure.

Typically, but not exclusively, the term "hydrophobic domain" is understood to mean blocks which are soluble or dispersible in fatty substances which are liquid at ambient temperature (e.g., 25 °C) or oils.

In some embodiments, the absorbent polymeric structure comprises a hydrophobic polymer.

In some embodiments, the polymer of the invention is a non-ionic polymer. In some embodiments, the polymer of the invention is an ionic polymer. In some embodiments, the polymer of the invention is a cross-linked polymer. The cross-linked polymer is, in some embodiments, composed of polymeric backbone and cross linkers.

In the context of the present invention, the term "polymeric backbone" refers to the main chains of polymeric skeleton together with chain branches projecting from the polymeric skeleton. The "crosslinked polymer" refers generally to a polymer which comprises the monomeric units, including the crosslinking bridges. According to some embodiments, the plurality of crosslinked polymeric backbones is prepared by co-polymerizing a plurality of the monomeric units.

As used herein, the phrase "co-polymer" refers to a polymer of at least two chemically distinct monomers.

According to some embodiments, the polymer or composition-of-matter is in form of dry powder. The polymer strands may be chemically crosslinked to various extents between groups in the backbone or side-chains, giving rise to a variety of properties (e.g., mechanical properties) of the polymer.

In some embodiments, the cross-linking is of covalent nature, i.e., covalent cross- linking, e.g., via a cross linking agent.

Herein, "covalent cross-linking" (also referred to herein as "chemical cross- linking") refers to a formation of a covalent bond ("cross-link") between two or more polymeric backbones (chains). A polymeric backbone may be attached to a plurality of other polymeric backbones, each other polymeric backbone being attached by a different covalent bond. Thus, a plurality of polymeric backbones (e.g., at least 5, at least 10, at least 20, at least 50, at least 100, at least 500, or at least 1000) may be linked together.

As used herein, the phrase "crosslinking agent" refers to a substance that promotes or regulates intermolecular covalent, ionic, hydrogen or other form of bonding between polymeric chains, linking them together to create a network of polymeric chains which result in increasing the rigidity of the structure. Crosslinking agents typically exhibit one or more, preferably two or more, bonding functionalities, for example, two double (vinyl) bonds (a functionality of four, or tetrafunctionality), three amines (a functionality of three, or trifunctionality), creating chemical bonds between two or more polymer chains. In some embodiments, the "crosslinking agent" may comprise one or more monomeric units having a same or similar chemical structure as the monomeric units in the backbone.

Comonomers constitute a type of crosslinking agents which are contemplated in some embodiments of the present invention. A comonomer is a monomer having at least three bonding functionalities (trifunctionality), which is incorporated into the backbone of a growing polymer in the course of the polymerization process. While two of its functionalities are used to form the polymeric backbone, the third (and more) functionality is free to form crosslinks with counterparts in other polymeric chains. In general, comonomers are used to generate crosslinking homogeneously along the polymeric chain. It is noted herein that the term "comonomer" is meant to encompass oligomers and short polymers, having at least three bonding functionalities, which can be incorporated into a growing polymer during the polymerization process.

In general, the crosslinking agent is selected according to the chemistry and polymerizing conditions used to form the polymeric backbone of the polymer. Non- limiting examples of crosslinking agents, according to some embodiments of the present invention, include ethylene glycol dimethylacrylate (EGDMA), poly(ethylene glycol) dimethacrylate (also known as polyethyleneoxide dimethacrylate, poly-EGDMA, nEGDMA or PEOdMA), Ν,Ν'-methylenebisacrylamide (MBA or MB Am), Ν,Ν'- methylenebis(2-methylacrylamide), methylene diacrylate, methylene bis(2- methylacrylate), diethylene glycol diacrylate, hexamethylene diacrylate, oxybis(methylene) bis(2-methylacrylate), oxybis(ethane-2,l-diyl) bis(2-methylacrylate), and glutaraldehyde.

In some embodiments, at least 0.01%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, or 50% of the polymer is cross-linked. In some embodiments, at most 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.1% of the polymer is cross-linked.

In some embodiments, about e.g., 0.01%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50% 60%, 70%, or 80% of the polymer is cross-linked with the cross-linking agent. Herein, the percent (%) refers to the number of monomeric units (of a polymeric backbone) having attached thereto a crosslink bond divided by the total number of the monomeric units of the polymeric backbone.

Typically, but not exclusively, the "degree of crosslinking" may be correlated to mechanical parameters which can be measured by several experimental methods, e.g., modulus. Other parameters can be the crosslink density (mol/cm³), the network chain density (mol/cm³) and the network chain molecular weight (gram/mol).

Thus, the composition, according to some embodiments of the present invention, can be characterized by the molar percent of the crosslinking agent used in its preparation, a measure that typically correlates with the degree of crosshnking. In the context of some embodiment of the present invention, the degree of crosshnking is defined as the molar ratio in percent's, or molar percentage, of the crosshnking agent relative to the polymeric backbone. In other words, in cases where the molar amount of monomeric unit in the polymeric backbone is 100 and the molar amount of the crosshnking agent is 2, the degree of crosshnking is 2 %. According to some of any of the embodiments of the present invention, the molar percentage of the crosshnking agent ranges from 0.00001 % to 50 % per polymer.

In some embodiments, the range of molar percentage of the crosshnking agent ranges from 0.01 % to 25 %, from 0.2 % to 25 %, from 1 % to 20 % or from 1 % to 15 %, including any subranges and intermediate values therebetween.

According to some of any of the embodiments described herein, various ranges and subranges of the molar percentage of the crosshnking agent are used in combination with various polymers, and various other parameters, as these are defined herein, and embodiments of the present invention encompass all of such combination. In some embodiments, the degree of crosshnking is selected according to the polymeric type and/or polymeric size, to maximize the capacity of the polymeric structure to incorporating the oil therein.

The polymerizable groups and polymerization according to any one of the embodiments described in this section may be used in the context of any one of the embodiments of any of the aspects of the inventions described herein, and may be incorporated within a polymer according to any one of the respective embodiments described herein.

The present inventors have designed and successfully prepared and practiced a composition which is characterized by a scaffold form.

As used herein, the term "scaffold" describes a two-dimensional or a three- dimensional supporting framework. The scaffold according to some embodiments of the present invention is composed of monomeric units which are cross-linked therebetween. The scaffold may further comprise compounds (e.g., oils) which are contained within the scaffold, without being cross -linked to the aforementioned monomeric units. In the context of the present invention, the amount of the volatile oil is governed, inter alia, by the chemical composition of the polymer chains, the degree of crosslinking, (number of interconnected links between the chains), the volatility of the oil, and temperature. In some embodiments, the cross-linked polymer is reinforced with other fibrous material to form a composite structure, which exhibits higher capability to absorb the oil.

In some embodiments, about e.g., 0.01%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of the polymer is incorporated with one or more volatile oils.

