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Title:
BIO-BASED FOOD PACKAGING COATINGS, AND DEVICES AND MATERIALS COMPRISING SAME
Document Type and Number:
WIPO Patent Application WO/2021/045928
Kind Code:
A1
Abstract:
Disclosed herein are bio-based additives such as enzymes and peptides for reducing microbial contamination of food packaging coatings and food that contacts the food packaging coating. Methods of preparing and using a food packaging coating having such bio-based preservatives is also disclosed. Articles of manufacture and devices such as food packaging comprising such a food packaging coating are described.

Inventors:
MCDANIEL CLAUDE (US)
Application Number:
PCT/US2020/047723
Publication Date:
March 11, 2021
Filing Date:
August 25, 2020
Export Citation:
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Assignee:
REACTIVE SURFACES LTD LLP (US)
International Classes:
A23B4/10; A01N25/10; A23B5/16; A23B7/16; A23B9/14; A23L3/3463; A23L3/3562; A23L3/3571; C09D5/14
Foreign References:
CA2293736C2012-07-31
US20030108648A12003-06-12
US20200299521A12020-09-24
Other References:
MCINNIS, BM ET AL.: "Bio-Based Antimicrobial Food Packaging", COATINGSTECH, vol. 15, no. 9, 2018, pages 36 - 43
Attorney, Agent or Firm:
SIMMONS, David (US)
Download PDF:
Claims:
CLAIMS

What is claimed is: 1. A method of forming an antibiological food packaging article, comprising: providing a food packaging article comprising a layer of polymeric material defining at least one food contacting surface; providing an antibiological coating composition comprising a water-soluble polymeric binder and a bio-based antibiological agent; and treating at least a portion of the at least one food contacting surface of the layer of polymeric material with the antibiological coating composition to form an antibiological food packaging article.

2. The method of claim 1 wherein the bio-based antibiological agent comprises an antibiological biopolymer.

3. The method of claim 2 wherein the antibiological biopolymers comprises chitosan.

4. The method of claim 1 wherein the bio-based antibiological agent comprises an antibiological biopolymer.

5. The method of claim 1 wherein the antibiological biopolymers consists of chitosan.

6. The method of claim 1 wherein the water-soluble polymeric binder comprises a polyvinyl alcohol (PVA) binder.

7. The method of claim 1 wherein: the water-soluble polymeric binder comprises a polyvinyl alcohol (PVA) binder; and the bio-based antibiological agent comprises chitosan.

8. The method of claim 7 wherein: the polyvinyl alcohol binder is provided in the antibiological coating composition as a 5% (w/w) PVA solution; the chitosan is provided in the antibiological coating composition as 1% (w/w) chitosan in 2% (v/v) acetic acid solution.

9. The method of claim 1 wherein the antibiological coating composition further comprises an antibiological peptide.

10. The method of claim 9 wherein the antibiological peptide comprises AMP7 peptide.

11. The method of claim 9 wherein the antibiological peptide is SEQ ID no. 40.

12. The method of claim 9 wherein: the water-soluble polymeric binder comprises a polyvinyl alcohol (PVA) binder the bio-based antibiological agent comprises chitosan; and the antibiological peptide comprises AMP7 peptide.

13. The method of claim 12 wherein: the polyvinyl alcohol binder is provided in the antibiological coating composition as a 5% (w/w) PVA solution; the chitosan is provided in the antibiological coating composition as 1% (w/w) chitosan in 2% (v/v) acetic acid solution; and the AMP7 peptide is provided in the antibiological coating composition as 10% (w/w) AMP7 in 5% (w/w) PVA solution.

14. The method of claim 1 wherein the antibiological coating composition further comprises at least one antibiological enzyme.

15. The method of claim 14 wherein the at least one antibiological enzyme comprises lysozyme, glucose oxidase, acylase or a combination thereof.

16. The method of claim 15 wherein the antibiological biopolymers consists of chitosan.

17. The method of claim 14 wherein the antibiological coating composition further comprises an antibiological peptide.

18. The method of claim 17 wherein: the water-soluble polymeric binder comprises a polyvinyl alcohol (PVA) binder; the bio-based antibiological agent comprises chitosan; the antibiological peptide comprises AMP7 peptide; and the antibiological coating composition further comprises at least one antibiological enzyme.

19. The method of claim 18 wherein: the at least one antibiological enzyme is selected from the group of antibiological enzyme consisting of lysozyme, glucose oxidase and acylase; the water-soluble polymeric binder and the bio-based antibiological agent are provided in the antibiological coating composition in approximately equal amounts; and each of the at least one antibiological enzymes is provided in the antibiological coating composition at a concentration of between 0.2% and 2.0%.

20. The method of claim 1 wherein the antibiological coating composition further comprises at least one antibiological enzyme.

21 .The method of claim 20 wherein the at least one antibiological enzyme is selected from the group consisting of lysozyme, glucose oxidase and acylase.

22. The method of claim 20 wherein: the at least one antibiological enzyme is selected from the group of antibiological enzyme consisting of lysozyme, glucose oxidase and acylase; the water-soluble polymeric binder and the bio-based antibiological agent are provided in the antibiological coating composition in approximately equal amounts; and each of the at least one antibiological enzymes is provided in the antibiological coating composition at a concentration of between 0.2% and 2.0%.

23. The method of claim 20 wherein: the water-soluble polymeric binder comprises a polyvinyl alcohol (PVA) binder; and the bio-based antibiological agent comprises chitosan.

24. The method of claim 23 wherein: the at least one antibiological enzyme is selected from the group of antibiological enzyme consisting of lysozyme, glucose oxidase and acylase; the chitosan and the PVA binder are provided in the antibiological coating composition in approximately equal amounts; and each of the at least one antibiological enzymes is provided in the antibiological coating composition at a concentration of between 0.2% and 2.0%.

25. The method of claim 20 wherein the antibiological coating composition further comprises an antibiological peptide.

26. The method of claim 25 wherein: the water-soluble polymeric binder comprises a polyvinyl alcohol (PVA) binder; the bio-based antibiological agent comprises chitosan; and the antibiological peptide comprises AMP7 peptide.

27. The method of claim 26 wherein: the at least one antibiological enzyme is selected from the group of antibiological enzyme consisting of lysozyme, glucose oxidase and acylase; the chitosan and the PVA binder are provided in the antibiological coating composition in approximately equal amounts; the AMP7 peptide is provided in the antibiological coating composition at a concentration of between 0.2% and 2.0%; and each of the at least one antibiological enzymes is provided in the antibiological coating composition at a concentration of between 0.2% and 2.0%.

28. An antibiological food packaging article, comprising: a food packaging article comprising a layer of polymeric material defining at least one food contacting surface; and an antibiological coating composition on at least a portion of the at least one food contacting surface of the layer of polymeric material, wherein the antibiological coating composition comprises a water-soluble polymeric binder, a bio-based antibiological agent, and at least one of an antibiological enzyme and an antibiological peptide.

Description:
BIO-BASED FOOD PACKAGING COATINGS, AND DEVICES AND MATERIALS

COMPRISING SAME

[0001] BACKGROUND OF THE INVENTION [0002] A. Field of the Invention

[0003]The composition may comprise a polymeric material such as a coating, a plastic, an elastomer, a composite, a laminate, an adhesive, or a sealant; a surface treatment such as a textile finish or a wax, that comprises a bio-based antibiological agent such as a biopolymer such as a chitosan, a peptide, and/or an active enzyme that reduces microbial growth on or in food contacting the composition. Articles of manufacture and devices that comprise the composition may be used in food contacting applications such as a food packaging article or device.

[0004] B. Description of the Related Art

[0005]A polymeric material such as a plastic, an elastomer, a composite, or a laminate, comprises a molecular polymer often to form a shaped material typically for a consumer or an industrial product. Antibiological chemicals (/. e. , biocides, fungicides, algaecides, mildewcides, etc.) are currently available and approved for use in the U.S. /NAFTA, Europe, and the Asia Pacific region for use with a material formulation such as a polymeric material, a surface treatment (e.g., a coating), etc. The surface of the polymeric material may be subject to addition of a surface treatment such as a coating, an adhesive, a sealant, a textile finish, and/or a wax, with a surface treatment typically used, for example, to protect, decorate, attach, and/or seal a surface and/or the underlying material. A polymeric material or surface treatment may comprise an antibiological chemical (e.g., a preservative, an antimicrobial chemical) for reducing microbial growth on or in the polymeric material or surface treatment.

[0006]A biomolecule comprises a molecule often produced and isolated from an organism, such as a biopolymer such as peptidoglycan, an enzyme which catalyzes a chemical reaction, or a non-enzymatic peptide, or other biomolecule may detrimentally affect the growth of a biological cell or virus. [0007] SUM MARY OF THE INVENTION

[0008] In general, the invention provides an antibiological food packaging coating, comprising: a polyvinyl alcohol binder; and at least two antibiological bio-based additives, wherein the at least two antibiological bio-based additives include chitosan and at least one antimicrobial peptide.

[0009] Further embodiments provide a method for reducing microbial growth on packaged food, comprising: preparing coating comprising a polyvinyl alcohol binder and at least two antibiological bio-based additives, wherein the at least two antibiological bio-based additives include chitosan and at least one antimicrobial peptide; applying the coating to a food packaging material, wherein said applying the coating produces at least one coated food packaging surface; and contacting the coated food packaging surface to food. Other embodiments provide an antibiological food packaging, comprising: a food packaging container and a coating upon a surface of the food packaging container, wherein the coating comprises a polyvinyl alcohol binder and at least two antibiological bio-based additives, wherein the at least two antibiological bio-based additives include chitosan and at least one antimicrobial peptide.

[0010] In certain embodiments, the antibiological peptide comprises SEQ ID no. 40, SEQ ID no. 41 , nisin, or a combination thereof. In certain aspects, the antibiological peptide comprises SEQ ID no. 40. In other embodiments, the antibiological peptide is SEQ ID no. 40. In some embodiments, the antibiological bio-based additives further comprise an antibiological enzyme. In specific aspects, the antibiological enzyme comprises lysozyme, alpha-chymotrypsin, cellulase, sulfatase, phosphodiesterase I, DNase I, phosphoric triester hydrolase, lipase, glucose oxidase, acylase, or a combination thereof. In other aspects, the antibiological peptide comprises SEQ ID no. 40, SEQ ID no. 41, nisin, or a combination thereof. In particular facets, the antibiological peptide comprises SEQ ID no. 40. In other facets, the antibiological peptide is SEQ ID no. 40. In specific facets, the antibiological enzyme comprises lysozyme. In other facets, the antibiological enzyme is lysozyme. In some facets, the antibiological peptide is SEQ ID no. 40. In additional aspects, the antibiological bio-based additives further comprise monolaurin. In other embodiments, each antibiological bio-based additive is between about 0.01% to about 10% by weight of the coating prior to cure. In certain embodiments, the coating cures into a solid film, and wherein each antibiological bio-based additive is between about 0.01% to about 10% by weight of the solid film.

[0011] BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1. Diagram of vacuum sealed food-simulant packaging system evaluation of plain films and peptide (e.g., ProteCoat™) coated films. The food simulant agar patty was contaminated with E. coli on the top surface. (Not drawn to scale.)

[0013] Figures 2A-2D: Dose responses of chitosan and AMP7, individually and in combination. The percent growth inhibition (as determined by reduced colony counts compared to polyvinyl alcohol (“PVA”) negative control) was used to calculate the dose response to coatings dosed with varying concentrations of AMP7 (FIG. 2A) and chitosan (FIG. 2B). Aheatmap shows the response (as % growth inhibition) to numerous combinations of different concentrations of AMP7 and chitosan (FIG. 2C). A contour map of synergy scores of the AMP7 and chitosan combinations, determined using the zero interaction potency method, by which the positive scores (red) indicate synergy, the negative scores (green) indicate antagonism, and zero scores (white) indicate additive responses (FIG. 2D).

[0014] Figure 3. Results for vacuum-sealed bags containing PVA coatings dosed with AMP7 and chitosan combinations. These data were used to calculate synergy scores using the Bliss model, which are visualized in a contour plot.

[0015] DETAILED DESCRIPTION OF THE EMBODIMENTS [0016] For a further understanding of the nature and function of the embodiments, reference should be made to the following detailed description. Detailed descriptions of the embodiments are provided herein, as well as, the best mode of carrying out and employing the present invention. It will be readily appreciated that the embodiments are well adapted to carry out and obtain the ends and features mentioned as well as those inherent therein. It is to be understood, however, that the present invention may be embodied in various forms. Therefore, specific details disclosed herein are not to be interpreted as limiting, but rather as a basis for the claims and as a representative basis for teaching to employ the present invention in virtually any appropriately detailed system, structure or manner. Other features will be readily apparent from the following detailed description; specific examples and claims; and various changes, substitutions, other uses and modifications that may be made to the embodiments disclosed herein without departing from the scope and spirit of the invention or as defined by the scope of the appended claims. [0017] It should be understood that a preservative(s), biomolecule composition(s), coating(s), paint(s), polymeric material(s), material formulation(s), compound(s), method(s), procedure(s), and technique(s) described herein are presently representative of various embodiments. These techniques are intended to be exemplary, are given by way of illustration only, and are not intended as limitations on the scope. All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

[0018] As used herein other than the claims, the terms "a," "an," "the," and/or "said" means one or more. As used herein in the claim(s), when used in conjunction with the words "comprise," "comprises" and/or "comprising," the words "a," "an," "the," and/or "said" may mean one or more than one. As used herein and in the claims, the terms "having," "has," "is," "have," "including," "includes," and/or "include" has the same meaning as "comprising," "comprises," and "comprise." As used herein and in the claims "another" may mean at least a second or more. As used herein and in the claims, "about" refers to any inherent measurement error or a rounding of digits for a value ( e.g a measured value, calculated value such as a ratio), and thus the term "about" may be used with any value and/or range. [0019] The phrase "a combination thereof" "a mixture thereof" and such like following a listing, the use of "and/or" as part of a listing, a listing in a table, the use of "etc" as part of a listing, the phrase "such as," and/or a listing within brackets with "e.g. " or “i.e.,’ refers to any combination {e.g., any sub-set) of a set of listed components, and combinations and/or mixtures of related species and/or embodiments described herein though not directly placed in such a listing are also contemplated. For example, composition(s) described as preservative(s) suitable for use in a coating described in different sections of the specification may be claimed individually and/or in a combination, as they are part of the same genera of preservative (e.g., a coating preservative). Such related and/or like genera(s), sub-genera(s), specie(s), and/or embodiment(s) described herein are contemplated both in the form of an individual component that may be claimed, as well as a mixture and/or a combination that may be described in the claims as, for example, "at least one selected from," "a mixture thereof" and/or "a combination thereof."

[0020] In various embodiments described herein, exemplary values are specified as a range, and all intermediate range(s), subrange(s), combination(s) of range(s) and individual value(s) within a cited range are contemplated and included herein. For example, citation of a range "0.03% to 0.07%" provides specific values within the cited range, such as, for example, 0.03%, 0.04%, 0.05%, 0.06%, and 0.07%, as well as various combinations of such specific values, such as, for example, 0.03%, 0.06% and 0.07%, 0.04% and 0.06%, and/or 0.05% and 0.07%, as well as sub-ranges such as 0.03% to 0.05%, 0.04% to 0.07%, and/or 0.04% to 0.06%, etc. In another example, a range of “0.0001 % to 20.0%” provides specific values and sub-ranges such as “8.5%,” and “11.3 to 18.9%.” Example 39 provides additional descriptions of specific numeric values within any cited range that may be used for an integer, intermediate range(s), subrange(s), combinations of range(s) and individual value(s) within a cited range, including in the claims.

[0021] Some terms often have different meanings for different material types and/or uses being described, and the meaning applicable to the material should be applied as appropriate in the context, as understood in the applicable art. For example, in the context of a polymeric material, other than a coating, a "film" ("polymeric film") of a polymeric material refers to a planar form (i.e., a large width and large length relative to thickness) capable of being flexed, creased without cracking, folded, or a combination thereof, while being self-supporting ( e.g ., a plastic wrap), though such a film may also be treated with a surface treatment (e.g., coated with a coating). A polymeric film comprises from about 5 pm to about 250 pm thick {e.g., about 10 pm to about 180 pm thick), while a plastic sheet ("sheeting") refers to a planar form having a thickness of about 250 pm to about 250 mm thick. Thus, a "film," for example, in the plastic art being described and/or claimed in the context of a plastic differs in composition, meaning, manufacture process, function and/or purpose than a "film" in a coating {e.g., a paint) art. In another example, a "cell" in a biological art refers to the smallest unit of living matter, while a "cell" in a polymeric material art {e.g., a plastic art, an elastomer art) refers to a void in a polymeric material to produce a solid foam material {e.g., a plastic foam, an elastomer foam material). A surface comprises the outer layer of any solid object. The term "substrate," in the context of a coating, may be synonymous with the term "surface." Flowever, as "substrate" has a different meaning in the art of enzymology, a chemical that undergoes an accelerated chemical reaction upon contact with an enzyme, the term "surface" may be preferentially used herein for clarity. In such instances, the appropriate definition and/or meaning for the term should be applied in accordance with the context of the term's use in light of the present disclosures. [0022] In many embodiments, the compositions and methods herein may produce materials ("material formulations") ( e.g a composition) with bioactivity as well as a manufactured article, a device, an apparatus, etc. comprising such a material formulation so that the manufactured article, a device, an apparatus, etc., has bioactivity. Examples of a material formulation that may comprise a biomolecule composition include a polymeric material, a surface treatment, a filler, or a combination thereof. For example, in many preferred embodiments, a biomolecule composition, may confer a property to, alter a property of and/or maintain a property of, a material formulation. Examples of a property that may be conferred, altered and/or maintained include resistance to a microorganism including conferring resistance to microorganism growth of materials (e.g., food) that contact the material formulation, resistance to biodegradation, enzymatic activity upon contact with a substrate of an enzyme, resistance to aging, consistent performance sustained over an extended period of time {e.g., service life), or a combination thereof. Numerous examples of component(s) (e.g., polymers, binders, liquid components, additives, coloring agents, etc.) of that are combined to prepared certain material formulations (e.g., a coating, a polymeric material) are described herein, and inclusion of a biomolecule composition may confer, alter and/or maintain a property to such a component, and may also confer, alter, and/or maintain such a property in later prepared material formulation comprising such a component. In the context of a biomolecule composition, active or bioactive refers to the effect of biomolecule, such as to retain function upon and/or within a material formulation. As used herein, a biomolecule (“biomolecule composition,” “biomolecular composition”) refers to a molecule (e.g., a compound) typically synthesized in living organisms, with examples of such biomolecules including but not limited to, a biopolymer (e.g., chitosan), an amino acid, a proteinaceous molecule (e.g., an enzyme, an antibody, a receptor, a transport protein, structural protein, an antibiological proteinaceous molecule), a nucleotide, a nucleic acid, a saccharide (e.g., a sugar), a polysaccharide, a lipid, a colorant (e.g., a chlorophyll), or a combination thereof. An effective amount refers to a concentration of component (e.g., a biomolecule composition) of a material formulation capable of exerting a desired effect (e.g., an antibiological activity). A biomolecule typically comprises one or more chemical moiety(s) ["specie(s)," "group(s)," "functionality(s)," "functional group(s)"] such as an amine, a carboxylic acid, a hydroxyl, an ester, a double bond, etc. that may be chemically reactive (e.g., reacted with a coupling agent) and/or acted upon contact with another biomolecule ( e.g ., an enzyme that acts on a hydroxyl moiety). An enzyme refers to a proteinaceous molecule that possesses the ability to accelerate a chemical reaction, typically acting one or more biomolecule(s) {e.g., a lipid, a proteinaceous molecule, a polysaccharide, etc.). As used herein a "proteinaceous molecule," proteinaceous composition," and/or "peptidic agent" comprises a polymer formed from one or more amino acid(s), such as a peptide {i.e., about 3 to about 100 amino acids), a polypeptide {i.e., about 101 or more amino acids, such as about 50,000 or more amino acids), and/or a protein. As used herein a "protein" may comprise a proteinaceous molecule comprising a contiguous molecular sequence three amino acids or greater in length, matching the length of a biologically produced proteinaceous molecule encoded by the genome of an organism. [0023] Various such biomolecules described herein or as would be known to one of ordinary skill in the art in light of the present disclosures, including those having an antibiological property, may be used. Various bio-based additives, polymeric materials, surface treatments, articles, devices, and methods of using and producing are described, for example, in U.S. Patent Application nos. 12/696,651, 12/474,921 and 12/882,563, each specifically incorporated herein by reference.

[0024] In some embodiments, a biomolecule composition comprises a biomolecule (e.g., a biopolymer, an enzyme, a peptide) that acts upon cellular component such as a lipid (e.g., a lipolytic enzyme), a protein (e.g., a peptidase, a protease), a polysaccharide (e.g., a polysaccharidase), a cell wall (e.g., a lysozyme), or a combination thereof, to produce an antibiological property (e.g., a biocidal effect, a biostatic effect, an antimicrobial effect, etc.). Such a biomolecule composition having an antibiological property (e.g., an antimicrobial property, an antifouling property, an anti-insect property, a biocidal property, a biostatic property, etc.) is known herein as an antibiological agent. A preferred bio-based antibiological agent is one that is able to confer to a material formulation (e.g., a polymeric material, a surface treatment, etc.) the antibiological property to a material formulation (e.g., a coating), a composition, etc., as well as an article, a device, an apparatus, etc. comprising such a material formulation upon incorporation of the antibiological agent upon the surface of and/or within the material body of the material formulation. Other chemical compositions having an antibiological property (“antibiological chemical”) such as an anti microbial agent typically incorporated in a polymeric material and/or a preservative traditionally incorporated in a coating (e.g., a paint) may be combined with a bio-based antibiological agent. An antibiological composition as used herein refers to an antibiological agent, an antibiological chemical, or a combination thereof. A preferred antibiological composition comprises a bio-based antibiological agent.

[0025] Preferred materials with bioactivity (e.g., antibiological activity such as antimicrobial activity, etc.) include those that compose all or part of food, a food packaging material, a food container, a food processing material, a coating upon food, a food packaging material, a food container, a food processing material, or a combination thereof. The disclosures herein describe various embodiments where a biomolecule's activity {e.g., a biopolymer’s antibiological activity, an enzyme's catalytic reaction, a peptide's antibiological activity) may be conferred to a material via incorporation of a biomolecule into and/or upon the surface of the material to confer a property to a material, alter a property of a material, and/or maintain a property of a material. In one example, a cured coating upon a food packaging material contains an active biomolecule such as a bioactive biopolymer (e.g., chitosan), a bioactive peptide (e.g., an antibiological peptide), and/or a bioactive enzyme to accelerate a chemical reaction (e.g., degrading a biomolecule from a living cell to produce an antibiological activity) within or upon the body of the coating differentiating such activity (e.g., antibiological activity, antimicrobial activity, catalytic activity of an enzyme, etc.) from a like ability of a coating that does not comprise an active biomolecule.

[0026] An antibiological composition may act by treating an infestation, preventing infestation, inhibiting infestation (e.g., preventing cell attachment), inhibiting growth, preventing growth, lysing, and/or killing; a biological entity such as a cell and/or a virus (e.g., one or more genera and/or species of a cell and/or a virus). Thus, some embodiments comprise a process for treating an infestation, preventing infestation, inhibiting infestation (e.g., preventing cell attachment), inhibiting growth, preventing growth, lysing, reducing the number of cell(s) and/or virus(s) to levels that no longer pose a threat {e.g., a threat to the health such as food poisoning and/or infection of a desired organism such as human), and/or killing a cell and/or a virus (e.g., a fungal cell, a bacterial cell); comprising contacting the cell and/or the virus with a material formulation and/or contacting a material that may have a cell and/or virus with the material formulation (e.g., a paint, a coating composition, a biomolecule composition) comprising at least one antibiological composition comprising an biomolecule antibiological agent (e.g., a biologically produced molecule and/or based on molecule based on biochemistry such as a chemically synthesized peptide). Possible modes of action by which an antibiological composition exert their effect(s) (e.g., an inhibitory effect, a fungicidal effect), may include, for example, destabilizing a cellular (e.g., a fungal cell) membrane (e.g., perturb membrane functions responsible for osmotic balance); a disruption of macromolecular synthesis (e.g., cell wall biosynthesis) and/or metabolism; disruption of appressorium formation; damage to one or more biomolecule component(s) of a biological cell or virus (e.g., via production of a toxin such as an reactive oxygen species), or a combination thereof (see, for example, Fiedler,

H. P., et. at., 1982; Isono, K. and S. Suzuki. 1979; Zasloff, M. 1987; US Patent Application 10/601,207).

[0027] An antibiological composition (e.g., an antibiological agent) may act on a biological entity such as a biological cell and/or a biological virus, such as for example a biological entity that contacts {e.g., a surface contact, an internal incorporation, an infiltration, an infestation) and/or contacting a material that may have a cell and/or virus with a material formulation. In various embodiments, a target cell and/or a target virus may be capable of infesting an object, and may, for example, infest, survive upon, survive within, grow on the surface, and/or grow within, an object. An object such as food, a coating, a film, a food container, a packaging material such as a food packaging material, etc.) may be susceptible ("prone") to infestation by a cell and/or a virus when it is capable of serving as a food source for a cell (e.g., the object comprises a substance that serves as a food source). Such a target cell and/or a target virus (include those that can infest and/or survive upon and/or within an object and may cause rancidity, spoilage, rot, defacement (e.g., deterioration or discoloration), odor, environment hazards, other undesirable effects, or a combination thereof. These changes may lead to the object becoming unsuitable for use. An antibiological composition activity (e.g., growth inhibition, biocidal activity) can provide and/or facilitate disinfection, decontamination and/or sanitization of an material and/or an object, which refer to the process of reducing the number of cell(s) and/or virus(s) to levels that no longer pose a threat {e.g., a threat to damaging food quality, a threat to the health of a desired organism such as human that consumes the food having a microorganism). Use of a bioactive antibiological composition can be accompanied by removal (e.g., manual removal such as washing, machine aided removal) of the cell(s) and/or the virus(s).

[0028] Examples of bio-based antibiological agents, some of which are antioxidants, include biopolymers such as chitosan, which is produced from chitin shells of crustaceans (e.g., shrimp) after treatment with an alkaline/base chemical (e.g., sodium hydroxide) and comprises N-acetyl-D-glucosamine and p-(1 4)-linked D-glucosamine. Chitosan and other antibiological biopolymers (e.g., k-carrageenan) may be polycationic (e.g., chitosan, K-carrageenan), polyanionic, or neutrally charged. In addition to biopolymers, an antibiological agent may be an antibiological peptide [e.g., ProteCoat ® (Reactive Surfaces, Ltd), nisin, etc.]; natamycin; sorbic acid; an essential oil, which comprise volatile aroma chemical(s) obtained from a plant (e.g., essential oil of cinnamon, lemongrass, citrus, clove, bergamot, mustard, thyme, tea tree, etc.) and/or an essential oil aroma chemical (e.g., citral, carvacrol, thymol, eugenol, cinnamic acid, cinnamaldehyde, etc.); lauric arginate ester (“ethyl lauroyl arginate,” “lauramide ethyl ester”); a citric acid ester of a diglyceride; a citric acid ester of monoglyceride; a tocopherol (e.g., alpha-tocopherol); acetic acid; ascorbic acid; ascorbyl palmitate; ascorbyl stearate; benzoic acid; calcium ascorbate; calcium propionate; calcium sorbate; Carnobacterium divergens M35; Carnobacterium maltaromaticum CB1; citric acid; erythorbic acid; gum guaiacum; iso-ascorbic acid; L- cysteine; L-cysteine hydrochloride; lecithin; lecithin citrate; Leuconostoc carnosum 4010; methyl paraben; monoglyceride citrate; monoisopropyl citrate; potassium acetate; potassium lactate; propionic acid; propyl paraben; sodium acetate; sodium ascorbate; sodium diacetate; sodium iso-ascorbate; sodium lactate; sodium propionate; tartaric acid; or a combination thereof. In preferred embodiments, an antibiological agent comprises a peptide such as ProteCoat ® which has been shown to involve synergy (see, for example, U.S. Patent Nos. U.S. Patent Nos. 6,020,312; 5,885,782; and 5,602,097, and Patent Application Nos. 10/884,355 and 11/368,086) between the peptides (e.g., antifungal peptides) and non-peptide antifungal composition(s) that may be useful in controlling growth of a Fusarium, a Rhizoctonia, a Ceratocystis, a Pythium, a Mycosphaerella, an Aspergillus and/or a Candida genera of fungi. These and other synergistic combinations of peptide agent(s) and non-peptide antibiological composition(s) may be used. Other food preservatives which have antibiological and/or antioxidant activity that may be included alone or in combination with a bio-based antibiological agent, including 4-hexylresorcinol; butylated hydroxyanisole (e.g., a mixture of 2-tertiarybutyl-4-hydroxyanisole and 3- tertiarybutyl-4-hydroxyanisole); butylated hydroxytoluene (3,5-ditertiarybutyl-4- hydroxytoluene); dimethyl decarbonate; ethyl lauroyl arginate; methyl-p-hydroxy benzoate; potassium benzoate; potassium bisulphite; potassium diacetate; potassium metabisulphite; potassium nitrate; potassium nitrite; potassium sorbate; propyl gallate; propyl-p-hydroxy benzoate; sodium benzoate; sodium bisulphite; sodium dithionite; sodium erythorbate; sodium metabisulphite; sodium nitrite; sodium salt of methyl-p-hydroxy benzoic acid; sodium salt of propyl-p-hydroxy benzoic acid; sodium sorbate; sodium sulphite; sulphurous acid; tertiary butyl hydroquinone; or a combination thereof.

