Melt process for preparing protein stabilized antimicrobial compositions

<u>Background of the Invention</u>

Certain types of botanical oils, such as thymol and carvacrol, are known to be environmentally friendly and effective in combating microorganisms. Unfortunately, however, the use of such oils has been limited in many commercial applications (e.g., wipes) due to their high volatility and instability in the presence of oxygen. Attempts to overcome this problem often involve the use of a larger amount of the botanical oils to prolong antimicrobial activity. Regrettably, this just leads to another problem in that high concentrations of essential oils can cause damage to certain types of food products, such as fruit. Other attempts have involved the encapsulation of the oil component with certain types of polymers, such as proteins. For example, an article entitled "<nplcit id="ncit0001" npl-type="s"><text>Encapsulation of Essential Oils in Zein Nanospherical Particles" (Parris, et al., J. Agric. Food Chem. 2005, 53, 4788-4792</text></nplcit>) broadly describes the encapsulation of thymol in zein nanospheres by mixing the oil with zein particles in the presence of a solvent (e.g., ethanol). The particles are said to be useful for oral or injectable administration of biological materials into the body. Another article entitled "<nplcit id="ncit0002" npl-type="s"><text>Controlled Release of Thymol from Zein Based Film" (Mastromatteo, et al., J. Innovative Food and Emerging Technologies 2009, 10, 222-227</text></nplcit>) broadly describes films formed by dissolving corn zein and glycerol into ethanol, and thereafter adding thymol to form a solution. The solution is poured into a Petri dish and dried to form the film.

One problem with the techniques described above is that they generally rely on solvents (e.g., ethanol) to help dissolve the botanical oil into a solution. A disadvantage of the use of solvents is that both the botanical oil and protein must be soluble in a common solvent system, which puts a limit on what type of components may be employed in the composition. Also, solvent-based solutions require a substantial amount of time, energy, and material for processing. Still further, a portion of the botanical oil may escape from the solution when the solvent is evaporated, which requires the use of a greater amount of the oil than would normally be needed. Notwithstanding the above, the ability to use a "solventless" process is complicated by the tendency of proteins to lose their flow properties when exposed to the intense shear and elevated temperature normally associated with melt processing. For example, proteins may undergo a conformational change ("denaturation") that causes disulfide bonds in the polypeptide to dissociate into sulfhydryl groups or thiyl radicals. Sulfhydryl groups form when disulfide bonds are chemically reduced while mechanical scission of disulfide bonds causes thiyl radicals to form. Once dissociated, however, free sulfhydryl groups randomly re-associate with other sulfhydryl groups to form new disulfide bond between polypeptides. Thiyl radicals can also randomly re-associate with other thiyl radicals forming new disulfide bonds or thiyl radicals can react with other amino acid functionality creating new forms of cross-linking between polypeptides. Because one polypeptide contains multiple thiol groups, random cross-linking between polypeptide leads to formation of an "aggregated" polypeptide network, which is relatively brittle and leads to a loss of flow properties.

As such, a need currently exists for a solventless process for forming a stable composition that contains an antimicrobially active botanical oil.

<u>Summary of the Invention</u>

From one aspect the present invention provides a method for forming an antimicrobial composition comprising dispersively blending an antimicrobially active botanical oil, a protein and a carrier fluid, and blending being within a melt blending device at a temperature of from 20°C to 100°C and an apparent shear rate of 100 to 500 seconds^-1, wherein botanical oils constitute from 0.1 wt.% to 30 wt.% of the composition, proteins constitute from 30 wt.% to 95 wt.% of the composition, and carrier fluids constitute from 1 wt.% to 50 wt.% of the composition, and wherein the protein is wheat gluten, and wherein the carrier fluid is glycerol or lactic acid detail below.

<u>Brief Description of the Figures</u>

A full and enabling disclosure of the present invention, including the best mode thereof, directed to one of ordinary skill in the art, is set forth more particularly in the remainder of the specification, which makes reference to the appended figures in which: <ul id="ul0001" list-style="none" compact="compact"><li><figref idref="f0001">Fig. 1</figref> is an SEM microphotograph of a control sample containing 70 wt.% gluten and 30 wt.% glycerol;</li><li><figref idref="f0001">Fig. 2</figref> is an SEM microphotograph of a control sample containing 70 wt.% gluten and 30 wt.% glycerol;</li><li><figref idref="f0002">Fig. 3</figref> is an SEM microphotograph of Sample 3 of Example 1;</li><li><figref idref="f0002">Fig. 4</figref> is an SEM microphotograph of Sample 3 of Example 1; and</li><li><figref idref="f0003">Fig. 5</figref> is a graph of the results obtained in Example 3, in which the % thymol retained in water is shown for a period of time ranging from 0 to 60 minutes.</li></ul>

<u>Detailed Description of Representative Embodiments</u>

Generally speaking, the present invention is directed to a method for forming an antimicrobial composition that includes mixing an antimicrobially active botanical oil (e.g., thymol or carvacrol) and protein within a melt blending device (e.g., extruder). Despite the problems normally associated with melt processing proteins, the present inventors have discovered that the processing conditions and components may be selectively controlled to allow for the formation of a stable, melt-processed composition that is able to exhibit good mechanical properties. For example, the extrusion temperature(s) and shear rate employed during melt blending are relatively low to help limit polypeptide dissociation, thereby minimizing the impact of aggregation and embrittlement. While the use of such low temperature/shear conditions often tend to reduce mixing efficiency, the present inventors have discovered that a carrier fluid may be employed to enhance the ability of the botanical oil to flow into the internal structure of the protein where it can be retained in a stable manner. The composition is also typically anhydrous and generally free of solvents. In this manner, the protein will not generally disperse before use and prematurely release the botanical oil.

Various embodiments of the present invention will now be described in more detail below.

I.

Components

A.

Botanical Oil

Botanical oils are employed in the composition produced by the method of the present invention as antimicrobial actives. The oil may be an "essential" oil that is extracted from a plant. Likewise, the botanical oil may also be isolated or purified from an essential oil, or it may simply be made synthetically to mimic a compound derived from a plant (e.g., synthetically made thymol). The botanical oils are generally soluble in lipids and believed to exhibit antimicrobial efficacy due to their ability to cause damage to the lipid component of the cell membrane in microorganisms, thereby inhibiting their proliferation. Essential oils are derived from herbs, flowers, trees, and other plants, and are typically present as tiny droplets between the cells of the plants and may be extracted by methods known to those of skill in the art (e.g., steam distillation, enfleurage (i.e., extraction using fat(s)), maceration, solvent extraction, or mechanical pressing). Examples of suitable essential oils for use in the present invention may include, for instance, anise oil, lemon oil, orange oil, oregano, rosemary oil, wintergreen oil, thyme oil, lavender oil, clove oil, hops, tea tree oil, citronella oil, wheat oil, barley oil, lemongrass oil, cedar leaf oil, cedar wood oil, cinnamon oil, fleagrass oil, geranium oil, sandalwood oil, violet oil, cranberry oil, eucalyptus oil, vervain oil, peppermint oil, gum benzoin, basil oil, fennel oil, fir oil, balsam oil, menthol, ocmea origanum oil, <i>Hydastis carradensis</i> oil, <i>Berberidaceae daceae</i> oil, Ratanhiae and <i>Curcuma longa</i> oil, sesame oil, macadamia nut oil, evening primrose oil, Spanish sage oil, Spanish rosemary oil, coriander oil, thyme oil, pimento berries oil, rose oil, bergamot oil, rosewood oil, chamomile oil, sage oil, clary sage oil, cypress oil, sea fennel oil, frankincense oil, ginger oil, grapefruit oil, jasmine oil, juniper oil, lime oil, mandarin oil, marjoram oil, myrrh oil, neroli oil, patchouli oil, pepper oil, black pepper oil, petitgrain oil, pine oil, rose otto oil, spearmint oil, spikenard oil, vetiver oil, or ylang ylang. Still other essential oils known to those of skill in the art are also contemplated as being useful within the context of the present invention (e.g., <nplcit id="ncit0003" npl-type="b"><text>International Cosmetic Ingredient Dictionary, 10th and 12th editions, 2004 and 2008</text></nplcit>).

In one embodiment, carvacrol and thymol-containing oils are purified from the species <i>Origanum vulgare</i> of a hirtum variety. Ideally this is a hybrid strain that produces high quality oils, but is not limited to this genus, species or strain. The oil extract may also be obtained from a plant of the genus <i>Nepeta</i> including, but not limited to species <i>Nepeta racemosa</i> (catmint), <i>Nepeta citriodora, Nepeta elliptica, Nepeta hindostoma, Nepeta lanceolata, Nepeta leucophylla, Nepeta longiobracteata, Nepeta mussinii, Nepeta nepetella, Nepeta sibthorpii, Nepeta subsessilis, Nepeta tuberosa,</i> Thymus glandulosus, Thymus hyemalis, Thymus vulgaris and Thymus zygis.

As indicated above, isolates and/or derivatives of essential oils may also be employed in the present invention. For example, monoterpene phenols are particularly suitable for use in the present invention, which may be isolated and purified from plant oil extracts, or made synthetically by known methods. Suitable monoterpene phenols may include, for instance, thymol, carvacrol and eucalyptol. Thymol (isopropyl-cresol) is one particularly suitable monoterpene phenol, which is a crystalline substance that has a boiling point of about 238°C at atmospheric pressure. Carvacrol (isopropyl-o-cresol), an isomer of thymol, is another suitable compound. Carvacrol is a liquid with a boiling point of about 233°C at atmospheric pressure. Thymol and carvacrol, as well as isomers thereof, may be derived from plant oil extracts or synthesized. For example, carvacrol may be synthesized by the reaction of nitrous acid with 1-methyl-2-amino-4-propyl benzene. In addition to being employed in an isolated or pre-synthesized form, essential oils containing the monoterpene phenols as major constituents may be employed, with the final concentrations of the monoterpene phenols being within the ranges provided herein. The term "major constituent" generally refers to those essential oils having monoterpene phenols in an amount of more than 50 wt.%. It is well-known in the art that such essential oils may also contain lesser amounts of other constituents, such as non-aromatic terpene compounds. Essential oils with organic phenolic compounds as the major constituent include, for example, anise oil, bay oil terpineless, clove bud, clove leaf, clove oil, clove stem, origanum oil, Peru balsam, pimento oil, eucalyptus oil, and thyme oil.

Due to the stability achieved by the antimicrobial composition produced by the method of the present invention, a relatively small amount of botanical oils is employed and still achieves the desired antimicrobial efficacy. The composition employs botanical oils in an amount of from 0.1 wt.% to 30 wt.%, in some embodiments from 0.5 wt.% to 20 wt.%, and in some embodiments, from 1 wt.% to 10 wt.%.

B.

Protein

The antimicrobial composition produced by the method of the present invention also contains a protein which is wheat gluten. Because the botanical oil tends to leach out during storage and before it is used in the desired application, the protein helps enhance the long term stability of the oil and, in turn, antimicrobial efficacy. Without intending to be limited by theory, it is believed that the physical structure of the protein can effectively encapsulate the botanical oil and inhibit its premature release. Nevertheless, when it is desired to release the botanical oil prior to and/or during use, the protein can disperse (e.g., disintegrate, dissolve or change physical form) when placed in an aqueous environment. The amount of time needed for dispersal of the protein so that it releases the desired antimicrobial active will depend at least in part upon the particular end-use design criteria. In most embodiments, the protein will begin to disperse and release the antimicrobial active within 5 minutes, suitably within 1 minute, more suitably within 30 seconds, and most suitably within 10 seconds.