The architecture of the polymer affects the performance of the packaging for preservation of food; that is, performance depends not only on the active agent (e.g., volatile oil) used, but where the active agents are placed in the polymer in relation to the food been preserved.

In some embodiments, scaling, or selecting, the proper amounts of active agents in the polymer is employed according to the amount of the polymer, as well as the type of food product being packaged. For example, if a polymeric-made absorbent patch architecture employs too much absorbent material relative to the amount of the food, then it might be insufficient to bring them together to initiate anti-microbial activity in a prolong manner. In addition, active agents placed in the polymer can themselves reduce the absorbency of the polymer. Therefore, scaling the amounts and ratios of active agents in relation to the amount of the polymer for the food packaging has a beneficial effect on preserving foods by the patches of this disclosure.

In some embodiments, the composition is characterized by the release of one or more volatile oils over a prolonged period of time. In some embodiments, the composition is characterized by retention of a portion of the volatile oil(s) within the polymeric structure for a prolonged period of time, such as at least 1, 2, 3, 4, 5, or 6 weeks.

In some embodiments, the composition is characterized by retention within a package on a surface thereof for a prolonged period of time. The term "prolonged period", or "long-lasting" is meant to refer to a time sufficient to allow preventing inhibiting or reducing or retarding the formation of load of a microorganism within the package and means anywhere from at least 30 minutes to one week or longer. Suitable non-limiting exemplary polymers used in the present subject matter include amphiphilic polymers.

Suitable non-limiting examples of cellulose polymers used in the present subject matter include cellulose ethers, such as hydroxyethyl cellulose, hydroxypropyl cellulose, microcrystalline cellulose, methylcellulose, carboxymethyl cellulose, sodium carboxymethylcellulose, methylhydroxyethyl cellulose, methylhydroxypropyl cellulose, hydroxypropylmethyl cellulose, hydroxyethylcarboxymethyl cellulose, carboxymethyl cellulose, carboxymethylhydroxyethyl cellulose, and mixtures thereof.

In some exemplary embodiments, the polymer is characterized by internal porous structure and/or large surface area BET e.g., microcrystalline cellulose (MCC). MCC, as a non-limiting example, is characterized by its internal porous structure and its large surface area. When MCC is dried via one of the drying methods; freeze-drying, spray drying, drum drying, oven drying, different kinds of MCC spongy powders are produced which differ from one another in their capacity.

In some embodiments, the polymer can also be compressed without losing its oil absorbing capabilities.

In some embodiments, the polymer is a synthetic polymer. The phrase "synthetic polymer" refers to polymers that are not found in nature, even if the polymers are made from naturally occurring biomaterials. Examples include, but are not limited to, aliphatic polyesters, poly(amino acids), copoly(ether-esters), polyalkylenes, oxalates, polyamides, tyrosine derived polycarbonates, poly(iminocarbonates), polyorthoesters, polyoxaesters, polyamidoesters, polyoxaesters containing amine groups, poly(anhydrides), polyphosphazenes, and combinations thereof.

Suitable synthetic polymers for use by the invention can also include biosynthetic polymers based on sequences found in naturally occurring proteins such as those of collagen, elastin, thrombin, fibronectin, or mutant or synthetic derivatives thereof or, starches, poly(amino acids), poly(propylene fumarate), gelatin, alginate, pectin, fibrin, oxidized cellulose, chitin, chitosan, tropoelastin, hyaluronic acid, polyethylene, polyethylene terephthalate, poly(tetrafluoroethylene), polycarbonate, polypropylene and poly(vinyl alcohol), ribonucleic acids, deoxyribonucleic acids, polypeptides, proteins, polysaccharides, polynucleotides and combinations thereof. In some embodiments, the polymer is a natural polymer. The phrase "natural polymer" refers to polymers that are naturally occurring. Non-limiting examples of such polymers include, silk, collagen-based materials, chitosan, hyaluronic acid, albumin, fibrinogen, and alginate.

In some embodiments, the polymer is a biocompatible polymer. The phrase

"biocompatible polymer" refers to any polymer (synthetic or natural) which when in contact with cells, tissues or body fluid of an organism does not induce adverse effects such as immunological reactions and/or rejections, cellular death, and the like. A biocompatible polymer can also be a biodegradable polymer. Non-limiting examples of biocompatible polymers include polyesters (PE), PCL, PLA, PGA, PEG, polyvinyl alcohol, polyvinyl pyrrolidone, polytetrafluoroethylene (PTFE, teflon), polypropylene (PP), polyvinylchloride (PVC), polymethylmethacrylate (PMMA), polyamides, segmented polyurethane, polycarbonate -urethane and thermoplastic polyether urethane, silicone- polyether-urethane, silicone -polycarbonate -urethane collagen, PEG-DMA, alginate, hydroxyapatite and chitosan, blends and copolymers thereof.

In another embodiment, the polymer is selected from: Allyl Methacrylates Crosspolymer (e.g., Polypore E200®, starch, cross-linked polyacrylic acid, and cross- linked cellulose (Crosscarmellose Sodium).

As known in the art, "croscarmellose (CMC)" is a derivative of carboxymethylcellulose sodium, and it's a cross-link polymer. CMC may be used to create an enteric coating for tablets, after blending with other polymers to create a dusty powder which is used then to form the tablet's coating.

In another exemplary embodiment, the polymer is poly( acrylic acid) (PAA).

As known in the art, PAA is a cross-linked anionic molecule with a high charged density. In aqueous solutions, PAA is able to absorb water and swell considerably, in addition to its ability of being a good stabilizer and emulsifier for oil/water interface.

A non-limiting example of a derivative of cross-linked PAA is Carbopol®. Carbopol® is known in the art as a polymer which is transferred from a liquid to a gel in high levels of pH, i.e., the gel's viscosity increases as the pH is raised. Hence, the polymer can be used to create matrix that contains oil. In still another exemplary embodiment, the polymer is starch. Starch is known in the art as a carbohydrate, composed of repetitive units of glucose being linked together by glycosidic bonds. In some embodiments, the starch is grafted i.e. consists of two molecules: amylase, and amylopectin. In some embodiments, the amylase is linear. In some embodiments, the amylopectin is branched. In some embodiments, the starch comprises 10-20% linear amylose, and 80-90% branched amylopectin. Comparing with the native starch, the grafted starch is more stable that's where its degradation temperature is lower with greater absorption ability at a neutral pH. Exemplary starches are, for example and without limitation, National Starch®, which is a modified corn starch and Eliane MD2®, which is a modified amylopectin potato starch which is composed of more than 99% amylopectin and less than 1% Amylose.

In some embodiments, the polymer used are both cross-linked and amphiphilic, which gives them the ability to absorb and release their oil content in a controlled release manner. The amounts of anti-bacterial oil and loading degree can be calculated by methods known in the art, e.g., gravimetrically. The term controlled release manner refers to the release of an active agent (e.g., oil) substance from the composition for the prolonged period of time.