[0029] In preferred embodiments, a material formulation comprising a biodegradable polymer (e.g., a biodegradable polymer used as a binder) may be used as (e.g., a thermoplastic) or upon (e.g., a coating, a surface treatment) an object, and in some instances the biodegradable polymer may be the bio-based antibiological agent (e.g., a chitosan), though other antibiological composition(s) (e.g., chemical biocide typically used in polymeric materials such as plastic), preservative(s), or other additives may be used in combination with the biodegradable polymer. A biodegradable polymer includes a natural polymer such as a polyamino acid/protein, a carbohydrate such as a polysaccharide (e.g., a chitin, an alginate, a starch, a glycosaminoglycan, an amylose, a Konjac ("glucomannan"), a cellulosic polymer, a dextrin, a xanthan gum, a welan gum, a pullulan, or a combination thereof. Examples of a biodegradable polymer includes a poly(alkylene oxalate), a polyamino acid [(e.g., a homopolymer of an amino acid, a copolymer of amino acids, a protein (e.g., casein, a whey protein, a soy protein, a zein; etc.)], a pseudo polyamino acid, a polyanhydride, a polycaprolactone, a polycyanoacrylate, a polydioxanone, a polyglycolide, a poly(hexamethylene-co-frans-1,4-cyclohexane dimethylene oxalate), a polyhydroxybutyrate, a polyhydroxyvalerate, a polylactide, a poly(ortho ester), a poly (p-dioxanone), a polyphosphazene, a polypropylene fumarate), a polyvinyl alcohol, a polyacrylate [e.g., a polymethacrylate, a poly(ethylene glycol- monomethacrylate)], a gelatin, a dextrin {e.g., a maltodextrin), an acacia, a polyaminotriazole, an albumin, a collagen, a fibrinogen, a fibrin, a gelatin, a polysaccharide (e.g., a chitosan, a carrageenan such as for example, k-carrageenan), pectin, a homogalacturonan, a rhamnogalacturonan, an amidated pectin, an alginate, b-cyclodextrin, amylose and amylopectin based starch, pullulan; etc.), or a combination thereof. In some instances, a wax (e.g., a beeswax, a carnauba wax; etc.) may be used as a substitute for a biodegradable polymer.

[0030] In preferred embodiments, a component of a polymeric material (e.g., a coating) is recognized by a governmental regulatory body as requiring less regulatory criteria for use in contacting food applications, such as a material that is generally recognized as safe (“GRAS”) by the United States Food and Drug Administration (“FDA”). In other preferred embodiments, such a material has had submitted a GRAS notification to the FDA. Various materials (e.g., a bio-based material) have submitted GRAS notifications, including polymer(s) (e.g., an antibiological enzyme); antibiological composition(s) (e.g., a chemical antimicrobial agent, a bio-based antibiological agent); an enzyme (e.g., an antibiological enzyme); a food perseverative who may increase shelf-life via a mechanism other than antimicrobial activity; a solvent; a surfactant; an antioxidant; a buffer; a dispersant; an emulsifier; a gum; a microencapsulant; a polymeric material filler; a water absorber; or a combination thereof. Examples of a polymer that may be used in a polymeric material (e.g., a polymer used as a binder) such as for a food contact material include a chemical polymer; a polysaccharide biopolymer; a protein biopolymer; a lipid biopolymer; a cell-wall, external environment interface (e.g., a cell wall) and/or exoskeleton biopolymer; or a combination thereof. Examples of a chemical polymer include polyvinyl alcohol; vinyl acetate-vinyl laurate copolymers; or a combination thereof. Examples of a polysaccharide biopolymer include an arabinogalactan; enzyme-modified dextrins (e.g., used as a thickener); ethyl cellulose; expanded substitution pattern hydroxypropyl methylcellulose; glucan; baker’s yeast b-glucan; b-glucans from oat bran; barley fiber; corn hull fiber; high- amylose cornstarch acetate; high-amylose cornstarch; citrus peel insoluble fiber; inulin; isomaltodextrin; isomalto-oligosaccharide; oat hull fiber; oligofructose and inulin; oligofructose; pea fiber; pecan shell fiber; polysaccharide complex of sodium alginate, konjac glucomannan (konjac), and xanthan gum; potato fiber; pullulan; rice bran fiber; rice bran wax; rice hull fiber; short-chain fructo-oligosaccharide; sugar beet fiber; tamarind seed polysaccharide; xylo- and arabinoxylo-oligosaccharides from wheat bran extract; xanthan gum; xylooligosaccharide; or a combination thereof. Examples of a protein biopolymer include hydrolyzed pork cartilage; polyglutamic acid; pork collagen; e-polylysine; or a combination thereof. Examples of a lipid biopolymer include polyglycerol fatty acid esters having of 11 to 40 polymerization of the polyglycerol (lipid biopolymer). Examples of a cell- wall, external environment interface, and/or exoskeleton biopolymer include chitosan from Aspergillus niger[ i.e. , a bio-based, antibiological (e.g., antimicrobial biopolymer)]; chitin- glucan from Aspergillus niger[ i.e., a bio-based, antibiological (e.g., antimicrobial biopolymer)]; or a combination thereof. Examples of an antibiological composition include sodium nitrite embedded up to 113 mg/square meter on a side of a food packaging film; sodium bisulfate; natamycin; lauramide arginine ethyl ester; chlorine dioxide up to 17.5 pg/square inch produced from (<30 pm) particles of magnesium sulfate, sodium silicate, sodium polyphosphate and sodium chlorite incorporated into low density polyethylene food packaging films; ferrous ammonium phosphate; ammonium, magnesium, and potassium chloride triple salt, hexahydrate; calcium propionate; chlorine dioxide, generated from sodium chlorite sulfated or in calcined kaolin clay; hydrogen gas; carbon monoxide; argon gas; allyl isothiocyanate; calcium disodium ethylenediaminetetraacetate (EDTA); white mineral oil, USP (viscosity ISO 100 ); sodium iron EDTA (molluscicide); chitosan from Aspergillus niger (bio-based, biopolymer); chitin-glucan from Aspergillus niger (bio-based, biopolymer); nisin (bio-based, peptide); sodium formate (buffer); six bacterial phages against shiga-toxin producing Escherichia coli preparation (bio-based); bacteriophage P100 preparation from Listeria innocua (bio-based); six bacterial monophages against Salmonella enterica (monophage cocktail) preparation (bio-based); five bacterial monophages against Shigella spp. preparation (bio-based); six bacterial monophages (LMSP-25, LIST-36, LMTA-34, LMTA-94, LMTA-57 and LMTA-148) against Listeria monocytogenes preparation (bio-based); bacterial monophages (BP-63 and BP-12) against Salmonella preparation (bio-based); bacterial monophages (Fo1a and S16) against Salmonella (bio-based); colicin (bio-based, enzyme/protein); lactoferrin (enzyme/protein); cow's milk-derived lactoferrin (enzyme/protein); volatile oil of mustard (bio-based); or a combination thereof. Examples of an enzyme include 1 ,4-a-glucan branching enzyme; acetolate decaroxylase; acid fungal protease; acid lactase; amylomaltase; asparaginase; aspartic protease; lactoferrin; branching glycosyltransferase; carbohydrase, catalase, colicin (bio-based, enzyme/protein); glucose oxidase, pectinase, and protease enzyme preparation; carbohydrase; carboxypeptidase; cellulase; chymosin; D-allulose 3-epimerase; dextranase; egg white lysozyme; endo-1 ,4-p-xylanase; glucoamylase; glucose oxidase; glycerophospholipid cholesterol acyltransferase; invertase and lactase enzyme preparation; isoamylase; laccase; lipase; lysophospholipase; maltotetraohydrolase; mannanase; milk- derived lactoferrin; pectate lyase; pectin esterase; pectin lyase; peroxidase; phosphodiesterase I; phospholipase A1 ; phospholipase A2; phospholipase C; phospholipase; polygalacturonase; protease; protein glutaminase; pullulanase; Saccharomyces cerevisiae expressing asparaginase II; Saccharomyces cerevisiae strain ECMoOI with enhanced expression of urea amidolyase; Saccharomyces cerevisiae strain ML01 carrying a gene encoding the malolactic enzyme from Oenococcus oeni and a gene encoding malate permease from Schizosaccharomyces pombe ; subtilisin; thermolysin; transglucosidase; transglutaminase; trehalase; xylanase; a-amylase; a-glucosidase; a-L- arabinofuranosidase; b-galactosidase; b-glucanase and xylanase enzyme preparation; b- glucanase and xylanase enzyme preparation; b-glucanase, cellulase, and xylanase enzyme; b-glucanase; or a combination thereof. Examples of a food perseverative who may increase shelf-life via a mechanism other than antibiological (e.g., antimicrobial) activity include 1-methylcyclopropene (fruit preservative); tasteless smoke (meat preservative); ice structuring protein preparation (bio-based); or a combination thereof. Examples of a solvent include methylsulfonylmethane; behenic acid; white mineral oil, USP (viscosity ISO 100); 1 ,3-propanediol; or a combination thereof. Examples of a surfactant include dioctyl sodium sulfosuccinate; alkyl polyglycoside (bio-based); lecithin from canola (bio-based, emulsifier); hydrogenated lecithin from soy (bio-based, emulsifier); soy lecithin with increased phosphatidylserine after enzyme modification (bio-based, emulsifier); sucrose fatty acid esters such as palmitic acid, lauric acid, and stearic acid sucrose monoesters produced by vinyl esters of palmitic, lauric, and stearic acids reacted with sucrose (bio-based, fruit preservative); sucrose fatty acid esters (bio-based, fruit preservative); or combination thereof. Examples of an antioxidant include ergothionine; phytic acid; hydroxytyrosol; or a combination thereof. Examples of a buffer include trisodium diphosphate; succinic acid; or a combination thereof. Examples of a dispersant include polyoxyethanyl-a-tocopheryl sebacate. Examples of an emulsifier include modified gum acacia (bio-based); citric acid esters of mono- and diglycerides (bio-based); ammonium phosphatide (bio-based); polyglycerol polyricinoleic acid; polyglycerol polyricinoleate; sodium potassium hexametaphosphate; sucrose acetate isobutyrate (bio based); stearyl alcohol (bio-based); b-cyclodextrin (bio-based, surfactant); a-cyclodextrin (bio-based, surfactant); or a combination thereof. Examples of a gum include maleated isoprenyl polymer with methoxy-polyethylene glycol; modified gum acacia (bio-based); or a combination thereof. Examples of a microencapsulant of other components in a material formation include erythritol fatty acid esters. Examples of a polymeric material filler include diatomaceous earth and perlite composite filtration media. Examples of a water absorber include synthetic amorphous silica (flow agent). [0031] Other bio-based additives include viruses ( e.g ., bacteriophages) that infect and lyse microorganism(s) (e.g., bacteria) that may be combined with polymeric materials (see, for example, U.S. Patent Publication no. 2016/0010077).

[0032] Examples of a preferred object include food, a food packaging material, a food container, a food processing material (e.g., a fork, a spoon, a knife, a cheese factory surface, an sausage grinder, etc.), a coating upon food, a food packaging material, a food container, a food processing material, or a combination thereof; with examples of such objects including meat (e.g., fish, crustaceans, poultry, eggs, etc.), edible liquids (e.g., oil, milk, juices, sauces, etc.), nuts, beans, grains, and any prepared food (e.g., a sandwich, cookies, lasagna, etc.), a packaging film, a container (e.g., a bottle, ajar, a box, a tray, etc.). A food packaging material, food container and/or food processing material, may comprise, for example, paper (e.g., wax paper), paperboard (e.g., Kraft paperboard), cardboard, foil/paper film, or plastic/paper film, metal (e.g., aluminum) foil, glass, ceramic, a pasteboard, metal, ceramic, wood, glass, solid foam (e.g., Styrofoam™), plastic (e.g., plastic film), or a combination thereof. The food packaging materials, food containers and/or food processing materials may be in various shapes, such as, for example, pan, a tray, a box, a bag, a bottle, a film (e.g., a plastic wrap), or a combination thereof. A food, food packaging materials, food containers and/or food processing materials, may comprise an antibiological composition (e.g., a bio-based antibiological agent) and/or one or more coatings of which one or more coatings may comprises an antibiological composition.

[0033] For example, plastic (e.g., thermoplastic) and other polymeric materials prepared from certain polymers are commonly used in food packaging materials, food containers and/or food processing materials, and may comprise (e.g., incorporate in the body of the material) an antibiological composition (e.g., an antibiological agent) and/or be coated with a coating comprising the antibiological composition. For example, a polyolefin plastomer, which comprises an olefin monomer, an oligomer, and/or a polymer and about 20% or less of a comonomer/copolymer {e.g., an octene, a butane, a hexane), and are typically processed {e.g., extrusion, blow molding) to produce a polymeric film {e.g., a cast film, a blowing film, a packaging film, a heat sealing film), a sealant {e.g., a multilayer bag sealant), a packaging pouch for food and/or a liquid, an overwrap, a container, and/or a lid. An ionomer typically may be used in a polymeric film and/or a sheet application such as a packaging application for a frozen food, a snack, a nut, a beverage {e.g., a juice, a wine), and/or a triglyceride ( e.g ., a margarine, an oil); or a combination thereof. A polylactide ( e.g ., a lactic acid-glycolic acid copolymer) generally may be used in a food packaging material. A polyamide ("nylon") may be used in a polymeric film and/or a sheet application such as a packaging {e.g., a food packaging). A polycarbonate may be used for a sheet and/or a polymeric film application {e.g., a packaging application, a food storage container). A glycol modified polycyclohexylenedimethylene terephthalate may be injection molded, and may be used in a polymeric film and/or a sheet application such as a packaging {e.g., a food packaging). An acid modified polycyclohexylenedimethylene terephthalate may be extruded and has use in a packaging film and/or a sheet {e.g., a food packaging) and/or as an oven cookware particularly when comprising a filler. A polyethylene terephthalate) may be used in a polymeric film and/or a sheet application such as a packaging application (e.g. a beverage bottle, a beverage bag, a food container). A low-density polyethylene may be used in a heat sealable material, a polymeric film and/or a sheet application such as a packaging film {e.g., a multilayer film, an automatic packaging thin film, a shrink film/wrap, a flexible film, a stretch wrap, a food packaging), a bag {e.g., a food bag), a container, a household product wrap, an agricultural polymeric film, and/or a vapor barrier {e.g., a multi layered polymeric film may be used as a vapor barrier for water); a seal layer; a foam application {e.g., an elastomeric foam). A high-density polyethylene may be used in a container {e.g., a milk container, a water container, a juice container), and/or a polymeric film and/or a sheet application such as a packaging application {e.g., a store bag, a produce bag, a delicatessen wrap, a garbage bag, a snack food packaging, a cereal packaging, a cracker packaging). An ethylene acrylic acid copolymer may be used in a polymeric film and/or a sheet application such as a packaging film {e.g., a food packaging, a medical produce packaging); an exterior heat seal layer for a composite; an extrusion coating {e.g., coating for a food package, a paperboard), a packaging material comprising aluminum foil bonded to an ethylene acrylic acid copolymer, such as a laminate, including, for example, a coating for a pouch of aluminum foil and/or an aluminum foil / ethylene acrylic acid copolymer / polyethylene laminate. An ethylene-methyl acrylate copolymer may be used in a polymeric film and/or a sheet application such as a heat-sealing layer in a laminate, an extrusion coating, a multiextrusion tie-layer between other polymer layers, and/or a heat seal. An ethylene-methyl acrylate copolymer comprising up to about 8% ethyl acrylate may be used in food packaging. An ethylene-ethyl acrylate copolymer may be used as a polymeric film, a sheet, a household application ( e.g ., a squeezable bottle, a spray bottle), and/or a food packaging. An ethylene-vinyl acetate copolymer may be used in a polymeric film and/or a sheet {e.g., a bag for ice, a stretch wrap, a food wrap, a food packaging material, a cling wrap). An ethylene-vinyl alcohol copolymer may be used in a polymeric film and/or sheet application such as a food packaging application {e.g., a package, a container, a bottle). An atactic polypropylene comprises a flexible material, and may be used in a laminating paper, an adhesive, a sealing strip, a bottle {e.g., a glass bottle); a polymeric film and/or a sheet application such as a packaging application {e.g., a frozen food wrap), particularly where an orientated polypropylene polymeric film may be used; a polymeric film application such as a bag in a box {e.g., a soup mix bag, a cracker bag, a cereal bag); a stand up pouch; a coated {e.g., acrylic coated) polymeric film with enhanced barrier and heat sealing properties; and/or a metallized polymeric film to reduce gas and vapor permeability. A polypropylene copolymer {e.g., comprising an ethylene monomer) typically comprises between about 1 % to about 7% weight percent of the comonomer, and may be used in a polymeric film and/or a sheet application such as a packaging application {e.g., a shrink wrap); a packaging for a food {e.g., a produce, a bakery item); and/or a heat-sealing layer in a food packaging. A medium flow polystyrene may be used in a bottle, a food packaging, a part, and/or a tumbler. A high heat resistance polystyrene may be used as a polymeric film {e.g., an orientated food packaging). A polystyrene/styrene-butadiene block copolymer blend typically may be used in a polymeric film and/or a sheet application such as a food packaging {e.g., a vegetable packaging), and a high-impact polystyrene may be used for a molded product such as a dairy product tub, a lid, a bowl, and/or a cup; a toy; a polymeric film and/or a sheet application {e.g., packaging application). A styrene-butadiene may be used in a wrapping material {e.g., a shrink-wrap); a disposable container for a food such as a bowl, and/or a lid. A polyterpene often may be used in a polymeric film and/or a sheet application {e.g., food packaging). A polyvinyl chloride polymeric film and/or a sheet typically may be used in a blister packaging {e.g., a produce packaging, a fish packaging); a bottle {e.g., an oil bottle, a dairy product bottle); and/or a food packaging {e.g., a vegetable packaging, a meat packaging). A polyvinylidene chloride copolymer {e.g., a vinylidene chloride-alkyl acrylate, a vinylidene chloride- methylacrylate, a vinylidene chloride-acrylonitrile, a vinylidene chloride-vinyl chloride) may be used in a container; a polymeric film and/or a sheet application such as a coextruded multi-layered film ( e.g ., co-extruded with a polypropylene, a polystyrene) and/or a food packaging material {e.g., a wrap, a shrinkable film, a heat sealing film); and/or used in a coating {e.g., a latex coating, a solvent-based coating) which may be used as a paper coating, a coating for use upon a polymeric film's, a plastic rigid container {e.g., a bottle) coating, and/or a paperboard coating. A styrenic thermoplastic elastomer may be used in a sheet; a polymeric film {e.g., a biomedical disposable glove, a pharmaceutical application, a food application, a household application). A styrene-ethylene-butylene may be blown and/or extruded molded into a polymeric film {e.g., a food application such as a food container). A nitrile butadiene rubber may be used in a polymeric film and/or a sheet application {e.g., a packaging); a material that contacts food {e.g., a creamery equipment); while a nitrile butadiene rubber blend with a thermoplastic elastomer may be used in a hose {e.g., a hose for water, food, oil).

[0034] A cellulose ester may be used to form a polymeric film and/or a sheet {e.g., a packaging application), and/or prepared as a solution/suspension as a coating. A cellulose acetate may be used as a polymeric film {e.g., a packaging film, a blister packaging), and/or a sheet. A cellulose triacetate may be used as a polymeric film {e.g., a packaging), and/or a sheet. A cellulose acetate propionate may be used in a sheet and/or a polymeric film {e.g., a packaging film). A cellulose methylcellulose may be used in a polymeric film, a packaging material, and/or a sheet. A fluoropolymer may be used in a polymeric film and/or a sheet such as for a packaging application. A polyvinylidene fluoride may be used in a polymeric film and/or sheet application {e.g., a packaging application), and/or a coating. A polychlorotrifluoroethylene may be used in a sheet and/or a polymeric film {e.g., packaging application) with very low vapor transmission, as well as a low and/or a non crystalline sheet. A polyvinyl fluoride may be used in a polymeric film and/or a sheet application {e.g., a packaging application). A polyoxyethylene may be used in a polymeric film and/or a sheet, often for a packaging application {e.g., a water-soluble packaging material, a heat sealable packaging material). An ethylene oxide-propylene oxide copolymer may be used similarly. A polyamide may be blended with another polymer such as a polyolefin {e.g., an ionomer, an EVA, a LDPE) and/or a polyvinylidene chloride; and such blends are typically used to extrude coat a paperboard; and/or produce a multilayered polymeric film for processed meat packaging. A nylon 11 may be used in a packaging film. A polyacrylonitrile copolymer often used for a packaging film, often selected for low gas permeability, includes styrene-acrylonitrile ("SAN") and/or vinylidene chloride-acrylonitrile.

A copolymer commonly referred to as Barex ® may be used for a beverage container. Another copolymer comprises acrylonitrile-butadiene-styrene; and a polymethacrylonitrile copolymer often comprises an acrylonitrile ( e.g ., styrene, a methyl methacrylate, a methacrylate, a butadiene, as well as an acrylonitrile) and may be used similarly. A polybutylene may be used in a polymeric film and/or a sheet application {e.g., a packaging material). A thermoplastic polyester may be used in a polymeric film and/or a sheet application {e.g., a packaging application). A polyethylene naphthalene may be used to produce a polymeric film and/or a sheet, often for a packaging application, such as a bottle {e.g., a beverage bottle). A polyethylene may be used in a polymeric film and/or a sheet {e.g., a packaging application, an agricultural sheet), a bottle, a container, and/or a foam application {e.g., a packaging material). A linear low-density polyethylene may be used in a polymeric film and/or a sheet application such as a packaging application {e.g., a stretch film, a cling film), a sack {e.g., a heavy-duty shipping sack, a grocery sack), and/or a bag {e.g., a shopping bag). A medium-density polyethylene may be used in a polymeric film and/or a sheet application such as a packaging application. An ultrahigh molecular weight polyethylene may be used as a polymeric film and/or a sheet application such as a packaging application. A polyimide may be used in a polymeric film and/or a sheet application {e.g., a packaging application). An acrylic plastic may be used as a polymeric film and/or a sheet application {e.g., a packaging application). A thermoplastic polyurethane may be used in a polymeric film and/or a sheet application {e.g., a packaging application); and/or a cellular plastic {e.g., a rigid foam, a semi-flexible foam, a flexible foam, an elastomeric foam). A polystyrene may be used in a polymeric film and/or a sheet application {e.g., a packaging application), and/or in a foam {e.g., a rigid foam) application {e.g., a flotation device, a packaging material such as a meat tray and/or egg container, a disposable cup). A styrene-acrylonitrile copolymer may be used in a connector for polyvinyl chloride tubing; a polymeric film and/or a sheet application such as a packaging. A rigid polyvinyl chloride generally comprises relatively little and/or no plasticizer, and may be used in a polymeric film and/or a sheet application {e.g., a packaging sheet); a plasticized ("flexible") polyvinyl chloride may be used in a polymeric film and/or a sheet application {e.g., a packaging application such as a shrink wrap). A phenoxy resin uses a high molecular weight {e.g., up to about 45,000 Daltons or more) epoxy resin, relative to a typical epoxy resin ( e.g ., about 8000 Daltons); and may be used in a polymeric film and/or a sheet application such as a packaging application {e.g., a bottle, a container). A polypropylene/ an ethylene propylene diene "M" thermoplastic vulcanizate blend may be used in a packaging application and/or a packaging seal.

[0035] Other material formulations, such as a coating, a surface treatment, and/or a polymeric material, may comprise an antibiological composition (e.g., an antibiological agent), and may be used in a food packaging, food container and/or food processing application. For example, an antibiological composition may be incorporated in a thermoplastic and/or applied to a surface of a thermoplastic. A thermoplastic comprises a thermoplastic polymer, and may be described as "plastics capable of being repeatedly softened or melted by increases in temperature and hardened by decreases in temperature. These changes are physical rather than chemical." [Handbook of Plastics, Elastomers, & Composites Fourth Edition" (Harper, C.A. Ed.) McGraw-Hill Companies, Inc, New York, p. 780, 2002] In certain embodiments, the thermoplastic polymeric material comprises a biodegradable polymer, a cellulosic polymer, a fluoropolymer, a polyether, a polyamide, a polyacrylonitrile, a polyamide-imide, a polyarylate, a polybenzimidazole, a polybutylene, a polycarbonate, a thermoplastic polyester, a polyetherimide, a polyethylene, a polyimide, a polyketone, an acrylic, a polymethylpentene, a polyphenylene oxide, a polyarylene sulphide, a polypropylene, a polyurethane, a polystyrene, a polysulfone resin, a polyterpene, a polyvinyl acetal, a polyvinyl acetate, a thermoplastic vinyl ester, a polyvinyl ether, a polyvinyl carbazole, a polyvinyl chloride, a polyvinylidene chloride, a polyimidazopyrrolone, a polyacrolein, a polyvinylpyridine, a polyvinylamide, a polyurea, a polyquinoxaline, or a combination thereof. In some aspects, the biodegradable polymer comprises a natural polymer, a synthetic polymer, a photodegradable polymer, a biomedical polymer, or a combination thereof. More examples of a thermoplastic polymeric material include a cellulosic polymer (e.g., a cellulose acetate, a cellulose triacetate, a cellulose acetate butyrate, a cellulose acetate propionate, a cellulose methylcellulose, a methylcellulose, a cellulosehydroxyethyl, an ethylcellulose, a hydroxypropylcellulose, a nitrocellulose, a regenerated cellulose, etc .); a fluoropolymer (e.g., an ethylene chlorotrifluoroethylene, an ethylene tetrafluoroethylene, a fluoridated ethylene propylene, a polyvinylidene fluoride, a polychlorotrifluoroethylene, a polytetrafluoroethylene, a tetrachloroethylene-perfluorovinyl ether copolymer, a polyvinyl fluoride, etc .); a polyether (e.g., a polyaryl ether, a chlorinated polyether, a polyoxymethylene, a polyoxyethylene, a polyoxypropylene, etc.); a polyamide (e.g., an aromatic polyamide, a polyphthalamide, etc.); a polyacrylonitrile; a polyamide-imide; a polyarylate; a polybenzimidazole; a polybutylene; a polycarbonate; a thermoplastic polyester (e.g., a liquid crystal polyester, a polybutylene terephthalate, a polycyclohexylenedimethylene terephthalate, a polyethylene terephthalate, a polyethylene naphthalene, etc.); a polyetherimide; a polyethylene (e.g., a very low-density polyethylene, a low-density polyethylene, a linear low-density polyethylene, a medium-density polyethylene, a high-density polyethylene, an ultrahigh molecular weight polyethylene, a chlorinated polyethylene, a phosphorylated polyethylene, an ethylene-acrylic acid copolymer, an ethylene-methyl acrylate copolymer, an ethylene- ethyl acrylate copolymer, an ethylene-n-butyl acrylate copolymer, an ethylene-vinyl acetate copolymer, an ethylene-vinyl alcohol copolymer, etc.); a polyimide; a polyketone; an acrylic; a polymethylpentene; a polyphenylene oxide; a polyarylene sulphide; a polypropylene; a polyurethane; a polystyrene (e.g., a styrene-acrylonitrile copolymer, a styrene-butadiene copolymer, an acrylonitrile butadiene styrene terpolymer, an acrylonitrile-chlorinated polyethylene-styrene terpolymer, an acrylic styrene acrylonitrile terpolymer, a styrene- acrylic copolymer, a styrene-divinylbenzene copolymer, a styrene-maleic anhydride copolymer, a reactive polystyrene, etc.); a polysulfone resin (e.g., a polysulfone, a polyaryl sulfone, a polyether sulfone, a polyphenyl sulfone, etc.); a polyterpene; a polyvinyl acetal; a polyvinyl acetate; a thermoplastic vinyl ester; a polyvinyl ether; a polyvinyl carbazole; a polyvinyl chloride (e.g., a chlorinated polyvinyl chloride, etc.); a polyvinylidene chloride; a polyimidazopyrrolone; a polyacrolein; a polyvinylpyridine; a polyvinylamide; a polyurea; a polyquinoxaline; or a combination thereof.