Any form of protein may be used, such as isolates, concentrates and flour. It may be desirable to use a wheat gluten that is relatively pure, such as those having a protein content of 75 wt.% or more, and in some cases, 85 wt.% or more. Gluten proteins may be purified by washing away any associated starch to leave a composite of gliadin and glutenin proteins. In one particular embodiment, a vital wheat gluten is employed. Such vital wheat gluten is commercially available as a creamy-tan powder produced from wheat flour by drying freshly washed gluten. For instance, vital wheat gluten can be obtained from Archer Daniels Midland ("ADM") of Decatur, Illinois under the designations WhetPro® 75 or 80.

If desired, the protein may also be modified using techniques known in the art to improve its ability to disperse in an aqueous solution, which may be applied to the composition to release the botanical oil during and/or just prior to use as described in more detail below. Suitable modification techniques may include pH modification, denaturation, hydrolysis, acylation, reduction and oxidation. Just as an example, gluten may sometimes absorb water until it begins to repel excess water. This results in gluten molecules that are associated closely together such that they resist dispersion in aqueous solutions. To counteract this tendency, the protein may be treated with a pH modifier to increase its solubility in aqueous environments. Typically, the pH modifier is a basic reagent that can raise the pH of the protein, thereby causing it to become more soluble in aqueous solutions. Monovalent cation-containing basic reagents (hereafter "monovalent basic reagents") are particularly suitable for use in the present invention. Examples of such monovalent basic reagents include, for instance, alkali metal hydroxides (e.g., sodium hydroxide and ammonium hydroxide) and ammonia. Of course, multivalent reagents, such as alkaline metal hydroxides (e.g., calcium hydroxide) and alkaline metal oxides (e.g., calcium oxide), may also be employed if desired. When employed, the pH modifier may be present in an amount such that the pH of the protein is from 7 to 14, and in some embodiments, from 8 to 12.

Hydrolysis of the protein material may also improve water solubility, and can be effected by treating the protein with a hydrolytic enzyme. Many enzymes are known in the art which hydrolyze protein materials, including, but not limited to, proteases, pectinases, lactases, and chymotrypsin. Enzyme hydrolysis is effected by adding a sufficient amount of enzyme to an aqueous dispersion of protein material, typically from 0.1% to 10% enzyme by weight of the protein material, and treating the enzyme and protein dispersion. After sufficient hydrolysis has occurred the enzyme may be deactivated by heating, and the protein material may be precipitated from the solution by adjusting the pH of the solution to about the isoelectric point of the protein material.

The antimicrobial composition produced by the method of the present invention employs proteins in an amount of from 30 wt.% to 95 wt.%, in some embodiments from 40 wt.% to 90 wt.%, and in some embodiments, from 50 wt.% to 80 wt.%.

C.

Carrier Fluid

A carrier fluid is also employed in the antimicrobial composition to help render the protein more flowable under melt processing conditions and able to receive the botanical oil within its internal structure. The carrier fluid is glycerol or lactic acid.

If desired, the carrier fluid may be selected to have a certain pH (refers to the pH prior to incorporation into the antimicrobial composition). For example, carrier fluids having a relatively low pH can reduce the tendency of gluten proteins to aggregate during melt processing. Thus, when gluten proteins are employed, a carrier fluid may be selected that has a pH of 6 or less, in some embodiments from 1 to 5, and in some embodiments, from 2 to 4. Examples of such carrier fluids may include lactic acid. In other embodiments, it may be desirable to use carrier fluids having a higher pH, such as when the plant protein is not generally sensitive to pH. One example of such a carrier fluid is glycerol, which has a pH of about 6.

The amount of the carrier fluids employed depends in part on the nature of the selected botanical oil and protein, and is from 1 wt.% to 50 wt.%, in some embodiments from 5 wt.% to 30 wt.%, and in some embodiments, from 10 wt.% to 20 wt.%.

D.

Other Components

In addition to those noted above, still other additives may also be incorporated into the composition. For example, starch polymers, which are often found in commercially available protein compositions, may also be employed in the present invention. When employed, such starch polymers typically constitute from 1 wt.% to 50 wt.% of the composition, in some embodiments from 10 wt.% to 45 wt.%, and in some embodiments, from 20 wt.% to 40 wt.% of the composition.

Although starch polymers are produced in many plants, typical sources includes seeds of cereal grains, such as corn, waxy corn, wheat, sorghum, rice, and waxy rice; tubers, such as potatoes; roots, such as tapioca (i.e., cassava and manioc), sweet potato, and arrowroot; and the pith of the sago palm. Chemically modified starches may also be employed as they typically possess a higher degree of water sensitivity, and therefore can help facilitate water sensitivity during use. Such chemically modified starches may be obtained through typical processes known in the art (e.g., esterification, etherification, oxidation, acid hydrolysis, enzymatic hydrolysis). Starch ethers and/or esters may be particularly desirable, such as hydroxyalkyl starches or carboxymethyl starches. The hydroxyalkyl group of hydroxylalkyl starches may contain, for instance, 2 to 10 carbon atoms, in some embodiments from 2 to 6 carbon atoms, and in some embodiments, from 2 to 4 carbon atoms. Representative hydroxyalkyl starches such as hydroxyethyl starch, hydroxypropyl starch, hydroxybutyl starch, and derivatives thereof. Starch esters, for instance, may be prepared using a wide variety of anhydrides (e.g., acetic, propionic or butyric) organic acids, acid chlorides, or other esterification reagents. The degree of esterification may vary as desired, such as from 1 to 3 ester groups per glucosidic unit of the starch. The starch polymer may contain different weight percentages of amylose and amylopectin or different polymer molecular weights. High amylose starches contain greater than 50% by weight amylose and low amylose starches contain less than 50% by weight amylose. Although not required, low amylose starches having an amylose content of from 10% to 40% by weight, and in some embodiments, from 15% to 35% by weight, are particularly suitable for use in the present invention. Examples of such low amylose starches include corn starch and potato starch, both of which have an amylose content of approximately 20% by weight.

Dispersion aids may also be employed to help create a uniform dispersion of the oil/protein/carrier fluid and retard or prevent separation of the antimicrobial composition into constituent phases. When employed, the dispersion aid(s) typically constitute from 0.01 wt.% to 10 wt.%, in some embodiments from 0.1 wt.% to 5 wt.%, and in some embodiments, from 0.5 wt.% to 4 wt.% of the antimicrobial composition. Although any dispersion aid may generally be employed in the present invention, surfactants having a certain hydrophilic/lipophilic balance may improve the long-term stability of the composition. As is known in the art, the relative hydrophilicity or lipophilicity of an emulsifier can be characterized by the hydrophilic/lipophilic balance ("HLB") scale, which measures the balance between the hydrophilic and lipophilic solution tendencies of a compound. The HLB scale ranges from 0.5 to approximately 20, with the lower numbers representing highly lipophilic tendencies and the higher numbers representing highly hydrophilic tendencies. In some embodiments of the present invention, the HLB value of the surfactants is from 1 to 15, in some embodiments from 1 to 12 and in some embodiments, from 2 to 10. If desired, two or more surfactants may be employed that have HLB values either below or above the desired value, but together have an average HLB value within the desired range.

One particularly suitable class of surfactants for use in the present invention are nonionic surfactants, which typically have a hydrophobic base (e.g., long chain alkyl group or an alkylated aryl group) and a hydrophilic chain (e.g., chain containing ethoxy and/or propoxy moieties). For instance, some suitable nonionic surfactants that may be used include, but are not limited to, ethoxylated alkylphenols, ethoxylated and propoxylated fatty alcohols, polyethylene glycol ethers of methyl glucose, polyethylene glycol ethers of sorbitol, ethylene oxide-propylene oxide block copolymers, ethoxylated esters of fatty (C₈ -C₁₈) acids, condensation products of ethylene oxide with long chain amines or amides, condensation products of ethylene oxide with alcohols, fatty acid esters, monoglyceride or diglycerides of long chain alcohols, and mixtures thereof. In one particular embodiment, the nonionic surfactant may be a fatty acid ester, such as a sucrose fatty acid ester, glycerol fatty acid ester, propylene glycol fatty acid ester, sorbitan fatty acid ester, pentaerythritol fatty acid ester or sorbitol fatty acid ester. The fatty acid used to form such esters may be saturated or unsaturated, substituted or unsubstituted, and may contain from 6 to 22 carbon atoms, in some embodiments from 8 to 18 carbon atoms, and in some embodiments, from 12 to 14 carbon atoms. In one particular embodiment, mono- and di-glycerides of fatty acids may be employed in the present invention.

The composition may also contain a preservative or preservative system to inhibit the growth of microorganisms over an extended period of time. Suitable preservatives may include, for instance, alkanols, disodium EDTA (ethylenediamine tetraacetate), EDTA salts, EDTA fatty acid conjugates, isothiazolinone, benzoic esters (parabens) (e.g., methylparaben, propylparaben, butylparaben, ethylparaben, isopropylparaben, isobutylparaben, benzylparaben, sodium methylparaben, and sodium propylparaben), benzoic acid, propylene glycols, sorbates and urea derivatives (e.g., diazolindinyl urea). Other suitable preservatives include those sold by Sutton Labs, such as "Germall 115" (amidazolidinyl urea), "Germall II" (diazolidinyl urea), and "Germall Plus" (diazolidinyl urea and iodopropynyl butylcarbonate). Another suitable preservative is Kathon CG®, which is a mixture of methylchloroisothiazolinone and methylisothiazolinone available from Rohm &amp; Haas; Mackstat H 66 (available from McIntyre Group, Chicago, IL). Still another suitable preservative system is a combination of 56% propylene glycol, 30% diazolidinyl urea, 11% methylparaben, and 3% propylparaben available under the name GERMABEN® II from International Specialty Products of Wayne, New Jersey.

To better enhance the benefits to consumers, other optional ingredients may also be used. For instance, some classes of ingredients that may be used include, but are not limited to: antioxidants (product integrity); anti-reddening agents, such as aloe extract; astringents-cosmetic (induce a tightening or tingling sensation on skin); colorants (impart color to the product); deodorants (reduce or eliminate unpleasant odor and protect against the formation of malodor on body surfaces); fragrances (consumer appeal); opacifiers (reduce the clarity or transparent appearance of the product); skin conditioning agents; skin exfoliating agents (ingredients that increase the rate of skin cell turnover such as alpha hydroxy acids and beta hydroxyacids); skin protectants (a drug product which protects injured or exposed skin or mucous membrane surface from harmful or annoying stimuli); and thickeners (to increase viscosity).

While a wide variety of different components may be employed, it is typically desired that the antimicrobial composition is formed without the use of solvents, particularly organic solvents, such as organic alcohols (e.g., ethanol). Not only does this enhance manufacturing efficiency, but it also limits the evaporation of the botanical oil that might otherwise be encountered during removal of the solvent. While the composition may be generally free of such solvents, it should of course be understood that a small amount may still be present in the resulting composition. Regardless, the composition typically contains solvents in an amount less than 20 wt.%, in some embodiments less than 10 wt.%, and in some embodiments, from 0.01 wt.% to 5 wt.%.

II.

Melt Processing Technique

As indicated above, the antimicrobial composition produced by the method of the present invention is formed by processing the components together in a melt blending device (e.g., extruder). The mechanical shear and heat provided by the device allows the components to be blended together in a highly efficient manner without the use of a solvent. Batch and/or continuous melt blending techniques may be employed in the present invention. For example, a mixer/kneader, Banbury mixer, Farrel continuous mixer, single-screw extruder, twin-screw extruder or roll mill, may be utilized. One particularly suitable melt-blending device is a co-rotating, twin-screw extruder (e.g., USALAB twin-screw extruder available from Thermo Electron Corporation of Stone, England or an extruder available from Werner-Pfleiderer from Coperion Ramsey, New Jersey). The raw materials (e.g., botanical oil, protein and carrier fluid) may be supplied to the melt blending device separately and/or as a blend. For example, the protein and/or botanical oil may be initially fed to a feeding port of the twin-screw extruder. Thereafter, a carrier fluid may be injected into the extruder downstream from the botanical oil and protein. Alternatively, the components may be simultaneously fed to the feed throat of the extruder or separately at a different point along its length.