In some embodiments, the polymer comprises (e.g., absorbed with) an oil. In some embodiments, the polymer comprising the oil is mixed with other patch-forming excipients. In some embodiments, the polymer is compressed by means known in the art e.g., by a laboratory-tableting machine including but not limited to laboratory Carver press (Carver Machine Works, Inc., Washington, NC). Reference is now made to Figure 1 which presents a schematic illustration of the fabrication process of a tablet or a patch. In some embodiments, the fabrication process includes a pressing step under a pressure of about 1- 10 tons, for at least 0.1 sec, 0.5 sec, or at least 1 second. As exemplified herein, the fabrication process includes a pressing step under a pressure of about 2 tons for at least 30 seconds.

The compositions can be in form of a single layer, double layer, or multilayer patch, which can be prepared using conventional methods known in the art, such as by compressing the patch. In one embodiment, the tablet or patch is a single layer table. In another embodiment, the tablet is a double layer tablet. In one embodiment, the composition is a one or double layer adhesive tablet or patch. The tablet may contain an acceptable plasticizer for the adhesive material.

Hereinthroughout adhesive tablet or patch are also referred to as "sticker".

As used herein, the term "patch" refers to the disclosed composition having any structural configuration of any shape, contour, depth, and dimension. A patch may have a uniform or nonuniform thickness, may be constructed of multiple components or singularly constructed. A patch may be configured to conform to an underlying structure. The total mass of the patch generally ranges from e.g., 50 mg to 100 g, depending on desired performance attributes such as the food and package size.

The patch can be of any suitable size for placement in a package (e.g., food package). In one embodiment, the surface area of the patch is from e.g., about 0.4 cm² to about 10 cm². In some embodiments, the patch has a suitable geometry for placement on the desired surface in the package's surface. For example, for placement on the package's surface, the patch may contain a compatible surface (e.g., convex, concave, etc.) designed to be placed adjacent to and adhere to the package's surface. In a double layer patch, this convex surface corresponds with the outer surface of the adhesive layer, as described hereinbelow, of the patch. Patches are typically round or oval, with a diameter up to e.g., 10 cm.

Reference is now made to Figures 2A-B which present images demonstrating the texture of the patch and the dimensions thereof (Figure 2A) and patches being adhered to a food package (Figure 2B).

The term "tablet" refers to the composition as disclosed herein, being in the form of a small, essentially solid pellet of any shape. Tablet shapes may be cylindrical, spherical, rectangular, capsular or irregular.

As used herein, the term "adhesive" refers to a substance, e.g., adhesive polymer, which can be utilized to join two surfaces.

The term "adhesive polymer" used herein means a polymer, which exhibits adhesion and can be used as a pressure sensitive adhesive.

In some embodiments, adhesive polymer exhibits adhesion at a defined range of temperature, e.g., room temperature. Polyacrylate, polyurethane, polyolefin, polyester, and the like, without being limited thereto, can be used as an adhesive polymer. A tackifier may be used in combination with the adhesive polymer, as in the case of conventional pressure sensitive adhesives. The adhesive polymer may be a polymer, which can be cured with heat or radiation after the uneven structure of the adhesive layer deforms and the appearance of the adhesive sheet is improved insofar as the preservative effects of the present invention are not impaired.

Suitable crosslinking agents include without limitation, an isocyanate compound, a melamine compound, a poly(meth)acrylate compound, an epoxy compound, an amide compound, and the like.

In either embodiment, the thickness of the adhesive layer is not limited insofar as the preservative effects of the present invention are not impaired.

In some embodiments, the polymer disclosed herein is further characterized as being an adhesive polymer. In some embodiments, the polymer disclosed herein further comprises an adhesive additive. In some embodiments, an adhesive additive is incorporated within the polymer.

In some embodiments, the composition of the invention further comprises a binder. In some embodiments, the concentration of the binder ranges from e.g., about 0.01% to about 20% by weight, of the composition. As exemplified herein, polyvinylpyrrolidone (PVP) is used as a binder, such as at a concentration of 10% by weight.

Binders impart cohesiveness to tablet formulation and ensures that the tablet remain intact after compression. Binders which may be used according to the invention include, but are not limited to hydroxy propyl methyl cellulose, hydroxy propyl cellulose, hydroxy ethyl cellulose, ethyl cellulose, cellulose derivatives, maize starch, polyvinylpyrrolidone alone or in combination with polyethylene glycols and the like. Binders may be used in the range of about 5% to about 30% by weight of total composition. Preferred binder being ethyl cellulose and polyvinylpyrrolidone. One or more binders may be used for preparing the wet granulation solution.

In some embodiments, the composition of the invention further comprises a lubricant. As exemplified herein, magnesium stearate is used as a lubricant, such as at a concentration of 1 % by weight. One skilled in the art will appreciate that the addition of a lubricant is advantageous for various manufacturing processes including pressing techniques. Lubricants that may be used as per the invention include, but are not limited to stearic acid, calcium stearate, glyceryl monostearate, glyceryl palmitostearate, zinc stearate, magnesium stearate, sodium stearyl fumarate, calcium stearyl fumarate, hydrogenated vegetable oil, mineral oil, polyethylene glycol, sodium lauryl sulphate and the like. Glidants which may be used include colloidal silicon dioxide, talc and the like. Lubricants and glidants may be used in the range of about 0.1% to about 5% by weight of total composition.

Flavoring agents may be present in the patch at e.g., 0.1% to 5% by weight, depending on the specific flavor and desired attributes of the specific food being packaged. Suitable flavoring agents include, but are limited to, oil of wintergreen, oil of peppermint, oil of spearmint, clove bud oil, menthol, anethole, methyl salicylate, eucalyptol, cassia, 1- menthyl acetate, sage, eugenol, parsley oil, oxanone, alpha-irisone, marjoram, lemon, orange, propenyl guaethol, cinnamon, vanillin, thymol, linalool, cinnamaldehyde glycerol acetal known as CGA, and combinations thereof.

In some embodiments, the concentration of the flavoring agent ranges from e.g., about 0.001% to about 10% by weight, of the composition.

Oils:

In some embodiments, the polymer absorbs oil in an amount of up to e.g., at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40 at least 45, at least 50, at least 55, at least 60, times of the polymeric weight.

The terms "absorb", "load", and any grammatical derivatives thereof, are used hereinthroughout interchangeably and are meant to refer to the incorporation of the oil within the polymeric composition as disclosed hereinthroughout.

In some embodiments, the polymeric structure (e.g., surface area and/or porosity) correlates to the concentration of the oil within the patch.

In some embodiments, the polymeric properties (e.g., being inert to the oil) correlates to the oil release concentration from the patch.

In some embodiments, the volatile oils of the invention are anti-microbial oils. In some embodiments, the composition of the invention is substantially devoid (e.g., less than 1%) of oil which does not have anti-microbial properties. It will be understood to one skilled in the art that oil which does not have anti-microbial properties may compete and disturb with the activity and/or efficacy of the antimicrobial volatile oil of the invention.