[0036] The antibiological composition (e.g., an antibiological agent) may be incorporated in a thermoset and/or applied to a surface of a thermoset. A thermoset ("thermoset plastic," "thermoset material") may be described as a "material that will undergo, and/or has undergone, a chemical reaction by the action of heat, catalysts, ultraviolet light, and the like, leading to a relatively infusible state that will not remelt after setting" [Handbook of Plastics, Elastomers, & Composites Fourth Edition" (Harper, C.A. Ed.) McGraw-Hill Companies, Inc, New York, 109, 2002] A thermoset material generally comprises a resin ("thermoset resin," "thermosetting resin"), often described as "any class of solid, semi-solid, or liquid organic material, generally the product of natural or synthetic origin with a high molecular weight and no melting point" [Handbook of Plastics, Elastomers, & Composites Fourth Edition" (Harper, C.A. Ed.) McGraw-Hill Companies, Inc, New York, 109, 2002] An example of a thermoset resin includes an alkyd resin, an allyl resin, an amino resin, a bismaleimide resin, an epoxy resin, a phenolic resin, a polyester resin, a polyimide resin, a polyurethane resin, a silicon resin, a vinyl ester resin, a casein, or a combination thereof. [0037] The antibiological composition (e.g., an antibiological agent) may be incorporated in an elastomer and/or applied to the surface of an elastomer. As used herein, an elastomer ("elastomeric material") comprises a "macromolecular material that returns rapidly to approximately the initial dimensions and shape after substantial deformation by a weak stress and release of the stress" while a rubber comprises a material "capable of recovering from a large deformation quickly and forcibly, and can be, and/or are already is, modified to a state in which it is essentially insoluble (but can swell) in a solvent." A rubber retracts within about one minute to less than about 1.5 times its original length after being held for about one minute at about twice its length at room temperature, while an elastomer retracts within about five minutes to within about 10% original length after being held for about five minutes at about twice its length at room temperature. In contrast, a plastic possesses plasticity, where application of a force that exceeds the material's yield value deforms the material continuously and permanently without rupture. An example of an elastomer includes a thermoplastic elastomer (e.g., an elastomeric polyolefin, a thermoplastic vulcanizate, a styrenic thermoplastic elastomer, a styrene-butadiene rubber, a thermoplastic polyurethane elastomer, a thermoplastic copolyester elastomer, a polyamide thermoplastic elastomer, etc.), a melt processable rubber, a synthetic rubber [e.g., a nitrile butadiene rubber, a butadiene rubber, a butyl rubber, a chlorosulfonated polyethylene, an epichlorohydrin, an ethylene propylene copolymer, a fluoroelastomer, a polyacrylate rubber, a poly(ethylene acrylic), a polychloroprene, a polyisoprene, a polysulfide rubber, a styrene butadiene rubber, a silicone rubber, etc.], a natural rubber, a propylene oxide elastomer, an ethylene-isoprene elastomer, an ethylene-vinyl acetate elastomer, a non-polymeric elastomer (e.g., a vulcanized oil), or a combination thereof. [0038] The antibiological composition (e.g., an antibiological agent) may be incorporated in an adhesive and/or a sealant; and/or applied to a surface of an adhesive and/or a sealant. An adhesive refers to a composition capable of attachment to one or more surfaces ("substrates") of one or more objects ("adherents"), wherein the composition comprises a solid or is capable of converting into the solid, wherein the solid is capable of holding a plurality of objects ("adherents") together by attachment to the surface of the objects while withstanding a normal operating stress load placed upon the objects and the solid. An adhesive typically comprises a solid or a liquid, but converts into a solid final form ("set") during normal use with desired attachment and material strength properties. A sealant comprises a composition capable of attachment to a plurality of surfaces to fill a space and/or a gap between the plurality of surfaces and form a barrier to a gas, a liquid, a solid particle, an insect, or a combination thereof. An adhesive generally functions to prevent movement of the adherents, while a sealant typically functions to seal adherents that move. An abhesive comprises a material ( e.g a coating such as a clear coating or a paint; or a mold release agent such a plastic release film) applied to a surface to inhibit adhesion/sticking of an additional material to the abhesive and/or a surface the abhesive covers. A "film adhesive" refers to a dry layer of an adhesive at the thickness of a polymeric film ("adhesive film") and/or a sheet ("adhesive sheet") generally capable of being cured by heat and/or pressure.

[0039] An adhesive may be classified by composition as a thermoplastic adhesive, a thermoset adhesive ("thermosetting adhesive"), an elastomeric adhesive, or a combination thereof {e.g., "alloy blend adhesive," "alloy adhesive," "blend adhesive"). Examples of adhesive include a thermoplastic adhesive, a thermoset adhesive, an elastomeric adhesive, an alloy adhesive, a non-polymeric adhesive, or a combination thereof.

Examples of an adhesive includes a cellulosic adhesive, a cyanoacrylate adhesive, a dextrin adhesive, an ethylene-vinyl acetate copolymer adhesive, a melamine formaldehyde adhesive, a natural rubber adhesive, a neoprene/phenolic adhesive, a neoprene rubber adhesive, a nitrile rubber adhesive, a nitrile/phenolic adhesive, a phenolic adhesive, a phenol/resorcinol formaldehyde adhesive, a phenoxy adhesive, a polyamide adhesive, a polybenzimidazole adhesive, a polyethylene adhesive, a polyester adhesive, a polyimide adhesive, a polyisobutylene adhesive, a polysulfide adhesive, a polyurethane adhesive, a polyvinyl acetal adhesive, a polyvinyl acetal/phenolic adhesive, a polyvinyl acetate adhesive, a polyvinyl alcohol adhesive, a reclaimed rubber adhesive, a resorcinol adhesive, a silicone adhesive, a styrenic TPE adhesive, a styrene butadiene adhesive, a vinyl phenolic adhesive, a vinyl vinylidene adhesive, an acrylic acid diester adhesive, an epoxy adhesive, an epoxy/phenolic adhesive, an epoxy/polysulfide adhesive, an urea formaldehyde adhesive, an urea formaldehyde/melamine formaldehyde adhesive, an urea formaldehyde/phenol resorcinol adhesive, or a combination thereof. Examples of a thermosetting adhesive comprise an acrylic adhesive, an acrylic acid diester adhesive, a cyanoacrylate adhesive, a cyanate ester adhesive, an epoxy adhesive, a melamine formaldehyde adhesive, a phenolic adhesive, a polybenzimidazole adhesive, a polyester adhesive, a polyimide adhesive, a polyurethane adhesive, a resorcinol adhesive, an urea formaldehyde adhesive, or a combination thereof. Examples of a thermoplastic adhesive comprise an acrylic adhesive, an ethylene-vinyl acetate copolymer adhesive, a carbohydrate adhesive ( e.g ., a dextrin adhesive, a starch adhesive, etc.), a cellulosic adhesive {e.g., a cellulose acetate adhesive, cellulose acetate butyrate adhesive, cellulose nitrate adhesive, etc.), a polyethylene adhesive, a phenoxy adhesive, a polyamide adhesive, a polyvinyl acetal adhesive, a polyvinyl acetate adhesive, a polyvinyl alcohol adhesive, a protein adhesive {e.g., an animal adhesive, a soybean adhesive, a blood adhesive, a fish adhesive, a casein adhesive, etc.), a vinyl vinylidene adhesive, or a combination thereof. Examples of an elastomeric adhesive comprise a butyl rubber adhesive, a natural rubber adhesive, a neoprene rubber adhesive, a nitrile rubber adhesive, a polyisobutylene adhesive, a polysulfide adhesive, a reclaimed rubber adhesive, a silicone adhesive, a styrenic TPE adhesive, a styrene butadiene adhesive, or a combination thereof. Examples of an alloy adhesive comprise an epoxy/polyamide adhesive, an epoxy/phenolic adhesive, an epoxy/polysulfide adhesive, a neoprene/phenolic adhesive, a nitrile/phenolic adhesive, a phenol/resorcinol formaldehyde adhesive, a polyvinyl acetal/phenolic adhesive, a vinyl/phenolic adhesive, an urea formaldehyde/phenol resorcinol adhesive, a urea formaldehyde/melamine formaldehyde adhesive, or a combination thereof. Examples of a non-polymeric adhesive include a mucilage adhesive. [0040] The antibiological composition (e.g., an antibiological agent) may be incorporated in a foamed material formulation and/or applied to a surface of a foamed material formulation. Foaming modifies a solid material formulation {e.g., a polymeric material) to comprise voids ("cells") by the action of a blowing agent, though mechanical action may be used to whip a gas {e.g., air) into a material formulation prior to curing and/or solidification. In context, a plastic that has undergone foaming may be referred to as a "cellular plastic," "foam," etc., an elastomer that has undergone foaming may be known herein as a "cellular elastomer," "foamed elastomer," etc., a polymeric material that has undergone foaming may be known herein as a "cellular polymeric material" "foamed polymeric material," etc., and so forth. [0041] The antibiological composition (e.g., an antibiological agent) may be incorporated in a polymeric material comprising a reinforcement and/or applied to surface of a polymeric material comprising a reinforcement. Examples of a polymeric material comprising a reinforcement include a reinforced polymeric material {e.g., a reinforced plastic, a reinforced thermoset, a reinforced thermoplastic, a reinforced elastomer, etc.), a composite, a laminate, a honeycomb, a coated fabric, or a combination thereof. A reinforced polymeric material comprises a polymer and a reinforcing filler. A reinforced polymeric material may be initially prepared in the form of a molding compound, which refers to a moldable solid and/or semisolid form of a reinforced polymeric material. Examples of a polymeric material comprising a reinforcement include a reinforced polymeric material {e.g., a reinforced plastic), a composite, a laminate, a honeycomb, a coated fabric, or a combination thereof.

A sheet molding compound ("SMC") may be prepared using a conveyor belt moving a plastic film {e.g., a PP film) covered with a layer of a molding compound resin {e.g., an unsaturated polyester resin, a vinyl ester resin, a polyurethane, etc.) being layered with a reinforcement {e.g., a fiberglass such as a roving, usually up to about 30% to about 40% glass fiber), and that layer of molding compound and reinforcement covered by another layer of molding compound and a plastic film. A sheet may be produced, for example, comprising layers of a plastic film, a molding compound, and a plastic film, often up to about 6.5 mm thick, that may be cut into a desired size. A bulk molding compound ("BMC," "high-strength compound") generally comprises a thermoset resin {e.g., an alkyd resin, an allyl resin, an amino resin, an epoxy resin, a phenolic resin, a polyester resin, a vinyl ester resin, a silicon resin, etc.) and a reinforcement {e.g., a fiber up to about 2.6 cm), a filler, an additive, or a combination thereof, and may be prepared by mixing at low intensity to reduce reinforcement degradation.

[0042] A composite ("composite material") comprises a polymer in the form of an infusible polymer matrix and a reinforcement, wherein the identities and properties of the polymer and the reinforcement are retained. The reinforcement may be held, bound, bonded, resides, and/or embedded within the matrix. A composite may be classified by the matrix material, and examples of a composite includes a polymer matrix composite, a metal {e.g., an aluminum, a titanium, etc.) composite, a ceramic {e.g., an alumina, a glass, a silicon carbide, etc.) composite, a carbon {e.g., an amorphous carbon) composite, or a combination thereof.

[0043] A type of composite comprises a laminate, which may be created by stacking and binding a plurality of layers of one or more materials, wherein each layer comprises a reinforcement and/or a polymer matrix material. A layer of material in a laminate may comprise a polymeric film and/or a sheet of a polymeric material ( e.g ., a composite, a plastic, an elastomer, etc.), a reinforcement {e.g., a metal, a wood, a glass, etc.), or a combination thereof. A multilayered plastic film and/or a multilayered plastic sheet may be produced by coextrusion rather than creation of a laminate, due to the ease of processing. [0044] Processing of a polymeric material refers to manipulation of the material into a desired form of shape, size, consistency {e.g., a solid), etc. Often a polymeric material undergoes drying to removed moisture and/or a volatile liquid component {e.g., water) prior to processing to allow production of a suitable product. A polymeric material may comprise an additive, such as one or more antibiological composition(s) [e.g., antibiological agent(s)], to confer and/or modify a property of the polymeric material formulation. An additive ("modifier") used in a polymeric material may be incorporated ("compounded"), such as by being admixed, absorbed, etc. into the polymeric material and/or a precursor material {e.g., a monomer, a prepolymer). It is contemplated that any additives described herein (e.g., a coating additive) or that would be known to one of ordinary skill in the art may be incorporated into a polymeric material, with non-limiting examples including an adhesion promoter, an anti-aging additive, an anti-blocking agent, an anti-fogging agent, an antioxidant, an antiozonant, an antistatic agent, a blowing agent, a coupling agent, a crosslinking agent, a curing agent {e.g., a catalyst), a colorant, a defoamer, a degrading agent, a deodorant, a diluent, a dispersant, a filler, a flame retardant, a flux {i.e., a processing flow enhancer such as a coumarone-indene resin for use in a vinyl polymer), an impact modifier, an inhibitor, an initiator, a low-profile additive, a lubricant, an antibiological composition (e.g., an antibiological agent), a plasticizer, a promoter, a slip agent, a processing aid, a thickening agent, a thinner, a mold release agent, a thixotrope, a nucleating agent, a stabilizer {e.g., a heat stabilizer, a light stabilizer such as an UV stabilizer also known as a "UV protector"), a surfactant, an odorant, a wetting agent, or a combination thereof.

[0045] It is contemplated that any processing technique for a polymeric material described herein or that would be known to one of ordinary skill in the art may be used to form an article of manufacture, a device, a material formulation, etc. which may be coated and/or incorporate an antibiological composition (e.g., an antibiological agent). Non-limiting examples of such processing techniques include: injection molding, injection compression that may be used to prepare a thin walled part, continuous chain injection molding, coinjection molding, reciprocating-screw injection molding, screw plasticating injection molding, transfer molding, injection blow molding, blow molding, compression molding, vacuum bag molding, pressure bag molding, autoclaved molding, calendaring, solvent casting, solution casting, solvent molding, dip casting ("dip molding"), fhermoforming, stretch forming, skiving, cold drawing, cold forming, forging ram extrusion, extrusion coextrusion, rotational molding, slush molding, spinning melt spinning, foam molding, integrated skin molding, steam molding, sandwich molding, in situ foam molding, in mold assembly, injection molding hybridization, potting encapsulation casting, or a combination thereof.

[0046] In some embodiments, polymeric material processing techniques that use lower temperatures are preferred (e.g., solvent casting, solution casting, cold drawing, cold forming, etc.) for use when incorporating and/or applying the antibiological composition (e.g., an antibiological agent) and/or a surface treatment {e.g., a coating, a textile finish) comprising an antibiological composition (e.g., an antibiological agent), during or immediately after the processing technique. For example, coating {e.g., clear coating, painting) of the device and/or the subdevice may occur as well in the in-mold assembly to improve efficiency of manufacture. For example, a surface of a laminate [e.g., a material layer ("substrate layer")-foam-skin laminate] may be coated as part of an in-mold assembly process. Reaction injection molding typically involves injecting a chemically reactive component {e.g., a prepolymer of a thermoset) into a mold to undergo production of a polymeric material, often as part of an in-mold assembly process {e.g., a reaction to produce a foam layer in association with a skin).

[0047] In some embodiments, an article of manufacture, a device, a material formulation, etc. such as one comprising a polymeric material such as a plastic, reinforced polymeric material, composite {e.g., a laminate), or a combination thereof, may be further processed by standard processing/manufacturing techniques known to those of ordinary skill in the art, after release from a mold and/or being fashioned {e.g., die cut, knife cut, etc.) into a desired shape, size, and/or material properties. A polymeric material object may be further altered through tooling and machining such as abrasion, grinding, grit blasting, drilling, threading, welding ( e.g ., friction welding, ultrasonic welding, heat welding, heated tool welding, resistance wire welding, induction welding, infrared welding, hot-gas welding, laser welding, vibration welding, spin welding, stitching, etc.), cutting, tapping, reaming, sawing, milling, turning, routing, wire brushing, etc., often to allow assembly with other component(s). For example, an article and/or a device comprising a polymeric material may be produced by fabrication, which involves machining a polymeric material, often in the form of a sheet, a tube, and/or a rod, into a desired form, and assembled as desired with other component(s) using such processes as ashing, blanking, buffing, cementing, drawing, drilling, filing, forming, flame treatment of a polymeric material surface, grinding, milling, piercing, polishing {e.g., flame polishing a thermoplastic), sanding, sawing, tumbling, routing, turning, trimming, or a combination thereof. An adhesive may be used to bind such items and/or components as desired. A polymeric film and/or a sheet may be cut to desired size to produce a tape, and combined with an adhesive. An insert may be incorporated in and/or upon the polymeric material, typically through welding.

[0048] Numerous assays for determining the properties of a polymeric material {e.g., a plastic) are available to aid in preparation, processing, post cure processing, and/or completion of manufacture of a polymeric material. An assay may be used to tailor one or more properties of a composition and/or an article made from a polymeric material as desired, particularly in formulating a polymeric material comprising a biomolecule composition that may confer and/or alter a property {e.g., rigidness/flexibility, service life). Examples of physical/mechanical properties and assays for a polymeric material include: abrasion resistance {e.g., ASTM D 1242, ASTM D 1044); barcol hardness {e.g., ASTM D 2583); Rockwell hardness of a plastic {e.g., ASTM D 785); bursting strength of a plastic film and/or a sheet {e.g., ASTM D 1599, ASTM D 774); blocking, which refers to the clinginess of a polymer film to itself {e.g., ASTM D 3354, ASTM D 1893); density and crystallinity {e.g., ASTM D 1505); weight-average molecular weight of a nonionic homopolymer {e.g., ASTM D 4001); coefficient of friction {e.g., ASTM D 3028, ASTM D 1894); coefficient of thermal expansion {e.g., ASTM D 696, ASTM E 228, ASTM E 831); compressive strength {e.g., ASTM D 575, ASTM D 649, ASTM D 695); fatigue endurance {e.g., ASTM D 671); tensile elongation at break, tensile elongation at yield, tensile strength at break, tensile strength at yield, tensile strength, ultimate tensile strength, and/or tensile modulus {e.g., ASTM D 412, ASTM D 638, ASTM D 1708); a tensile/modulus property of a plastic film and/or a sheeting ( e.g ., ASTM D 882); stiffness/apparent modulus of rigidity of a plastic ( e.g ., ASTM D 1043); folding endurance of a polymeric film {e.g., ASTM D 2176); rigidity of a polyolefin polymeric film and/or a sheeting {e.g., ASTM D 2923); bearing strength {e.g., ASTM D 953); tear strength/tear resistance of a plastic film and/or a sheet {e.g., ASTM D 1922, ASTM D 1004; ASTM D 1938, ASTM D 2582); track/erosion resistance {e.g., ASTM D 2303); a flexural property such as flexural modulus, flexural strength {e.g., ASTM D 790, ASTM D 747,

ASTM D 650); brittleness of a polymeric material/impact resistance loss {e.g., ASTM D 1790, ASTM D 746); tensile/impact breakage of a plastic, such as a plastic container {e.g., ASTM D 1822, ASTM D 2463); impact strength/resistance of a plastic {e.g., a plastic film; see e.g., ASTM D 256 REV A, ASTM D 4272, ASTM D 1709, ASTM D 3029, ASTM D 3420); impact strength such as chip impact strength {e.g., ASTM D 4508); package yield of a plastic film {e.g., ASTM D 4321); puncture resistance of a plastic {e.g., ASTM D 3763); Poisson's ratio {e.g., ASTM E 132); tensile creep, flexural creep, compressive creep, and/or creep-rupture of a plastic {e.g., ASTM D 2990); fracture resistance {e.g., ASTM E 399); dynamic mechanical properties of a plastic at various temperatures and vibration frequencies {e.g., ASTM D 4065); biaxial orientation {e.g., ASTM D 2673, ASTM D 3664); shrinkage from mold dimensions of a thermoplastic {e.g., ASTM D 955); vapor transmission of a heat-sealing package {e.g., ASTM D 3079); and/or specific gravity and specific volume {e.g., ASTM D 792).

[0049] A surface treatment {e.g., a coating, a textile finish) may be added to the surface of a material (e.g., a polymeric material, glass, a cellulosic material (e.g., cardboard, paper, etc.), a surface already treated or coated with a surface treatment, etc.), and in some embodiments the surface treatment comprises an upper layer of material (e.g., a top-coat). The antibiological composition (e.g., an antibiological agent) and/or a surface treatment comprising the antibiological composition (e.g., an antibiological agent) may also be applied to the surface of a material that has not been treated with another surface treatment. For example, various coating techniques, including machine-based coating techniques, as described herein or as would be known to one of ordinary skill in the art may be used. For example, roll coating may use a roll to move a polymeric sheet and/or a polymeric film through a coating in a pan to coat the polymeric sheet and/or the polymeric film. Vapor curing may be used to coat a material, and involves contacting an uncrosslinked coating with a vaporized curing agent in an enclosed chamber to produce a cured coating upon the material.

[0050] Numerous assays described herein or as would be known to one of ordinary skill in the art for determining the properties of a manufactured article, a device, etc. comprising a polymeric material ( e.g a plastic) are available to aid in preparation, processing, post cure processing, and/or completion of manufacture of the manufactured article, a device, etc.

For example, an assay may be used to tailor one or more properties of an article, a device, a composition, etc. made from a polymeric material as desired, particularly in formulating a polymeric material comprising an antibiological composition (e.g., an antibiological agent) [e.g., via incorporation, applying the antibiological composition (e.g., an antibiological agent) to the surface, applying a surface treatment comprising an antibiological composition (e.g., an antibiological agent) to a surface, etc.].

[0051] It is contemplated that a coating comprising an antibiological composition (e.g., an antibiological agent) may be applied as a coating layer upon another coating, including clear coatings and paints as described herein or as would be known in the art for various applications. In some embodiments, particularly for a commonly used clear coating or paint, a thin layer for such a coating comprises about 5 pm to about 1500 pm thick (e.g., about 15 pm to about 150 pm thick), though the coating comprising an antibiological composition (e.g., an antibiological agent) may also be of this thickness in some embodiments. However, in certain embodiments, an antibiological food packaging coating may be applied in an uncured layer on a surface, or form a cured film upon a surface, of about between 5 nanometers to 1500 pm thick (e.g., about 5 nanometers to about 15 pm). Further, the coating comprising an antibiological composition (e.g., an antibiological agent) may comprise any component for a coating described herein or as would be known to one of ordinary skill in the art in light of the present disclosures. In an alternative embodiment, a material formulation (e.g., a paint or clear coating) may be prepared that lacks sufficient antibiological composition (e.g., an antibiological agent) to produce a desired amount of antibiological composition (e.g., an antibiological agent) properties, but still possesses other properties suitable for use in other applications.

[0052] A clear-coating refers to a coating that is not opaque (e.g., transparent, semi transparent, translucent) and/or does not produce an opaque solid film after application and cure, but may be colored or non-colored. Hiding power refers to the ability of a coating and/or a film ( e.g ., an opaque coating or film) to prevent light from being reflected from a surface. Examples of a clear-coating include, a lacquer, a varnish, a shellac, a stain, a water repellent coating, or a combination thereof. A paint refers to a "pigmented liquid, liquefiable or mastic composition designed for application to a substrate in a thin layer which is converted to an opaque solid film after application used for protection, decoration or identification, or to serve some functional purpose such as the filling or concealing of surface irregularities, the modification of light and heat radiation characteristics, etc." ["Paint and Coating Testing Manual, Fourteenth Edition of the Gardner-Sward Handbook"

(Koleske, J. V. Ed.), p. 696, 1995] It is contemplated that various formulations of paints known in the art may be converted to suitable clear coating by removal of components, such as colorants, that produce hiding power, opacity, or a combination thereof. Standard techniques for determining the hiding power of a coating and/or a film {e.g., paint, a powder coating) are described, for example, in "ASTM Book of Standards, Volume 06.01 , Paint -- Tests for Chemical, Physical, and Optical Properties; Appearance," E284-02b, D344-97, D2805-96a, D2745-00 and D6762-02a 2002; "ASTM Book of Standards, Volume 06.02,

Paint -- Products and Applications; Protective Coatings; Pipeline Coatings," D5007-99,

D5150-92 and D6441-99, 2002; and "Paint and Coating Testing Manual, Fourteenth Edition of the Gardner-Sward Handbook" (Koleske, J. V. Ed.), pp. 481-506, 1995.

[0053] A coating generally comprises one or more component materials that contribute to the properties of the coating, the ability of a coating to be applied to a surface, the ability of the coating to undergo film formation, and/or the properties of the produced film. Examples of such a coating component include a binder, a liquid component, a colorant, an additive, or a combination thereof, and such materials are contemplated for used in a coating.

[0054] A binder ("polymer," "resin," "film former") comprises a molecule capable of film formation. Film formation refers to a physical and/or a chemical change of a binder in a coating, wherein the change converts the coating into a film. Often, a binder converts into a film through a polymerization reaction, wherein a first binder molecule (e.g., a monomer) covalently bonds with at least a second binder molecule (e.g., a monomer) to form a polymer. A thermoplastic binder and/or a coating reversibly softens and/or liquefies when heated. Film formation for a thermoplastic coating generally comprises a physical process, typically the loss of the volatile {e.g., liquid) component from a coating. A thermosetting binder undergoes film formation by a chemical process, typically the crosslinking of a binder into a network polymer. Examples of a binder include an oil-based binder ( e.g ., an oil; an alkyd resin such as an oil length alkyd binder, a high solid alkyd, an uralkyd, a waterborne alkyd; an oleoresinous binder, a fatty acid epoxy ester, etc.), a polyester resin, a modified cellulose binder, a polyamide binder, an amidoamine binder, an amino resin, an urethane binder (e.g., a waterborne urethane, an urethane powder, etc.), a phenolic resin (e.g., a resole, a novolac, an epoxy resin, an ambient condition curing epoxy, a bake curing epoxy, an electrodeposition epoxy, a powder coating epoxy, an cycloaliphatic epoxy, etc.), a polyhydroxyether binder, an acrylic resin (e.g., a thermoplastic acrylic resin, a waterborne thermoplastic acrylic, a thermosetting acrylic resin such as an acrylic-epoxy combination, an acrylic-amino combination, an acrylic-urethane combination, a waterborne thermosetting acrylic, etc.), a polyvinyl binder (e.g., a plastisol, an organosol, etc.), a rubber resin (e.g., chlorinated rubber resin, a synthetic rubber resin, etc.), a bituminous binder, a polysulfide binder, a silicone binder, or a combination thereof.

[0055] A liquid component comprises a chemical composition in a liquid state {e.g., a liquid state while comprised in a coating, a film, a material formulation, etc.). A liquid component may be added to a coating formulation, for example, to improve a rheological property for ease of application, alter the period of time that thermoplastic film formation occurs, alter an optical property {e.g., color, gloss, etc.) of a film, alter a physical property of a coating {e.g., reduce flammability) and/or a film {e.g., increase flexibility), participate in a chemical reaction of a coating component (e.g., a water molecule hydrolyzing a silane molecule into a silanol molecule), dissolve and/or suspend a coating component (e.g., water dissolving a biomolecule composition into a coating formulation), or a combination thereof. In general aspects, the liquid component comprises a solvent, a thinner, a diluent, a plasticizer, or a combination thereof. A solvent comprises a liquid component used to dissolve one or more components of a material {e.g., a coating). A thinner comprises a liquid component used to reduce the viscosity of a coating, and may aid dissolving a coating component (e.g., act as a cosolvent) and/or increase miscibility of two or more coating component. A diluent comprises a liquid component that does not dissolve a binder. In other aspects, the liquid component comprises a liquid organic compound, an inorganic compound, water, or a combination thereof. Examples of a liquid organic compound includes a hydrocarbon (e.g., an aliphatic hydrocarbon, a cycloaliphatic hydrocarbon, a terpene, an aromatic hydrocarbon, etc.), an oxygenated compound (e.g., an alcohol, a ketone, an ester, a glycol ether, a ketone, an ether, etc.), a chlorinated hydrocarbon, a nitrated hydrocarbon, a miscellaneous organic liquid component, a plasticizer, or a combination thereof. In some embodiments, water may not be used to in a coating formulation, such as wherein a particular biomolecule composition can be dissolved and/or suspended in a non-aqueous liquid component.