Regardless, the materials are dispersively blended under low shear/pressure and at a low temperature to minimize protein dissociation associated with aggregation. Nevertheless, the temperature is still typically slightly at or above the softening point of the protein. Thus the melt blending occurs at a temperature of from 20°C to 100°C, in some embodiments, from 30°C to 80°C, and in some embodiments, from 40°C to 70°C. Likewise, the apparent shear rate during melt blending ranges from 100 seconds^-1 to 5,000 seconds^-1, in some embodiments from 200 seconds^-1 to 2,000 seconds^-1, and in some embodiments, from 400 seconds^-1 to 1,200 seconds^-1. The apparent shear rate is equal to <i>4Q</i>/<i>πR³</i>, where Q is the volumetric flow rate ("m³/s") of the polymer melt and R is the radius ("m") of the capillary (e.g., extruder die) through which the melted polymer flows. The apparent melt viscosity of the resulting antimicrobial composition may be relatively low, such as from 1 to 100 Pascal seconds (Pa·s), in some embodiments from 5 to 60 Pa·s, and in some embodiments, from 20 to 50 Pa·s, as determined at a temperature of 160°C and a shear rate of 1000 sec^-1. The melt flow index (190°C, 2.16 kg) of the composition may also range from 0.05 to 50 grams per 10 minutes, in some embodiments from 0.1 to 15 grams per 10 minutes, and in some embodiments, from 0.5 to 5 grams per 10 minutes.

Once formed, the antimicrobial composition produced by the method of the present invention may be used in a variety of forms, such as particles, lotion, cream, jelly, liniment, ointment, salve, oil, foam, gel, film, wash, coating, liquid, capsule, tablet or concentrate. In one particular embodiment, for example, the antimicrobial composition may be formed into a film, either alone or in conjunction with an additional film-forming material. The film may be used in a wide variety of applications, such as in the packaging of items (e.g., food products, medical products, garments, garbage, or absorbent articles (e.g., diapers). The film may have a mono-layered or multi-layered structure. Multilayer films normally contain at least one base layer and at least one skin layer, but may contain any number of layers desired. The base layer and/or the skin layer may contain the antimicrobial composition produced by the method of the present invention. Any known technique may be used to form a film from the compounded material, including blowing, casting or flat die extruding. In one particular embodiment, the film may be formed by a blown process in which a gas (e.g., air) is used to expand a bubble of the extruded polymer blend through an annular die. The bubble is then collapsed and collected in flat film form. Processes for producing blown films are described, for instance, in <patcit id="pcit0001" dnum="US3354506A"><text>U.S. Patent No. 3,354,506 to Raley</text></patcit>; <patcit id="pcit0002" dnum="US3650649A"><text>U.S. Patent No. 3,650,649 to Schippers</text></patcit>; and <patcit id="pcit0003" dnum="US3801429A"><text>U.S. Patent No. 3,801,429 to Schrenk et al.</text></patcit>, as well as <patcit id="pcit0004" dnum="US20050245162A" dnum-type="L"><text>U.S. Patent Application Publication Nos. 2005/0245162 to McCormack, et al.</text></patcit> and <patcit id="pcit0005" dnum="US20030068951A"><text>2003/0068951 to Boggs, et al.</text></patcit>. In yet another embodiment, however, the film is formed using a casting technique.

Besides being formed into a film, the antimicrobial composition produced by the method of the present invention may also be formed into particles and applied to other types of articles. Powderization may be accomplished using any of a variety of known techniques. Suitable pulverizing techniques may include, for instance, cryogenic disk mill or hammer mill, solid state shear pulverization using cold extrusion technology, double stream mills(e.g., Type PSKM or PPSM mills available from Pallmann Industries), and other known powderization methods. Cryogenic downsizing techniques or cold extrusion pulverization techniques may be particularly suitable as such techniques limit the degree to which the volatile botanical oil is heated and lost during powder formation. Examples of such techniques are described in more detail, for instance, in <patcit id="pcit0006" dnum="US5395055A"><text>U.S. Patent No. 5,395,055 to Shutov, et al.</text></patcit> The shape of the particles may vary as desired, such as spherical, nodular or flake. The average size of the particles may also be selected to optimize the ability of the botanical oil to be released during use. More particularly, the present inventors have discovered that smaller particle sizes can generally result in a greater release rate of the oil when dispersed in an aqueous solution due to their high surface area to volume ratio. However, at too small of a size, the botanical oil may become unstable during storage and actually begin to leach out of the particles prior to use. In this regard, the present inventors have discovered that an average size of from 10 to 3,000 micrometers, in some embodiments from 50 to 800 micrometers, and in some embodiments, from 100 to 600 micrometers, can help achieve a good balance between stability and releasibility.

Regardless of its particular form, the antimicrobial particles may be applied to a wide variety of different articles for imparting antimicrobial efficacy. In one particular embodiment, the composition is applied to a wipe. Such wipes may be used to reduce microbial or viral populations on a hard surface (e.g., sink, table, counter or sign) or surface on a user/patient (e.g., skin, mucosal membrane, such as in the mouth, nasal passage, stomach, or vagina, wound site or surgical site). The wipe may provide an increased surface area to facilitate contact of the composition with microorganisms. In addition, the wipe may also serve other purposes, such as providing water absorption, or barrier properties. The wipe may also eliminate microorganisms through frictional forces imparted to the surface.

The wipe may be formed from any of a variety of materials as is well known in the art. Typically, however, the wipe includes a fibrous web that contains absorbent fibers. For example, the wipe may be a paper product containing one or more paper webs, such as facial tissue, bath tissue, paper towels or napkins. The paper product may be single-ply in which the web forming the product includes a single layer or is stratified (i.e., has multiple layers), or multi-ply, in which the webs forming the product may themselves be either single or multi-layered. Normally, the basis weight of such a paper product is less than 120 grams per square meter ("gsm"), in some embodiments less than 80 gsm, in some embodiments less than 60 grams per square meter, and in some embodiments, from 10 to 60 gsm. Any of a variety of materials can also be used to form the paper web(s) of the product. For example, the material used to make the paper product may include absorbent fibers formed by a variety of pulping processes, such as kraft pulp, sulfite pulp or thermomechanical pulp. The pulp fibers may include softwood fibers having an average fiber length of greater than 1 mm and particularly from 2 to 5 mm based on a length-weighted average. Such softwood fibers can include, but are not limited to, northern softwood, southern softwood, redwood, red cedar, hemlock, pine (e.g., southern pines), spruce (e.g., black spruce) and combinations thereof. Exemplary commercially available pulp fibers suitable for the present invention include those available from Kimberly-Clark Corporation under the trade designations "Longlac-19". Hardwood fibers, such as eucalyptus, maple, birch and aspen, can also be used. In certain instances, eucalyptus fibers may be particularly desired to increase the softness of the web. Eucalyptus fibers can also enhance the brightness, increase the opacity, and change the pore structure of the web to increase its wicking ability. Moreover, if desired, secondary fibers obtained from recycled materials may be used, such as fiber pulp from sources such as, for example, newsprint, reclaimed paperboard, and office waste. Further, other natural fibers can also be used in the present invention, such as abaca, sabai grass, milkweed floss, pineapple leaf, bamboo and algae. In addition, in some instances, synthetic fibers can also be utilized.

If desired, the absorbent fibers (e.g., pulp fibers) may be integrated with synthetic fibers to form a composite. Synthetic thermoplastic fibers may also be employed in the nonwoven web, such as those formed from polyolefins, e.g., polyethylene, polypropylene and polybutylene; polytetrafluoroethylene; polyesters, e.g., polyethylene terephthalate; polyvinyl acetate; polyvinyl chloride acetate; polyvinyl butyral; acrylic resins, e.g., polyacrylate, polymethylacrylate and polymethylmethacrylate; polyamides, e.g., nylon; polyvinyl chloride; polyvinylidene chloride; polystyrene; polyvinyl alcohol; polyurethanes; polylactic acid; polyhydroxyalkanoate and copolymers thereof. Because many synthetic thermoplastic fibers are inherently hydrophobic (i.e., non-wettable), such fibers may optionally be rendered more hydrophilic (i.e., wettable) by treatment with a surfactant solution before, during, and/or after web formation. Other known methods for increasing wettability may also be employed, such as described in <patcit id="pcit0007" dnum="US5057361A"><text>U.S. Patent No. 5,057,361 to Savovitz, et al</text></patcit>. The relative percentages of such fibers may vary over a wide range depending on the desired characteristics of the composite. For example, the composite may contain from 1 wt.% to 60 wt.%, in some embodiments from 5 wt.% to 50 wt.%, and in some embodiments, from 10 wt.% to 40 wt.% synthetic polymeric fibers. The composite may likewise contain from 40 wt.% to 99 wt.%, in some embodiments from 50 wt.% to 95 wt.%, and in some embodiments, from 60 wt.% to 90 wt.% absorbent fibers.

Composites, such as described above, may be formed using a variety of known techniques. For example, a nonwoven composite may be formed that is a "coform material" that contains a mixture or stabilized matrix of thermoplastic fibers and a second non-thermoplastic material. As an example, coform materials may be made by a process in which at least one meltblown die head is arranged near a chute through which other materials are added to the web while it is forming. Such other materials may include, but are not limited to, fibrous organic materials such as woody or non-woody pulp such as cotton, rayon, recycled paper, pulp fluff and also superabsorbent particles, inorganic and/or organic absorbent materials and treated polymeric staple fibers. Some examples of such coform materials are disclosed in <patcit id="pcit0008" dnum="US4100324A"><text>U.S. Patent Nos. 4,100,324 to Anderson, et al.</text></patcit>; <patcit id="pcit0009" dnum="US5284703A"><text>5,284,703 to Everhart, et al.</text></patcit>; and <patcit id="pcit0010" dnum="US5350624A"><text>5,350,624 to Georger, et al</text></patcit>. Alternatively, the nonwoven composite may be formed be formed by hydraulically entangling staple length fibers and/or filaments with high-pressure jet streams of water. Various techniques for hydraulically entangling fibers are generally are disclosed, for example, in <patcit id="pcit0011" dnum="US3494821A"><text>U.S. Patent Nos. 3,494,821 to Evans </text></patcit>and <patcit id="pcit0012" dnum="US4144370A"><text>4,144,370 to Bouolton</text></patcit>. Hydraulically entangled nonwoven composites of continuous filaments (e.g., spunbond web) and natural fibers (e.g., pulp) are disclosed, for example, in <patcit id="pcit0013" dnum="US5284703A"><text>U.S. Patent Nos. 5,284,703 to Everhart, et al.</text></patcit> and <patcit id="pcit0014" dnum="US6315864B"><text>6,315,864 to Anderson, et al</text></patcit>. Hydraulically entangled nonwoven composite of staple fiber blends (e.g., polyester and rayon) and natural fibers (e.g., pulp), also known as "spunlaced" fabrics, are described, for example, in <patcit id="pcit0015" dnum="US5240764A"><text>U.S. Patent No. 5,240,764 to Haid, et al</text></patcit>.

Regardless of the materials or processes utilized to form the wipe, the basis weight of the wipe is typically from 20 to 200 grams per square meter ("gsm"), and in some embodiments, between 35 to 100 gsm. Lower basis weight products may be particularly well suited for use as light duty wipes, while higher basis weight products may be better adapted for use as industrial wipes.