In some embodiments, as will be understood to one skilled in the art, a mixture of volatile oils can be more efficacious when each volatile oil has an ability to inhibit (prevent), reduce or retard different types of bacterial growth, fungal, yeast and/or mold growth. In some embodiments, the molar percent of the crosslinking bonding within the composition correlates to the concentration of the oil within the patch.

As used herein, the terms "volatile oil", "essential oil", or, for simplicity, "oil", which are used hereinthroughout interchangeably, mean a volatile oil derived from, for example and without limitation, leaves, stem, flower or twigs of plants or synthetically- made compounds that have the same chemical attributes. Chemically, the plant essential oil or derivative thereof, which may be extracted from natural sources or synthetically made, typically comprises, without limitation, an acyclic monoterpene alcohol or aldehyde, a benzenoid aromatic compound comprising at least one oxygenated substituent or side chain, or a monocarbocyclic terpene generally having a six-membered ring bearing one or more oxygenated substituents. The term "volatile oil" is also meant to include derivatives thereof, including racemic mixtures, enantiomers, diastereomers, hydrates, salts, solvates, metabolites, analogs, and homologs. The term "volatile oil" also encompasses variants or mimics of such compounds that share one or more of their characteristics or functions.

Volatile oils can be pure single compounds. However, other volatile oils are mixtures of compounds, for example turpentine oil (pinene and dipentene). Other volatile oils, their chemistry and plant families are known in the art. When the volatile oil is a mixture of compounds, the present invention encompasses each of the constituent compounds of the essential oil.

As plant-volatile oil compounds are known and used for other uses, they may be routinely prepared by a skilled artisan by employing known methods. Exemplary methods for deriving an essential oil include, but are not limited to, steam distillation, pressing fruit rinds, solvent extraction, macerating the flowers and leaves in fat and treating the fat with solvent, enfleurage and synthetically.

Exemplary volatile oils or their constituents include, but are not limited to, eucalyptus oil, geranium oil, lemongrass oil, petitgrain oil, rosemary oil, thyme oil (white and red), lavender oil, tea tree oil, Tagete minuta oil, lovage oil, Lippia javanica oil, lemon oil, orange oil, grapefruit oil, oil of bergamot, galbanun oil, acetophenone, allyl caprate, a- amylcinnamic aldehyde, amyl salicylate, trans-anethole, anisaldehyde, benzyl alcohol, benzyl acetate, benzyl propionate, bomeol, β-caryophyllene, caryophyllene, cinnamyl acetate, cinnamaldehyde, cinnamic alcohol, cinnamyl alcohol, carvacrol, carveol, citral, citronellal, citronellol, cumin aldehyde, cyclamen aldehyde, almond (Benzaldehyde). decanol, dimethyl salicylate, ethyl butyrate, ethyl caprate, ethyl cinnamate, eucalyptol (cineole), eugenol, iso-eugenol, galaxolide, geranial, geraniol, germacrene D, guaiacol, hexenol, a-hexylcinnamic aldehyde, hydroxycitrolnellal, ionone, ipsdienone, isopropenyl acetophenone, linalol, linalyl acetate, d-limonene, menthol, p- methylacetophenone, methyl anthranilate, methyl dihydrojasmonate, methyl eugenol, methyl ionone, methyl salicylate, neral, a-phellandrene, pennyroyal oil, perillaldehyde, 1- or 2-phenyl ethyl alcohol, 1- or 2- phenyl ethyl propionate, piperonal, piperitenone, piperonyl acetate, piperonyl alcohol, o- isopropenyl anisole, D-pulegone, terpinen-4-ol, terpinyl acetate, A-tert- butylcyclohexyl acetate, a-terpineol, thymol, trans-tagetenone, myrcenone, linalool, octyl gallate carvone, ipsenone, a-phellandrene, piperitenone, gamma-undecalactone, undecenal, vanillin, vitamin E, and ethyl vanillin.

In exemplary embodiments, the volatile oil is selected from carvacrol, thymol, oregano, lemon grass oil, or a combination thereof.

In some embodiments, the one or more volatile oils act in synergy.

The term "synergy" and any grammatical derivative thereof, as used herein, describe a cooperative action encountered in combinations of two or more biologically active compounds in which the combined effect exhibited by the two or more compounds when used together exceeds the sum of the effect of each of the compounds when used alone. "Synergy" is therefore often determined when a value representing an effect of a combination of two or more volatile oils is greater than the sum of the same values obtained for each of these agents when acting alone.

Typically, but not exclusively, the term "volatile" in the context of oils is meant to refer to having a low melting point i.e. less than about 70 °C. It is therefore noteworthy that in the room temperature (i.e. about 25 °C) some of the oils may be in a solid state (e.g., thymol) and some in a liquid (e.g., carvacrol). Additionally, or alternatively, the term "volatile" refers to materials that are liquid under ambient conditions and have a vapor pressure as measured at 4-25° C, or 4-7° C of e.g., at least about 0.001 mmHg, or, in some embodiments, from about 2.0 mmHg to about 6.0 mmHg, or, in some embodiments, from about 0.002 mmHg to about 2.0 mmHg.

In some embodiments, the volatile oils are characterized by vapor pressure of at least 0.001 mmHg, about 0.002 mmHg to about 2.0 mmHg at about 4-7 ° C.

Exemplary vapor pressure values (at 20° C -25 °C) of oils used in the context of the present disclosure are: carvacrol: 0.023 mmHg, and Thymol: 0.002 mmHg.

Additionally, or alternatively, the term "volatile" refers to materials having a boiling point at one atmosphere of pressure (1 atm) of less than 250° C.

Since the oils are very distinct in their fragrance, and therefore, each may be selected for a different type of food according to effectiveness and customer preferences (e.g., citrus oil for fruits and carvacrol oil for meat and vegetables).

In exemplary embodiments, the oil(s) may be analyzed by one or more methods known in the art, e.g., GC-MS and/or by UV-Vis before and after loading thereto in the polymer to verify the chemical integrity or possible damage to the polymeric structure during loading.

Microorganisms :

According to another aspect of some embodiments of the present invention there is provided a method of inhibiting, reducing or retarding the formation of load of a microorganism and/or the formation of a biofilm, in and/or on an article e.g., food package. The method comprises incorporating in and/or on the article any one of the compositions- of-matter as described herein, including any of the respective embodiments thereof.

As demonstrated hereinbelow, a composition of matter as described herein has shown to exhibit anti-biofilm formation activity and can thus prevent, retard or reduce the formation of a mass of a biofilm.

In some embodiments, the antimicrobial dosage is adjusted. In some embodiments, the antimicrobial dosage is adjusted by e.g., altering the number of the disclosed patches within the package. For example, in one package the number of patches may be e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and even more, according to parameters, selected from, without limitation, food type, environmental conditions, and the size or the structure of the package. In some embodiments, the antimicrobial dosage is adjusted by altering the amount of the oil absorbed within the polymeric composition.