[0056] A colorant ("colorizing agent") comprises a composition that confers an optical property to a coating. Examples of an optical property, depending upon the application, include a reflection property, a light absorption property, a light scattering property, or a combination thereof. A colorant that increases the reflection of light may increase gloss. A colorant that increased light scattering may increase the opacity and/or confer a color to a coating and/or a film. Light scattering of a broad spectrum of wavelengths may confer a white color to a coating and/or a film. Scattering of a certain wavelength may confer a color associated with the wavelength to a coating and/or a film. Light absorption also affects opacity and/or color, such as broad-spectrum light absorption conferring a black color to a coating and/or a film. Absorbance of a certain wavelength may eliminate the color associated with the wavelength from the appearance of a coating and/or a film. Examples of a colorant include a pigment, a dye, an extender, or a combination thereof. A colorant ( e.g a pigment, a dye) and procedures for determining the optical properties and physical properties {e.g., hiding power, transparency, light absorption, light scattering, tinting strength, color, particle size, particle dispersion, pigment content, color matching) of a colorant, a coating component, a coating and/or a film are described in, for example, (in "Industrial Color Testing, Fundamentals and Techniques, Second, Completely Revised Edition," 1995; "Colorants for Non-Textile Applications," 2000; "Colour Index International," 3 rd Ed. Society of Dyers and Colourists American Association of Textile Chemists and Colorists, 1971; and "Colour Index International," 3 rd Ed. Pigment and Solvent Dyes,

Society of Dyers and Colourists American Association of Textile Chemists and Colorists, 1997). Pigments possess a variety of properties in addition to color, with examples including a tinctorial property (/. e. , the ability to produce a color), an insolubility property {i.e., the ability to remain a solid upon contact with a coating component such as, a liquid component), a corrosion resistance property {i.e., the ability to reduce the damage of a chemical such as water that contacts a metal), a durability property, a heat resistance property, an opacity property, a transparency property, or a combination thereof. A camouflage pigment refers to a pigment typically selected to camouflage a surface ( e.g ., a military surface) from visual and, in specific facets, infrared detection. A color property refers to the ability of a composition to confer a visual color and/or metallic appearance, with examples of a color property pigment includes a black pigment, a brown pigment, a white pigment, a pearlescent pigment, a violet pigment, a blue pigment, a green pigment, a yellow pigment, an orange pigment, a red pigment, a metallic pigment, an extender pigment, or a combination thereof. A dye comprises a composition that is soluble in the other component(s) of a coating, and further confers a color property to the coating. In some embodiments, a colorant may comprise a biomolecule composition (e.g., a bio-based antibiological agent).

[0057] A coating additive comprises any material added to a coating to confer a property other than that described for a binder, a liquid component, a colorizing agent, or a combination thereof. Examples of a coating additive include a biomolecule composition (e.g., a bio-based antibiological agent), an accelerator, an adhesion promoter, an antioxidant, an antiskinning agent, an antifloating agent, an antiflooding agent, an antifoaming agent, an antisettling agent, an antiskinning agent, an anti-insect additive, a buffer, a catalyst (e.g., a drier, an acid, a base, a urethane catalyst, etc.), a coalescing agent, a corrosion inhibitor, a defoamer, a dehydrator, a dispersant, a drier, an electrical additive, an emulsifier, a film-formation promoter, a fire retardant, a flow control agent, a gloss aid, a leveling agent, a light stabilizer (e.g., a UV absorber, a radical scavenger, etc.), a marproofing agent, a matting agent, a neutralizing agent, an antibiological composition (e.g., a bio-based antibiological agent, a chemical preservative, etc.), a rheology modifier, a slip agent, a thickening agent, a wetting agent, a viscosity control agent, or a combination thereof. The content for an individual coating additive in a coating may be about 0.000001 % to about 20.0%. However, in many embodiments, the concentration of a single additive in a coating may comprise between 0.000001% and about 10.0%. In some embodiments, a polymeric material additive may be used in a coating.

[0058] A coating may be applied to a surface using any technique described herein or as would be known to one of ordinary skill in the art. In the context of a coating, "application," "apply," or "applying" refers to the process of transferring of a coating to a surface to produce a layer of coating upon the surface. As known herein in the context of a coating, an "applicator" refers to a devise that may be used to apply the coating to a surface. Examples of an applicator include a machine or device, such as a coating machine (e.g., an optical coating application machine), a brush, a roller, a pad, a rag, a spray applicator, etc. Examples of application techniques include dipping, pouring, siphoning, brushing, rolling (e.g., roll coating), padding, ragging, spraying (e.g., spraying by a robot), anodizing, deposition (e.g., electrodeposition), an electrostatic technique, electroplating, vapor curing, layer by layer assembly, photografting, and/or laminating of a film onto a surface, or a combination thereof. In certain embodiments, the layer of coating undergoes film formation ("curing," "cure"), which refers to the physical and/or chemical change of a coating to a solid when in the form of a layer upon the surface. In certain aspects, a coating may be prepared, applied and/or cured at an ambient condition, a baking condition, or a combination thereof, within the ability of biomolecule composition (e.g., a bio-based antibiological agent) to retain bioactivity. An ambient condition comprises a temperature range between about -10°C to about 40°C, while a "baking condition" or "baking" comprises contacting a material formulation with a temperature above about 40°C and/or raising the temperature of a material formulation above about 40°C (e.g., about 40°C to about 300°C, about 40°C to about 130°C).

[0059] A coating may comprise a volatile coating component (e.g., a coalescing agent, a solvent such as water or a non-water solvent, a thinner, a diluent, etc.), a non-volatile coating component (/. e. , a component that remains upon a surface after cure such as a binder, a colorizing agent, a plasticizer, a coating additive, etc.), or a combination thereof.

A coating component may undergo a chemical change to form a film, such as binder undergoes a crosslinking and/or a polymerization reaction (e.g., an oxidative and/or free radical chemical reaction) to produce a film. A chemical film formation reaction may be promoted by irradiating the coating (e.g., irradiating the coating to electromagnetic radiation such as UV radiation, visible light, infrared radiation; particle radiation such as electron- beam radiation, etc.), heating the coating, or a combination thereof. In some alternate embodiments, a coating known herein as a non-film forming coating undergoes a reduced amount of film formation than such a solid film is not produced during the period of time it may be used on a surface. In other alternative embodiments, a coating may undergo film formation, but produce a film whose properties makes it more suited for a temporary use, and is known herein as a temporary film. [0060] A plurality of coating layers ( e.g ., 1 to about 30 layers), known herein as a "multicoat system" ("multicoating system"), may be applied upon a surface, and one or more of the coating layers may differ in composition and/or properties, with examples of coating layers including a sealer, a water repellent, a primer, an undercoat, a topcoat, or a combination thereof. A topcoat is the uppermost coating layer, regardless of whether it is part of a multicoat system or a single coating layer upon a surface, and in many embodiments a topcoat comprising an antibiological composition (e.g., an antibiological agent).

[0061] A coating may be classified by its end use, including, for example, as an architectural coating, an industrial coating, a specification coating, or a combination thereof, and such coatings are described, for example, in "Paint and Surface Coatings: Theory and Practice" 2 nd Edition, pp. 190-192, 1999; in "Paints, Coatings and Solvents" 2 nd Edition, pp. 330-410, 1998; in "Organic Coatings: Science and Technology, Volume 1: Film Formation, Components, and Appearance" 2 nd Edition, pp. 138 and 317-318. An architectural coating refers to "an organic coating intended for on-site application to interior or exterior surfaces of residential, commercial, institutional, or industrial buildings, in contrast to industrial coatings. They are protective and decorative finishes applied at ambient conditions" ["Paint and Coating Testing Manual, Fourteenth Edition of the Gardner-Sward Flandbook"

(Koleske, J. V. Ed.), p. 686, 1995)]. Examples of an architectural coating includes a wood coating, a masonry coating, an artist’s coating, a plastic coating, a metal coating, a glass coating, or a combination thereof. Artist coatings refer to a coating used by artists for a decorative purpose.

[0062] An industrial coating refers to a coating applied to a surface of a manufactured product in a factory setting, typically for a protective and/or aesthetic purpose. Examples of an industrial coating comprises an automotive coating, a can coating, a sealant coating, or a combination thereof. Can coatings refer to coatings used on a container {e.g., an aluminum container, a steel container), such as for a food, a chemical, or a combination thereof. Sealant coatings refer to coatings used to fill a joint (e.g., a coating that bridges by contact a gap between two or more surfaces) to reduce or prevent passage of a gas {e.g., air), water, a small material {e.g., dust), a temperature change, or a combination thereof. A marine coating comprises a coating used on a surface that contacts water and/or a surface that comprises part of a structure continually near water {e.g., a ship vehicle, a dock, a drilling platform for fossil fuels, etc.). [0063] Can coatings refer to coatings used on a container ( e.g ., an aluminum container, a steel container), such as for a food, a chemical, or a combination thereof. The manufacturers of a can typically require that a coating conform to specific properties of corrosion resistance, inertness {e.g., to prevent flavor alterations in food, a chemical reaction with a container’s contents, etc.), appearance, durability, or a combination thereof. Typically, a can coating comprises an acrylic-coating, an alkyd-coating, an epoxy-coating, a phenolic-coating, a polyester-coating, a poly(vinyl chloride)-coating, or a combination thereof. Though a can may be made of the same or similar material, different surfaces of a can may require coating(s) of differing properties of inertness, durability and/or appearance. For example, a coating for a surface of the interior of a can that contacts the container’s contents may be selected for a chemical inertness property, a coating for a surface at the end of a can may be selected for a physical durability property, or a coating for a surface on the exterior of a can may be selected for an aesthetic property. To meet the varying can's surface requirements, a can coating may comprise a multicoat system. In specific embodiments, a can multicoat system comprises a primer, a topcoat, or a combination thereof. In certain embodiments, an epoxy-coating, a poly(vinyl chloride-coating), or a combination thereof may be selected as a primer for a surface at the end of a can. In other embodiments, an oleoresinous-coating, a phenolic-coating, or a combination thereof may be selected as a primer for a surface in the interior of a can. In some aspects, a waterborne epoxy and acrylic coating may be selected as a topcoat for a surface of an interior of a can. In additional embodiments, an acrylic-coating, an alkyd-coating, a polyester-coating, or a combination thereof may be selected as an exterior coating. In certain facets, a can coating {e.g., a primer, a topcoat) may comprise an amino resin, a phenolic resin, or a combination thereof for crosslinking in a thermosetting film formation reaction. In certain embodiments, a can coating may be applied to a surface by spray application. In other embodiments, a can coating undergoes film formation by UV irradiation. Specific procedures for determining the suitability of a coating and/or a film for use as a can coating, have been described, for example, in "Paint and Coating Testing Manual, Fourteenth Edition of the Gardner-Sward Handbook," (Koleske, J. V. Ed.), pp. 717-724, 1995.

[0064] A specification coating ("specification finish coating") refers to a coating formulated to a "precise statement of a set of requirements to be satisfied by a material, produce, system, or service that indicates the procedures for determining whether each of the requirements are satisfied" with various sources for specification coating requirements and procedures for determining the suitability of a coating ( e.g ., an aircraft coating) described in ["Paint and Coating Testing Manual, Fourteenth Edition of the Gardner-Sward Handbook" (Koleske, J. V. Ed.), pp. 683-695, 891-893, 1995] Examples of a specification finish coating include a military specified coating (e.g., a chemical agent resistant coating ("CARC"), a camouflage coating), a Federal agency {e.g., Department of Transportation) specified coating, a state specified coating, a pipeline coating, a traffic marker coating, an aircraft coating, a nuclear power plant coating, or a combination thereof. A camouflage coating comprises a coating that may be formulated with a material {e.g., a pigment) that reduces the differences in visible light or non-visible light {e.g., infrared radiation) detection between a coated surface relative to the surrounding environment. A pipeline coating is applied to pipes, such as ones used to convey a fossil fuel. A traffic marker coating comprises a coating {e.g., a paint) used to visibly convey information on a surface usually subjected to weathering and abrasion {e.g., a pavement). An aircraft coating protects and/or decorates a surface of an aircraft vehicle. A nuclear power plant, which generally possesses particular properties {e.g., gamma radiation resistance, chemical resistance, etc.).

[0065] The selection of a biomolecule composition for incorporation into a material formulation to confer an antibiological property, a manufactured article having a surface treatment and/or surface with such an antibiological property, a device having a surface treatment and/or surface with such an antibiological property, etc. may be based on the bioactivity of the biomolecule composition. Methods for assaying and/or selecting an antibiological biomolecule composition are described herein as well as in U.S. Patent Nos. 6,020,312; 5,885,782; and 5,602,097, and Patent Application Nos. 10/884,355 and 11/368,086, such as, for example, contacting a material formulation (e.g., a coating) comprising a proteinaceous molecule with a biological cell and measuring growth over time relative to a like material formulation comprising less or no selected proteinaceous molecule content. For example, an in vitro method to determine bioactivity of a peptide, such as a peptide from a synthetic peptide combinational library, may be used (Furka, A., et. al., 1991; Houghten, R. A., et. al., 1991; Houghten, R. A., et. al., 1992).

[0066] Examples of a biological cell include a prokaryotic cell and/or a eukaryotic cell. In some embodiments, an antibiological composition (e.g., an antibiological agent) functions by binding a biomolecule of a biological entity to disrupt the function of the biomolecule, such as, for example an enzyme cleaving a cellular biomolecule important in adherence to a surface; a peptide associating with and disrupting a cellular membrane important in metabolic function and/or the physical integrity of a cell; etc. In many embodiments, a cellular component such as component of a cell wall, a viral component (e.g., a viral proteinaceous molecule), and/or a cellular membrane may comprise a target of an antibiological composition (e.g., an antibiological agent). Examples of such cellular component includes a cell wall, a viral proteinaceous molecule, and/or a cellular membrane biomolecule component includes a peptidoglycan, a pseudopeptidoglycan, a teichoic acid, a teichuronic acid, a cellulose, a neutral polysaccharide, a chitin, an argarose, a mannin, a glucan, a proteinaceous molecule, a lipid {e.g., a phospholipid), or a combination thereof. For example, many proteinaceous molecule(s) have antibiological properties, such as an antibiological peptide, antibiological polypeptide, an antibiological enzyme, and/or an antibiological protein. In a further example, a lipolytic enzyme such as a phospholipase and/or an antimicrobial peptide that acts to compromise the integrity of a cell membrane, may allow ease of access for one or more enzyme(s) that degrade a cell wall component, and/or allow ease of access for non-biomolecule antibiological composition (e.g., a preservative) to act as well. However, for the purposes of preparing and using an active antibiological composition (e.g., an antibiological agent), used in a material formulation (e.g., a paint, a coating composition), it may not be necessary to understand the mechanism by which the desired antibiological effect is exerted on a cell and/or a virus. In some aspects, such an antibiological composition (e.g., an antibiological agent) may possess a biocidal and/or a biostatic activity. For example, an antimicrobial and/or an antifouling enzyme may act as a biocide and/or a biostatic. In some embodiments, an antibiological composition (e.g., an antibiological agent) may inhibit growth of a cell and/or a virus, which refers to cessation and/or reduction of cell (e.g., a fungal cell) and/or viral proliferation, and can also include inhibition of expression of cellularly produced proteins in a static cell colony.

[0067] In an example, a biomolecule composition possessing an antibiological activity (e.g., biocidal activity, biostatic activity, etc.) may be incorporated separately into one or more components of surface treatment (e.g., a coating, a textile finish) and confer antibiological activity to those components prior to, during, and/or after admixing to form a surface treatment. In another example, a biomolecule composition possessing an antibiological property may be admixed with the material formulation with or without prior incorporation of a biomolecule composition to one or more components of the material formulation. In an additional example, the surface of a manufactured article, a device, a material formulation, etc., having a material formulation comprising a biomolecule composition (e.g., a surface treatment) applied to the surface would also possess such property(s), such as for example a cured film possessing antibiological property to protect the film and/or material the film whose surface the film is adhered. Thus, in many preferred embodiments, a material formulation may comprise an antibiological composition (e.g., an antibiological agent) by being formulated, prepared, processed, post-cured processed, manufactured, and/or applied {e.g., applied to a surface), in a fashion to be suitable to possess an antibiological activity and/or function (e.g., an antimicrobial activity, an antifouling activity).

[0068] In other embodiments, a biomolecule composition may possess a self-cleaning bioactivity, and may confer such a self-cleaning property to a material formulation comprising such a biomolecule composition. As used herein, self-cleaning bioactivity includes conversion of a chemical (e.g., a biomolecule) into a product that is less adherent (e.g., easier to remove with washing), and/or retards infestation (e.g., adherence to a surface, growth upon a surface) of a biological entity (e.g., a biological cell, a virus). In many embodiments, a biomolecule composition possesses one of or both antibiological and self-cleaning property(s).

[0069] An antibiological agent may be combined with any other antibiological composition described herein and/or known in the art, such as a preservative (e.g., a chemical biocide, a chemical biostatic) traditionally used in a surface treatment (e.g., a coating, a paint) and/or an antimicrobial agent (e.g., a chemical biocide, a chemical biostatic) and/or technique (see for example, Baldridge, G.D. et. at., 2005; Hancock, R.E.W. and Scott,

M.G., 2000) traditionally used in a polymeric material (e.g., a plastic, a composite, etc.).

For example, one or more antibiological agent(s) may be used in combination with and/or as a substitute for one or more existing antibiological composition (e.g., a preservative, an antimicrobial agent, a fungicide, a fungistatic, a bactericide, an algaecide, etc.) identified herein and/or in the art. Examples of an antibiological composition that an antibiological agent may substitute for and/or be combined include, but are not limited to those non- peptidic antimicrobial compounds (/. e. , biocides, fungicides, algaecides, mildewcides, etc.) which have been shown to be of utility and are currently available and approved for use in the U.S. /NAFTA, Europe, and the Asia Pacific region, and numerous examples are described herein for use with a material formulation such as a polymeric material, a surface treatment (e.g., a coating), etc. Some such combinations of antibiological agent(s) and/or combinations with another antibiological compositions may provide an advantage such as a broader range of activity against various organisms (e.g., a bacterium, an alga, a fungus, etc.), a synergistic antibiological and/or preservative effect, a longer duration of effect, or a combination thereof. In some aspects, an antibiological composition (e.g., an antibiological agent) and/or technique comprises a detergent {e.g., a nonionic detergent, a zwitterionic detergent, an ionic detergent), such as CHAPS (zwitterionic), a Triton X series detergent (nonionic), and/or a SDS (ionic); a basic protein such as a protamine; a cationic polysaccharide such as chitosan; a metal ion chelator such as EDTA; or a combination thereof, all of which have may have effectiveness against a lipid cellular membrane, and may be incorporated into a material formulation and/or used in a washing composition {e.g., a washing solution, a washing suspension, a washing emulsion) applied to a material formulation. In another example, an additional preservative, a biocide, a biostatic composition, or a combination thereof, comprises a non-peptidic antimicrobial composition, a non-amino based antimicrobial agent, a compounded peptide antimicrobial agent, an enzyme-based antimicrobial agent, or a combination thereof, such as those described in U.S. Patent Application 11/865,514 filed 10/1/07, incorporated by reference. In some embodiments, the concentration of any individual antibiological composition’s component(s) (e.g., an antimicrobial agent, an antibiological chemical) comprises about 0.000000001% to about 20% {e.g., about 0.000000001% to about 4%) or more, of a material formulation, an antibiological composition (e.g., an antibiological agent), a washing composition, or a combination thereof.

[0070] In some aspects, a biological entity that may be a target of an antibiological composition (e.g., an antibiological agent) comprises an Animalia (“animal”) cell, a Plantae (“plant”) cell, an Archaea ('Archaebacteria") cell, a Eubacteria ("bacteria") cell, a Fungi (“fungi”) cell, a Protista (“protists”) cell, a virus {e.g., an enveloped virus), or a combination thereof. An Archaea typically comprises a cell wall comprising a pseudopeptidoglycan, a peptide, a polypeptide, a protein (e.g., a glycoprotein), or a combination thereof. A Eubacteria typically comprises a cell wall comprising a peptidoglycan, a peptide, a polypeptide, a protein (e.g., a glycoprotein), a lipid, or a combination thereof. A Gram positive bacterium generally have a cell wall composed of a thick layer of peptidoglycan overlaid by a thinner layer of teichoic acid. A "Gram -negative Eubacteria" ("Gram negative bacteria”) refers to Eubacteria comprising a cell wall that typically stains negative with Gram stain reaction and may be surrounded by a second lipid bilayer ("outer cell membrane"). Gram negative bacteria have a thinner layer of peptidoglycan. Organisms of the eukaryotic Fungi Kingdom ("fungi," fungus") include organisms commonly referred to as a molds, morels, mildews, mushrooms, puffballs, rusts, smuts, truffles, and yeasts. A fungi cell wall typically comprises a beta-1 ,4-linked homopolymers of N-acetylglucosamine (“chitin”) and a glucan. The glucan is usually an alpha-glucan, such as a polymer comprising an alpha-1 ,3- and alpha-1 ,6- linkage (Griffin, 1993). Some Ascomycota species (e.g., Ophiostomataceae) comprise a cell wall comprising a cellulose. Certain species of Chytridiomycota {e.g., Coelomomycetales) do not possess a cell wall (Alexopoulos et. at., 1996). Organisms of the Kingdom Protista ("protists") refer to a heterogenous set of eukaryotic unicellular, oligocellular and/or multicellular organisms that may not have been classified as belonging to the other eukaryotic Kingdoms, though they typically have features related to the Plant Kingdom {e.g., an alga, which generally are photosynthetic), the Fungi Kingdom {e.g., an Oomycota ) and/or the Animal Kingdom {e.g., a protozoa). Organisms of certain Protista Phyla, particularly those organisms commonly known as "algae," comprise a cell wall, silica-based shell and/or exoskeleton {e.g., a test, a frustule), or other durable material at the cell-external environment interface. A diatom refers to a unicellular alga that possess a cell wall comprising silicon. An Euglenophyta (“euglenoids”) generally is unicellular, aquatic algae and comprises a pellicle, which comprises an outer membrane reinforced by proteins, rather than a cell wall. A Chlorophyta (“green algae”) typically forms unicellular to oligocellular cluster(s), and comprises a cell wall comprising a cellulose. A Rhodophyta (“red algae”) generally is multicellular and comprises a cell wall comprising a sulfated polysaccharide, such as, for example, an agar, a carrageenan, a cellulose, or a combination thereof. A Pyrrophyta (“fire algae,” “dinoflagellate”) generally is a unicellular marine organism possessing a cell wall comprising cellulose. A Ciliophora (“ciliate”) generally is unicellular and comprises a pellicle. An Oomycota (“oomycete,” “water mold”) is a fungi-like organism, and is often listed in the fungal sections of biological culture collections. An Oomycota is typically unicellular but differ from a fungus by possessing a cell wall that comprises a cellulose and/or a glycan. Examples of a virus (e.g., an enveloped virus) that may be a target of an antibiological composition (e.g., an antibiological agent) includes a DNA virus.

[0071] A polymeric material (e.g., a plastic) will often comprise an antimicrobial agent (i.e. , an antimicrobial chemical). An antimicrobial agent typically comprises a biocide {e.g., a fungicide, a bactericide, a herbicide a mildewcide, an algaecide, a viricide, a germicide, a microbiocide, a slimicide) and/or a biostatic {e.g., a fungistatic, a bacteriostatic, a mildewstatic, an algaestatic, a viristatic, a herbistatic, a germistatic, a microbiostatic, a slimistatic) to inhibit the growth of an organism such as a bacteria, a fungi, a mildew, an algae, a virus, a microorganism, or a combination thereof, on and/or within a material formulation. An antimicrobial agent within a polymeric material typically diffuses and/or travels to the surface of the polymeric material during normal service life to provide a more continuous activity at the surface in reducing microbial growth. Often, an antimicrobial agent comprises a carrier such as a liquid component {e.g., a solvent, a plasticizer), a resin, or a combination thereof. Specific examples of a carrier typically used as an antimicrobial agent carrier includes plasticizer {e.g., a diisodecyl phthalate, an epoxidized soybean oil), an oil, or a combination thereof. Examples of an antimicrobial agent commonly used in a polymeric material includes 2-n-octyl-4-ixothiazonin-3-1 ; 10,10- oxybisphenoxarsine ("OBPA"); zinc 2-pyridinethiol-1 -oxide ("zinc-omadine"), trichlorophenoloxyphenol ("triclosan"), or a combination thereof, though a preservative used in a coating as well as an antibiological composition (e.g., an antibiological agent) are contemplated for use in a polymeric material, and such an antimicrobial agent may be used either alone or in combination with another antibiological composition in any composition, article, method, machine, etc. described herein in light of the present disclosures. An antimicrobial agent generally comprises about 0.000001% to about 1% of a polymeric material, and about 2% to about 10% of an anti-microbial agent and a carrier mixture, respectively, though given the inclusion of a biomolecule composition as part of a polymeric material and other compositions described herein, the content of an antimicrobial agent may be increased from about 0.000001% to about 10% or more. An antimicrobial agent often acts as a deodorant by reducing the growth of odor producing microorganism, particularly in a fiber {e.g., a textile) and/or a polymeric film application for packaging of food and/or trash. [0072] A coating may comprise a preservative to reduce and/or prevent the deterioration of an object, a coating and/or a film by an organism such as a microorganism. A microorganism may be considered a contaminant capable damaging an object, a film and/or a coating to the point of suitable usefulness in a given embodiment. The amount of preservative added to a coating comprising a biomolecule composition may be increased relative to a preservative content of a similar coating lacking such an added biomolecule composition. In certain aspects, the amount of preservative may be increased about 1.01 to about 10-fold or more, the amount of an example of a preservative content described herein or used in the art, in light of the present disclosures.

[0073] Examples of preservatives include a biocide (e.g., a microbiocide, a bactericide, a fungicide, an algaecide, a mildewcide, a mulluscicide, a viricide, etc.), which reduces and/or prevents the growth of an organism by killing the organism (e.g., a microorganism, a spore), a biostatic (e.g., a microbiostatic, a bacteriostatic, a fungistatic, an algaestatic, a mildewstatic, a mulluscistatic, a viristatic, etc.), which reduces and/or prevents the growth of an organism (e.g., a microorganism, a spore) but generally does not necessarily kill the organism, or a combination thereof (e.g., a combination of the effects).

[0074] Techniques for determining microbial contamination of a coating and/or a coating component have been described (see, for example, 'ASTM Book of Standards, Volume 06.01, Paint -- Tests for Chemical, Physical, and Optical Properties; Appearance," D5588- 97, 2002).

[0075] Determination of whether damage to a coating and/or a film may be due to a microorganism (e.g., a film algal defacement, a film fungal defacement), as well as the efficacy of addition of a preservative to a coating and/or a film composition in reducing microbial damage to a coating and/or a film, may be empirically determined [see, for example, Flick, E. W. “Handbook of Paint Raw Materials, Second Edition,” 263-285 and 879-998, 1989; in “Paint and Coating Testing Manual, Fourteenth Edition of the Gardner- Sward Handbook,” (Koleske, J. V. Ed.), pp 261-267 and 654-661, 1995; in “Paint and Surface Coatings, Theory and Practice, Second Edition,” (Lambourne, R. and Strivens, T. A., Eds.), pp. 193-194, 371-382 and 543-547, 1999; Wicks, Jr., Z. W., Jones, F. N.,

Pappas, S. P. “Organic Coatings, Science and Technology, Volume 1: Film Formation, Components, and Appearance,” pp. 318-320, 1992; Wicks, Jr., Z. W., Jones, F. N.,

Pappas, S. P. “Organic Coatings, Science and Technology, Volume 2: Applications, Properties and Performance,” pp. 145, 309, 319-323 and 340-341, 1992; in “Paints, Coatings and Solvents, Second, Completely Revised Edition,” (Stoye, D. and Freitag, W., Eds.) pp 6, 127 and 165, 1998; In “Waterborne Coatings and Additives,” 202-216, 1995; in “Handbook of Coatings Additives,” pp. 177-224, 1987; and in “PCI Paints & Coatings Industry,” pp. 56, 58, 60, 62, 64, 66-68, 70, 72 and 74, July 2003] In conducting such procedures, microorganisms such as, for example, Gram-negative Eubacteria including Alcaligenes faecalis (ATCC No. 8750), Pseudomonas aeruginosa (ATCC Nos. 10145 and 15442), Pseudomonas fluorescens (ATCC No. 13525), Enterobacter aerogenes (ATCC No. 13048), Escherichia coli (ATCC No. 11229), Proteus vulgaris (ATCC No. 8427), Oscillatoria sp. (ATCC No. 29135), and Calothrix sp. (ATCC No. 27914); Gram-positive Eubacteria including Bacillus subtilis (ATCC No. 27328), Brevibacterium ammoniagenes (ATCC No. 6871), and Staphylococcus aureus (ATCC No. 6538); filamentous fungi including Aspergillus oryzae (ATCC No. 10196), Aspergillus flavus (ATCC No. 9643), Aspergillus niger (ATCC Nos. 9642 and 6275), Aureobasidium pullulans (ATCC No. 9348), Penicillium sp. (ATCC No. 12667), Penicillium citrinum (ATCC No. 9849), Penicillium funiculosum (ATCC No. 9644), Cladosporium cladosporoides (ATCC No. 16022), Trichoderma viride (ATCC No. 9645), Ulocladium atrum (ATCC No. 52426), Alternaria alternate (ATCC No. 52170), and Stachybotrys chartarum (ATCC No. 16026); yeast including Candida albicans (ATCC No. 11651); and Protista including Chlorella sp. (ATCC No. 7516), Chlorella vulgaris (ATCC No. 11468), Chlorella pyrenoidosa (UTEX No. 1230), Chlorococcum oleofaciens (UTEX No. 105), Ulothrix acuminata (UTEX No. 739), Ulothrix gigas (ATCC No. 30443), Scenedesmus quadricauda (ATCC No. 11460), Trentepohlia aurea (UTEX No. 429), and Trentepohlia odorata (CCAP No. 483/4); have been used as positive control contaminants of a coating. [0076] In additional aspects, a poor and/or a low microorganism/biological resistance rating for a coating may be denoted as a colony recovery/growth rating of 2 to 4, a discoloration/disfigurement rating of 0 to 5, a fouling resistance ("F.R.") or antifouling film ('A.F") rating of 0 to 70, and observed growth ( e.g fungal growth) on specimens of 2 to 4, respectively, as described in 'ASTM Book of Standards, Volume 06.01 , Paint -- Tests for Chemical, Physical, and Optical Properties; Appearance," D3274-95, D2574-00, D3273-00, D5589-97 and D5590-00, 2002; and in "ASTM Book of Standards, Volume 06.02, Paint -- Products and Applications; Protective Coatings; Pipeline Coatings," D3623-78a, 2002. An additional example of a standard microorganism/biological resistance assay may be described in "ASTM Book of Standards, Volume 06.01 , Paint -- Tests for Chemical,

Physical, and Optical Properties; Appearance," D4610-98 and D3456-86, 2002; in "ASTM Book of Standards, Volume 06.02, Paint -- Products and Applications; Protective Coatings; Pipeline Coatings," D4938-89, D4939-89, D5108-90, D5479-94, D6442-99, D6632-01, □4940-98 and D5618-94, 2002; and "ASTM Book of Standards, Volume 06.03, Paint -- Pigments, Drying Oils, Polymers, Resins, Naval Stores, Cellulosic Esters, and Ink Vehicles," D912-81 and D964-65, 2002.