The wipe may assume a variety of shapes, including but not limited to, generally circular, oval, square, rectangular, or irregularly shaped. Each individual wipe may be arranged in a folded configuration and stacked one on top of the other to provide a stack of wet wipes. Such folded configurations are well known to those skilled in the art and include c-folded, z-folded and quarter-folded configurations. For example, the wipe may have an unfolded length of from 2.0 to 80.0 centimeters, and in some embodiments, from 10.0 to 25.0 centimeters. The wipes may likewise have an unfolded width of from 2.0 to 80.0 centimeters, and in some embodiments, from 10.0 to 25.0 centimeters. The stack of folded wipes may be placed in the interior of a container, such as a plastic tub, to provide a package of wipes for eventual sale to the consumer. Alternatively, the wipes may include a continuous strip of material which has perforations between each wipe and which may be arranged in a stack or wound into a roll for dispensing. Various suitable dispensers, containers, and systems for delivering wipes are described in <patcit id="pcit0016" dnum="US5785179A"><text>U.S. Patent Nos. 5,785,179 to Buczwinski, et al.</text></patcit>; <patcit id="pcit0017" dnum="US5964351A"><text>5,964,351 to Zander</text></patcit>; <patcit id="pcit0018" dnum="US6030331A"><text>6,030,331 to Zander</text></patcit>; <patcit id="pcit0019" dnum="US6158614A"><text>6,158,614 to Haynes, et al.</text></patcit>; <patcit id="pcit0020" dnum="US6269969B"><text>6,269,969 to Huang, et al.</text></patcit>; <patcit id="pcit0021" dnum="US6269970B"><text>6,269,970 to Huang, et al.</text></patcit>; and <patcit id="pcit0022" dnum="US6273359B"><text>6,273,359 to Newman, et al.</text></patcit>

The composition may be incorporated into the wipe in a variety of different ways. For example, the composition may be applied to a surface of the wipe using known techniques, such as printing, dipping, spraying, melt extruding, coating (e.g., solvent coating, powder coating or brush coating) or foaming. If desired, the composition may be applied in a pattern that covers from 5% to 95%, in some embodiments from 10% to 90%, and in some embodiments, from 20% to 75% of a surface of the wipe. Such patterned application may have various benefits, including enhanced aesthetic appeal or improved absorbency. The particular type or style of the pattern is not a limiting factor of the invention, and may include, for example, any arrangement of stripes, bands, dots, or other geometric shape. The pattern may include indicia (e.g., trademarks, text, and logos), floral designs, abstract designs or any configuration of artwork. It should be appreciated that the "pattern" may take on virtually any desired appearance. The composition may also be blended with the fibers used to form the wipe. This may be particularly useful when the composition is in the form of particles. For example, such particles may be blended with the absorbent fibers (e.g., pulp fibers or staple fibers) during hydraulic entanglement or coforming. The particles may also be incorporated into the thermoplastic material of the wipe (e.g., meltblown web) using known techniques.

The amount of the antimicrobial composition on the wipe may vary depending on the nature of the substrate and its intended application. For example, the add-on level of the composition may be from 5% to 100%, in some embodiments from 10% to 80%, and in some embodiments, from 20% to 70%. The "add-on level" is determined by subtracting the weight of the untreated substrate from the weight of the treated substrate, dividing this calculated weight by the weight of the untreated substrate, and then multiplying by 100%. Lower add-on levels may provide optimum functionality of the substrate, while higher add-on levels may provide optimum antimicrobial efficacy.

To use the composition, an aqueous solution may simply be added, thereby dispersing the protein and releasing the botanical oil. The aqueous solution may contain only water, or it may contain water in combination with other components. For example, a weak acid may be employed to help disperse the protein and facilitate the release of the oil upon contact with the aqueous solution. Suitable acids for this purpose may include, for instance, organic carboxylic acids, such as citric acid, oxalic acid, lactic acid and acetic acid. Regardless, the present inventors have surprisingly discovered that the amount of the botanical oil released into the aqueous solution can be even greater than the normal solubility limit of the oil in water. Without intending to be limited by theory, it is believed that this can be achieved because the physical structure of the protein is able to effectively "carry" the volatile into the released solution. For example, the solubility limit of thymol in water (at 25°C) is typically about 0.1 wt.%. When released from the composition produced by the method of the present invention, however, the concentration of thymol in the released solution can be greater than 0.1 wt.%, in some embodiments greater than 0.5 wt.%, in some embodiments from 1 wt.% to 10 wt.%., and in some embodiments, from 2 wt.% to 8 wt.%.

The present inventors have discovered that the composition produced by the method of the present invention may inhibit (e.g., reduce by a measurable amount or to prevent entirely) the growth of one or more microorganisms when exposed thereof. Examples of microorganisms that may be inhibited include bacteria, protozoa, algae, and fungi (e.g., molds and yeast). Furthermore is possible to use this invention to inactivate viruses, prions and other infectious particles. For example, the composition may inhibit the growth of several medically significant bacteria groups, such as Gram negative rods (e.g., <i>Entereobacteria</i>); Gram negative curved rods (e.g., <i>Heliobacter</i> and <i>Campylobacter</i>); Gram negative cocci (e.g., <i>Neisseria</i>); Gram positive rods (e.g., <i>Bacillus</i> and <i>Clostridium</i>, etc.); Gram positive cocci (e.g., <i>Staphylococcus</i> and <i>Streptococcus</i>); obligate intracellular parasites (e.g,. <i>Ricckettsia</i> and <i>Chlamydia</i>); acid fast rods (e.g., <i>Myobacterium</i> and <i>Nocardia</i>); spirochetes (e.g., <i>Treponema</i> and <i>Borellia</i>); and mycoplasmas (i.e., tiny bacteria that lack a cell wall). Particularly species of bacteria that may be inhibited with the composition produced by the method of the present invention include <i>Escherichia coli</i> (Gram negative rod), <i>Klebsiella pneumonia</i> (Gram negative rod), <i>Streptococcus</i> (Gram positive cocci), <i>Salmonella choleraesuis</i> (Gram negative rod), <i>Staphyloccus aureus</i> (Gram positive cocci), and <i>P. aeruginosa</i> (Gram negative rod). In addition to bacteria, other microorganisms of interest include fungi (e.g., <i>Aspergillus niger</i>) and yeasts (e.g., <i>Candida albicans</i>).

Upon exposure for a certain period of time, the composition may provide a log reduction of at least 2, in some embodiments at least 3, in some embodiments at least 4, and in some embodiments, at least 5 (e.g., 6). Log reduction, for example, may be determined from the % population killed by the composition according to the following correlations: <tables id="tabl0001" num="0001"><table frame="none"><tgroup cols="2" colsep="0"><colspec colnum="1" colname="col1" colwidth="24mm" /><colspec colnum="2" colname="col2" colwidth="25mm" /><thead><row><entry valign="top">% Reduction</entry><entry valign="top">Log Reduction</entry></row></thead><tbody><row rowsep="0"><entry>90</entry><entry>1</entry></row><row rowsep="0"><entry>99</entry><entry>2</entry></row><row rowsep="0"><entry>99.9</entry><entry>3</entry></row><row rowsep="0"><entry>99.99</entry><entry>4</entry></row><row rowsep="0"><entry>99.999</entry><entry>5</entry></row><row rowsep="0"><entry>99.9999</entry><entry>6</entry></row></tbody></tgroup></table></tables>

Such a log reduction may be achieved in accordance with the present invention after only a relatively short exposure time. For example, the desired log reduction may be achieved after exposure for only 30 minutes, in some embodiments 15 minutes, in some embodiments 10 minutes, in some embodiments 5 minutes, in some embodiments 1 minute, and in some embodiments, 30 seconds.

The present invention may be better understood with reference to the following examples.

Materials Employed

<ul id="ul0002" list-style="bullet" compact="compact"><li>Thymol (99.5% purity) was obtained from Sigma-Aldrich.</li><li>Carvacrol (98% purity) was obtained from Sigma-Aldrich.</li><li>Eucalyptol (C80601) was obtained from Sigma-Aldrich.</li><li>Soy Protein Flour (50% protein, 50% starch) was obtained from ADM.</li><li>WhetPro® 75 vital wheat gluten (75% protein, 25% starch) was obtained from ADM.</li><li>Emery 917 Glycerine (or Glycerol) was obtained from Cognis Oleochemicals.</li><li>L-Lactic Acid (Purac)</li></ul>

Test Methods

Thymol Stability

Samples were placed in an oven at 40°C, 50°C, or 55°C for a certain number of days. The residual thymol level was determined through "High Performance Liquid Chromatography (HPLC) analysis." More particularly, the thymol level in each sample was determined by generating a thymol calibration curve by the following method. Approximately 70 mg thymol was weighed into a 100-mL volumetric flask. Approximately 50-mL of a 0.1% acetic acid:IPA mixture (50:50) was added to the flask and the contents swirled to promote dissolution. The volume was diluted with a 0.1% acetic acid:IPA mixture (50:50) and subsequent dilutions were performed to generate a calibration curve with a concentration range of approximately 700 µg/mL to 70 µg/mL. Samples were prepared as follows. Approximately 100 mg of sample was used for each code, where each code was analyzed in duplicate at every pull point. The measured material was cut up into small pieces and placed into a 40-mL vial. To each vial, 10.0 mL 0.1% acetic acid was added and the contents were shaken and sonicated for 30 minutes periods until the sample is dispersed. To each vial, 10.0 mL IPA was added and the contents were sonicated for 10 minutes to promote mixing and extraction of thymol. The resulting solutions were filtered through nylon filters prior to injection. Thymol levels were calculated using the thymol calibration curve described above. <tables id="tabl0002" num="0002"><table frame="none"><title><u>HPLC Equipment &amp; Conditions</u></title><tgroup cols="2" colsep="0" rowsep="0"><colspec colnum="1" colname="col1" colwidth="37mm" /><colspec colnum="2" colname="col2" colwidth="46mm" /><tbody><row><entry>Column:</entry><entry>Phenomenex NH₂</entry></row><row><entry>Column Temperature:</entry><entry>Ambient</entry></row><row><entry>Mobile Phase:</entry><entry>50:50 (IPA:0.1 % acetic acid)</entry></row><row><entry>Flow rate:</entry><entry>0.6 mL/min.</entry></row><row><entry>Injection volume:</entry><entry>15 microliters</entry></row><row><entry>ELS detection:</entry><entry>280 nm</entry></row></tbody></tgroup></table></tables>

Thymol Concentrations in Extractions

An aliquot of a sample was centrifuged at approximately 5000 rpm until visible settling occurred (approximately 30 minutes). The solution was filtered using two (2) different types of syringe filters: (1) Pall Life Acrodisc 13 mm 0.2 micron nylon membrane and (2) Whatman Puradisc-0.2 micron polyethersulfone membrane with polypropylene housing. 1.0 mL of the centrifuged solution was pipetted into a 10-mL flask. The contents were dissolved and diluted with a 0.1% AA:IPA (50:50) solution to volume. The solution was then filtered with Pall Acrodisc 0.45 micron nylon membrane. The thymol concentration was determined through "High Performance Liquid Chromatography (HPLC) analysis" according to the following conditions: <tables id="tabl0003" num="0003"><table frame="none"><title><u>HPLC Equipment &amp; Conditions</u></title><tgroup cols="2" colsep="0" rowsep="0"><colspec colnum="1" colname="col1" colwidth="30mm" /><colspec colnum="2" colname="col2" colwidth="87mm" /><tbody><row><entry>HPLC:</entry><entry>Agilent 1100 HPLC system.</entry></row><row><entry>Column:</entry><entry>Phenomenex Luna NH₂ (5 µm, 250 mm x 4.6 mm)-ambient</entry></row><row><entry>Detector:</entry><entry>UV/Vis at 280 nm</entry></row><row><entry>Mobile Phases:</entry><entry>(75:25) (IPA: 0.1% acetic acid)</entry></row><row><entry>Flow Rate:</entry><entry>approximately 0.6 mL/min.</entry></row><row><entry>Injection Volume:</entry><entry>approximately 15 µL</entry></row><row><entry>Run Time:</entry><entry>6 minutes</entry></row></tbody></tgroup></table></tables>

Zone of Inhibition

To determine antimicrobial efficacy, a zone of inhibition test was performed. More specifically, a 0.05 g sample was placed on a freshly spreading lawn of test microorganism on TSA (Trypticase Soy Agar). Two microorganisms were used, <i>Staphylococcus aureus</i> (ATCC #27660) as a Gram positive bacteria and <i>Escherichia coli</i> as a Gram negative bacteria (ATCC #25922). After 24 hours incubation at 37°C, plates were measured for clear zones of inhibition surrounding each sample (Clear zone(mm) = diameter of clear zone - sample (wipe) diameter).