Herein "antimicrobial activity" is referred to as an ability to inhibit (prevent), reduce or retard bacterial growth, fungal, yeast and/or mold growth, biofilm formation or eradicate living bacterial cells, or their spores, or fungal cells or viruses in a suspension, on a surface or in a moist environment.

Herein, inhibiting or reducing or retarding the formation of load of a microorganism refers to inhibiting reducing or retarding growth of microorganisms and/or eradicating a portion or all of an existing population of microorganisms.

Thus, the composition-of matter comprising one or more volatile oils as described herein can be used both in reducing the formation of microorganisms on or in an article, and in killing microorganisms in or on an article e.g., food package.

The inhibition or reduction or retardation of formation of a biofilm assumes that the biofilm has not yet been formed, and hence the presence of the composition-of matter of the present invention is required also in cases where no biofilm is present or detected.

As used herein, the term "preventing" in the context of the anti-biofilm or antimicrobial activity, indicates that the formation of a microbial load, e.g., biofilm and/or microorganism cells is essentially nullified or is reduced by e.g., at least 20 %, at least 30 %, at least 40 %, at least 50 %, at least 60 %, at least 70 %, at least 80 %, at least 90 %, including any value therebetween, of the appearance of the load in a comparable situation lacking the presence of the composition-of matter of the present invention or an article containing same. Alternatively, preventing means a reduction to e.g., at least 15 %, 10 % or 5 % of the appearance of the microbial load in a comparable situation, lacking the presence of the composition-of matter of the present invention or an article containing same. Methods for determining a level of appearance of a microbial load or biofilm are known in the art.

In some embodiments, at least 90 % of antimicrobial activity of the composition- of matter of the present invention is maintained for at least e.g., 1 day, 2 days, 5 days, 10 days, 15 days, 20 days, 25 days, 30, days 35 days, 40 days, 45 days, 50 days, 55 days, 60 days of storage duration. In some embodiments, the phrase "inhibiting the growth of a microorganism" refers to an effect of a compound, a composition or a combination of a compound with another active agent, which stops and/or reverses the propagation of a microorganism, such that at least one cell or a culture thereof is no longer multiplying or growing and/or is killed as a result of coming in contact with, or being near to, the polymeric composition comprising oil compounds. In embodiments of the present invention where the microorganism is a pathogenic microorganism, the effect of inhibiting the growth thereof is oftentimes beneficial.

In some embodiments, the composition has an indirect antimicrobial activity. "indirect" or "near", as used herein, refers to a short distance from an object (e.g., package or the location of the composition, patch, or tablet as disclosed hereinthroughout). The location may be on top of or outside of, or inside of the article (e.g., food package) comprising the disclosed composition, at any direction therefrom.

The microorganism can be, for example, a unicellular microorganism (prokaryotes, archaea, bacteria, eukaryotes, protists, fungi, algae, euglena, protozoan, dinoflagellates, apicomplexa, trypanosomes, amoebae and the likes), or a multicellular microorganism. As used herein, the terms "bacteria", or "bacterial cells" may refer to either Gram-positive bacteria and/or to Gram-negative bacteria and archae, including pathogenic and multi-drug resistant (MDR) bacteria.

In some embodiments of the present invention, the composition-of-matter in any embodiment as described hereinthroughout, may be characterized by high affinity to a specified bacteria type, species, or genus. Therefore, in some embodiments of the present invention the composition-of-matter in any embodiment as described hereinthroughout, may be used an effective way for selectively targeting bacteria.

In some embodiments, the inhibiting or reducing or retarding the formation of load of a microorganism is referred to as being capable of exerting "anti-biofouling activity". Herein "anti-biofouling activity" or "antifouling activity" is referred to as an ability to inhibit (prevent), reduce or retard biofilm formation on a substrate's surface.

The term "biofilm", as used herein, refers to an aggregate of living cells which are stuck to each other and/or immobilized onto a surface as colonies. The cells are frequently embedded within a self-secreted matrix of extracellular polymeric substance (EPS), also referred to as "slime", which is a polymeric sticky mixture of nucleic acids, proteins and polysaccharides.

In the context of the present embodiments, the living cells forming a biofilm can be cells of a unicellular microorganism (prokaryotes, archaea, bacteria, eukaryotes, protists, fungi, algae, euglena, protozoan, dinoflagellates, apicomplexa, trypanosomes, amoebae and the likes), or cells of multicellular organisms in which case the biofilm can be regarded as a colony of cells (like in the case of the unicellular organisms) or as a lower form of a tissue.

In the context of the present embodiments, the cells are of microorganism origins, and the biofilm is a biofilm of microorganisms, such as bacteria and fungi. The cells of a microorganism growing in a biofilm are physiologically distinct from cells in the "planktonic form" of the same organism, which by contrast, are single-cells that may float or swim in a liquid medium. Biofilms can go through several life-cycle steps which include initial attachment, irreversible attachment, one or more maturation stages, and dispersion.

The phrases "anti-biofilm formation activity" refers to the capacity of a substance to affect the prevention of formation of a biofilm of bacterial, fungal and/or other cells; and/or to affect a reduction in the rate of buildup of a biofilm of bacterial, fungal and/or other cells, on a surface of a substrate. In some embodiments, the biofilm is formed of bacterial cells.

In some embodiments, a biofilm is formed of bacterial cells of bacteria selected from the group consisting of all Gram-positive and Gram-negative bacteria and archae.

The terms "bacterium" or "bacteria", as used herein, refer to all prokaryotic organisms, including those within all of the phyla in the Kingdom Procaryotae. It is intended that these terms encompass all microorganisms considered to be bacteria including Mycoplasma, Chlamydia, Actinomyces, Streptomyces, and Rickettsia. All forms of bacteria are included within this definition including cocci, bacilli, spirochetes, spheroplasts, protoplasts, etc. Also, included within these terms are prokaryotic organisms that are Gram-negative or Gram-positive as described hereinabove. "Gram-negative" and "Gram-positive" refer to staining patterns with the Gram-staining process, which is well known in the art. (See e.g., Finegold and Martin, Diagnostic Microbiology, 6th Ed., CV Mosby St. Louis, pp. 13-15 (1982)). Non-limiting examples of bacteria include bacteria of a genus selected from the group including Salmonella, Shigella, Escherichia, Enterobacter, Serratia, Proteus, Yersinia, Citrobacter, Edwardsiella, Providencia, Klebsiella, Hafnia, Ewingella, Kluyvera, Morganella, Planococcus, Stomatococcus, Micrococcus, Staphylococcus, Vibrio, Aeromonas, Plessiomonas, Haemophilus, Actinobacillus, Pasteurella, Mycoplasma, Ureaplasma, Rickettsia, Coxiella, Rochalimaea, Ehrlichia, Streptococcus, Enter ococcus, Aerococcus, Gemella, Lactococcus, Leuconostoc, Pedicoccus, Bacillus.