[0077] As used herein, a coating ("coat," "surface coat," "surface coating") refers to "a liquid, liquefiable or mastic composition that is converted to a solid protective, decorative, or functional adherent film after application as a thin layer" ("Paint and Coating Testing Manual, Fourteenth Edition of the Gardner-Sward Handbook" (Koleske, J. V. Ed.), p. 696, 1995; and in "ASTM Book of Standards, Volume 06.01, Paint -- Tests for Chemical, Physical, and Optical Properties; Appearance," D16-00, 2002). A coating comprising a preservative described herein in embodiments is generally thinner than many common types of coatings (e.g., a typical architectural paint), and in many embodiments, may be from about a molecular layer (e.g., about 32 picometer (“pm”) to about 10,000 pm) to about 5 pm thick.

[0078] A biomolecule composition may be incorporated upon and/or within {e.g., embedded) a material formulation used in the manufacture of an article, a device, a composition, etc. via methods described herein or as would be known to one of ordinary skill in the art in light of the present disclosure. These methods include, for example, application of a surface treatment (e.g., a coating) to the surface of a manufactured article, a device, and/or a composition, etc .; direct addition to a material formulation, incorporation as a component of a de novo formulation during preparation, post preparation absorption, in situ incorporation, post-polymerization incorporation, or a combination thereof, and may be used a substitute for, or in combination with, the other techniques described herein for processing and incorporation of such a composition into a material formulation. For a purpose such as ease of production, a material formulation may be provided as a single premixed formulation. In some embodiments, the components of a material formulation may be stored separately prior to combining for use. For example, for a purpose such as to optimize the initial activity {e.g., the activity of a biomolecule composition component) and/or extend the useful lifetime of the material formulation and/or the activity of a biomolecule composition, a biomolecule composition may be packaged separately from the other components of a material formulation into which the biomolecule composition may be added/incorporated/admixed. Thus, in certain embodiments, one or more components of a material formulation may be stored separately ( e.g a kit of components) prior to combining. The components may be stored in two or more containers (“pot”) {e.g., about 2 to about 20 containers) in a multipack kit. In preferred embodiments, the biomolecule composition is incorporated into a surface treatment (e.g., a coating) that is applied to the surface of a material that will be at least a part or all of a manufactured article, a device, a composition, etc.

[0079] The properties of an antibiological composition (e.g., an antibiological agent) in and/or upon a manufactured article, a device, a material formulation, etc. may be empirically determined by techniques described herein or as would be known to one of ordinary skill in the art. For example, a coating, such as one comprising an antibiological composition (e.g., an antibiological agent) described herein, with a desired set of properties for a particular use may be prepared by varying the ranges and/or combinations of coating component(s), including a biomolecule composition (e.g., a bio-based antibiological agent) described herein, and such coating selection and preparation may be done in light of the present disclosures. For example, a variety of assays are available to measure various properties of a coating, a coating application technique, and/or a film to determine the degree of suitability of a coating composition for use in a particular use (see, for example, in "Fless’s Paint Film Defects: Their Causes and Cure," 1979). In a further example, the physical properties (e.g., purity, density, solubility, volume solids and/or specific gravity, rheology, viscometry, and particle size) of the resulting a liquid coating product (e.g., on comprising a biomolecule composition), can be assessed using standard techniques of the art and/or as described in PAINT AND COATING TESTING MANUAL, 14 th ed. of the Gardner- Sward Flandbook, J.V. Koleske, Editor (1995), American Society for Testing and Materials (ASTM), Ann Arbor, Ml, and applicable published ASTM assay methods (e.g., "ASTM Book of Standards, Volume 06.01 , Paint -- Tests for Chemical, Physical, and Optical Properties; Appearance" (2002) ASTM International, West Conshohocken, Pennsylvania, U.S.A.; "ASTM Book of Standards, Volume 06.02, Paint -- Products and Applications; Protective Coatings; Pipeline Coatings" (2002) ASTM International, West Conshohocken, Pennsylvania, U.S.A.; "ASTM Book of Standards, Volume 06.03, Paint -- Pigments, Drying Oils, Polymers, Resins, Naval Stores, Cellulosic Esters, and Ink Vehicles" (2002) ASTM International, West Conshohocken, Pennsylvania, U.S.A.). Alternatively, any other suitable assay method of the art, may be employed for assessing physical properties of the coating mixture comprising an above-described antibiological composition (e.g., an antibiological agent). A paint and/or a coating comprising an antibiological composition (e.g., an antibiological agent) may then be assayed and used as described elsewhere herein, or the product may be employed for any other suitable purpose in the art in light of this disclosure. [0080] For example, weathering resistance refers to a film’s ability to endure and/or protect a surface from an external environmental condition. Examples of environmental conditions that may damage a film and/or a surface include contact with varying conditions of temperature, moisture, sunlight {e.g., UV resistance), pollution, biological organisms, or a combination thereof. Examples of a standard technique for determining the weathering resistance of a film {e.g., a film on a manufactured article) by evaluating the degree of damage {e.g., fungal growth, color alteration, dirt accumulation, gloss loss, chalking, cracking, blistering, flaking, erosion, surface rust), are described in "ASTM Book of Standards, Volume 06.01, Paint -- Tests for Chemical, Physical, and Optical Properties; Appearance," D4141-01, D1729-96, D660-93, D661-93, D662-93, D772-86, D4214-98, D3274-95, D714-02, D1654-92, D2244-02, D523-89, D1006-01, D1014-95, and D1186-01, 2002; "ASTM Book of Standards, Volume 06.02, Paint -- Products and Applications; Protective Coatings; Pipeline Coatings," D3719-00, D610-01, D1641-97, D2830-96, and D6763-02, 2002; and "Paint and Coating Testing Manual, Fourteenth Edition of the Gardner-Sward Flandbook," (Koleske, J. V. Ed.), pp. 619-642, 1995. Additionally, standard techniques in the art for determining the resistance of a film to artificial weathering {e.g., heat, moisture, light, UV irradiation) at an accelerated timetable are described in "ASTM Book of Standards, Volume 06.01 , Paint -- Tests for Chemical, Physical, and Optical Properties; Appearance," D822-01, D4587-01, D5031-01, D6631-01, D6695-01, D5894-96, and D4141 -01 , 2002; "ASTM Book of Standards, Volume 06.02, Paint -- Products and Applications; Protective Coatings; Pipeline Coatings," D5722-95, D3361-01 and D3424-01, 2002; and "Paint and Coating Testing Manual, Fourteenth Edition of the Gardner-Sward Handbook" (Koleske, J. V. Ed.), pp. 643-653, 1995.

[0081] Standard procedures for determining the visual appearance of a coating component, a coating and/or a film ( e.g ., reflectance, retroreflectance, fluorescence, photoluminescent light transmission, color, tinting strength, whiteness, gloss, measurement instruments, computerized data analysis) have been described, for example, in "ASTM Book of Standards, Volume 06.01 , Paint -- Tests for Chemical, Physical, and Optical Properties; Appearance," E284-02b, E312-02, E805-01a, E179-96, E991-98, E1247-92, E308-01 , E313-00, E808-01, E1336-96, E1341-96, E1347-97, E1360-90, D332-87, D387- 00, E1455-97, E1477-98a, E1478-97 E1164-02, E1331-96, E1345-98, E1348-02, E1349- 90, D5531-94, D3964-80, E1651-94, E1682-96, E1708-95, E1767-95, E1808-96, E1809- 01, E2022-01 , E2072-00, E2073-02, E2152-01, E2153-01, D1544-98, E259-98, D3022-84, D1535-01, E2175-01, E2214-02, D4449-90, E167-96, E430-97, D4039-93, D5767-95, and E2222-02, 2002; "ASTM Book of Standards, Volume 06.02, Paint -- Products and Applications; Protective Coatings; Pipeline Coatings," D4838-88, D3928-00a, and D5326- 94a, 2002; and "ASTM Book of Standards, Volume 06.03, Paint -- Pigments, Drying Oils, Polymers, Resins, Naval Stores, Cellulosic Esters, and Ink Vehicles," D2090-98, D2090-98 and D6166-97, 2002. Specific techniques for matching two or more colored coatings and/or coating components to reduce differences {e.g., metamerism) have been described, for example, in "ASTM Book of Standards, Volume 06.01 , Paint -- Tests for Chemical, Physical, and Optical Properties; Appearance," D4086-92a, E1541-98 D2244-022002. Specific techniques for determining differences in the color of a coating and/or a coating component, particularly to insure color consistency of a coating composition, have been described, for example, in "ASTM Book of Standards, Volume 06.01 , Paint -- Tests for Chemical, Physical, and Optical Properties; Appearance," D1729-96, D2616-96, E1499-97, and D3134-97, 2002.

[0082] As used herein, "bioactivity resistance" refers to the ability of a biomolecule composition to confer a desired property during and/or after contact with a stress condition normally assayed for in a standard assay procedure for a material formulation. Examples of such a stress condition includes, for example, a temperature (e.g., a baking condition), contact with a material formulation component (e.g., an organic liquid component), contact with a chemical reaction (e.g., thermosetting film formation), contact with damaging agent to a material formulation (e.g., weathering such as UV irradiation via sunlight, detergents, and/or solvents such as automotive chemicals and/or petroleum products), etc. In specific facets, wherein a biomolecule composition comprises a desired biomolecule (e.g., a bio- based antibiological agent), a biomolecule may possess a greater bioactivity resistance such as determined with such an assay procedure, and a particular coating formulation may be optimized for a desired range of bioactivity resistance.

[0083] Such bioactivity resistance may be determined using a standard procedure for material formulation described herein or in the art, in light of the present disclosures. For example, any assay described herein or in the art in light of the present disclosures may be used to determine the bioactivity resistance wherein an enzyme retains detectable enzymatic activity upon contact with a condition typically encountered in a standard assay. Additionally, in certain aspects, it is contemplated that a material formulation comprising an enzyme may lose part or all of a detectable, desirable bioactivity during the period of time of contact with standard assay condition, but regain part or all of the enzymatic bioactivity after return to non-assay conditions. An example of this process is the thermal denaturation of an enzyme at an elevated temperature range into a configuration with lowered or absent bioactivity, followed by refolding of an enzyme, upon return to a more suitable temperature range for the enzyme, into a configuration possessing part or all of the enzymatic bioactivity detectable prior to contact with the elevated temperature. In another example, an enzyme may demonstrate such an increase in bioactivity upon removal of a solvent, a chemical, etc.

[0084] In some embodiments, an enzyme identified as having a desirable enzymatic property for one or more target substrates may be selected for incorporation into a material formulation. The determination of an enzymatic property may be conducted using any technique described herein or in the art, in light of the present disclosures. For example, the determination of the rate of cleavage of a substrate, with or without a competitive or non-competitive enzyme inhibitor, can be utilized in determining the enzymatic properties of an enzyme, such as Vmax, K m , K C at/K m and the like, using analytical techniques such as Lineweaver-Burke analysis, Bronsted plots, etc. Brockerhoff, Flans and Jensen, Robert G. "Lipolytic Enzymes", Academic Press, Inc., New York, New York, pp 10-24, 1974; Dumas,

D. P. et. at., Biotech. Appl. Biochem. 11:235-243, 1989a; Dumas, D. P. et. at., The Journal of Bio. Chem. 264(33): 19659-19665, 1989b; Dumas, D. P. et. al., Arch. Biochem. Biophys. 277:155-159, 1990; Caldwell, S. R. and Raushel, F. M., Biochem. 30:744-7450, 1991;

Donarski, W. J. et. al., Biochemistry 28:4650-4655, 1989; Raveh, L. et. al., 1992; Shim, H. et. al., J. Biol. Chem. 273(28): 17445-17450, 1998; Watkins, L. M. et. al., J. Biol. Chem. 272(41 ):25596-25601 , 1997; Hill, C. M., Bioorganic Medicinal Chemistry Letters 10:1285- 1288, 2000; Hartleib, J. and Ruterjans, H., Biochim et Biophys Acta 1546:312-324, 2001 ; Lineweaver, H. and Burke, D., J. Am. Chem. Soc. 56:658-666, 1934; Segel, I. H., Biochemical Calculations: How to Solve Mathmatical Problems in General Biochemistry 2 nd Edition, John Wiley & Sons, Inc., New York, 1976). Such analysis may be used to identify an enzyme with a specifically enzymatic property for one or more substrates, given that use of an assay for an enzyme's activity may be incorporated with identification of a proteinaceous molecule as having enzymatic activity.

[0085] In some cases, these techniques may be modified by replacement a biomolecule composition prepared by different techniques with another (e.g., a purified enzyme replace with an immobilized enzyme) in the material formulation, and assaying the bioactivity of such a material formulation. Such measurements of the enzymatic activity of compositions may be used to select a material formulation with the desired activity properties of stability, activity, and such like, in different environmental conditions {e.g., weathering, pressure, interfacial characteristics, the effects of an inhibitor, temperature, detergent, organic solvent, etc.) and/or after contact with different substrate(s) {e.g., contact with substrates mimicking vegetable oil properties vs. those for a sterol when assaying for a lipolytic enzyme) to assess properties such as the antibiological activity, substrate preference, enantiomeric specificity, kinetic properties, etc. of a material formulation.

[0086] Enzymes are identified by a numeric classification system such as the International Union of Biochemistry and Molecular Biology ("IUBMB") which identifies enzymes by the type of reaction catalyzed and enumerates a sub-class by a designated enzyme commission number ("EC"). Thus, an enzyme may comprise an oxidoreductase (EC 1 ), a transferase (EC 2), a hydrolase (EC 3), a lyase (EC 4), an isomerase (EC 5), a ligase (EC 6), or a combination thereof. An enzyme may be able to catalyze multiple reactions, and thus have activities of multiple EC classifications.

[0087] An oxidoreductase catalyzes an oxido-reduction of a substrate, wherein the substrate comprises either a hydrogen donor and/or an electron donor. An oxidoreductase may be classified by the substrate moiety of the donor and/or the acceptor. Examples of an oxidoreductase include an oxidoreductase that acts on a donor CH-OH moiety, (EC 1.1 ); a donor aldehyde or a donor oxo moiety, (EC 1 .2); a donor CH-CH moiety, (EC 1.3); a donor CH-NH2 moiety, (EC 1 .4); a donor CH-NH moiety, (EC 1 .5); a donor nicotinamide adenine dinucleotide ("NADH") or a donor nicotinamide adenine dinucleotide phosphate ("NADPH"), (EC 1.6); a donor nitrogenous compound, (EC 1.7); a donor sulfur moiety, (EC 1.8); a donor heme moiety, (EC 1.9); a donor diphenol and/or a related moiety as donor, (EC 1.10); a peroxide as an acceptor, (EC 1.11); a donor hydrogen, (EC 1.12); a single donor with incorporation of molecular oxygen ("oxygenase"), (EC 1.13); a paired donor, with incorporation or reduction of molecular oxygen, (EC 1.14); a superoxide radical as an acceptor, (EC 1.15); an oxidoreductase that oxidizes a metal ion, (EC 1.16); an oxidoreductase that acts on a donor CH2 moiety, (EC 1.17); a donor iron-sulfur protein, (EC 1.18); a donor reduced flavodoxin, (EC 1.19); a donor phosphorus or donor arsenic moiety, (EC 1.20); an oxidoreductase that acts on an X-H and an Y-H to form an X-Y bond, (EC 1.21 ); as well as an other oxidoreductase, (EC 1.97); or a combination thereof.

[0088] A transferase catalyzes the transfer of a moiety from a donor compound to an acceptor compound. A transferase may be classified based on the chemical moiety transferred. Examples of a transferase include a transferase that catalyzes the transfer of an one-carbon moiety, (EC 2.1); an aldehyde and/or a ketonic moiety, (EC 2.2); an acyl moiety, (EC 2.3); a glycosyl moiety, (EC 2.4); an alkyl and/or an aryl moiety other than a methyl moiety, (EC 2.5); a nitrogenous moiety, (EC 2.6); a phosphorus-containing moiety, (EC 2.7); a sulfur-containing moiety, (EC 2.8); a selenium-containing moiety, (EC 2.9); or a combination thereof.

[0089] A hydrolase catalyzes the hydrolysis of a chemical bond. A hydrolase may be classified based on the chemical bond cleaved or the moiety released or transferred by the hydrolysis reaction. Examples of a hydrolase include a hydrolase that catalyzes the hydrolysis of an ester bond, (EC 3.1); a glycosyl released/transferred moiety, (EC 3.2); an ether bond, (EC 3.3); a peptide bond, (EC 3.4); a carbon-nitrogen bond, other than a peptide bond, (EC 3.5); an acid anhydride, (EC 3.6); a carbon-carbon bond, (EC 3.7); a halide bond, (EC 3.8); a phosphorus-nitrogen bond, (EC 3.9); a sulfur-nitrogen bond, (EC 3.10); a carbon-phosphorus bond, (EC 3.11); a sulfur-sulfur bond, (EC 3.12); a carbon- sulfur bond, (EC 3.13); or a combination thereof.

[0090] Examples of an esterase (EC 3.1 ) include a carboxylic ester hydrolase (EC 3.1.1 ); a thioester hydrolase (EC 3.1.2); a phosphoric monoester hydrolase (EC 3.1.3); a phosphoric diester hydrolase (EC 3.1.4); a triphosphoric monoester hydrolase (EC 3.1.5); a sulfuric ester hydrolase (EC 3.1.6); a diphosphoric monoester hydrolase (EC 3.1.7); a phosphoric triester hydrolase (EC 3.1.8); an exodeoxyribonuclease producing a 5'-phosphomonoester (EC 3.1.11 ); an exoribonuclease producing a 5'-phosphomonoester (EC 3.1.13); an exoribonuclease producing a 3'-phosphomonoester (EC 3.1.14); an exonuclease active with a ribonucleic acid and/or a deoxyribonucleic acid and producing a 5'- phosphomonoester (EC 3.1.15); an exonuclease active with a ribonucleic acid and/or a deoxyribonucleic acid and producing a 3'-phosphomonoester (EC 3.1.16); an endodeoxyribonuclease producing a 5'-phosphomonoester (EC 3.1.21); an endodeoxyribonuclease producing a 3'-phosphomonoester (EC 3.1.22); a site-specific endodeoxyribonuclease specific for an altered base (EC 3.1.25); an endoribonuclease producing a 5'-phosphomonoester (EC 3.1.26); an endoribonuclease producing a 3'- phosphomonoester (EC 3.1.27); an endoribonuclease active with a ribonucleic acid and/or a deoxyribonucleic acid and producing a 5'-phosphomonoester (EC 3.1.30); an endoribonuclease active with a ribonucleic acid and/or a deoxyribonucleic acid and producing a 3'-phosphomonoester (EC 3.1.31 ); or a combination thereof. [0091] Examples of a carboxylic ester hydrolase (EC 3.1.1) include a carboxylesterase (EC

3.1.1.1); an arylesterase (EC 3.1.1.2); a triacylglycerol lipase (EC 3.1.1.3); a phospholipase A2 (EC 3.1.1.4); a lysophospholipase (EC 3.1.1.5); an acetylesterase (EC 3.1.1.6); an acetylcholinesterase (EC 3.1.1.7); a cholinesterase (EC 3.1.1.8); a tropinesterase (EC 3.1.1.10); a pectinesterase (EC 3.1.1.11 ); a sterol esterase (EC 3.1.1.13); a chlorophyllase (EC 3.1.1.14); a L-arabinonolactonase (EC 3.1.1.15); a gluconolactonase (EC 3.1.1.17); an uronolactonase (EC 3.1.1.19); a tannase (EC 3.1.1.20); a retinyl-palmitate esterase (EC 3.1.1.21); a hydroxybutyrate-dimer hydrolase (EC 3.1.1.22); an acylglycerol lipase (EC 3.1.1.23); a 3-oxoadipate enol-lactonase (EC 3.1.1.24); a 1 ,4-lactonase (EC 3.1.1.25); a galactolipase (EC 3.1.1.26); a 4-pyridoxolactonase (EC 3.1.1.27); an acylcarnitine hydrolase (EC 3.1.1.28); an aminoacyl-tRNA hydrolase (EC 3.1.1.29); a D- arabinonolactonase (EC 3.1.1.30); a 6-phosphogluconolactonase (EC 3.1.1.31); a phospholipase A1 (EC 3.1.1.32); a 6-acetylglucose deacetylase (EC 3.1.1.33); a lipoprotein lipase (EC 3.1.1.34); a dihydrocoumarin hydrolase (EC 3.1.1.35); a limonin-D-ring- lactonase (EC 3.1.1.36); a steroid-lactonase (EC 3.1.1.37); a triacetate-lactonase (EC 3.1.1.38); an actinomycin lactonase (EC 3.1.1.39); an orsellinate-depside hydrolase (EC

3.1.1.40); a cephalosporin-C deacetylase (EC 3.1.1.41); a chlorogenate hydrolase (EC 3.1.1.42); a a-amino-acid esterase (EC 3.1.1.43); a 4-methyloxaloacetate esterase (EC 3.1.1.44); a carboxymethylenebutenolidase (EC 3.1.1.45); a deoxylimonate A-ring- lactonase (EC 3.1.1.46); a 1-alkyl-2-acetylglycerophosphocholine esterase (EC 3.1.1.47); a fusarinine-C ornithinesterase (EC 3.1.1.48); a sinapine esterase (EC 3.1.1.49); a wax-ester hydrolase (EC 3.1.1.50); a phorbol-diester hydrolase (EC 3.1.1.51); a phosphatidylinositol deacylase (EC 3.1.1.52); a sialate O-acetylesterase (EC 3.1.1.53); an acetoxybutynylbithiophene deacetylase (EC 3.1.1.54); an acetylsalicylate deacetylase (EC 3.1.1.55); a methylumbelliferyl-acetate deacetylase (EC 3.1.1.56); a 2-pyrone-4,6- dicarboxylate lactonase (EC 3.1.1.57); a N-acetylgalactosaminoglycan deacetylase (EC 3.1.1.58); a juvenile-hormone esterase (EC 3.1.1.59); a bis(2-ethylhexyl)phthalate esterase (EC 3.1.1.60); a protein-glutamate methylesterase (EC 3.1.1.61); a 11-cis-retinyl-palmitate hydrolase (EC 3.1.1.63); an all-trans-retinyl-palmitate hydrolase (EC 3.1.1.64); a L- rhamnono-1 ,4-lactonase (EC 3.1.1.65); a 5-(3,4-diacetoxybut-1-ynyl)-2,2'-bithiophene deacetylase (EC 3.1.1.66); a fatty-acyl-ethyl-ester synthase (EC 3.1.1.67); a xylono-1 ,4- lactonase (EC 3.1.1.68); a cetraxate benzylesterase (EC 3.1.1.70); an acetylalkylglycerol acetylhydrolase (EC 3.1.1.71); an acetylxylan esterase (EC 3.1.1.72); a feruloyl esterase (EC 3.1.1.73); a cutinase (EC 3.1.1.74); a poly(3-hydroxybutyrate) depolymerase (EC 3.1.1.75); a poly(3-hydroxyoctanoate) depolymerase (EC 3.1.1.76); an acyloxyacyl hydrolase (EC 3.1.1.77); a polyneuridine-aldehyde esterase (EC 3.1.1.78); a hormone- sensitive lipase (EC 3.1.1.79); an acetylajmaline esterase (EC 3.1.1.80); a quorum- quenching N-acyl-homoserine lactonase (EC 3.1.1.81); a pheophorbidase (EC 3.1.1.82); a monoterpene e-lactone hydrolase (EC 3.1.1.83); or a combination thereof.