Microplate Assay

To determine the germicidal efficacy of an extracted thymol solution, a microplate germicidal assay was performed. In this method, the test solution was brought into contact with 60 wells of test microorganisms (4*10⁶CFU (colony forming unit)/well) coated on the bottom of 96 well flat plates for 4½ minutes. At the end of the contact time, 200 µL of a "Letheen" neutralizing broth (included 0.5% Tween 80) was added to each well to deactivate the active ingredients. After addition of the neutralizer, 50 µL of TSB (Tryptic Soy Broth) was added and then the microplate was incubated to allow for out-growth of survivors. After incubation, the number of wells showing growth of the target microorganism was recorded. If the media in the well was turbid, then the well was counted as a failure to disinfect. If the well was not turbid after incubation, then the well was recorded as achieving disinfection. All tests were performed against two different microorganisms, <i>Staphylococcus aureus</i> (ATCC #6538) as Gram positive bacteria and <i>Pseudomonas aeruginosa</i> (ATCC #15442) as Gram negative bacteria.

<u>EXAMPLE 1</u>

A "PRISM USALAB 16" lab scale twin screw extruder was employed to melt process five (5) different samples of protein (WhetPro® 75 or soy flour), glycerol, and thymol. The extruder contained eleven (11) different zones, although zones 1 through 5 were not utilized in this Example. Temperature zone 11 was a strand die. The protein and thymol were pre-blended (6.6 wt% thymol and 93.4 %) and subsequently added to the extruder at zone 6 at a feed rate of 0.5 Ibs/hr (0.06 g/s). Glycerol was then added at zone 7 at a feed rate of 0.21 Ib/hr (0.026 g/s). The screw configuration was composed of conveying elements at zones 6 and 7, kneading blocks at zones 8 and 9, and conveying elements at zone 10. The screw speed was 50 rpm. The resulting strand was pelletized to form a pellet with a size of about 3 mm in diameter. The temperature profile for each of the five (5) blend samples is set forth below. <tables id="tabl0004" num="0004"><table frame="all"><tgroup cols="9"><colspec colnum="1" colname="col1" colwidth="24mm" /><colspec colnum="2" colname="col2" colwidth="23mm" /><colspec colnum="3" colname="col3" colwidth="30mm" /><colspec colnum="4" colname="col4" colwidth="15mm" /><colspec colnum="5" colname="col5" colwidth="15mm" /><colspec colnum="6" colname="col6" colwidth="15mm" /><colspec colnum="7" colname="col7" colwidth="15mm" /><colspec colnum="8" colname="col8" colwidth="17mm" /><colspec colnum="9" colname="col9" colwidth="17mm" /><thead><row><entry morerows="1" align="center" valign="middle">Sample</entry><entry morerows="1" align="center" valign="middle">Protein Type</entry><entry morerows="1" align="center" valign="middle">Target Temp. (°C)</entry><entry namest="col4" nameend="col9" align="center" valign="middle">Temperature Profile (°C)</entry></row><row><entry align="center" valign="middle">Zone 6</entry><entry align="center" valign="middle">Zone 7</entry><entry align="center" valign="middle">Zone 8</entry><entry align="center" valign="middle">Zone 9</entry><entry align="center" valign="middle">Zone 10</entry><entry align="center" valign="middle">Zone 11</entry></row></thead><tbody><row><entry align="center" valign="middle">1</entry><entry align="center" valign="middle">Gluten</entry><entry align="center" valign="middle">Ambient</entry><entry align="center" valign="middle">28</entry><entry align="center" valign="middle">31</entry><entry align="center" valign="middle">32</entry><entry align="center" valign="middle">34</entry><entry align="center" valign="middle">32</entry><entry align="center" valign="middle">33</entry></row><row><entry align="center" valign="middle">2</entry><entry align="center" valign="middle">Gluten</entry><entry align="center" valign="middle">50°C</entry><entry align="center" valign="middle">27</entry><entry align="center" valign="middle">31</entry><entry align="center" valign="middle">34</entry><entry align="center" valign="middle">40</entry><entry align="center" valign="middle">50</entry><entry align="center" valign="middle">41</entry></row><row><entry align="center" valign="middle">3</entry><entry align="center" valign="middle">Gluten</entry><entry align="center" valign="middle">90°C</entry><entry align="center" valign="middle">29</entry><entry align="center" valign="middle">33</entry><entry align="center" valign="middle">37</entry><entry align="center" valign="middle">49</entry><entry align="center" valign="middle">90</entry><entry align="center" valign="middle">48</entry></row><row><entry align="center" valign="middle">4*</entry><entry align="center" valign="middle">Gluten</entry><entry align="center" valign="middle">120°C</entry><entry align="center" valign="middle">35</entry><entry align="center" valign="middle">42</entry><entry align="center" valign="middle">51</entry><entry align="center" valign="middle">68</entry><entry align="center" valign="middle">120</entry><entry align="center" valign="middle">67</entry></row><row><entry align="center" valign="middle">5 (Reference)</entry><entry align="center" valign="middle">Soy flour</entry><entry align="center" valign="middle">90°C</entry><entry align="center" valign="middle">25</entry><entry align="center" valign="middle">28</entry><entry align="center" valign="middle">31</entry><entry align="center" valign="middle">42</entry><entry align="center" valign="middle">90</entry><entry align="center" valign="middle">61</entry></row></tbody></tgroup><tgroup cols="9" rowsep="0"><colspec colnum="1" colname="col1" colwidth="24mm" /><colspec colnum="2" colname="col2" colwidth="23mm" /><colspec colnum="3" colname="col3" colwidth="30mm" /><colspec colnum="4" colname="col4" colwidth="15mm" /><colspec colnum="5" colname="col5" colwidth="15mm" /><colspec colnum="6" colname="col6" colwidth="15mm" /><colspec colnum="7" colname="col7" colwidth="15mm" /><colspec colnum="8" colname="col8" colwidth="17mm" /><colspec colnum="9" colname="col9" colwidth="17mm" /><tbody><row><entry namest="col1" nameend="col9" align="justify">* Sample processed outside the scope of claim 1</entry></row></tbody></tgroup></table></tables>

After processing, it was determined that Samples 1-5 retained 90.8 wt.%, 88.2 wt.%, 88.4 wt.%, 85.4 wt.%, and 99.4 wt.%, respectively, of their initial thymol levels. Once formed, the samples were put into an air tight bag and placed in - 10°C freezer. The resulting samples were tested for thymol stability and zone of inhibition using the test methods described above. The results are set forth below in Tables 1 and 2. <tables id="tabl0005" num="0005"><table frame="all"><title><b>Table 1: Thymol Level After Aging at 40°C</b></title><tgroup cols="7"><colspec colnum="1" colname="col1" colwidth="24mm" /><colspec colnum="2" colname="col2" colwidth="14mm" /><colspec colnum="3" colname="col3" colwidth="14mm" /><colspec colnum="4" colname="col4" colwidth="16mm" /><colspec colnum="5" colname="col5" colwidth="16mm" /><colspec colnum="6" colname="col6" colwidth="16mm" /><colspec colnum="7" colname="col7" colwidth="16mm" /><thead><row><entry morerows="1" align="center">Sample</entry><entry namest="col2" nameend="col7" align="center" valign="middle">Thymol level (wt.%)</entry></row><row><entry align="center" valign="middle">1 day</entry><entry align="center" valign="middle">4 days</entry><entry align="center" valign="middle">10 days</entry><entry align="center" valign="middle">21 days</entry><entry align="center" valign="middle">39 days</entry><entry align="center" valign="middle">60 days</entry></row></thead><tbody><row><entry align="center" valign="middle">1</entry><entry align="center" valign="middle">4.07</entry><entry align="center" valign="middle">3.75</entry><entry align="center" valign="middle">4.09</entry><entry align="center" valign="middle">3.83</entry><entry align="center" valign="middle">3.69</entry><entry align="center" valign="middle">3.44</entry></row><row><entry align="center" valign="middle">2</entry><entry align="center" valign="middle">4.04</entry><entry align="center" valign="middle">3.95</entry><entry align="center" valign="middle">3.78</entry><entry align="center" valign="middle">3.91</entry><entry align="center" valign="middle">4.37</entry><entry align="center" valign="middle">3.97</entry></row><row><entry align="center" valign="middle">3</entry><entry align="center" valign="middle">4.42</entry><entry align="center" valign="middle">4.4</entry><entry align="center" valign="middle">4.14</entry><entry align="center" valign="middle">4.37</entry><entry align="center" valign="middle">3.99</entry><entry align="center" valign="middle">3.88</entry></row><row><entry align="center" valign="middle">4 *</entry><entry align="center" valign="middle">4.16</entry><entry align="center" valign="middle">4.49</entry><entry align="center" valign="middle">4.57</entry><entry align="center" valign="middle">4.72</entry><entry align="center" valign="middle">4.52</entry><entry align="center" valign="middle">3.99</entry></row><row><entry align="center" valign="middle">5 (Reference)</entry><entry align="center" valign="middle">4.21</entry><entry align="center" valign="middle">4.51</entry><entry align="center" valign="middle">4.42</entry><entry align="center" valign="middle">4.2</entry><entry align="center" valign="middle">4.27</entry><entry align="center" valign="middle">4.58</entry></row></tbody></tgroup></table></tables><tables id="tabl0006" num="0006"><table frame="all"><title><b>Table 2: Zone of Inhibition Area After Aging at 40°C</b></title><tgroup cols="15"><colspec colnum="1" colname="col1" colwidth="24mm" /><colspec colnum="2" colname="col2" colwidth="14mm" /><colspec colnum="3" colname="col3" colwidth="18mm" /><colspec colnum="4" colname="col4" colwidth="14mm" /><colspec colnum="5" colname="col5" colwidth="18mm" /><colspec colnum="6" colname="col6" colwidth="14mm" /><colspec colnum="7" colname="col7" colwidth="18mm" /><colspec colnum="8" colname="col8" colwidth="14mm" /><colspec colnum="9" colname="col9" colwidth="18mm" /><colspec colnum="10" colname="col10" colwidth="14mm" /><colspec colnum="11" colname="col11" colwidth="18mm" /><colspec colnum="12" colname="col12" colwidth="14mm" /><colspec colnum="13" colname="col13" colwidth="18mm" /><colspec colnum="14" colname="col14" colwidth="14mm" /><colspec colnum="15" colname="col15" colwidth="18mm" /><thead><row><entry morerows="2" align="center" valign="middle">Sample</entry><entry namest="col2" nameend="col15" align="center" valign="middle">Area of ZOI (mm)</entry></row><row><entry namest="col2" nameend="col3" align="center" valign="middle">0 days</entry><entry namest="col4" nameend="col5" align="center" valign="middle">1 day</entry><entry namest="col6" nameend="col7" align="center" valign="middle">4 days</entry><entry namest="col8" nameend="col9" align="center" valign="middle">10 days</entry><entry namest="col10" nameend="col11" align="center" valign="middle">20 days</entry><entry namest="col12" nameend="col13" align="center" valign="middle">40 days</entry><entry namest="col14" nameend="col15" align="center" valign="middle">60 days</entry></row><row><entry align="center" valign="middle"><i>E. coli</i></entry><entry align="center" valign="middle"><i>S</i>. <i>aureus</i></entry><entry align="center" valign="middle"><i>E. coli</i></entry><entry align="center" valign="middle"><i>S</i>. <i>aureus</i></entry><entry align="center" valign="middle"><i>E. coli</i></entry><entry align="center" valign="middle"><i>S</i>. <i>aureus</i></entry><entry align="center" valign="middle"><i>E. coli</i></entry><entry align="center" valign="middle"><i>S</i>. <i>aureus</i></entry><entry align="center" valign="middle"><i>E. coli</i></entry><entry align="center" valign="middle"><i>S</i>. <i>aureus</i></entry><entry align="center" valign="middle"><i>E. coli</i></entry><entry align="center" valign="middle"><i>S</i>. <i>aureus</i></entry><entry align="center" valign="middle"><i>E. coli</i></entry><entry align="center" valign="middle"><i>S</i>. <i>aureus</i></entry></row></thead><tbody><row><entry align="center" valign="middle">1</entry><entry align="center" valign="middle">5</entry><entry align="center" valign="middle">6</entry><entry align="center" valign="middle">6</entry><entry align="center" valign="middle">6</entry><entry align="center" valign="middle">7</entry><entry align="center" valign="middle">6</entry><entry align="center" valign="middle">6</entry><entry align="center" valign="middle">5</entry><entry align="center" valign="middle">7</entry><entry align="center" valign="middle">4</entry><entry align="center" valign="middle">6</entry><entry align="center" valign="middle">4</entry><entry align="center" valign="middle">6</entry><entry align="center" valign="middle">4</entry></row><row><entry align="center" valign="middle">2</entry><entry align="center" valign="middle">5</entry><entry align="center" valign="middle">6</entry><entry align="center" valign="middle">6</entry><entry align="center" valign="middle">6</entry><entry align="center" valign="middle">6</entry><entry align="center" valign="middle">6</entry><entry align="center" valign="middle">6</entry><entry align="center" valign="middle">5</entry><entry align="center" valign="middle">7</entry><entry align="center" valign="middle">4</entry><entry align="center" valign="middle">7</entry><entry align="center" valign="middle">4</entry><entry align="center" valign="middle">6</entry><entry align="center" valign="middle">4</entry></row><row><entry align="center" valign="middle">3</entry><entry align="center" valign="middle">6</entry><entry align="center" valign="middle">6</entry><entry align="center" valign="middle">6</entry><entry align="center" valign="middle">6</entry><entry align="center" valign="middle">6</entry><entry align="center" valign="middle">6</entry><entry align="center" valign="middle">5</entry><entry align="center" valign="middle">5</entry><entry align="center" valign="middle">7</entry><entry align="center" valign="middle">4</entry><entry align="center" valign="middle">6</entry><entry align="center" valign="middle">3</entry><entry align="center" valign="middle">5</entry><entry align="center" valign="middle">4</entry></row><row><entry align="center" valign="middle">4_*</entry><entry align="center" valign="middle">5</entry><entry align="center" valign="middle">6</entry><entry align="center" valign="middle">6</entry><entry align="center" valign="middle">6</entry><entry align="center" valign="middle">6</entry><entry align="center" valign="middle">5</entry><entry align="center" valign="middle">5</entry><entry align="center" valign="middle">4</entry><entry align="center" valign="middle">6</entry><entry align="center" valign="middle">5</entry><entry align="center" valign="middle">5</entry><entry align="center" valign="middle">3</entry><entry align="center" valign="middle">5</entry><entry align="center" valign="middle">4</entry></row><row><entry align="center" valign="middle">5 (Reference)</entry><entry align="center" valign="middle">10</entry><entry align="center" valign="middle">10</entry><entry align="center" valign="middle">10</entry><entry align="center" valign="middle">10</entry><entry align="center" valign="middle">10</entry><entry align="center" valign="middle">11</entry><entry align="center" valign="middle">5</entry><entry align="center" valign="middle">3</entry><entry align="center" valign="middle">2</entry><entry align="center" valign="middle">2</entry><entry align="center" valign="middle">1.5</entry><entry align="center" valign="middle">2.5</entry><entry align="center" valign="middle">0</entry><entry align="center" valign="middle">0</entry></row></tbody></tgroup></table></tables>