Corymb acterium, Arcanobacterium, Actinomyces, Rhodococcus, Listeria, Erysipelothrix, Gardnerella, Neisseria, Campylobacter, Arcobacter, Wolinella, Helicobacter, Achromobacter, Acinetobacter, Agrobacterium, Alcaligenes, Chryseomonas, Comamonas, Eikenella, Flavimonas, Flavobacterium, Moraxella, Oligella, Pseudomonas, Shewanella, Weeksella, Xanthomonas, Bordetella, Franciesella, Brucella, Legionella, Afipia, Bartonella, Calymmatobacterium, Cardiobacterium, Streptobacillus, Spirillum, Peptostreptococcus, Peptococcus, Sarcinia, Coprococcus, Ruminococcus, Propionibacterium, Mobiluncus, Bifidobacterium, Eubacterium, Lactobacillus, Rothia, Clostridium, Bacteroides, Porphyromonas, Prevotella, Fusob acterium, Bilophila, Leptotrichia, Wolinella, Acidaminococcus, Megasphaera, Veilonella, Norcardia, Actinomadura, Norcardiopsis, Streptomyces, Micropolysporas, Thermoactinomycetes, Mycobacterium, Treponema, Borrelia, Leptospira and Chlamydiae.

In some embodiments of the present invention the bacteria are of one or more of the following species: Acinetobacter baumanii, Belicobacter pylori, Burkholderia multivorans, Campylobacter jejuni, Deinococcus radiodurans , E. coli, Enterobacter cloacae, Enterococcus faecalis, Haemophilus influenzae, Klebsiella pneumoniae, Klebsiella oxytoca,, Mycobacterium tuberculosis, Neisseria gonorrhoeae, Neisseria meningitidis, Pseudomonas aeruginosa, Pseudomonas phosphoreui, Escherichia coli, Bacillus Subtifis, Borrelia burgfrferi, Neisseria meningitidis, Neisseria gonorrhoeae, Yersinia pestis, Campylobacter jejuni, Deinococcus radiodurans, Mycobacterium tuberculosis, Enterococeus faecalis, Streptococcus pneumoniae, Streptococcus pyogenes and Staphylococcus aureus, Salmonella typhimuriunim, Serratia marcescens, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus pneumoniae, Staphylococcus sanguis, Staphylococcus viridans, Vibrio harveyi, Vibrio cholerae, Vibrio parahaeniolyticus, Vibrio alginolyticus, Yersinia enterocolitica or Yersinia pestis, including any strain or mutant thereof.

A wide-spectrum activity range is oftentimes indicative of activity in inhibiting the growth of eukaryotic microorganisms such as fungi, as well as prokaryotic microorganisms such as bacteria.

Representative examples of pathogenic fungi, against which a compound having general Formula A as described herein can be efficiently used according to the present embodiments include, without limitation, fungi of the genus Absidia: Absidia corymbifera; genus Ajellomyces: Ajellomyces capsulatus, Ajellomyces dermatitidis; genus Arthroderma: Arthroderma benhamiae, Arthroderma fulvum, Arthroderma gypseum, Arthroderma incurvatum, Arthroderma otae, Arthroderma vanbreuseghemii; genus Aspergillus: Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger; genus Blastomyces: Blastomyces dermatitidis; genus Candida: Candida albicans, Candida glabrata, Candida guilliermondii, Candida krusei, Candida parapsilosis, Candida tropicalis, Candida pelliculosa; genus Cladophialophora: Cladophialophora carrionii; genus Coccidioides: Coccidioides immitis; genus Cryptococcus: Cryptococcus neoformans; genus Cunninghamella: Cunninghamella sp.; genus Epidermophyton: Epidermophyton floccosum; genus Exophiala: Exophiala dermatitidis; genus Filobasidiella: Filobasidiella neoformans; genus Fonsecaea: Fonsecaea pedrosoi; genus Fusarium: Fusarium solani; genus Geotrichum: Geotrichum candidum; genus Histoplasma: Histoplasma capsulatum; genus Hortaea: Hortaea werneckii; genus Issatschenkia: Issatschenkia orientalis; genus Madurella: Madurella grisae; genus Malassezia: Malassezia furfur, Malassezia globosa, Malassezia obtusa, Malassezia pachydermatis, Malassezia restricta, Malassezia slooffiae, Malassezia sympodialis; genus Microsporum: Microsporum canis, Microsporum fulvum, Microsporum gypseum; genus Mucor: Mucor circinelloides; genus Nectria: Nectria haematococca; genus Paecilomyces: Paecilomyces variotii; genus Paracoccidioides: Paracoccidioides brasiliensis; genus Penicillium: Penicillium marneffei; genus Pichia, Pichia anomala, Pichia guilliermondii; genus Pneumocystis: Pneumocystis carinii; genus Pseudallescheria: Pseudallescheria boydii; genus Rhizopus: Rhizopus oryzae; genus Rhodotorula: Rhodotorula rubra; genus Scedosporium: Scedosporium apiospermum; genus Schizophyllum: Schizophyllum commune; genus Sporothrix: Sporothrix schenckii; genus Trichophyton: Trichophyton mentagrophytes, Trichophyton rubrum, Trichophyton verrucosum, Trichophyton violaceum; and of the genus Trichosporon:

Trichosporon asahii, Trichosporon cutaneum, Trichosporon inkin, Trichosporon mucoides.

Representative examples of parasites and protozoa, against which a compound having general Formula A as described herein may be used according to the present embodiments include, without limitation, various types of amoeba, Leishmania spp, Plasmodium falciparum Trypanosoma cruzi (causing Chagas' disease), Trypanosoma bucei (causing "sleeping sickness"), Plasmodium vivax (causing malaria), Cryptosporidium parvum (causing cryptosporidiosis), Cyclospora cayetanensis, Giardia lamblia (causing giardiasis) and many others.

The substrate:

In some embodiments of the present invention, the polymeric composition (e.g., in form of a patch) is incorporated on a substrate's surface e.g., via an adhesive layer of the polymeric composition.

Substrate's surfaces usable according to some embodiments of the present invention can therefore be hard or soft, organic or inorganic surfaces, including, but not limited to, glass surfaces; porcelain surfaces; ceramic surfaces; polymeric surfaces such as, for example, plastic surfaces, rubbery surfaces, and surfaces comprising or made of polyolefins and any derivative thereof, polymers such as polypropylene (PP), polycarbonate (PC), polyethylene (PE), high-density polyethylene (HDPE), low-density polyethylene (LDPE), unplasticized polyvinyl chloride (PVC), polyamides (e.g., nylon), polyamine, polypropylene, polyesters (e.g., polyethylene terephthalate) and fluoropolymers including but not limited to polytetrafluoroethylene (PTFE, Teflon®), metallic substrates and surfaces thereof (e.g., gold surfaces); or can comprise or be made of silicon, organosilicon, stainless steel, gold, polymers as described herein or include any combination of the above. In some embodiments, the substrate is or a part of an article.