[0092] Examples of an enzyme that acts on a carbon-nitrogen bond, other than a peptide bond (EC 3.5) include an enzyme acting on a linear amide (EC 3.5.1); a cyclic amide (EC 3.5.2); a linear amidine (EC 3.5.3); a cyclic amidine (EC 3.5.4); a nitrile (EC 3.5.5); an other compound (EC 3.5.99); or a combination thereof. Examples of an enzyme that catalyzes a reaction on a carbon-nitrogen bond of a non-peptide linear amide (EC 3.5.1) include an asparaginase (EC 3.5.1.1); a glutaminase (EC 3.5.1.2); a w-amidase (EC 3.5.1.3); an amidase (EC 3.5.1.4); a urease (EC 3.5.1.5); a b-ureidopropionase (EC 3.5.1.6); a ureidosuccinase (EC 3.5.1.7); a formylaspartate deformylase (EC 3.5.1.8); an arylformamidase (EC 3.5.1.9); a formyltetrahydrofolate deformylase (EC 3.5.1.10); a penicillin amidase (EC 3.5.1.11); a biotinidase (EC 3.5.1.12); an aryl-acylamidase (EC 3.5.1.13); an aminoacylase (EC 3.5.1.14); an aspartoacylase (EC 3.5.1.15); an acetylornithine deacetylase (EC 3.5.1.16); an acyl-lysine deacylase (EC 3.5.1.17); a succinyl-diaminopimelate desuccinylase (EC 3.5.1.18); a nicotinamidase (EC 3.5.1.19); a citrullinase (EC 3.5.1.20); a N-acetyl-p-alanine deacetylase (EC 3.5.1.21); a pantothenase (EC 3.5.1.22); a ceramidase (EC 3.5.1.23); a choloylglycine hydrolase (EC 3.5.1.24); a N- acetylglucosamine-6-phosphate deacetylase (EC 3.5.1.25); a N4-(P-N-acetylglucosaminyl)- L-asparaginase (EC 3.5.1.26); a N-formylmethionylaminoacyl-tRNA deformylase (EC 3.5.1.27); a N-acetylmuramoyl-L-alanine amidase (EC 3.5.1.28); a 2- (acetamidomethylene)succinate hydrolase (EC 3.5.1.29); a 5-aminopentanamidase (EC 3.5.1.30); a formylmethionine deformylase (EC 3.5.1.31); a hippurate hydrolase (EC 3.5.1.32); a N-acetylglucosamine deacetylase (EC 3.5.1.33); a D-glutaminase (EC 3.5.1.35); a N-methyl-2-oxoglutaramate hydrolase (EC 3.5.1.36); a glutamin-(asparagin- )ase (EC 3.5.1.38); an alkylamidase (EC 3.5.1.39); an acylagmatine amidase (EC 3.5.1.40); a chitin deacetylase (EC 3.5.1.41); a nicotinamide-nucleotide amidase (EC 3.5.1.42); a peptidyl-glutaminase (EC 3.5.1.43); a protein-glutamine glutaminase (EC 3.5.1.44); a 6-aminohexanoate-dimer hydrolase (EC 3.5.1.46); a N-acetyldiaminopimelate deacetylase (EC 3.5.1.47); an acetylspermidine deacetylase (EC 3.5.1.48); a formamidase (EC 3.5.1.49); a pentanamidase (EC 3.5.1.50); a 4-acetamidobutyryl-CoA deacetylase (EC 3.5.1.51); a peptide-N4-(N-acetyl-p-glucosaminyl)asparagines amidase (EC 3.5.1.52); a N- carbamoylputrescine amidase (EC 3.5.1.53); an allophanate hydrolase (EC 3.5.1.54); a long-chain-fatty-acyl-glutamate deacylase (EC 3.5.1.55); a N,N-dimethylformamidase (EC 3.5.1.56); a tryptophanamidase (EC 3.5.1.57); a N-benzyloxycarbonylglycine hydrolase (EC 3.5.1.58); a N-carbamoylsarcosine amidase (EC 3.5.1.59); a N-(long-chain- acyl)ethanolamine deacylase (EC 3.5.1.60); a mimosinase (EC 3.5.1.61); an acetylputrescine deacetylase (EC 3.5.1.62); a 4-acetamidobutyrate deacetylase (EC 3.5.1.63); a Na-benzyloxycarbonylleucine hydrolase (EC 3.5.1.64); a theanine hydrolase (EC 3.5.1.65); a 2-(hydroxymethyl)-3-(acetamidomethylene)succinate hydrolase (EC 3.5.1.66); a 4-methyleneglutaminase (EC 3.5.1.67); a N-formylglutamate deformylase (EC 3.5.1.68); a glycosphingolipid deacylase (EC 3.5.1.69); an aculeacin-A deacylase (EC 3.5.1.70); a N-feruloylglycine deacylase (EC 3.5.1.71); a D-benzoylarginine-4-nitroanilide amidase (EC 3.5.1.72); a carnitinamidase (EC 3.5.1.73); a chenodeoxycholoyltaurine hydrolase (EC 3.5.1.74); a urethanase (EC 3.5.1.75); an arylalkyl acylamidase (EC 3.5.1.76); a N-carbamoyl-D-amino acid hydrolase (EC 3.5.1.77); a glutathionylspermidine amidase (EC 3.5.1.78); a phthalyl amidase (EC 3.5.1.79); a N-acetylgalactosamine-6- phosphate deacetylase (EC 3.5.1.80); a N-acyl-D-amino-acid deacylase (EC 3.5.1.81); a N- acyl-D-glutamate deacylase (EC 3.5.1.82); a N-acyl-D-aspartate deacylase (EC 3.5.1.83); a biuret amidohydrolase (EC 3.5.1.84); a (S)-N-acetyM-phenylethylamine hydrolase (EC 3.5.1.85); a mandelamide amidase (EC 3.5.1.86); a N-carbamoyl-L-amino-acid hydrolase (EC 3.5.1.87); a peptide deformylase (EC 3.5.1.88); a N- acetylglucosaminylphosphatidylinositol deacetylase (EC 3.5.1.89); an adenosylcobinamide hydrolase (EC 3.5.1.90); a N-substituted formamide deformylase (EC 3.5.1.91); a pantetheine hydrolase (EC 3.5.1.92); a glutaryl-7-aminocephalosporanic-acid acylase (EC 3.5.1.93); a y-glutamyl-y-aminobutyrate hydrolase (EC 3.5.1.94); a N-malonylurea hydrolase (EC 3.5.1.95); a succinylglutamate desuccinylase (EC 3.5.1.96); an acyl- homoserine-lactone acylase (EC 3.5.1.97); a histone deacetylase (EC 3.5.1.98); or a combination thereof. Examples of an enzyme that catalyzes a reaction on a carbon- nitrogen bond of a non-peptide cyclic amide (EC 3.5.2) include a barbiturase (EC 3.5.2.1); a dihydropyrimidinase (EC 3.5.2.2); a dihydroorotase (EC 3.5.2.3); a carboxymethylhydantoinase (EC 3.5.2.4); an allantoinase (EC 3.5.2.5); a b-lactamase (EC 3.5.2.6); an imidazolonepropionase (EC 3.5.2.7); a 5-oxoprolinase (ATP-hydrolysing) (EC 3.5.2.9); a creatininase (EC 3.5.2.10); a L-lysine-lactamase (EC 3.5.2.11 ); a 6- aminohexanoate-cyclic-dimer hydrolase (EC 3.5.2.12); a 2,5-dioxopiperazine hydrolase (EC 3.5.2.13); a N-methylhydantoinase (ATP-hydrolysing) (EC 3.5.2.14); a cyanuric acid amidohydrolase (EC 3.5.2.15); a maleimide hydrolase (EC 3.5.2.16); a hydroxyisourate hydrolase (EC 3.5.2.17); an enamidase (EC 3.5.2.18); or a combination thereof.

[0093] Examples of an enzyme that acts on an acid anhydride (EC 3.6) include an enzyme acting on: a phosphorus-containing anhydride (EC 3.6.1); a sulfonyl-containing anhydride (EC 3.6.2); an acid anhydride catalyzing transmembrane movement of a substance (EC 3.6.3); an acid anhydride involved in cellular and/or subcellular movement (EC 3.6.4); a GTP involved in cellular and/or subcellular movement (EC 3.6.5); or a combination thereof. [0094] A lyase catalyzes the cleavage of a chemical bond by reactions other than hydrolysis and/or oxidation. A lyase may be classified based on the chemical bond cleaved. Examples of a lyase include a lyase that catalyzes the cleavage of a carbon- carbon bond, (EC 4.1); a carbon-oxygen bond, (EC 4.2); a carbon-nitrogen bond, (EC 4.3); a carbon-sulfur bond, (EC 4.4); a carbon-halide bond, (EC 4.5); a phosphorus-oxygen bond, (EC 4.6); an other lyase, (EC 4.99); or a combination thereof.

[0095] An isomerase catalyzes a change within one molecule. Examples of an isomerase include a racemase and/or an epimerase, (EC 5.1); a cis-trans- isomerase, (EC 5.2); an intramolecular isomerase, (EC 5.3); an intramolecular transferase, (EC 5.4); an intramolecular lyase, (EC 5.5); an other isomerases, (EC 5.99); or a combination thereof. [0096] A ligase catalyzes the formation of a chemical bond between two substrates with the hydrolysis of a diphosphate bond of a triphosphate such as ATP. A ligase may be classified based on the chemical bond created. Examples of a lyase include a ligase that form a carbon — oxygen bond, (EC 6.1); a carbon — sulfur bond, (EC 6.2); a carbon — nitrogen bond, (EC 6.3); a carbon — carbon bond, (EC 6.4); a phosphoric ester bond, (EC 6.5); or a combination thereof.

[0097] A lipolytic enzyme comprises an enzyme that catalyzes a reaction or series of reactions on a lipid substrate. In many embodiments, a lipolytic enzyme produces one or more products that are more soluble in a polar liquid component ( e.g ., an aqueous media, water, a water comprising detergent) than the substrate, which may promote ease of removal such as from a surface of a material formulation (e.g., a coated surface, a polymeric material incorporating an enzyme). Examples of a lipid include a triglyceride; a diglyceride; a monoglyceride; a phospholipid; a glycolipid {e.g., galactolipid); a steroid {e.g., cholesterol); a wax; a fat-soluble vitamin {e.g., vitamin A, D, E, K, etc .); a petroleum -based material, such as, for example, a hydrocarbon composition such as gasoline, a crude petroleum oil, a petroleum grease, etc .; or a combination thereof. Lipolytic enzymes have been identified in cells across the phylogenetic categories, and purified for analysis and/or use in commercial applications (Brockerhoff, Hans and Jensen, Robert G. "Lipolytic Enzymes," 1974). Further, numerous nucleotide sequences for lipolytic enzymes have been isolated, the encoded protein sequence determined, and in many cases the nucleotide sequences recombinantly expressed for high level production of a lipolytic enzyme {e.g., a lipase), particularly for isolation, purification and subsequent use in an industrial/commercial application such as laundry detergents ["Lipases their Structure, Biochemistry and Application" (Paul Woolley and Steffen B. Peterson, Eds.) Cambridge University Press, Great Britain, 1994] [0098] Examples of a lipolytic esterase and a ceramidase include a carboxylesterase (EC 3.1.1.1), a lipase (EC 3.1.1.3), a lipoprotein lipase (EC 3.1.1.34), an acylglycerol lipase (EC 3.1.1.23), a hormone-sensitive lipase (EC 3.1.1.79), a phospholipase Ai (EC 3.1.1.32), a phospholipase A2 (EC 3.1.1.4), a phosphatidylinositol deacylase (EC 3.1.1.52), a phospholipase C (EC 3.1.4.3), a phospholipase D (EC 3.1.4.4), a phosphoinositide phospholipase C (EC 3.1.4.11), a phosphatidate phosphatase (EC 3.1.3.4), a lysophospholipase (EC 3.1.1.5), a sterol esterase (EC 3.1.1.13), a galactolipase (EC 3.1.1.26), a sphingomyelin phosphodiesterase (EC 3.1.4.12), a sphingomyelin phosphodiesterase D (EC 3.1.4.41), a ceramidase (EC 3.5.1.23), a wax-ester hydrolase (EC 3.1.1.50), a fatty-acyl-ethyl-ester synthase (EC 3.1.1.67), a retinyl-palmitate esterase (EC 3.1.1.21), a 11-c/s-retinyl-palmitate hydrolase (EC 3.1.1.63), an all-trans- retinyl- palmitate hydrolase (EC 3.1.1.64), a cutinase (EC 3.1.1.74), an acyloxyacyl hydrolase (EC 3.1.1.77), a petroleum lipolytic enzyme, or a combination thereof. In some aspects, a series of enzyme reactions releases a fatty acid and/or degrades a lipid, such as in the case of a combination of a sphingomyelin phosphodiesterase that produces a N- acylsphingosine from a sphingomyelin phospholipid, followed by a ceramidase hydrolyzing an amide bond in a /V-acylsphingosine to produce a free fatty acid and a sphingosine.

[0099] Carboxylesterase catalyzes reactions on a fatty acid of about 10 or less carbons, to differentiate its preferred substrate and classification from a lipase), with structural and sequence information is known in the art (e.g., PDB entries: 1AUO, 1AUR, etc.). Lipase structural and sequence information is known in the art (e.g., PDB entries: 1AKN, 1BU8, etc.). Phospholipase Ai structural and sequence information is known in the art (e.g., PDB entries: 1FW2, 1FW3, etc.). Phospholipase A2 structural and sequence information is known in the art (e.g., PDB entries 1A2A, 1A3F, etc.). Phospholipase C structural and sequence information is known in the art (e.g., PDB entries: 1AH7, 1CA1, etc.).

Phospholipase D structural and sequence information is known in the art (e.g., PDB entries: 1F0I, IVOR, etc.). Phosphoinositide phospholipase C structural and sequence information is known in the art (e.g., PDB entries: 1DJG, 1DJH, etc.). Lysophospholipase structural and sequence information is known in the art (e.g., PDB entries: 1G86, 1HDK, etc.). Sterol esterase structural and sequence information is known in the art (e.g., PDB entries: 1AQL, 2BCE, etc.).

[0100] A peptidase catalyzes a reaction on a peptide bond (e.g., a proteinaceous molecule’s peptide bond to degrade the proteinaceous molecule for antibiological activity and/or ease of removal from a surface, though other secondary reactions ( e.g an esterase activity) may also be catalyzed in some cases. A peptidase generally may be categorized as either an exopeptidase (EC 3.4.11-19) or an endopeptidase (EC 3.4.21-24 and EC 3.4.99). Examples of a peptidase include an alpha-amino-acyl-peptide hydrolase (EC 3.4.11), a peptidyl-amino-acid hydrolase (EC 3.4.17), a dipeptide hydrolase (EC 3.4.13), a peptidyl peptide hydrolase (EC 3.4), a peptidylamino-acid hydrolase (EC 3.4), an acylamino-acid hydrolase (EC 3.4), an aminopeptidase (EC 3.4.11), a dipeptidase (EC 3.4.13), a dipeptidyl-peptidase (EC 3.4.14), a tripeptidyl-peptidase (EC 3.4.14), a peptidyl- dipeptidase (EC 3.4.15), a serine-type carboxypeptidase (EC 3.4.16), a metallocarboxypeptidase (EC 3.4.17), a cysteine-type carboxypeptidase (EC 3.4.18), an omega peptidase (EC 3.4.19), a serine endopeptidase (EC 3.4.21), a cysteine endopeptidase (EC 3.4.22), an aspartic endopeptidase (EC 3.4.23), a metalloendopeptidase (EC 3.4.24), a threonine endopeptidase (EC 3.4.25), an endopeptidase of unknown catalytic mechanism (EC 3.4.99), or a combination thereof. Examples of a serine endopeptidase (EC 3.4.21 ) includes a chymotrypsin (EC 3.4.21.1 ); a chymotrypsin C (EC 3.4.21.2); a metridin (EC 3.4.21.3); a trypsin (EC 3.4.21.4); a thrombin (EC 3.4.21.5); a coagulation factor Xa (EC 3.4.21.6); a plasmin (EC 3.4.21.7); an enteropeptidase (EC 3.4.21.9); an acrosin (EC 3.4.21.10); an a-Lytic endopeptidase (EC 3.4.21.12); a glutamyl endopeptidase (EC 3.4.21.19); a cathepsin G (EC 3.4.21.20); a coagulation factor Vila (EC 3.4.21.21 ); a coagulation factor IXa (EC 3.4.21.22); a cucumisin (EC 3.4.21.25); a prolyl oligopeptidase (EC 3.4.21.26); a coagulation factor Xla (EC 3.4.21.27); a brachyurin (EC 3.4.21.32); a plasma kallikrein (EC 3.4.21.34); a tissue kallikrein (EC 3.4.21.35); a pancreatic elastase (EC 3.4.21.36); a leukocyte elastase (EC 3.4.21.37); a coagulation factor Xlla (EC 3.4.21.38); a chymase (EC 3.4.21.39); a complement subcomponent C (EC 3.4.21.41); a complement subcomponent C (EC 3.4.21.42); a classical-complement-pathway C3/C5 convertase (EC 3.4.21.43); a complement factor I (EC 3.4.21.45); a complement factor D (EC 3.4.21.46); an alternative- complement-pathway C3/C5 convertase (EC 3.4.21.47); a cerevisin (EC 3.4.21.48); a hypodermin C (EC 3.4.21.49); a lysyl endopeptidase (EC 3.4.21.50); an endopeptidase La (EC 3.4.21.53); a g-renin (EC 3.4.21.54); a venombin AB (EC 3.4.21.55); a leucyl endopeptidase (EC 3.4.21.57); a tryptase (EC 3.4.21.59); a scutelarin (EC 3.4.21.60); a kexin (EC 3.4.21.61 ); a subtilisin (EC 3.4.21.62); an oryzin (EC 3.4.21.63); a peptidase K (EC 3.4.21 .64); a thermomycolin (EC 3.4.21 .65); a thermitase (EC 3.4.21 .66); an endopeptidase So (EC 3.4.21.67); a t-plasminogen activator (EC 3.4.21.68); a protein C (activated) (EC 3.4.21.69); a pancreatic endopeptidase E (EC 3.4.21.70); a pancreatic elastase II (EC 3.4.21.71 ); an IgA-specific serine endopeptidase (EC 3.4.21.72); a u- plasminogen activator (EC 3.4.21 .73); a venombin A (EC 3.4.21 .74); a furin (EC 3.4.21 .75); a myeloblastin (EC 3.4.21 .76); a semenogelase (EC 3.4.21 .77); a granzyme A (EC 3.4.21.78); a granzyme B (EC 3.4.21.79); a streptogrisin A (EC 3.4.21.80); a streptogrisin B (EC 3.4.21.81 ); a glutamyl endopeptidase II (EC 3.4.21.82); an oligopeptidase B (EC 3.4.21 .83); a limulus clotting factor (EC 3.4.21 .84); a limulus clotting factor (EC 3.4.21 .85); a limulus clotting enzyme (EC 3.4.21.86); a repressor LexA (EC 3.4.21.88); a signal peptidase I (EC 3.4.21 .89); a togavirin (EC 3.4.21.90); a flavivirin (EC 3.4.21 .91 ); an endopeptidase Clp (EC 3.4.21.92); a proprotein convertase 1 (EC 3.4.21.93); a proprotein convertase 2 (EC 3.4.21 .94); a snake venom factor V activator (EC 3.4.21 .95); a lactocepin (EC 3.4.21.96); an assemblin (EC 3.4.21.97); a hepacivirin (EC 3.4.21.98); a spermosin (EC 3.4.21.99); a sedolisin (EC 3.4.21.100); a xanthomonalisin (EC 3.4.21.101 ); a C- terminal processing peptidase (EC 3.4.21.102); a physarolisin (EC 3.4.21.103); a mannan- binding lectin-associated serine protease-2 (EC 3.4.21.104); a rhomboid protease (EC 3.4.21.105); a hepsin (EC 3.4.21.106); a peptidase Do (EC 3.4.21.107); a HtrA2 peptidase (EC 3.4.21 .108); a matriptase (EC 3.4.21 .109); a C5a peptidase (EC 3.4.21 .110); an aqualysin 1 (EC 3.4.21 .111 ); a site-1 protease (EC 3.4.21 .112); a pestivirus NS3 polyprotein peptidase (EC 3.4.21.113); an equine arterivirus serine peptidase (EC 3.4.21.114); an infectious pancreatic necrosis birnavirus Vp4 peptidase (EC 3.4.21.115); a SpolVB peptidase (EC 3.4.21.116); a stratum corneum chymotryptic enzyme (EC 3.4.21.117); a kallikrein 8 (EC 3.4.21.118); a kallikrein 13 (EC 3.4.21.119); an oviductin (EC 3.4.21 .120); or a combination thereof.

[0101] Trypsin (EC 3.4.21.4) structural and sequence information is known in the art [e.g.; PDB entries: 1A0J; 1AND; etc.]. Chymotrypsin (EC 3.4.21.1 ) structural and sequence information is known in the art [e.g., PDB entries: 1AB9, 1ACB, etc.]. Chymotrypsin C (EC 3.4.21.2) hydrolyzes a peptide bond, particularly those comprising a Leu, a Tyr, a Phe, a Met, a Trp, a Gin, and/or an Asn, with structural and sequence information is known in the art [e.g., KEGG sequences: HSA * - * 11330(CTRC); PTR * - * 739685(CTRC); etc.]. Subtilisin (EC 3.4.21.62) structural and sequence information is known in the art [e.g., PDB entries:

1 A2Q, 1 AF4, etc.].

[0102] In specific embodiments, an antibiological enzyme comprises a glycosylase (EC 3.2). In more specific embodiments, the enzyme comprises a glycosidase (EC 3.2.1 ), which comprises an enzyme that hydrolyses an O- glycosyl compound, a S-glycosyl compound, or a combination thereof. In particular aspects, the glycosidase acts on an O- glycosyl compound, and examples of such an enzyme include a lysozyme, an agarase, a cellulase, a chitinase, or a combination thereof. In other embodiments, an antibiological enzyme include a lysozyme, a lysostaphin, a libiase, a lysyl endopeptidase, a mutanolysin, a cellulase, a chitinase, an a-agarase, an b-agarase, a /V-acetylmuramoyl-L-alanine amidase, a lytic transglycosylase, a glucan endo-1 ,3-p-D-glucosidase, an endo-1,3(4)-p- glucanase, a b-lytic metalloendopeptidase, a 3-deoxy-2-octulosonidase, a peptide-N4-(N- acetyl^-glucosaminyl)asparagine amidase, a mannosyl-glycoprotein endo-b-N- acetylglucosaminidase, a i-carrageenase, a k-carrageenase, a l-carrageenase, an a- neoagaro-oligosaccharide hydrolase, an endolysin, an autolysin, a mannoprotein protease, a glucanase, a mannose, a zymolase, a lyticase. a lipolytic enzyme, or a combination thereof. Such antibiological enzymes are described, for example, in U.S. Patent Application nos. 12/696,651, 12/474,921 and 12/882,563, each specifically incorporated herein by reference. An antibiological biomolecule composition may be combined with any other antibiological composition described herein and/or known in the art, such as a preservative and/or an anti-microbial agent (e.g., a chemical biocide, a chemical biostatic, a fungicide, a fungistatic, a bactericide, an algaecide, etc.) (see, for example, U.S. Patent no. 8,618,066).

[0103] Lysozyme (EC 3.2.1.17) structural and sequence information is known in the art [e.g., PDB entries 1021, 1031, etc.). Lysostaphin (EC 3.4.24.75) structural and sequence information is known in the art [e.g., PDB entries 1QWY, 2B0P, etc.]. Lysyl endopeptidase (EC 3.4.21.50) structural and sequence information is known in the art (e.g., PDB entries 1arb, 1arc; etc.).

[0104] Cellulase (EC 3.2.1.4) structural and sequence information is known in the art [e.g., PDB entries 1A39; 1A3H; etc.]. Chitinase (EC 3.2.1.14) structural and sequence information is known in the art [e.g., PDB entries: 1CNS; 1CTN; etc.] b-agarase (EC 3.2.1.81) structural and sequence information is known in the art [e.g., PDB entries 104Y, 104Z, etc.].

[0105] N-acetylmuramoyl-L-alanine amidase (EC 3.5.1.28) structural and sequence information is known in the art [ e.g ., PDB entries 1ARO, 1GVM, etc.]. A lytic transglycosylase ("lytic murein transglycosylase," EC 3.2.1.-) structural and sequence information is known in the art [e.g., PDB entries 1Q2R, 1Q2S, etc.]. Glucan endo-1,3-p-D- glucosidase (EC 3.2.1.39) structural and sequence information is known in the art [e.g.,

PDB entries 1GHS, 2CYG, etc.]. Endo-1,3(4)-p-glucanase (EC 3.2.1.6) structural and sequence information is known in the art [e.g., PDB entries 1UP4, 1UP6, etc.]. Peptide-/V 4 - (A/-acetyl-p-glucosaminyl)asparagine amidase (EC 3.5.1.52) structural and sequence information is known in the art [e.g., PDB entries 1PGS, 1PNF, etc.]. Mannosyl- glycoprotein endo-pW-acetylglucosaminidase (EC 3.2.1.96) structural and sequence information is known in the art [e.g., PDB entries 1C3F, 1C8X, etc.].

[0106] i-carrageenase (EC 3.2.1.157) structural and sequence information is known in the art {e.g., PDB entries 1 H80, 1KTW, etc.) k-carrageenase (EC 3.2.1.83) structural and sequence information is known in the art {e.g., PDB entries 1DYP; etc.).

[0107] In certain embodiments, a material formulation comprises an esterase identified by enzyme commission number EC 3.1.8, the phosphoric triester hydrolases, such as a phosphoric triester hydrolase includes an aryldialkylphosphatase (EC 3.1.8.1 ) and/or a diisopropyl-fluorophosphatase (EC 3.1.8.2). An aryldialkylphosphatase (EC 3.1.8.1) ("organophosphorus hydrolase,” “paraoxonase”) structural and sequence information is known in the art with examples including PDB entries [e.g., 1JGM, 1DPM, etc.). A diisopropyl-fluorophosphatase (EC 3.1.8.2) structural and sequence information is known in the art (e.g., PDB entries 1E1A, 1PJX, etc.). Organophosphorus acid anhydrolases ("OPAA”) are also included in E.C.3.1.8.2, with structural and sequence information is known in the art (e.g., GenBank accession nos. U29240, U56398; etc.).

[0108] In certain aspects, a biomolecule (e.g., a proteinaceous molecule) for a biomolecule composition may be biologically produced in a cell, a tissue and/or an organism including but not limited to being endogenously and/or recombinantly produced and/or synthesized (e.g., a chemically synthesized peptide) using any method or technique in the art. [In "Molecular Cloning" (Sambrook, J., and Russell, D.W., Eds.) 3rd Edition, Cold Spring Flarbor, New York: Cold Spring Flarbor Laboratory Press, 2001 ; In "Current Protocols in Molecular Biology" (Chanda, V. B. Ed.) John Wiley & Sons, 2002; In "Current Protocols in Cell Biology" (Morgan, K. Ed.) John Wiley & Sons, 2002; In "Current Protocols in Nucleic Acid Chemistry" (Harkins, E. W. Ed.) John Wiley & Sons, 2002; In "Current Protocols in Protein Science" (Taylor, G. Ed.) John Wiley & Sons, 2002; In "Current Protocols in Pharmacology" (Taylor, G. Ed.) John Wiley & Sons, 2002; In "Current Protocols in Cytometry" (Robinson, J. P. Ed.) John Wiley & Sons, 2002; In "Current Protocols in Immunology" (Coico, R. Ed.) John Wiley & Sons, 2002]

[0109] Selection of certain cell(s) and/or virus(s) are capable of growth in environmental conditions typically harmful to many other types of cells ("extremophiles"), such as conditions of extreme temperature, salt and/or pH. A biomolecule derived from such a cell and/or a virus may be useful in certain embodiments for durability, activity, or other property of a material formulation comprising a biomolecule composition (e.g., a bio-based antibiological agent) that undergoes conditions similar to {e.g., the same or overlapping ranges) as those found in the cell’s and/or the virus's growth environment. A "hyperthermophile" or "thermophile" typically grows in temperatures greater than about 40°C, often up to about 120°C or more. A "psychrophile" typically grows at about -10°C to about 20°C, and a "mesophile" typically grows at about 20°C to about 40°C, and an "extreme halophile" may be capable of living in salt-water conditions of about 1.5 M (8.77% w/v) sodium chloride to about 2.7 M (15.78% w/v) or more sodium chloride. An "extreme acidophile" may be capable of growing in about pH 1 to about pH 6, while an "extreme alkaliphile" may be capable of growing in about pH 8 to about pH 14.

[0110] In some embodiments, after production of a biomolecule by a living cell, the composition comprising the biomolecule may undergo one or more processing procedure(s) to prepare a biomolecule composition (e.g., a bio-based antibiological agent). Examples of such procedures include concentrating, drying, applying physical force, extracting, resuspending, controlling temperature, permeabilizing, disrupting, chemically modifying, encapsulating, proteinaceous molecule purification, immobilizing, or a combination thereof. Sterilizing ("inactivating") kills living matter {e.g., a cell, a virus), while attenuation reduces the virulence of a living matter. In further embodiments, sterilizing and/or attenuation of a material formulation may be accomplished by any method known in the art, and are commonly applied in the food, medical, and pharmaceutical arts to sterilize and/or attenuate pathogenic microorganisms [see, for example, "Food Irradiation:

Principles and Applications," 2001; "Manual of Commercial Methods in Clinical Microbiology" (Truant, A. L, Ed.), 2002; "Manual of Clinical Microbiology 8 th Edition Volume 1" (Murray P. R., Baron, E. J., Jorgensen, J. H., Pfaller, M. A., Yolken, R. H., Eds.), 2003; "Manual of Clinical Microbiology 8 th Edition Volume 2" (Murray P. R., Baron, E. J., Jorgensen, J. H., Pfaller, M. A., Yolken, R. H., Eds.), 2003; and "Biological Safety Principles and Practice 3 rd Edition" (Fleming, D. O. and Hunt, D. L., Eds.), 2000]

Examples of sterilizing and/or attenuating may include contacting the living matter with a toxin, a solvent and/or a chemically reactive material formulation component, irradiating the living matter [e.g., infrared ("IR") radiation, ionizing radiation, microwave radiation, ultra violet ("UV") radiation, particle radiation such as alpha radiation, electron beam/beta radiation, neutron radiation, proton radiation], heating the living matter above a temperature suitable for life {e.g., 100°C in many cases, more for an extremophile), or a combination thereof or a combination thereof. Concentrating refers to any process reducing the volume of a composition, an article, etc. An undesired component that comprises the excess volume is removed and/or the desired composition may be localized to a reduced volume. Concentrating may be by any method known in the art, including, for example, washing, filtrating, a gravitational force, a gravimetric force (e.g., centrifugation), or a combination thereof. Drying (e.g., freeze-drying, lyophilizing, spray drying) may remove an undesired liquid, and may produce a material that is more stable (e.g., enzymatic activity retention during storage) than an undried material. Physical force {e.g., grinding, milling, shearing) may alter the average particle size of a material, such as producing a powder form of a material (e.g., a powdered enzyme). An underside or desired material (e.g., an enzyme) may be partly or fully removed, a cellular material may be permeabilized (e.g., cell wall permeabilized), a materials solubility may be altered, or a combination thereof, by extraction with appropriate solvents. Permeabilization [e.g., contacting with a permeabilizing agent such as DMSO, ethylenediaminetetraacetic acid ("EDTA"), tributyl phosphate, contact with a solvent, pressure such as processing through a French press, sonication, mechanical shearing, homogenization, sonication, freeze drying, spray drying, freezing then thawing, contact with a porin and/or an enzyme such as a lysozyme, etc.] of a biomolecule composition comprising a cell membrane and/or a cell wall may promote the separation of cells, reduce the average particle size of the material, allow greater access to a biomolecule in a cell {e.g., to promote ease of extraction), or a combination thereof.