As indicated in Table 1, the thymol levels remained high even after approximately 60 days of aging at 40°C. Table 2 also reveals that after extrusion, thymol in Samples 1-4 still maintained antimicrobial activity, even after 60 days of aging at 40°C. Reference Example 5 (soy flour at 90°C) did not exhibit activity after 60 days, but was still effective after 40 days. Without intending to be limited by theory, it is believed that this difference may stem from the difference in protein concentrations between the WhetPro® 75 (75% protein) and the soy flour (50% protein).

SEM microphotographs were taken of Sample 3 and a control sample containing no thymol (70 wt.% gluten and 30 wt.% glycerol). The results are shown in <figref idref="f0001 f0002">Figs. 1-4</figref>. As shown, there is no structural or morphological difference between the sample containing thymol (Sample 3, <figref idref="f0002">Figs. 3-4</figref>) and the control sample (<figref idref="f0001">Figs. 1-2</figref>). This indicates that thymol is homogenously compatible and retained within the protein structure. Also, there is no phase separation between the thymol and protein/glycerol matrix, which helps to providing for the prolonged release of the thymol.

<u>EXAMPLE 2 (Reference)</u>

WhetPro® 75 was placed in desiccant glass jar containing ammonium hydroxide solution (20-35% ammonia, 65-80% water) for 10 days. The mixture was then extruded with thymol at a concentration of 85 wt.% treated gluten and 15 wt.% thymol. Specifically, a PRISM USALAB 16" lab scale twin screw extruder was employed to melt process the ammonium hydroxide treated WhetPro® 75 and thymol. The extruder contained eleven (11) different zones, although zones 1 through 5, and 11 were not utilized in this Example. The extruder was used without the die system (zone 11) to allow for ease of material to exit extruder. The treated protein and thymol were pre-blended (17 wt% thymol and 83 wt.% protein) and subsequently added to the extruder at zone 6 at a feed rate of 0.4 Ibs/hr (0.05 g/s). The screw configuration was composed of conveying elements at zones 6 and 7, kneading blocks at zones 8 and 9, and conveying elements at zone 10. The screw speed was 100 rpm. The temperature profile for zones 6-10 was 35°C, 44°C, 56°C, 70°C, 70°C respectively.

The material was pelletized and cooled by placing in -32°C for minimum of 24 hrs, resulting cooled material was powderized via Brickmann/Retsch lab scale grinding mill (set speed =1) and collected at a size of 250 to 425 |jm by sieving. Sample was tested for thymol stability after aging at 50°C for 1, 4, 15, and 18 days using the test methods described above. The results indicated that when gluten was treated with NH₄OH, the rate loss of thymol decreased by approximately one half (0.01% per hour) as compared to a control of untreated gluten (0.02% per hour).

<u>EXAMPLE 3</u>

A "PRISM USALAB 16" lab scale twin screw extruder was employed to melt process WhetPro® 75, lactic acid, and thymol. The extruder contained eleven (11) different zones, although zones 1 through 5, and 11 were not utilized in this Example. The extruder was used without the die system (zone 11) to allow for ease of material to exit extruder. The protein and thymol were pre-blended (17.3 wt% thymol) and subsequently added to the extruder at zone 6 at a feed rate of 0.5 Ibs/hr (0.06 g/s). Lactic acid was then added at zone 7 at a feed rate of 0.087 Ib/hr (0.011 g/s) to give a composition of 70.4% WhetPro®, 14.8% lactic acid, 14.7% thymol. The screw configuration was composed of conveying elements at zones 6 and 7, kneading blocks at zones 8 and 9, and conveying elements at zone 10. The screw speed was 50 rpm. The temperature profile for zones 6-10 was 24°C, 32°C, 42°C, 70°C, 70°C respectively. The resulting material was contained in plastic bag and stored at -32°C. Cooled material was downsized via Brickmann/Retsch lab scale grinding mill (set speed =1) and collected at a size of 250 to 425 µm by sieving. The samples were tested for thymol stability after aging at 50°C for 1, 4, 15, 18, 56, 104 days using the test methods described above. The results are set forth in Table 3. <tables id="tabl0007" num="0007"><table frame="all"><title><b>Table 3: Thymol Level After Aging at 50°C</b></title><tgroup cols="8"><colspec colnum="1" colname="col1" colwidth="18mm" /><colspec colnum="2" colname="col2" colwidth="14mm" /><colspec colnum="3" colname="col3" colwidth="14mm" /><colspec colnum="4" colname="col4" colwidth="14mm" /><colspec colnum="5" colname="col5" colwidth="16mm" /><colspec colnum="6" colname="col6" colwidth="16mm" /><colspec colnum="7" colname="col7" colwidth="16mm" /><colspec colnum="8" colname="col8" colwidth="18mm" /><thead><row><entry morerows="1" align="center" valign="middle">Example</entry><entry namest="col2" nameend="col8" align="center" valign="middle">Thymol Level (wt%)</entry></row><row><entry align="center" valign="middle">0 days</entry><entry align="center" valign="middle">1 days</entry><entry align="center" valign="middle">4 days</entry><entry align="center" valign="middle">15 days</entry><entry align="center" valign="middle">18 days</entry><entry align="center" valign="middle">56 days</entry><entry align="center" valign="middle">104 days</entry></row></thead><tbody><row><entry align="center" valign="middle">3</entry><entry align="center" valign="middle">14.3</entry><entry align="center" valign="middle">14</entry><entry align="center" valign="middle">12.4</entry><entry align="center" valign="middle">9.1</entry><entry align="center" valign="middle">8.2</entry><entry align="center" valign="middle">7.55</entry><entry align="center" valign="middle">7.09</entry></row></tbody></tgroup></table></tables>

<u>EXAMPLE 4 (Reference)</u>

A "PRISM USALAB 16" lab scale twin screw extruder was employed to melt process WhetPro® 75 Gluten and thymol. The extruder contained eleven (11) different zones, although zones 1 through 5, and 11 were not utilized in this Example. The extruder used a 0.75-inch (1.9 cm)die system (zone 11) to allow for ease of material to exit extruder. The protein and thymol were pre-blended (17 wt% thymol and 83% protein) and subsequently added to the extruder at zone 6 at a feed rate of 0.5 Ibs/hr (0.06 g/s) to give a composition of 83% WhetPro® and 17% thymol. The screw configuration was composed of conveying elements at zones 6 and 7, kneading blocks at zones 8 and 9, and conveying elements at zone 10. The screw speed was 50 rpm. The temperature profile for zones 6-11 was 37°C, 47°C, 60°C, 70°C, 70°C, and 70°C, respectively. The resulting material was contained in plastic bag and stored at -32°C. Cooled material was downsized via Brickmann/Retsch lab scale grinding mill (set speed =1) and collected at a size &lt;250 µm. The samples were tested for thymol stability after aging at 55°C for 0, 14, 19 days. The results are set forth in Table 4. <tables id="tabl0008" num="0008"><table frame="all"><title><b>Table 4: Thymol Level After Aging at 55°C</b></title><tgroup cols="4"><colspec colnum="1" colname="col1" colwidth="21mm" /><colspec colnum="2" colname="col2" colwidth="15mm" /><colspec colnum="3" colname="col3" colwidth="15mm" /><colspec colnum="4" colname="col4" colwidth="17mm" /><thead><row><entry morerows="1" align="center" valign="middle">Example</entry><entry namest="col2" nameend="col4" align="center" valign="middle">Thymol Level (wt%)</entry></row><row><entry align="center" valign="middle">0 days</entry><entry align="center" valign="middle">7 days</entry><entry align="center" valign="middle">19 days</entry></row></thead><tbody><row><entry align="center" valign="middle">4</entry><entry align="center" valign="middle">14.3</entry><entry align="center" valign="middle">14</entry><entry align="center" valign="middle">12.4</entry></row></tbody></tgroup></table></tables>

100 milliliters of deionized water was then added to 5.88 grams of the non-aged particles, as well as to 1 gram of neat thymol. The concentration of thymol in water was determined for samples extracted for 2, 10, and 60 minutes by the method entitled "<i>Thymol Concentrations in Extractions</i>." The results are shown in <figref idref="f0003">Fig. 5</figref>. As indicated, the amount of thymol released into water from the thymol/protein particle was approximately four (4) times greater than a sample extracted from neat thymol.