Articles:

According to an aspect of some embodiments of the present invention there is provided an article which comprises any one of the composition-of-matter as described herein. Any article that may benefit from a compositions-of-matter comprising the polymeric patch and one or more volatile oils described herein is contemplated.

Exemplary article includes packages or containers, for example, food packages and containers, beverage packages and containers, medical device packages, other biological sample packages and containers, and any other packages or containers of various articles.

The term "food package" as used herein is defined as a package designed to contain food inside of it. In its broadest sense, the "package" portion of the food package generally comprises at least one wall and at least one seal. The food may be, for example and without being limited thereto, fresh foods such as vegetables, fruits, meat, seafood, and ground beef.

In the food package the food may be in both direct and indirect contact with the packaging material.

Typically, but not exclusively, the food package would be of a size and configuration which is commonly used to contain food sold in a retail situation.

In some embodiments of the present invention, the patch is a disposable patch that can be attached to the interior of any type of package and release anti-microbial agents within the package.

It is to be recognized that the versatility of the patch as disclosed herein and the simple handing may allow using thereof for imparting currently used packages in all types thereof, a preservative activity.

As used herein the term "preservative" and any grammatical derivative thereof refers to as used herein, "preservative" is used to prevent the growth of bacteria and/or fungi and/or molds in the environment within and/or near the package.

The preservative effect further includes, without limitation, extending the shelf life of foods, reduce the risk from pathogens and provide an extra margin of safety for health.

General:

As used herein the term "about" refers to ± 10 %.

The terms "comprise", "comprising", "includes", "including", "having" and their conjugates mean "including but not limited to".

The term "consisting of means "including and limited to". The term "consisting essentially of" means that the composition, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.

The word "exemplary" is used herein to mean "serving as an example, instance or illustration". Any embodiment described as "exemplary" is not necessarily to be construed as preferred or advantageous over other embodiments and/or to exclude the incorporation of features from other embodiments.

The word "optionally" is used herein to mean "is provided in some embodiments and not provided in other embodiments". Any particular embodiment of the invention may include a plurality of "optional" features unless such features conflict.

As used herein, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.

Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases "ranging/ranges between" a first indicate number and a second indicate number and "ranging/ranges from" a first indicate number "to" a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.

As used herein the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.

As used herein, the term "treating" includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical or aesthetical symptoms of a condition or substantially preventing the appearance of clinical or aesthetical symptoms of a condition.

It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.

EXAMPLES

Reference is now made to the following examples which, together with the above descriptions, illustrate the invention in a non-limiting fashion.

EXAMPLE 1

Sample characterization

Materials and Experimental Methods

Patch fabrication: A method for effectively absorbing volatile anti -bacterial oil in an amphiphilic polymer is designed with two layers. One layer provides the adhesiveness of the patch and the other is the active layer. In exemplary procedures, different polymers are used: Polypore®, starch, crosslinked polyacrylic acid (Carbopol®), hydroxypropyl cellulose (HPC), crosslinked crosscarmellose cellulose (CMC), maltodextrin and microcrystalline cellulose (MCC). These polymers are amphiphilic, which gives them the ability to absorb and release their content in a controlled pattern. All tested polymers are absorbed with known amounts of anti -bacterial oil and the loading degree is calculated gravimetrically. Then, each tested polymer absorbed with the oil is mixed with other patch forming excipients and pressed by a laboratory-tableting machine to obtain an adhesive patch as described below.

Tablet fabrication: In exemplary procedures, for the fabrication of an anti -bacterial patch, a super absorbent polymer is absorbed with anti-bacterial volatile oil before mixing with other food-grade polymers using a mortar and pestle. The active layer was composed of 89% polymer+oil, 10% Polyvinylpyrrolidone (PVP) (used as a binder) and 1% Magnesium stearate (used as a lubricant). Figure 1 presents a schematic illustration of the fabrication process of tablet or patch.

Adhesiveness: The adhesiveness to several types of surfaces used in the food industry (e.g., Styrofoam, nylon and polystyrene) is measured by means of tensiometry (Instron universal machine) while the friability of the patches is measured by a pharmaceutical disintegration apparatus.

Anti-bacterial oils: Carvacrol or lemon grass oils are very effective against a range of different pathogenic and spoilage microorganisms including Gram-negative and Gram- positive bacteria, yeast and molds. These oils are very distinct in their fragrance, and therefore, each can be selected for a different type of food according to effectiveness and customer preferences (e.g., citrus oil for fruits and carvacrol oil for meat and vegetables). In exemplary embodiments, each tested oil is analyzed by GC-MS and by UV-Vis before and after loading to verify the chemical integrity or possible damage during loading.

Tablets saturation: In order to determine the maximum oil capacity of each polymer in the tablets and to choose the most suitable polymer for the tablet, different percentage were tested including 25%, 50%, 75% and 100%. The polymers were examined for their saturation and absorption of carvacrol and lemon grass oil. Polymers were absorbed with oil and their flowability was measured to insure this process is suitable to scale-up process. Seven different polymers were examined: National starch^®, Eliane^®, maltodextrin, Carbopol^®, HPC, MCC and CMC. Each polymer (5 gr) was poured into mortar and oil was added dropwise (each cycle was of 200 μΐ) while pounding with pestle. The mixture (polymer + 200 μΐ oil) was transferred into a funnel to check whether the polymer is flowable or not. Process was repeated until the polymer became saturated with oil which was determined by the limited ability to passed through funnel. The ratio between the oil weight (gr) and the polymer's dry weight (gr) was tested.

Release kinetics: The release kinetics of the oil from the patch is analyzed by GC- MS at different temperatures (25°C and 4°C). Fabricating a patch that releases antimicrobial oil very slowly (e.g., over a period of a few weeks) is not effective in case the microbes have a very short lag period. On the other hand, the an ti -microbial agent should not be incompatible with the package in a way that it gets released within minutes. The ambient conditions are also considered since release kinetics of volatile oils depends highly on temperature. Thus, the release kinetics of the tablet's containing Carvacrol oil with 50- 75% saturation within the polymers: Eliane, maltodextrin, Carbopol, and MCC was examined over 80 minutes at 4°C and 25°C by gas chromatography FID. The two temperatures tested represent the different maintenance for food products- at room temperature and at 4°C (refrigerator). The relative fumes concentrations were obtained by the instrument represent the relative concentration of the oil fumes in the vial and were normalized by the tablet weight.

EXAMPLE 2

Antimicrobial activity

Antibacterial activity:

In vitro effectiveness of the tested oils was evaluated in by examination of sandwiches (provided by Lord Sandwich; e.g., yellow cheese, tuna, Schnitzel sandwiches). Considering that in any active packaging containing volatile oils, most ant i- microbial activity is produced by the generated vapors, oils are also being evaluated in the vapor phase. Decrease concentration (DC) and inhibitory concentration (IC) is used to evaluate the activity of the tested anti-bacterial oils. The evaluation is conducted on various combinations (different polymers, two oils, various number of patches for each food package), so as to the select optimal microbial agents for improving fresh food preservation.