[0111] The form of a biomolecule composition (e.g., a bio-based antibiological agent) may varied through processing techniques to optimize a desired property such as bioactivity and/or bioactivity resistance upon and/or within a particular material formulation. In certain aspects, the total content of desired biomolecule may range from about 0.0000001% to about 99.9999% of a material prepared from a biological cell, by volume and/or dry weight. A whole cell material refers to particulate material the majority {i.e., greater than 50% by weight or volume) resembles an intact living cell upon microscopic examination, while if less than the majority is does not resemble intact living cells such material is referred to as cell-fragment material. In some cases, the presence of cellular material (e.g., a cell wall biomolecule) other than the desired biomolecule (e.g., an enzyme) may provide a protective effect from a material formulation's component {e.g., a solvent, a binder, a polymer, a crosslinking agent, a reactive chemical such as a peroxide, an additive, etc .); a material formulation related chemical reaction {e.g., thermosetting reaction); a potentially damaging agent that a material formulation may contact {e.g., a chemical, a solvent, a detergent, etc .); or a combination thereof. A purified biomolecule composition (e.g., a purified enzyme, a purified peptide, etc.) comprises a desired biomolecule that has been removed in any degree from other extraneous materials {e.g., cellular material, nutrient or culture medium used in growth and/or expression, etc.), such as wherein the concentration of a desired biomolecule has been enhanced about 2 to about 1 ,000,000-fold or more, from its original concentration in a material {e.g., a recombinant cell, a nutrient or culture medium, a chemical synthesis reaction composition, etc.). In other embodiments, a purified biomolecule may comprise about 0.0000001% to about 100% of a composition comprising a biomolecule. Non-limiting techniques for purification in addition to other techniques described herein or as would be known to one of ordinary skill in the art include ammonium sulfate precipitation, ultrafiltration, polyethylene glycol suspension, hexanol extraction, methanol precipitation, Triton X-100 extraction, acrinol treatment, isoelectric focusing, alcohol treatment, acid treatment, acetone precipitation, affinity chromatography {e.g., antibody affinity chromatography, lectin affinity chromatography), fast protein liquid chromatography, high performance liquid chromatography "HPLC"), ion-exchange chromatography, exclusion chromatography; and/or electrophoretic {e.g., polyacrylamide gel electrophoresis, isoelectric focusing) methods, precipitation using antibodies, salts, heat denaturation, centrifugation, dialyzing, etc. Commercially available preparations of a purified biomolecule composition often comprise about 90% to about 100% of a specific biomolecule.

[0112] Chemical modification of a biomolecule composition (e.g., a bio-based antibiological agent) may be used to alter a physical {e.g., hydrophobicity, hydrophilicity, dispersal of particulate material, etc.) and/or a chemical property {e.g., reactivity with a material formulation's component) to enhance suitability in a material formulation. Non-limiting examples of such modifications include acylatylation; amination; hydroxylation; phosphorylation; methylation; adding a detectable label such as a fluorescein isothiocyanate; covalent attachment of a poly ethylene glycol; a derivation of an amino acid by a sugar moiety, a lipid, a phosphate, a farnysyl group; or a combination thereof, as well as others in the art [see, Greene, T. W. and Wuts, P. G. M. "Productive Groups in Organic Synthesis," Second Edition, pp. 309-315, John Wiley & Sons, Inc., USA, 1991; and co pending U.S. Patent Application 10/655,345 "Biological Active Coating Components, Coatings, and Coated surfaces, filed Sept 4, 2003; in "Molecular Cloning," 2001; "Current Protocols in Molecular Biology," 2002] Encapsulation (e.g., microencapsulation) of a biomolecule composition may enhance and/or confer the particulate nature of the biomolecule composition; provide protection to the biomolecule composition; stabilize a biomolecule composition; increase the average particle size to a desired range; allow slow and/or controlled release from the encapsulating material of a component of a biomolecule composition {e.g., a an enzyme, an antimicrobial peptide, etc.) and/or an additional encapsulated material {e.g., a chemical preservative/pesticide, an isolated biomolecule, etc.); alter surface charge, hydrophobicity, hydrophilicity, solubility and/or disperability of a biomolecule composition {e.g., a particulate material) and/or an additional encapsulated material; or a combination thereof. Examples of microencapsulation {e.g., microsphere) compositions (e.g., a gelatin, a hydrogenated vegetable oil, a maltodextrin, a polyurea, a sucrose, an acacia, an amino resin, an ethylcellulose, a polyester, etc.) and techniques are described in, for example, U.S. Patent Nos. 4,324,683, 4,839,046, 4,988,623, 5,026,650, 5153,131, 6,485,983, 5,627,021 and 6,020,312. Other microencapsulation methods which may be employed are those described in U.S. Patent Nos. 5,827,531; 6,103,271; and 6,387,399. A biomolecule composition may be resuspended (e.g., converted into a suspension, an emulsion, etc.) and/or dissolved in a liquid component (e.g., one comprising a cryopreservative, a xeroprotectant, a biomolecule stabilizer, etc.), typically for storage, further processing, and/or addition to a material formulation. During processing, the temperature may be maintained at or less than the optimum temperature for the activity of a living organism and/or a biomolecule that may detrimentally affect a biomolecule. Immobilization refers to attachment (i.e., by covalent and/or non-covalent interactions) of a biomolecule ( e.g ., an enzyme, a peptide) to a solid support ("carrier") and/or crosslinking an enzyme {e.g., a CLEC). A method of immobilization includes, for example, absorption, ionic binding, covalent attachment, crosslinking, entrapment into a gel, entrapment into a membrane compartment, or a combination thereof (Kurt Faber, "Biotransformations in Organic Chemistry, a Textbook, Third Edition." Springer-verlag Berlin Heidelberg, pp. 345- 356, 1997). For example, immobilization of an enzyme to a material formulation’s surface at the molecular level or scale, to limit conformational changes in the presence of a solvent that result in loss of activity, prevent enzyme aggregation, improve enzyme resistance to proteolytic digestion by limiting conformational change(s) and/or exposure of cleavage site(s), to increase the surface area of an exposed enzyme to a substrate for catalytic activity, or a combination thereof. Various reactive moieties of a proteinaceous molecule may be used in immobilization (e.g., covalent immobilization), such as lysine amino moiety, an aspartate carboxyl moiety a glutamate carboxyl moiety, the C-terminal carboxylic acid, the N-terminal amine, etc. A surface may be modified to comprise a reactive moiety, such as by a polymer comprising a monomer with a reactive moiety and/or creating a reactive moiety (e.g., a hydroxyl group on glass or polymeric materials via chemical reactions, contact with flame or plasma, etc.), wherein the reactive moiety acts to immobilize a biomolecular composition (e.g., via a direct covalent bond, via a crosslinker or tethering agent, etc.). Absorption may be used, for example, to attach a proteinaceous molecule onto a material where it may be held by a non-covalent {e.g., hydrogen bonding, Van der Waals forces) interaction. In some embodiments, a proteinaceous molecule may be stabilized in a material formulation (e.g., a polymeric material, a surface treatment) by immobilization to another molecule (“carrier molecule”) within the material formulation.

[0113] A biomolecule may be derived from a non-biological source, such as the case of a proteinaceous and/or a nucleotide sequence engineered by the hand of man. For example, a nucleotide sequence encoding a synthetic peptide sequence from a peptide library. In some aspects, one or more peptides may be prepared as a peptide library, which typically comprises a plurality (e.g., about 2 to about 10 10 peptides). A peptide library may comprise a D-amino acid, an L-amino acid, a cyclic amino acid, a common amino acid, an uncommon amino acid ( e.g ., a non-naturally occurring amino acid), a stereoisomer ( e.g ., a D-amino acid stereoisomer, an L-amino acid stereoisomer), or a combination thereof. A peptide library may comprise a synthetically produced peptide and/or a biologically produced peptide (e.g., a recombinantly produced peptide, see for example U.S. Patent No. 4,935,351).

[0114] It is possible to alter a proteinaceous molecule (e.g., an enzyme, an antibody, a receptor, a peptide, a polypeptide) with a defined amino acid sequence and/or length for one or more properties. Examples of a property, in the context of a proteinaceous molecule, includes, but is not limited to, a ligand binding property, a catalytic property, a stability property, a property related to environmental safety, a charge property, or a combination thereof. As used herein, a wild-type proteinaceous molecule refers to an amino acid sequence that functions as an enzyme and matches the sequence encoded by an isolated gene from a natural source. A proteinaceous molecule (e.g., an enzyme, an antibody, a receptor, a peptide, a polypeptide) comprising a chemical modification and/or a sequence modification that functions the same or similar (e.g., a modified enzyme of the same EC classification as the unmodified enzyme) comprises a "functional equivalent" to, and "in accordance" with, an un-modified proteinaceous molecule. For example, various amino acids have been given a numeric quantity based on the characteristics of charge and hydrophobicity, called the hydropathic index (Kyte, J. and Doolittle, R. F. J. Mol. Biol., 157:105-132, 1982), as well as a value has based on hydrophilicity (seem for example,

U.S. Pat. No. 4,554,101), and an amino acid may be substituted for a different amino acid having a similar hydropathic and/or hydrophobicity value (e.g., generally within +/- 2, within +/- 1, and/or within +/-0.5), and retain similar if not identical biological activity. In some cases, a proteinaceous molecule may be 70%, 80%, 90%, up to 100% similar in sequence identity and/or length and retain similar if not identical biological activity. Functional equivalents are that may be used are described, for example, in U.S. Patent Application nos. 12/696,651, 12/474,921 and 12/882,563, each specifically incorporated herein by reference.

[0115] In addition to the sources described herein for a biomolecule, a reagent, a living cell, etc., such a material and/or a chemical formula thereof may be obtained from convenient source such as a public database, a biological depository, and/or a commercial vendor.

For example, various nucleotide sequences, including those that encode amino acid sequences, may be obtained at a public database, such as the Entrez Nucleotides database, which includes sequences from other databases including GenBank ( e.g ., CoreNucleotide), RefSeq, and protein database bank (“PDB”). Another example of a public databank for nucleotide and amino acid sequences includes the Kyoto Encyclopedia of Genes and Genomes ("KEGG") (Kanehisa, M.ef. a/., Nucleic Acids Res. 36:D480-D484, 2008; Kanehisa, M. et. al., Nucleic Acids Res. 34:D354-357, 2006; Kanehisa, M. and Goto, S., Nucleic Acids Res. 28:27-30, 2000). In another example, various amino acid sequences may be obtained at a public database, such as the Entrez databank, which includes sequences from other databases including SwissProt, PIR, PRF, PDB, Gene, GenBank, and RefSeq. Numerous nucleic acid sequences and/or encoded amino acid sequences can be obtained from such sources. In a further example, a biological material comprising, or are capable of comprising such a biomolecule {e.g., a living cell, a virus), may be obtained from a depository such as the American Type Culture Collection ('ATCC"), P.O. Box 1549 Manassas, VA 20108, USA. Many chemical compositions may be further identified by a Chemical Abstracts Service registration number ("CAS No."). For example, a lipase may be obtained from a commercial vendor, such as a type VII lipase from Candida rugosa (Sigma-Aldrich product no. L1754; >700 unit/mg solid; CAS No. 9001- 62-1); a Lipoase (Novozymes; Lipolase 100 L, Type EX), which typically comprises about 2% (w/w) lipase from Thermomyces lanuginosus (CAS No. 9001-62-1).

[0116] Of course, an antibiological agent (e.g., an enzyme, a peptide) may be combined with another biomolecule composition (e.g., an enzyme, a cell based particulate material), for the purpose to confer an additional property {e.g., a catalytic activity, a binding property) other than one related to an antibiological (e.g., an antimicrobial and/or antifouling) function. Examples of another biomolecule composition include an enzyme such as a lipolytic enzyme, though some lipolytic enzymes may have antibiological activity; a phosphoric triester hydrolase; a sulfuric ester hydrolase; a peptidase, some of which may have an antibiological activity; a peroxidase, or a combination thereof. Alternatively, in several embodiments, a biomolecule composition may be used with little or no antibiological function. For example, a material formation may comprise a combination of active enzymes with little or no active antibiological (e.g., antimarine, antifouling, antimicrobial, etc.) enzyme present.

[0117] In various embodiments, an article, a device, a composition, a method, etc. may comprise one or more selected biomolecules ( e.g ., 1 to 1000 or more different selected biomolecules of interest), in various combinations thereof {e.g., an enzyme, an antibody, a peptide, etc.). In some embodiments, the concentration of any individual selected biomolecule comprises about 0.000000001 % to about 100%, of the material formulation {e.g., about 0.000000001 % to about 50%, about 1 % to about 50%) by weight or number of molecules (e.g., cured 1 :1 coupling agent to enzyme in a cured molecular coating after loss of volatile liquid components).

[0118] In addition to the disclosures herein, a preservative and use of a preservative in a coating is known in the art, and all such materials and techniques for using a preservative in a coating may be used (see, for example, Flick, E. W. "Handbook of Paint Raw Materials, Second Edition," 263-285 and 879-998, 1989; in "Paint and Coating Testing Manual, Fourteenth Edition of the Gardner-Sward Handbook," (Koleske, J. V. Ed.), American Society for Testing and Materials, Philadelphia, PA, U.S.A., pp 261 -267 and 654-661 , 1995; in "Paint and Surface Coatings, Theory and Practice, Second Edition," (Lambourne, R. and Strivens, T. A., Eds.), Andrew Publishing, Woodhead Publishing Ltd, Abington Hall, Abington, Cambridge CB1 6AH, England, pp. 193-194, 371-382 and 543-547, 1999; Wicks, Jr., Z. W., Jones, F. N., Pappas, S. P. "Organic Coatings, Science and Technology, Volume 1 : Film Formation, Components, and Appearance," 2 nd edition, by Zeno W. Wicks Jr., Frank N. Jones, S. Peter Pappas, Publisher: Wiley-lnterscience (John Wiley & Sons, Inc. 605 Third Avenue, New York, NY), pp. 318-320, 1992; Wicks, Jr., Z. W., Jones, F. N., Pappas, S. P. "Organic Coatings, Science and Technology, Volume 2: Applications, Properties and Performance," pp. 145, 309, 319-323 and 340-341 , 1992; and in "Paints, Coatings and Solvents, Second, Completely Revised Edition," (Stoye, D. and Freitag, W., Eds.) pp 6, 127 and 165, 1998; and in "Handbook of Coatings Additives," Wiley-Vch, New York, U.S.A., pp. 177-224, 1987).

[0119] For example, the quality of a liquid coating mixture may suffer markedly if a microorganism (e.g., a mold) degrades one or more of the components during storage (e.g., in-can). In certain embodiments, a preservative may comprise an in-can preservative, an in-film preservative, or a combination thereof. An in-can preservative comprises a composition that reduces and/or prevents the growth of a microorganism prior to film formation. Addition of an in-can preservative during a waterborne coating production typically occurs with the introduction of water to a coating composition. Typically, an in-can preservative may be added to a coating composition for function during coating preparation, storage, or a combination thereof. An in-film preservative comprises a composition that reduces or prevents the growth of a microorganism after film formation. In many embodiments, an in-film preservative comprises the same chemical as an in-can preservative, but added to a coating composition at a higher (e.g., about two-fold or more) concentration for continuing activity after film formation.

[0120] Examples of a preservative used in a coating include a metal compound (e.g., an organo-metal compound) biocide, an organic biocide, or a combination thereof. Examples of a metal compound biocide include a barium metaborate (CAS No. 13701-59-2), which may function as a fungicide and/or a bactericide; a copper(ll) 8-quinolinolate (CAS No. 10380-28-6), which may function as a fungicide; a phenylmercuric acetate (CAS No. 62-38- 4), a tributyltin oxide (CAS No. 56-35-9), which may be less selected for use against Gram negative bacteria; a tributyltin benzoate (CAS No. 4342-36-3), which may function as a fungicide and a bactericide; a tributyltin salicylate (CAS No. 4342-30-7), which may function as a fungicide; a zinc pyrithione ("zinc 2-pyridinethiol-N-oxide"; CAS No. 13463-41-7), which may function as a fungicide; a zinc oxide (CAS No. 1314-13-2), which may function as a fungistatic, a fungicide and/or an algaecide; a combination of zinc- dimethyldithiocarbamate (CAS No. 137-30-4) and a zinc 2-mercaptobenzothiazole (CAS No. 155-04-4), which acts as a fungicide; a zinc pyrithione (CAS No. 13463-41-7), which may function as a fungicide; a metal soap; or a combination thereof. Examples of a metal comprised in a metal soap biocide include a copper, a mercury, a tin, a zinc, or a combination thereof. Examples of an organic acid comprised in a metal soap biocide include a butyl oxide, a laurate, a naphthenate, an octoate, a phenyl acetate, a phenyl oleate, or a combination thereof.

[0121] An example of an organic biocide that acts as an algaecide includes a 2-methylthio- 4-tert-butylamino-6-cyclopropylamino-s-triazine (CAS No. 28159-98-0). Examples of an organic biocide that acts as a bactericide include a combination of a 4,4-dimethyl- oxazolidine (CAS No. 51200-87-4) and a 3,4,4-trimethyloxazolidine (CAS No. 75673-43-7); a 5-hydroxy-methyl-1-aza-3,7-dioxabicylco (3.3.0.) octane (CAS No. 59720-42-2); a 2(hydroxymethyl)-aminoethanol (CAS No. 34375-28-5); a 2-(hydroxymethyl)-amino-2- methyl-1 -propanol (CAS No. 52299-20-4); a hexahydro-1 ,3,5-triethyl-s-triazine (CAS No. 108-74-7); a 1-(3-chloroallyl)-3,5,7-triaza-1-azonia-adamantane chloride (CAS No. 51229- 78-8); a 1 -methyl-3, 5, 7-triaza-1-azonia-adamantane chloride (CAS No. 76902-90-4); a p- chloro-m-cresol (CAS No. 59-50-7); an alkylamine hydrochloride; a 6-acetoxy-2, 4-dim ethyl- 1, 3-dioxane (CAS No. 828-00-2); a 5-chloro-2-methyl-4-isothiazolin-3-one (CAS No. 26172- 55-4); a 2-methyl-4-isothiazolin-3-one (CAS No. 2682-20-4); a 1 ,3-bis(hydroxymethyl)-5,5- dimethylhydantoin (CAS No. 6440-58-0); a hydroxymethyl-5, 5-dimethylhydantoin (CAS No. 27636-82-4); or a combination thereof. Examples of an organic biocide that acts as a fungicide include a parabens; a 2-(4-thiazolyl)benzimidazole (CAS No. 148-79-8); a N- trichloromethyl-thio-4-cyclohexene-1 ,2-dicarboximide (CAS No. 133-06-2); a 2-n-octyl-4- isothiazoline-3-one (CAS No. 26530-20-1); a 2,4,5,6-tetrachloro-isophthalonitrile (CAS No. 1897-45-6); a 3-iodo-2-propynyl butyl carbamate (CAS No. 55406-53-6); a N- (trichloromethyl-thio)phthalimide (CAS No. 133-07-3); a tetrachloroisophthalonitrile (CAS No. 1897-45-6); a potassium N-hydroxy-methyl-N-methyl-dithiocarbamate (CAS No. 51026- 28-9); a sodium 2-pyridinethiol-1 -oxide (CAS No. 15922-78-8); or a combination thereof. Examples of a parabens include a butyl parahydroxybenzoate (CAS No. 94-26-8); an ethyl parahydroxybenzoate (CAS No. 120-47-8); a methyl parahydroxybenzoate (CAS No. 99- 76-3); a propyl parahydroxybenzoate (CAS No. 94-13-3); or a combination thereof. Examples of an organic biocide that acts as a bactericide and fungicide include a 2- mercaptobenzo-thiazole (CAS No. 149-30-4); a combination of a 5-chloro-2-methyl-3(2H)- isothiazoline (CAS No. 26172-55-4) and a 2-methyl-3(2H)-isothiazolone (CAS No. 2682-20- 4); a combination of a 4-(2-nitrobutyl)-morpholine (CAS No. 2224-44-4) and a 4,4’-(2- ethylnitrotrimethylene dimorpholine (CAS No. 1854-23-5); a tetra-hydro-3,5-di-methyl-2H- 1 ,3,5-thiadiazine-2-thione (CAS No. 533-74-4); a potassium dimethyldithiocarbamate (CAS No. 128-03-0); or a combination thereof. An example of an organic biocide that acts as an algaecide and fungicide includes a diiodomethyl-p-tolysulfone (CAS No. 20018-09-1). Examples of an organic biocide that acts as an algaecide, a bactericide and a fungicide include a glutaraldehyde (CAS No. 111-30-8); a methylenebis(thiocyanate) (CAS No. 6317- 18-6); a 1 ,2-dibromo-2,4-dicyanobutane (CAS No. 35691-65-7); a 1 ,2-benzisothiazoline-3- one ("1 ,2-benzisothiazolinone"; CAS No. 2634-33-5); a 2-(thiocyanomethyl- thio)benzothiazole (CAS No. 21564-17-0); or a combination thereof. An example of an organic biocide that acts as an algaecide, a bactericide, a fungicide and a mollusicide includes a 2-(thiocyanomethyl-thio)benzothiozole (CAS No. 21564-17-0) and/or a methylene bis(thiocyanate) (CAS No. 6317-18-6). [0122] In some embodiments, an antifungal agent (e.g., a fungicide, a fungistatic) may comprise a copper (II) 8-quinolinolate (CAS No. 10380-28-6); a zinc oxide (CAS No. 1314- 13-2); a zinc-dimethyl dithiocarbamate (CAS No. 137-30-4); a 2-mercaptobenzothiazole, zinc salt (CAS No. 155-04-4); a barium metaborate (CAS No. 13701-59-2); a tributyl tin benzoate (CAS No. 4342-36-3); a bis tributyl tin salicylate (CAS No. 22330-14-9), a tributyl tin oxide (CAS No. 56-35-9); a parabens: ethyl parahydroxybenzoate (CAS No. 120-47-8), a propyl parahydroxybenzoate (CAS No. 94-13-3); a methyl parahydroxybenzoate (CAS No. 99-76-3); a butyl parahydroxybenzoate (CAS No. 94-26-8); a methylenebis(thiocyanate) (CAS No. 6317-18-6); a 1 ,2-benzisothiazoline-3-one (CAS No. 2634-33-5); a 2-mercaptobenzo-thiazole (CAS No. 149-30-4); a 5-chloro-2-methyl-3(2H)- isothiazolone (CAS No. 57373-19-0); a 2-methyl-3(2H)-isothiazolone (CAS No. 57373-20- 3); a zinc 2-pyridinethiol-N-oxide (CAS No. 13463-41-7); a tetra-hydro-3,5-di-methyl-2H- 1 ,3,5-thiadiazine-2-thione (CAS No. 533-74-4); a N-trichloromethyl-thio-4-cyclohexene-1 ,2- dicarboximide (CAS No. 133-06-2); a 2-n-octyl-4-isothiazoline-3-one (CAS No. 26530-20- 1); a 2,4,5,6-tetrachloro-isophthalonitrile (CAS No. 1897-45-6); a 3-iodo-2-propynyl butylcarbamate (CAS No. 55406-53-6); a diiodomethyl-p-tolylsulfone (CAS No. 20018-09- 1); a N-(trichloromethyl-thio)phthalimide (CAS No. 133-07-3); a potassium N-hydroxy- methyl-N-methyl-dithiocarbamate (CAS No. 51026-28-9); a sodium 2-pyridinethiol-1 -oxide (CAS No. 15922-78-8); a 2-(thiocyanomethylthio) benzothiazole (CAS No. 21564-17-0); a 2-4(-thiazolyl) benzimidazole (CAS No. 148-79-8); or a combination thereof [see, for example, V.M. King, "Bactericides, Fungicides, and Algicides," Ch. 29, pp. 261-267; and D.L. Campbell, "Biological Deterioration of Paint Films," Ch. 54, pp. 654-661; both in PAINT AND COATING TESTING MANUAL, 14th ed. of the Gardner-Sward Handbook, J.V. Koleske, Editor (1995), American Society for Testing and Materials, Ann Arbor, Ml] Additional biological products that may possess antifungal activity are described in the background discussion of U.S. Patent Nos. 6,020,312; 5,602,097; and 5,885,782. U.S. Patent No. 5,882,731 (Owens) describes a number of common and proprietary chemical mildewcide-comprising products that have been investigated as additives for water-based latex mixtures.

[0123] In certain embodiments an environmental law or regulation may encourage the selection of an organic biocide such as a benzisothiazolinone derivative. An example of a benzisothiazolinone derivative comprises a Busan™ 1264 (Buckman Laboratories, Inc.), a Proxel™ GXL (BIT), a Proxel™ TN (BIT/Triazine), a Proxel™ XL2 (BIT), a Proxel™ BD20 (BIT) and a Proxel™ BZ (BIT/ZPT) (Avecia Inc.), a Preventol® VP OC 3068 (Bayer Corporation), and/or a Mergal® K10N (Troy Corp.) which comprises a 1,2- benzisothiazoline-3-one (CAS No. 2634-33-5). In the case of a Busan™ 1264, the primary use may be function as a bactericide and/or a fungicide at about 0.03% to about 0.5% in a waterborne coating, though a Busan™ may be used as a wood and/or a packaging preservative (e.g., a biocide, a mold inhibitor, a bactericide). A Proxel™ TN comprises a 1 ,2-benzisothiazoline-3-one (CAS No. 2634-33-5) and a hexahydro-1,3,5-tris(2- hydroxyethyl)-s-triazine ("triazine"; CAS No. 4719-04-4), a Proxel™ GXL, a Proxel™ XL2 and a Proxel™ BD20 comprises a 1 ,2-benzisothiazoline-3-one (CAS No. 2634-33-5), a Proxel™ BZ comprises a 1 ,2-benzisothiazoline-3-one (CAS No. 2634-33-5) and a zinc pyrithione (CAS No. 13463-41-7), and are typically used in an industrial coating and/or a water-based coating as a bactericide and/or a fungicide. A Mergal® K10N comprises a 1 ,2-benzisothiazoline-3-one (CAS No. 2634-33-5), and may be used in a waterborne coating as a bactericide and/or a fungicide.

[0124] Often, a preservative comprises a proprietary commercial formulation and/or a compound sold under a tradename. Examples include an organic biocide under the tradename Nuosept® (International Specialty Products, "ISP"), which are typically used in a waterborne coating, often as an antimicrobial preservative. Specific examples of a Nuosept® biocide include a Nuosept® 95, which comprises a mixture of bicyclic oxazolidines, and may be added to about 0.2% to about 0.3% concentration to a coating; a Nuosept® 145, which comprises an amine reaction product, and may be added to about 0.2% to about 0.3% concentration to a coating; a Nuosept® 166, which comprises a 4,4- dimethyloxazolidine (CAS No. 51200-87-4), and may be added to about 0.2% to about 0.3% concentration to a basic pH waterborne coating; or a combination thereof. A further example comprises a Nuocide® (International Specialty Products) biocide(s), which are typically used fungicide(s) and/or algaecide(s). Examples of a Nuocide® biocide comprises Nuocide® 960, which comprises about 96% tetrachlorisophthalonitrile (CAS No. 1897-45- 6), and may be used at about 0.5% to about 1.2% in a waterborne and/or a solvent based coating as a fungicide; a Nuocide® 2010, which comprises a chlorothalonil (CAS No. 1897- 45-6) and an IPBC (CAS No. 55406-53-6) at about 30%, and may be used at about 0.5% to about 2.5% in a coating as a fungicide and/or an algaecide; a Nuocide® 1051 and a Nuocide® 1071, each which comprises about 96% N-cyclopropyl-N-(1-dimethylethyl)-6- (methylthio)-l ,3, 5-triazine-2, 4-diamine (CAS No. 28159-98-0), and may be used as an algaecide in an antifouling coating at about 1.0% to about 6.0% or a water-based coating at about 0.05% to about 0.2%, respectively; and a Nuocide® 2002, which comprises a chlorothalonil (CAS No. 1897-45-6) and a triazine compound at about 30%, and may be used at about 0.5% to about 2.5% in a coating and/or a film as a fungicide and/or an algaecide; or a combination thereof.