<u>EXAMPLE 5</u>

A "PRISM USALAB 16" lab scale twin screw extruder was employed to melt process two (2) different samples of WhetPro® 75 (gluten), glycerol, and volatile oil (carvacrol or eucalyptol). The extruder contained eleven (11) different zones, although zones 1 through 5 were not utilized in this Example. Temperature zone 11 was a strand die. WhetPro® 75 was added to the extruder at zone 6 via a drop feeder at a feed rate of 0.5 Ibs/hr (0.06 g/s). Glycerol and the volatile oil were then added via a syringe pump at zone 7 at a feed rate of 0.24 Ib/hr (0.030 g/s). The formulation was set to contain 81 wt.% WhetPro® 75, 11 wt.% glycerol, and 5 wt.% of volatile oil. The screw configuration was composed of conveying elements at zones 6 and 7, kneading blocks at zones 8 and 9, and conveying elements at zone 10. The screw speed was 100 rpm. The temperature profile for the samples are set forth below. <tables id="tabl0009" num="0009"><table frame="all"><tgroup cols="10"><colspec colnum="1" colname="col1" colwidth="15mm" /><colspec colnum="2" colname="col2" colwidth="19mm" /><colspec colnum="3" colname="col3" colwidth="26mm" /><colspec colnum="4" colname="col4" colwidth="29mm" /><colspec colnum="5" colname="col5" colwidth="13mm" /><colspec colnum="6" colname="col6" colwidth="13mm" /><colspec colnum="7" colname="col7" colwidth="13mm" /><colspec colnum="8" colname="col8" colwidth="13mm" /><colspec colnum="9" colname="col9" colwidth="15mm" /><colspec colnum="10" colname="col10" colwidth="15mm" /><thead><row><entry morerows="1" align="center" valign="middle">Sample</entry><entry morerows="1" align="center" valign="middle">Volatile Oil</entry><entry morerows="1" align="center" valign="middle">Pellet Size (mm)</entry><entry morerows="1" align="center" valign="middle">Target Temp. (°C)</entry><entry namest="col5" nameend="col10" align="center" valign="middle">Temperature Profile (°C)</entry></row><row><entry align="center" valign="middle">Zone 6</entry><entry align="center" valign="middle">Zone 7</entry><entry align="center" valign="middle">Zone 8</entry><entry align="center" valign="middle">Zone 9</entry><entry align="center" valign="middle">Zone 10</entry><entry align="center" valign="middle">Zone 11</entry></row></thead><tbody><row><entry align="center" valign="middle">7</entry><entry align="center" valign="middle">Carvacrol</entry><entry align="center" valign="middle">3</entry><entry align="center" valign="middle">70</entry><entry align="center" valign="middle">∼35</entry><entry align="center" valign="middle">∼40</entry><entry align="center" valign="middle">∼55</entry><entry align="center" valign="middle">70</entry><entry align="center" valign="middle">70</entry><entry align="center" valign="middle">70</entry></row><row><entry align="center" valign="middle">8</entry><entry align="center" valign="middle">Eucalyptol</entry><entry align="center" valign="middle">3</entry><entry align="center" valign="middle">70</entry><entry align="center" valign="middle">∼35</entry><entry align="center" valign="middle">∼40</entry><entry align="center" valign="middle">∼55</entry><entry align="center" valign="middle">70</entry><entry align="center" valign="middle">70</entry><entry align="center" valign="middle">70</entry></row></tbody></tgroup></table></tables>

Once formed, the samples were put into an air tight bag and placed in -10°C freezer. The resulting samples were tested for volatile oil stability using the test method described above. The results are set forth below in Table 5. <tables id="tabl0010" num="0010"><table frame="all"><title><b>Table 5: Volatile Oil Level After Aging at 50°C</b></title><tgroup cols="6"><colspec colnum="1" colname="col1" colwidth="17mm" /><colspec colnum="2" colname="col2" colwidth="14mm" /><colspec colnum="3" colname="col3" colwidth="14mm" /><colspec colnum="4" colname="col4" colwidth="16mm" /><colspec colnum="5" colname="col5" colwidth="16mm" /><colspec colnum="6" colname="col6" colwidth="16mm" /><thead><row><entry morerows="1" align="center" valign="middle">Sample</entry><entry namest="col2" nameend="col6" align="center" valign="middle">Volatile Oil level (wt.%)</entry></row><row><entry valign="middle">0 days</entry><entry valign="middle">5 days</entry><entry align="center" valign="middle">10 days</entry><entry align="center" valign="middle">20 days</entry><entry align="center" valign="middle">40 days</entry></row></thead><tbody><row><entry valign="middle">7</entry><entry valign="middle">3.70</entry><entry valign="middle">3.80</entry><entry valign="middle">3.00</entry><entry valign="middle">3.10</entry><entry valign="middle">2.95</entry></row><row><entry valign="middle">8</entry><entry valign="middle">1.66</entry><entry valign="middle">1.54</entry><entry valign="middle">1.53</entry><entry valign="middle">0.43</entry><entry valign="middle">0.59</entry></row></tbody></tgroup></table></tables>

<u>EXAMPLE 6</u>

A "PRISM USALAB 16" lab scale twin screw extruder was employed to melt process WhetPro® 75, glycerol, and thymol. The extruder contained eleven (11) different zones, although zones 1 through 5, and 11 were not utilized in this Example. The extruder was used with a 0.75-inch (1.9 cm) die system (zone 11) to allow for ease of material to exit extruder. The protein and thymol were pre-blended (17.3 wt% thymol) and subsequently added to the extruder at zone 6 at a feed rate of 0.5 Ibs/hr (0.06 g/s). Glycerol was then added at zone 7 at a feed rate of 0.087 Ib/hr (0.011 g/s) check process rounds to give a approximate composition of 71% WhetPro®, 14% glycerol, 15% thymol. The screw configuration was composed of conveying elements at zones 6 and 7, kneading blocks at zones 8 and 9, and conveying elements at zone 10. The screw speed was 50 rpm. The temperature profile for zones 9-11 was 70°C. The resulting material was contained in plastic bag and stored at -32°C. Cooled material was downsized via Brickmann/Retsch lab scale grinding mill (set speed =1) and collected at a size of: &lt;250, 250-425, 425-710, 710-1000 µm by sieving. The resulting samples were tested for thymol stability at 55°C using the test method described above. The results are set forth below in Table 6. <tables id="tabl0011" num="0011"><table frame="all"><title><b>Table 6: Rate Loss of Thymol for Various Particle Size Ranges</b></title><tgroup cols="3"><colspec colnum="1" colname="col1" colwidth="53mm" /><colspec colnum="2" colname="col2" colwidth="67mm" /><colspec colnum="3" colname="col3" colwidth="47mm" /><thead><row><entry align="center" valign="top">Particle Size (microns)</entry><entry align="center" valign="top">% Thymol (w/w) after 7 day aging</entry><entry align="center" valign="top">Rate loss (%/day)</entry></row></thead><tbody><row><entry align="center">&lt;250*</entry><entry align="center">2.3</entry><entry align="center">12.1</entry></row><row><entry align="center">250-425</entry><entry align="center">5.7</entry><entry align="center">8.9</entry></row><row><entry align="center">425-710</entry><entry align="center">6.7</entry><entry align="center">7.9</entry></row><row><entry align="center">710-1000</entry><entry align="center">8.0</entry><entry align="center">6.7</entry></row></tbody></tgroup><tgroup cols="3" rowsep="0"><colspec colnum="1" colname="col1" colwidth="53mm" /><colspec colnum="2" colname="col2" colwidth="67mm" /><colspec colnum="3" colname="col3" colwidth="47mm" /><tbody><row><entry namest="col1" nameend="col3" align="justify">*An example calculation for the "rate loss" is as follows: <i>[(15</i>% <i>initial thymol</i> - <i>2.3% thymol aged)</i> ÷ <i>15% initial thymol]</i> ÷ <i>7 days *100</i> = <i>12.1 %</i>/<i>day</i></entry></row></tbody></tgroup></table></tables>

As indicated, the amount of thymol lost from the thymol/protein particle was dependent on particle size in that the loss was significantly increased for particles having a smaller particle size.

<u>EXAMPLE 7</u>

A composition of 71% WhetPro®, 14% glycerol, 15% thymol was prepared and downsized as described in Example 6. Particle size ranges of &lt;250, 250-425, and &gt;425 microns were collected via seiving. Once formed, 100 milliliters of deionized water was added to 6.67 grams of the non-aged particles. The concentration of thymol in water was determined after 10 minutes of extraction in water by the method described above. The results are shown in Table 7. <tables id="tabl0012" num="0012"><table frame="all"><title><b>Table 7: Thymol Released for Various Particle Sizes</b></title><tgroup cols="2"><colspec colnum="1" colname="col1" colwidth="42mm" /><colspec colnum="2" colname="col2" colwidth="39mm" /><thead><row><entry align="center" valign="top">Particle Size (microns)</entry><entry align="center" valign="top">% Thymol in water (%wt)</entry></row></thead><tbody><row><entry align="center">&lt;250</entry><entry align="center">0.062</entry></row><row><entry align="center">250-425</entry><entry align="center">0.0296</entry></row><row><entry align="center">&gt;425</entry><entry align="center">0.017</entry></row></tbody></tgroup></table></tables>

As indicated, the amount of thymol released into water from the thymol/protein particle was dependent on particle size in that the concentration of thymol was increase for reduced particle sizes.

<u>EXAMPLE 8</u>

A composition of 71% WhetPro®, 14% glycerol, 15% thymol was prepared and downsized as described in Example 6. The particle size range of &lt;250 was collected via seiving. Once formed, 50 milliliters of deionized water was added to 6.67 grams of the non-aged particles. The concentration of thymol in water was determined at 2, 10, and 60 minutes by the method described above giving thymol concentrations by weight of 0.058%, 0.057%, 0.072% respectively.

<u>EXAMPLE 9</u>

A composition of 71% WhetPro®, 14% glycerol, 15% thymol was prepared and downsized as described in Example 6. The particle size range of &lt;250 was collected via seiving. Once formed, 100 milliliters of ethanol was added to 6.67 grams of the non-aged particles. The concentration of thymol in ethanol was determined at 2, 10, and 60 minutes by the method described above giving thymol concentrations by weight of 0.255, 0.260%, 0.292% respectively.

<u>EXAMPLE 10</u>

A composition of 71% WhetPro®, 14% glycerol, 15% thymol was prepared by extrusion and downsized as described in Example 6. The particle size range of 250-425 collected via seiving. Once formed, 100 milliliters of 7.7 X 10^-6 M citric acid in deionized water was added to 6.67 grams of the non-aged particles. The concentration of thymol in solution was determined after extraction for 10 minutes by the method described above giving a thymol concentration by weight of 0.032%.