In exemplary procedures, the tested sandwiches were inserted into their original packages with or without using the patches. Sandwiches were kept in refrigerator for 10 days before analysis and their total bacterial counts were evaluated by the institute for food microbiology and consumer goods LTD, (Nesher, Israel).

In exemplary procedures, tested foods are inserted into their original packages with or without using the patches. The condition of the tested foods is evaluated subjectively and objectively during storage.

In exemplary procedures, a sensory panel composed of at least 4 to 6 members is used to assess each tested food. The same trained persons participate in each evaluation cycle, which is carried out in a double-blind study. Special attention is given to the color and the presence of exudate in the package prior to opening and the assessment of abnormal odors during the opening of the package. Each attribute is scored on a three -point hedonic scale where: l=acceptable; 2=marginal; and 3=unacceptable. Assessment is designed to identify spoilage conditions exclusively. Other assessments are more objective and include pH, gas assays (GC-MS) and microbial analysis (cell culture). In exemplary procedures, tests are performed on sliced strawberries, tomato, sliced salami or Gouda cheese. A food is placed in a package containing 0 (control), I or 2 patches for several weeks. Samples are taken at pr -determined times followed by analysis of fungal spore production.

Indirect antibacterial activity

Antibacterial activity of Tea tree, Carvacrol and mixture of both oils released from MCC polymer patches was tested on the growth of coliform., following incubation for 10 days at 7°C.

Results:

Tablets saturation

As shown in Figures 3, MCC showed the highest ability to absorb oil while CMC and Potato starch absorbed increased amounts of oil as well. Carbopol and maltodextrin absorbed efficiently while corn starch and HPC showed low absorbability.

MCC, CMC and Potato starch absorbed high amounts of oil due to their high cross- linking and porosity structure. When comparing corn to potato starch, the corn starch swallowed low amounts of oil due to its low levels of amylopectin. Maltodextrin absorbed less oil in comparison to potato starch, because it has less cross linking and its structure is shorter. Carbopol is highly cross-linked and can absorb oil efficiently due to its structure and porosity that allows to trap large amount of oil.

Release kinetics

All polymers showed a release rate that remains constant even as the concentration of the chemical decreases, i.e. zero rate release, which is preferable due to the ability to control the concentration of the antimicrobial agents in the package. Plateau was observed after 40 minutes at 25°C (Fig. 4A) compared to 50 minutes at the 4 °C (Fig. 4B) and the slop indicates that the release rate at 25 °C is higher than at 4°C, because 4°C is a better environment.

The optimal polymers for antimicrobial activity in fresh food packages are MCC and Potato starch, because as shown in Figures 4, their tablets have higher release rate than Carbopol and Maltodextrin tablets. This presents a correlation between the absorbability and the polymer release rate. The results indicate that increased porosity in polymers enlarge the amount of oil being released. Vapor pressures of monoterpenes is dependent on temperature. Therefore, the amount of oil released at 25 °C is higher than at 4°C for all tablets.

Antibacterial activity

As shown in Figures 5, in all cases, the sticker reduced total bacterial count by 2 to 6 fold.

As shown in Figure 6, all patches reduced total coliforms count. The tea tree oil had the most efficacious antibacterial activity. A mixture with less effective anti-microbial properties as the carvacrol oil, as showed in Figure 6, may compete and disturb with the overall antibacterial activity. Therefore, a mixture of both oils was less effective patch than the patch with tea tree oil.

Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A composition-of-matter comprising a polymer having incorporated within an antimicrobial effective amount of one or more volatile oils, wherein said polymer has surface area of 0.1 to 4 m²/g.

2. The composition of claim 1 , wherein said polymer has surface area of 0.4 to 2 m²/g.

3. The composition of claim 1, wherein said polymer has a pore volume of 0.001 to 0.01 cm³/g.

4. The composition of claim 1 , wherein said antimicrobial effective amount of one or more volatile oils is at least 1% by total weight of said composition.

5. The composition-of-matter of claim 1, wherein a weight ratio of said oil and said polymer is in a range of between 5 to 60.

6. The composition of claim 1, wherein at least 1% of said oil is non-covalently incorporated within said polymer.

7. The composition of claim 1, wherein at least 1% of said oil does not undergo oxidization within said polymer for a pre-determined time.

8. The composition-of-matter of claim 1, wherein said one or more volatile oils are characterized by vapor pressure of at least about 0.001 mmHg at about 4-7 °C.

9. The composition-of-matter of claim 1, wherein said one or more volatile oils are selected from the group consisting of oregano, carvacrol and thymol, lemon grass, or isomers, derivatives and mixtures thereof.

10. The composition-of-matter of claim 1, wherein said one or more volatile oils have an antimicrobial effect.

11. The composition of claim 1 , wherein said polymer is an amphiphilic polymer.

12. The composition of claim 1, wherein said polymer is a hydrophobic polymer.

13. The composition of claim 1 wherein said polymer is a cross-linked polymer.

14. The composition-of-matter of claim 1, wherein said polymer is selected from the group consisting of polyacrylic acid, carbohydrate, polyallyl methacrylates, cellulose, starch, maltodextrin and mixtures, copolymers or derivatives thereof.

15. The composition-of-matter of any claim 14, wherein said cellulose is selected from the group consisting of: microcrystalline cellulose and croscarmellose.

16. The composition-of-matter of any one the proceeding claims, being in a form of a patch or a tablet.

17. The composition-of-matter of claim 16, wherein the patch or the tablet are in the form of a single layer.

18. The composition-of-matter of claim 16, wherein the patch or the tablet are in the form of a double layer.

19. The composition-of-matter of any one of claims 17 and 18, comprising at least one layer comprising one or more adhesive materials.

20. The composition-of-matter of any one of claim 16, wherein said polymer and said oil are compressed thereby forming the patch or tablet.

21. The composition-of-matter of any one of claims 1 to 20 further comprising any one of a flavoring agent, a binder, a lubricant, or a combination thereof.

22. The composition-of-matter of any one of claims 1 to 21, for use as a preservative activity.

23. An article comprising the composition-of-matter of any one of claims 1-22.

24. The article of claim 22, being selected from the group consisting of a food package, and medical device package.

25. A method of inhibiting or reducing a formation of load of a microorganism and/or a formation of a biofilm, within an article, the method comprising incorporating the composition-of-matter of any one of claims 1-20 within said article.

26. The method of claim 25, wherein said microorganism is selected from bacteria, molds and fungi.

27. A method for the preparation of an antimicrobial composition, the method comprising:

(i) providing a polymer having a surface area of 0.1 to 4 m²/g; (ii) providing an antimicrobial effective amount of one or more volatile oils; and

(iii) saturating said polymer with the antimicrobial effective amount of one or more volatile oils.

28. The method of claim 27, further comprising a step of pressing the saturated polymer under a pressure of at least 1 ton.