<u>EXAMPLE 11</u>

A composition of 71% WhetPro®, 14% glycerol, 15% thymol was prepared by extrusion and downsized as described in Example 6. The particle size range of 250-425 was collected via seiving. Once formed, 100 milliliters of 7.5% citric acid by weight in deionized water was added to 6.67 grams of the non-aged particles. The concentration of thymol in solution was determined after extraction for 10 minutes by the method described above giving thymol concentration by weight of 0.046%.

<u>EXAMPLE 12</u>

A composition of 81% WhetPro®, 14% glycerol, 5% thymol was prepared by extrusion and downsized as described in Example 6. The particle size range of &lt;250 micron was collected via seiving. Once formed, 100 milliliters of 0.1% aqueous acetic acid was added to 20 grams of the non-aged particles. The concentration of thymol in acetic acid solution was determined after extraction for 2, 10, and 60 minutes by the method described above giving thymol concentrations by weight of 0.069%, 0.080%, 0.083% respectively.

<u>EXAMPLE 13</u>

Various compositions containing different thymol concentrations were extruded and downsized to particle size of &lt;250 microns. The resulting samples were tested for thymol stability at 55°C using the test method described above. The results are set forth in Table 8. <tables id="tabl0013" num="0013"><table frame="all"><title><b>Table 8: Thymol Level After Aging at 55°C</b></title><tgroup cols="6"><colspec colnum="1" colname="col1" colwidth="21mm" /><colspec colnum="2" colname="col2" colwidth="24mm" /><colspec colnum="3" colname="col3" colwidth="30mm" /><colspec colnum="4" colname="col4" colwidth="14mm" /><colspec colnum="5" colname="col5" colwidth="14mm" /><colspec colnum="6" colname="col6" colwidth="16mm" /><thead><row><entry namest="col1" nameend="col3" align="center" valign="middle">Composition</entry><entry namest="col4" nameend="col6" align="center" valign="middle">Thymol Level (wt%)</entry></row><row><entry align="center" valign="middle">Protein</entry><entry align="center" valign="middle">Carrier Fluid</entry><entry align="center" valign="middle">% Thymol Add-On</entry><entry align="center" valign="middle">0 days</entry><entry align="center" valign="middle">7 days</entry><entry align="center" valign="middle">19 days</entry></row></thead><tbody><row><entry align="center" valign="middle">71% Gluten</entry><entry align="center" valign="middle">14% glycerol</entry><entry align="center" valign="middle">15</entry><entry align="center" valign="middle">14.3</entry><entry align="center" valign="middle">2.3</entry><entry align="center" valign="middle">1.3</entry></row><row><entry align="center" valign="middle">81% Gluten</entry><entry align="center" valign="middle">14% glycerol</entry><entry align="center" valign="middle">5</entry><entry align="center" valign="middle">4.6</entry><entry align="center" valign="middle">3</entry><entry align="center" valign="middle">2.6</entry></row><row><entry align="center" valign="middle">85% Gluten</entry><entry align="center" valign="middle">14% glycerol</entry><entry align="center" valign="middle">1</entry><entry align="center" valign="middle">1</entry><entry align="center" valign="middle">0.7</entry><entry align="center" valign="middle">0.7</entry></row></tbody></tgroup></table></tables>

Table 8 illustrates that thymol loss was dependent on thymol concentration and time. For example, higher concentrations resulted in a higher thymol loss. Likewise, a longer aging time resulted in a lower rate loss of thymol. Table 9 summarizes the thymol rate loss for each composition at each aging time interval of 0 days to 7 and 7 days to 19 days. <tables id="tabl0014" num="0014"><table frame="all"><title><b>Table 9: Thymol Rate Loss</b></title><tgroup cols="5"><colspec colnum="1" colname="col1" colwidth="21mm" /><colspec colnum="2" colname="col2" colwidth="24mm" /><colspec colnum="3" colname="col3" colwidth="19mm" /><colspec colnum="4" colname="col4" colwidth="27mm" /><colspec colnum="5" colname="col5" colwidth="29mm" /><thead><row><entry namest="col1" nameend="col3" align="center" valign="top">Particle Composition</entry><entry namest="col4" nameend="col5" align="center" valign="top">Thymol Rate loss (%/day)</entry></row><row><entry align="center" valign="top">Protein</entry><entry align="center" valign="top">Carrier Fluid</entry><entry align="center" valign="top">%Thymol</entry><entry align="center" valign="top">From 0 to 7 days</entry><entry align="center" valign="top">From 7 to 19 days</entry></row></thead><tbody><row><entry align="center">71% Gluten</entry><entry align="center">14% glycerol</entry><entry align="center">15</entry><entry align="center">1.71</entry><entry align="center">0.08</entry></row><row><entry align="center">81% Gluten</entry><entry align="center">14% glycerol</entry><entry align="center">5</entry><entry align="center">0.23</entry><entry align="center">0.03</entry></row><row><entry align="center">85% Gluten</entry><entry align="center">14% glycerol</entry><entry align="center">1</entry><entry align="center">0.04</entry><entry align="center">0.00</entry></row></tbody></tgroup></table></tables>

<u>EXAMPLE 14</u>

A composition of 71% WhetPro®, 14% glycerol, 15% thymol was prepared by extrusion and downsized as described in Example 6. The particle size range of 250-425 micron was collected via sieving.

A series of thymol extractions were carried out from the particles prepared in Example 14 that involved various amounts of thymol/protein particles, various amounts of water, addition of citric acid particles, and extracting time. The extracting method involved adding specified amount of water to protein/thymol particles and citric acid particles, waiting a specified amount of time while shaking, and centrifuging to collect supernatant. In addition, water was added to neat thymol as a control, shaken for specified amount of time, and centrifuged to collect supernatant. The thymol concentration in supernatant was determined by the method described above. The composition of the solutions and the resulting thymol concentration are set forth in Table 10. <tables id="tabl0015" num="0015"><table frame="all"><title><b>Table 10: Thymol Extractions from Particles</b></title><tgroup cols="9"><colspec colnum="1" colname="col1" colwidth="16mm" /><colspec colnum="2" colname="col2" colwidth="17mm" /><colspec colnum="3" colname="col3" colwidth="17mm" /><colspec colnum="4" colname="col4" colwidth="19mm" /><colspec colnum="5" colname="col5" colwidth="17mm" /><colspec colnum="6" colname="col6" colwidth="17mm" /><colspec colnum="7" colname="col7" colwidth="23mm" /><colspec colnum="8" colname="col8" colwidth="20mm" /><colspec colnum="9" colname="col9" colwidth="25mm" /><thead><row><entry namest="col1" nameend="col9" align="center" valign="middle">Particle Composition</entry></row><row><entry align="center" valign="middle">Protein</entry><entry align="center" valign="middle">Carrier Fluid</entry><entry align="center" valign="middle">% Thymol</entry><entry align="center" valign="middle">Particle Size (microns)</entry><entry align="center" valign="middle">Amount (grams)</entry><entry align="center" valign="middle">Citric acid (grams)</entry><entry align="center" valign="middle">Deionized water (milliliters)</entry><entry align="center" valign="middle">Extracting time (mins)</entry><entry align="center" valign="middle">% Thymol (wt/wt) in extract solution</entry></row></thead><tbody><row><entry morerows="4" align="center" valign="middle">71% Gluten</entry><entry morerows="4" align="center" valign="middle">14% glycerol</entry><entry morerows="4" align="center" valign="middle">15%</entry><entry morerows="4" align="center" valign="middle">250-425</entry><entry align="center" valign="middle">0.75</entry><entry align="center" valign="middle">0.38</entry><entry align="center" valign="middle">12.4</entry><entry align="center" valign="middle">30</entry><entry align="center" valign="middle">0.557</entry></row><row><entry align="center" valign="middle">1.5</entry><entry align="center" valign="middle">0.38</entry><entry align="center" valign="middle">12.4</entry><entry align="center" valign="middle">30</entry><entry align="center" valign="middle">1.115</entry></row><row><entry align="center" valign="middle">0.75</entry><entry align="center" valign="middle">0.38</entry><entry align="center" valign="middle">12.4</entry><entry align="center" valign="middle">10</entry><entry align="center" valign="middle">0.509</entry></row><row><entry align="center" valign="middle">0.75</entry><entry align="center" valign="middle">0</entry><entry align="center" valign="middle">12.4</entry><entry align="center" valign="middle">30</entry><entry align="center" valign="middle">0.048</entry></row><row><entry align="center" valign="middle">2.25</entry><entry align="center" valign="middle">1.14</entry><entry align="center" valign="middle">37.2</entry><entry align="center" valign="middle">30</entry><entry align="center" valign="middle">0.610</entry></row><row><entry morerows="1" align="center" valign="middle">n/a</entry><entry morerows="1" align="center" valign="middle">n/a</entry><entry morerows="1" align="center" valign="middle">100 (control)</entry><entry morerows="1" align="center" valign="middle">&lt;250</entry><entry align="center" valign="middle">2</entry><entry align="center" valign="middle">1.14</entry><entry align="center" valign="middle">97</entry><entry align="center" valign="middle">30</entry><entry align="center" valign="middle">0.081</entry></row><row><entry align="center" valign="middle">2</entry><entry align="center" valign="middle">0</entry><entry align="center" valign="middle">100</entry><entry align="center" valign="middle">30</entry><entry align="center" valign="middle">0.084</entry></row></tbody></tgroup></table></tables>

The reported solubility limit of thymol in water is normally is 0.1 grams per 100 grams of water (0.1%) at 25°C. As illustrated above, however, the concentrations of thymol in the extract solutions were up to 11 times greater than the reported limited solubility. Two of the solutions above (0.557% thymol and 1.115% thymol) were also tested for antimicrobial efficacy according to the microplate assay described above. The results are set forth below in Table 11. <tables id="tabl0016" num="0016"><table frame="all"><title><b>Table 11: Efficacy of Thymol Extractions</b></title><tgroup cols="8"><colspec colnum="1" colname="col1" colwidth="18mm" /><colspec colnum="2" colname="col2" colwidth="16mm" /><colspec colnum="3" colname="col3" colwidth="17mm" /><colspec colnum="4" colname="col4" colwidth="16mm" /><colspec colnum="5" colname="col5" colwidth="23mm" /><colspec colnum="6" colname="col6" colwidth="17mm" /><colspec colnum="7" colname="col7" colwidth="30mm" /><colspec colnum="8" colname="col8" colwidth="32mm" /><thead><row><entry align="center" valign="middle">Example</entry><entry align="center" valign="middle">Protein</entry><entry align="center" valign="middle">Carrier Fluid</entry><entry align="center" valign="middle">% Thymol</entry><entry align="center" valign="middle">Particle Size (microns)</entry><entry align="center" valign="middle">Amount (grams)</entry><entry align="center" valign="middle"># of wells showing growth after test for <i>S</i>. <i>aureus</i></entry><entry align="center" valign="middle"># of wells showing growth after test for <i>P</i>. <i>aeruginosa</i></entry></row></thead><tbody><row><entry align="center" valign="middle">15</entry><entry morerows="1" align="center" valign="middle">71% Gluten</entry><entry morerows="1" align="center" valign="middle">14% glycerol</entry><entry morerows="1" align="center" valign="middle">15</entry><entry morerows="1" align="center" valign="middle">250-425</entry><entry align="center" valign="middle">0.75</entry><entry align="center" valign="middle">1</entry><entry align="center" valign="middle">1</entry></row><row><entry align="center" valign="middle">16</entry><entry align="center" valign="middle">1.5</entry><entry align="center" valign="middle">3</entry><entry align="center" valign="middle">1</entry></row></tbody></tgroup></table></tables>