[0125] An additional example of a tradename biocide for a coating includes a Vancide® (R. T. Vanderbilt Company, Inc.). Examples of a Vancide® biocide include a Vancide® TH, which comprises a hexahydro-1,3,5-triethyl-s-triazine (CAS No. 108-74-7), and may be used in a waterborne coating; a Vancide® 89, which comprises a N-trichloromethylthio-4- cyclohexene-1 ,2-dicarboximide (CAS No. 133-06-2) and related compounds such as a captan (CAS No. 133-06-2), and may be used as a fungicide in a coating; or a combination thereof. A bactericide and/or a fungicide for a coating, particularly a waterborne coating, comprises a Dowicil™ (Dow Chemical Company). Examples of a Dowicil™ biocide include a Dowicil™ QK-20, which comprises a 2,2-dibromo-3-nitrilopropionamide (CAS No. 10222- 01-2), and may be used as a bactericide at about 100 ppm to about 2000 ppm in a coating; a Dowicil™ 75, which comprises a 1-(3-chloroallyl)-3,5,7-triaza-1-azoniaadamantane chloride (CAS No. 51229-78-8), and may be used as a bactericide at about 500 ppm to about 1500 ppm in a coating; a Dowicil™ 96, which comprises a 7-ethyl bicyclooxazolidine (CAS No. 7747-35-5), and may be used as a bactericide at about 1000 ppm to about 2500 ppm in a coating; a Bioban™ CS-1135, which comprises a 4,4-dimethyloxazolidine (CAS No. 51200-87-4), and may be used as a bactericide at about 100 ppm to about 500 ppm in a coating, or a combination thereof the forgoing. An additional example of a tradename preservative (e.g., a biocide) for a coating includes a Kathon® (Rohm and Haas Company). An example of a Kathon® biocide includes a Kathon® LX, which typically comprises a 5- chloro-2-methyl-4-isothiazolin-3-one (CAS no 26172-55-4) and a 2-methyl-4-isothiazolin-3- one (CAS no 2682-20-4) at about 1.5%, and may be added from about 0.05% to about 0.15% in a coating. Examples of tradename fungicide and/or an algaecide include those described for a Fungitrol® (International Specialty Products), which typically may be used as fungicide(s), and a Biotrend® (International Specialty Products), which often is used as biocide(s); and are often formulated for a solvent based and/or a waterborne coating, an in- can and/or a film preservation. An example comprises a Fungitrol® 158, which comprises about 15% tributyltin benzoate (CAS No. 4342-36-3) and about 21.2% alkylamine hydrochlorides, and may be used at about 0.35% to about 0.75% in a waterborne coating for in-can and/or a film preservation. An additional example comprises a Fungitrol® 11 , which comprises a N-(trichloromethylthio) phthalimide (CAS No. 133-07-3), and may be used at about 0.5% to about 1.0% as a fungicide for solvent based coating. A further example comprises a Fungitrol® 400, which comprises about 98% a 3-iodo-2-propynl N- butyl carbamate ("IPBC") (Cas No. 55406-53-6), and may be used at about 0.15% to about 0.45% as a fungicide for a waterborne and/or a solvent based coating.

[0126] Further examples of a tradename preservative (e.g., a biocide) for a coating includes various Omadine® and/or Triadine® product(s) (Arch chemicals, Inc.), a Densil™ P, Densil™ C404 (e.g., a chlorthalonil), a Densil™ DN (BUBIT), a Densil™ DG20 and a Vantocil™ IB (Avecia Inc.), a Polyphase® 678, a Polyphase® 663, a Polyphase® CST, a Polyphase® 641 , a Troysan® 680 (Troy Corp.), a Rocima® 550 (i.e. , a preservative), a Rocima® 607 (i.e., a preservative), a Rozone® 2000 (i.e., a dry film fungicide), and a Skane™ M-8 (i.e., a dry film fungicide; Rohm and Flaas Company) and a Myacide™ GDA, a Myacide™ GA 15, a Myacide™ Ga 26, a Myacide™ 45, a Myacide™ AS Technical, a Myacide™ AS 2, a Myacide™ AS 30, a Myacide™ AS 15, a Protectol™ PE, a Daomet™ Technical and/or a Myacide™ FIT Technical (BASF Corp.). A zinc omadine® ("zinc pyrithione"; CAS No. 13463-41-7) may function as a fungicide and/or an algaecide typically used as an in-film preservative and/or an antifouling preservative; a sodium omadine® ("sodium pyrithione"; CAS No. 3811-73-2) may be used as a fungicide and/or an algaecide in-film preservative; a copper omadine® ("copper pyrithione"; CAS No. 14915-37-8) may be used as a fungicide and/or an algaecide in-film preservative and/or an antifouling preservative; a Triadine® 174 ("triazine," "1 ,3,5-triazine-(2FH,4FH,6FH)-triethanol"; "hexahydro- 1 ,3,5-tris(2-hydroxyethyl)-s-triazine"; Cas No. 4719-04-4) may function as a bacteria biostatic and/or a bactericide typically used in a waterborne coating; an omacide IPBC ("lodopropynyl-butyl carbomate") may function as a fungicide; a Densil™ P comprises a dithio-2,2-bis(benzmethylamide) (CAS No. 2527-58-4) and may be used in an industrial coating, a water-based coating and/or a film as a fungicide and/or a bactericide; a Densil™ C404 comprises a 2,4,5,6-tetrachloroisophthalonitrile ("chlorothalonil"; CAS No. 1897-45-6) and may be used as a fungicide; a Densil™ DN and a Densil™ DG20 comprise a N-butyl- 1 ,2-benzisothiazolin-3-one (CAS No. 4299-07-4), and each may be used as a fungicide; a Vantocil™ IB comprises a poly(hexamethylene biguanide) hydrochloride ("PHMB"; CAS No. 27083-27-8) and may function as a microbiocide; a Polyphase® 678 comprises carbendazim (CAS No. 10605-21-7) and a 3-iodo-2-propynyl butyl carbamate (CAS No. 55406-53-6), and may be used as an antimicrobial biocide for an exterior coating and/or a surface treatment; a Polyphase® 663 comprises a 3-iodo-2-propynyl butyl carbamate (CAS No. 55406-53-6), a carbendazim (CAS No. 10605-21-7) and a diuron (CAS No. 330-54-1) and may be used as a fungicide and/or an algaecide in an exterior coating; a Rocima® 550 comprises a 2-methyl-4-isothiazolin-3-one (CAS No. 2682-20-4), and may be used as a bactericide and/or a fungicide for a waterborne coating; a Rozone® 2000 comprises a 4,5- dichloro-2-N-octyl-3(2H)-isothiazolone (CAS No. 64359-81-5) and may be used as a microbiocide for a latex coating; a Skane™ M-8 comprises a 2-Octyl-4-isothiazolin-3-one (CAS No. 26530-20-1), and may be used as an in-film fungicide; a Myacide™ GDA Technical (50% Glutaraldehyde), a Myacide™ GA 15, a Myacide™ Ga 26 and a Myacide™ 45 each comprise a glutaraldehyde solution (CAS No. 111-30-8), and are typically used as an algaecide, a bactericide, and/or a fungicide; a Myacide™ AS Technical (Bronopol, solid), a Myacide™ AS 2, Myacide™ AS 30, a Myacide™ AS 15 each comprise a 2-bromo- 2-nitropropane-1 ,3-diol solution ("bronopol"; Cas No. 52-51-7) and are typically used as an algaecide; a Protectol™ PE comprises a phenoxyethanol liquid (CAS No. 122-99-6) and may be used as a microbiocide and/or a fungicide; a Dazomet™ Technical comprises a

3.5-dimethyl-2H-1,3,5-thiadiazinane-2-thione solid ("dazomet"; CAS No. 533-74-4) and may be used as a microbiocide and/or a fungicide; a Myacide™ HT Technical comprises a

1.3.5-tris-(2-hydroxyethyl)-1 ,3,5-hexahydrotriazine liquid ("Triazine," CAS No. 4719-04-4) and may be used as a microbiocide and/or a fungicide. Additional examples of tradename preservatives (all from Cognis Corp., Ambler, PA) includes a Nopcocide® N400, which comprises a Cholorthalonil-40% solution; a Nopcocide® N-98, which comprises a Chlorothalonil-100%; a Nopcocide® P-20, which comprises an IPBC-20% solution; a Nopcocide® P-40, which comprises an IPBC-40% solution; a Nopcocide® P-100, which comprises an IPBC-100% active; or a combination thereof.

[0127] The general effectiveness of various embodiments is demonstrated in the following Examples. Some methods for preparing compositions are illustrated, and starting materials are made according to procedures known in the art or as illustrated herein. The following Examples are provided so that the embodiments might be more fully understood. These Examples are illustrative only and should not be construed as limiting in any way, as various material formulation(s) comprising biomolecule composition(s) ( e.g a coating formulation comprising a bio-based antibiological agent applied to a manufactured article or device) may be prepared in light of the disclosures herein.

[0128] Example 1 : This Example demonstrates the ability of a lysozyme to survive the incorporation process into a coating, demonstrates lysozyme hydrolytic activity in a coating environment, and demonstrates the ability of lysozyme to survive in-can conditions for 48 hours. A Sherwin-Williams Acrylic Latex paint was used. Materials, reagents and equipment used are shown in the tables below.

[0129] The reagents prepared included a Micrococcus cell suspension comprising 9 mg M. lysodeikticus in 25 mL sodium phosphate buffer, and a lysozyme solution comprising a 5 mg/mL stock solution. The paint formulations used are shown in the table below.

[0130] The paint was mixed with a glass stirring rod and a paint mixer. Each film was immediately drawn onto polypropylene surfaces with a thickness of 8 mil. Cure time for the Sherwin-Williams was 72 hrs. To demonstrate in-can durability, the Sherwin-Williams Acrylic Latex comprising lysozyme wet paint was sealed and shelf stored at ambient temperature. After 48 hrs in-can, films were drawn onto polypropylene surfaces with a thickness of 8 mils and were allowed to cure 72 hrs prior to assay. Coupons were generated as free films from the polypropylene surface. Films were generated in three sizes: 2 cm 2 : 1 cm by 2 cm; 4 cm 2 : 1 cm by 4 cm; or 6 cm 2 : 1 cm by 6 cm. [0131] For qualitative assessment, individual films were placed into labeled 15 mL tubes. Films of each size (2, 4 and 6 cm 2 ) were evaluated in triplicate. In addition to a control paint with no additive, two other controls were utilized, a positive control and a negative control. The positive control comprised: lysozyme in buffer added to each of three 15 mL tubes in concentrations approximating the amount of lysozyme in the films (i.e., 40 pg, 80 pg, and 120 pg). Each amount was assayed in triplicate. The negative control comprised: 5 mL of 0.36 mg/mL M. lysodeikticus cell suspension pipetted into a single 15 mL tube. 5 mL 0.36 mg/mL Micrococcus lysodeikticus cell suspension was added to all reaction tubes to begin the reaction. The tubes were placed on a rocker at ambient conditions for approximately 22 hours. Where possible, the films were removed from the suspension and determine opacity using the Klett-Summerson Colorimeter (turbidity unit: Klett Unit or KU). [0132] Particulate matter in the samples interfered with quantitation; photographs of each set of 2 cm 2 paint films and controls following 22-hour contact to M. lysodeikticus cell suspension were taken, and observations recorded in the Tables below.

1 Each evaluation was performed in triplicate

2 Thinned in opacity, with some suspended particulate matter

[0133] The strips comprising lysozyme of all three sizes of coupons cleared the M. lysodeikticus suspension, indicating that the lysozyme maintains activity in the coating environment. Cleared suspensions (lysozyme comprising coupons and controls) comprised large particles which interfere with the quantitation of the cleared suspensions. The particulate matter was less detectable in the 2 cm 2 set comprising lysozyme, so this size coupon was used for the quantitative demonstrations.

KU = Klett Units, measure of turbidity at 540 nm.

[0134] A lysozyme in Sherwin-Williams Acrylic Latex was able to lyse about 88% of the M. lysodeikticus culture over 4 hours, relative to the control which exhibited about a 15% drop in opacity. After in-can shelving for 48 hrs (i.e., the lysozyme was mixed into the Sherwin- Williams Acrylic Latex, capped and shelved for 48 hrs prior to drawing down the films), the lysozyme remained active, lysing about 64% of the M. lysodeikticus culture relative to the about 21% lysis exhibited by the control panels.

[0135] Example 2: This Example demonstrates the retention of lysozyme activity after in- can storage in a paint coating for 48 hours; followed by film formation and activity measurements after enzyme loss due to leaching in a paint film in a saturated condition at 1 , 2 and 24 hours after submersion. Materials, reagents and equipment used are shown in the tables below.

[0136] The reagents prepared included a Micrococcus cell suspension comprising 9 mg M. lysodeikticus in 25 mL sodium phosphate buffer, and a lysozyme solution comprising a 5 mg/mL stock solution.

[0137] The paint formulations that were prepared included a Sherwin-Williams Acrylic Latex Control (no additive), and a Sherwin-Williams Acrylic Latex comprising 1 mg/mL lysozyme. Each paint was mixed with a glass stirring rod and a paint mixer. Each film was immediately drawn onto polypropylene surfaces with a thickness of 8 mil. Cure time was 120 hrs. The Sherwin-Williams Acrylic Latex comprising a lysozyme wet paint was sealed and shelf stored at ambient temperature. After 48 hrs in-can storage, films were drawn onto polypropylene surfaces with a thickness of 8 mils and were allowed to cure 72 hrs prior to assay. Materials for assay were generated from the polypropylene surface as a 2 cm 2 (1 x 2 cm) free film. [0138] The assay procedure included placing individual films into labeled 15 ml_ tubes. 24 hours prior to addition of Micrococcus lysodeikticus cell suspension, 5 ml_ KPO4 buffer was added to the 24-hour control and coupon comprising a lysozyme tube, as well as one tube comprising 41 pg lysozyme solution (positive control) and one tube comprising 5 ml_ of the M. lysodeikticus cell suspension (negative control). These tubes were placed on the shaker for 24 hrs.

[0139] Two hours prior to addition of M. lysodeikticus, 5 ml_ potassium phosphate buffer was added to the 2-hour control and lysozyme tubes each comprising a coupon, as well as one tube comprising 41 pg lysozyme solution (positive control) and one tube comprising 5 mL of the M. lysodeikticus cell suspension (negative control). These tubes were placed on the shaker for 2 hrs.

[0140] One hour prior to addition of M. lysodeikticus cell suspension, 5 mL potassium phosphate buffer was added to 1-hour control and coupon comprising a lysozyme tubes, as well as one tube comprising 41 pg lysozyme solution (positive control) and one tube comprising 5 mL of the M. lysodeikticus cell suspension (negative control). These tubes were placed on the shaker for one hour.

[0141] The paint coupons were then transferred from each tube to a second reaction tube.

5 mL of the M. lysodeikticus cell suspension was added to both film and KPO4 buffer incubation buffer. The tubes were placed on the rotating shaker horizontally and shaken for approximately 4 hours, at which time each tube was measured in a Klett-Summerson Photoelectric Colorimeter to determine opacity.

KU = Klett Unit, measure of turbidity at 540 nm

[0142] At the three time points assayed, lysozyme leached out of films that comprised a lysozyme. The ability of the films comprising a lysozyme to lyse M. lysodeikticus was inversely related to the time the coupon was submerged. Over the first 2 hrs the films lost approximately 21 % ± 3 % of the lytic activity per hour. This loss decreased substantially over the following 22 hrs, with the loss slowing to approximately 3% per hour. After 24 hours of liquid submersion, approximately one-third of the activity of a coupon comprising a lysozyme was retained. Though reduction of activity due to leaching may continue, activity may also be permanently retained in the films. The total percentage lysis by coupon and buffer pairs decreased with increasing leaching time.

[0143] Example 3: This Example demonstrates the surface efficacy of paint films comprising a lysozyme in actively lyse M. lysodeikticus in a minimally hydrated environment. Materials, reagents and equipment used are shown in the tables below.

[0144] The reagents prepared included a Micrococcus cell suspension comprising 9 mg Micrococcus lysodeikticus in 25 mL sodium phosphate buffer, and a lysozyme solution comprising a 5 mg/mL stock solution. [0145] The paint formulations prepared for the assay included a Sherwin-Williams Acrylic

Latex Control (no additive), and a Sherwin-Williams Acrylic Latex with 1 mg/mL lysozyme. Each paint was mixed with a glass stirring rod and a paint mixer. Each film was immediately drawn onto polypropylene surfaces with a thickness of 8 mil. Cure time was 72 hrs. Assay materials were generated from the polypropylene surface as a 2 cm 2 (1 x 2 cm) free film.

[0146] The assay procedure included placing individual coupons into separate Petri dishes. Each set of control coupons and coupons comprising a lysozyme was assayed in triplicate. Two controls were set up for this experiment: a M. lysodeikticus suspension control comprising 90 pL 20 mg/mL M. lysodeikticus cell suspension that was pipetted into a petri dish; and a 1 mg/mL lysozyme control comprising 40.64 pL 1 mg/mL lysozyme solution (an amount approximately equal to the amount of lysozyme in the 2 cm 2 coupon comprising a lysozyme) that was pipetted into a petri dish. M. lysodeikticus cell suspension was distributed onto the surface of each individual coupon in a minimal volume (90 pL). Petri dishes were kept on a flat surface. After 4 hours, KPO4 buffer was added to all samples to recover the unlysed portion of the M. lysodeikticus cell suspension. The suspension was removed from each dish with a pipette and placed into individual test tubes. Each suspension was read in the Klett-Summerson Photoelectric Colorimeter, using potassium phosphate buffer as a control.

*KU = Klett units, measure of turbidity at 540 nm. [0147] The paint comprising a lysozyme contacted with 0.18 mg of a M. lysodeikticus suspension for 4 hours lysed 65% ± 10% of the Micrococcus cells, compared to only 7% ± 5% of cells lysed by the paint controls. This demonstrated that lysozyme can function in the low water (i.e., a minimally hydrated) environment of a coating. It is contemplated that a biological assay including a spray application of an assay organism would also demonstrate biostatic and/or biocidal activity.

[0148] Example 4: This Example demonstrates the effectiveness of lysozyme in lysing the bacterium Micrococcus lysodeikticus. M. lysodeikticus was used as a lysozyme substrate in a liquid suspension in the assay. The assay measured the rate of decrease in the absorbance as a relative measure of the amount/availability/activity of a lysozyme present in a material. As cell lysis occurs, the turbidity of a cell suspension decreased, and therefore, the absorbance of a cell suspension decreased. Materials and reagents that were used are shown in the table below.

[0149] The reagents that were prepared included a M. lysodeikticus cell suspension comprising 9 mg M. lysodeikticus in 25 mL sodium phosphate buffer, and a lysozyme solution comprising a 5 mg/mL stock solution.

[0150] The assay procedure included diluting the lysozyme stock solution with buffer to create the following samples: 5 mg/mL(undiluted); 2.5 mg/mL; 1 mg/mL; 0.5 mg/mL; 0.1 mg/mL; 0.05 mg/mL; 0.01 mg/mL; 0.005 mg/mL; 0.001 mg/mL; 0.0005 mg/mL; 0.0001 mg/mL; and 0.00005 mg/mL. Control samples included: 3 replicates of 200 pL M. lysodeikticus cell suspension and 3 replicates of 200 pL buffer that were pipetted into 6 wells total in a 96-well microplate. A 194 pL Micrococcus cell suspension was pipetted into 3 rows of 12 wells each. 6 pl_ of each lysozyme concentration assayed was then added to the M. lysodeikticus cell suspension using a multi-pipette and mixed. The plate was immediately placed into the Thermo Multiskan Ascent Plate Reader; each well was read every 10 seconds for 30 minutes to determine the absorbance at 450 nm.

[0151] The results for the lysozyme assay under the conditions as described: 1 mg of lysozyme was able to lyse 0.047 mg of M. lysodeikticus per sec. The lysozyme was effective in lysing M. lysodeikticus cells, and these results were consistent under both conditions evaluated (Tris vs NahtePC )

[0152] Example 5: This Example demonstrates the ability of a chymotrypsin to survive the incorporation process into a coating and demonstrates chymotrypsin activity in a coating environment. A chymotrypsin free film assay was used for determining the activity of chymotrypsin, as measured by ester hydrolysis (esterase) activity of a p-nitrophenyl acetate substrate, in free-films using a plate reader. A functioning vent hood was used for the assay when appropriate for material handling. A Sherwin-Williams Acrylic Latex paint was used. Equipment and reagents that were used are shown in the tables below.

[0153] Sample preparation included: 14.5mM p-nitrophenyl acetate (66mg/25ml) in isopropyl alcohol, and 200mM TRIS; pH 7.1 (adjust to pH 7.1 with HCI). [0154] The paint formulations that were prepared included a Sherwin-Williams Acrylic Latex control (no additive), and a Sherwin-Williams Acrylic Latex comprising 200 mg/mL a- Chymotrypsin. Each paint was mixed with a glass stirring rod and a paint mixer. Each film was immediately drawn onto polypropylene surfaces with a thickness of 8 mil. Cure time was 24 days. Materials for assay were generated from the polypropylene surface as 1 cm 2 , 2 cm 2 and 3 cm 2 free films.

[0155] The plate reader assay comprised: cutting free films into appropriate size pieces; adding 600 pL ddH20 into a 2 ml microtube; then adding 750 pL 200 mM TRIS to each microtube; adding 150 pL of 14.5 mM p-nitrophenyl acetate to each tube; and taking the 0- time sample, then adding the free film to the tube (control sample is free film with no chymotrypsin).

[0156] The analysis included: taking out 100 pi and reading the absorbance at 405 nm, at the appropriate time points; and determining the initial rate slope by plotting absorbance vs. time to calculate chymotrypsin activity.

[0157] A chymotrypsin in Sherwin-Williams Acrylic Latex was able to hydrolyze the model substrate at rate 20x faster than the control. The test coupons demonstrate a dose response which corresponds to a hydrolytic capacity of 0.86 pmol/min/cm 2 , as formulated in this demonstration. [0158] Quality control included reading and become familiar with the operating instructions for equipment used in the analysis. Operating instructions and preventive maintenance records were placed near the relevant equipment, and kept in a labeled central binder in the work area. Working solutions which are out of date or prepared incorrectly were disposed of and not used.

[0159] Safety procedures and precautions included wearing a full-length laboratory coat; and not eating, drinking, smoking, use of tobacco products or application of cosmetics near the procedure. Consumables and disposable items that come in contact with or are used in conjunction with samples disposal were in the proper hazard containers. This includes, but is not limited to, pipette tips, bench-top absorbent paper, diapers, Kim wipes, test tubes, etc. Biohazard containers were considered full when their contents reach three-quarters of the way to the top of the bag or box. Bench-top biohazard bags were placed into a large biohazard burn box when full. Biohazard containers were not filled to overflowing. Biohazard bags were disposed of by closing with autoclave tape, and autoclaving immediately. Spills and spatters were immediately cleaned from durable surfaces by applying 70% ethanol (for bacteriological spills) to the spill, followed by wiping or blotting.

All equipment used in sample analyses were wiped down on a daily basis or whenever tests were performed. Absorbent pads were placed under samples when useful. Hands were washed with antibacterial soap before exiting the room, when a test was finished, and before the end of the day. The Material Safety Data Sheet ("MSDS") applicable to each chemical was read. MSDS documents have been prominently posted in the laboratory. During a fire alarm during laboratory operations, evacuation procedures were followed. Nitrile protective gloves were worn whenever handling organophosphates. All organophosphate waste was disposed of properly. [0160] Example 6: This Example demonstrates the ability of a cellulase to survive the incorporation process into a coating and demonstrates cellulase activity in a coating environment. A Glidden Latex paint was used. A plate reader was used to assay a free- film comprising a cellulase for the enzyme's activity. Equipment and reagents that were used are shown in the table below.

[0161] Sample preparation included: 14.5 mM 4-Nitrophenyl b-D-cellobioside in ddH20; 50 mM sodium acetate buffer; pH 5.0 (adjust to pH 5.0 with HCI); and 2 N NaOH in ddH20. [0162] The plate reader assay comprised: placing free films into 2 ml microtubes; add 1.2 ml 50 mM sodium acetate buffer, 0.15 ml 14.5 mM 4-Nitrophenyl b-D-cellobioside and 0.15 ml ddH20, in the 2 ml microtube; placing tubes on rocker; taking out 100 pi from the tubes into a 96-well plate at desired time points; adding 200 mI of 2 N NaOH and reading the absorbance at 405 nm; and determining the initial rate slope by plotting absorbance vs. time to calculate cellulase activity. [0163] The paint formulations that were prepared included a Sherwin-Williams Acrylic Latex control (no additive), and a Sherwin-Williams Acrylic Latex comprising 100 g/gal, 200 g/gal and 300 g/gal cellulase. Each paint was mixed with a glass stirring rod and a paint mixer. Each film was immediately drawn onto polypropylene surfaces with a thickness of 8 mil. Cure time was 24 hrs. Materials for assay were generated from the polypropylene surface as a 3 cm 2 free film.

[0164] A cellulase in a Glidden Latex was able to hydrolyze the model substrate at a rate approximately 100x faster than the control. Quality control and safety procedures were as described in Example 5.

[0165] Example 7: This Example demonstrates preparation of technical papers coated with a latex coating comprising an antimicrobial enzyme additive, an antimicrobial peptide additive, or a combination thereof. The additives may be embedded in the coating. The antimicrobial enzyme additive comprised lysozyme, and the antimicrobial peptide additive comprised ProteCoat ® (Reactive Surfaces, Ltd.; also described in U.S. Patent application nos. 10/884,355; 11/368,086; and 11/865,514, each incorporated by reference). Materials that were used are shown in the tables below.

[0166] Paint formulations comprising enzyme were prepared as follows: 1 g lysozyme per 100 g coating; 0.5 g lysozyme per 100 g coating; 0.1 9 lysozyme per 100 g coating; and a negative control (no additive). Paint formulations comprising a peptide additive were prepared as follows: 125 mg ProteCoat® per 1 g coating; 250 mg ProteCoat® per 1 g coating; 375 mg ProteCoat® per 1 g coating; or a negative control (no additive). Paint formulations comprising peptide and lysozyme were prepared as follows: 375 mg ProteCoat® per 1 g lysozyme (1 g) coating; 250 mg ProteCoat® per 1 g lysozyme (0.5 g) coating; 375 mg ProteCoat® per 1 g lysozyme (0.1 g) coating, and a negative Control (no additive). All paint formulations were mixed well. The paper was cut into quarters, coatings drawn onto paper surfaces with a spreader, and wet weight determined. The coated paper was dried at about 37.8°C for approximately 10 min, and dry weight determined. [0167] A single coating material and one paper stock was evaluated. The paper comprised celluosic fibers typically used in technical paper applications, and had an acrylic latex coating added to the fibers. [0168] To prepare the antimicrobial paper ('AM-Paper"), the antimicrobial additives were formulated for each coating on percentage dry weight to standardize the coating for comparison. The antimicrobial additives are listed in the table below.

[0169] The antimicrobial additives were weighed out, added to pre-weighed coating suspensions and mixed by hand for 10 to 20 minutes. After mixing, the coating was applied by draw down, in which approximately 3-5 ml_s of coating was applied along one 8.5" edge of an 8.5" x 11 " pre-weighed paper, and then spread evenly over the surface of the paper with a calibrated rod by drawing the rod down the full length of the paper. The coated paper was then placed into a 100°C oven for 10 to 15 minutes to dry. After drying, the coated paper was weighed to determine the amount of coating on each sheet.

[0170] To conduct an assay to qualitatively assess antimicrobial activity, a paper strip of approximately 1 cm x 5 cm was cut from the control and each antimicrobial paper. 5 ml_ of the M. lysodeikticus suspension was poured into each of 4 x 15 mL conical tubes. The prepared strip was dropped into the suspension, and mixed occasionally by inversion. Clearing changes were observed. [0171] Example 8: This Example demonstrates and provides a standard spectrophotometric assay procedure for lysozyme activity in a plate reader. Equipment and reagents that were used are shown in the table below.

[0172] Micrococcus lysodeikticus cell suspension was made by adding 9 mg Micrococcus lysodeikticus to 25 mL 10 mM Tris-HCI, pH 8.0 and mixing well. Lysozyme solution was prepared by adding 10 mg lysozyme in 1 mL 10 mM Tris-HCI, pH 8.0, and mixing well. Reaction buffer was 10 mM Tris-HCI, pH 8.0, with an alternative reaction buffer being 0.1 M KPO4 pH 6.4.

[0173] A standard curve of the M. lysodeikticus was prepared. The lysozyme stock solution was diluted with the reaction buffer to create the following series: 10 mg/mL (undiluted); 5.0 mg/mL; 2.5 mg/mL; 1 mg/mL; 0.5 mg/mL; 0.1 mg/mL; 0.05 mg/mL; 0.01 mg/mL; 0.005 mg/mL; 0.001 mg/mL; 0.0005 mg/mL; 0.0001 mg/mL, and 0 mg/mL. The controls included 3 replicates of 194 pL M. lysodeikticus cell suspension plus 6 pl_ buffer; and 3 replicates of 200 pL buffer.

[0174] Analysis of samples included determining activity by monitoring the clearing of the cell suspension at 570 nm and determining the best fit to a standard curve. For a 200 mI_ assay, 180 mI_ M. lysodeikticus in reaction buffer was added to each well 1 to 12 of 3 rows. The reaction was started by adding 20 mI_ of each lysozyme dilution to each well in the triplicate series. The plate was immediately placed into the reader, and the changes in absorbance at 570 nm (OD570) recorded. The number of reads may be 10-20 with second intervals. The plate reader's velocity table contained data for reaction rate in mOD/min. This assay can be scaled by increasing each suspension proportionately ( e.g a 2 mL reaction is used for material strip analysis).

[0175] Analysis of the data included calculating the initial velocities for the recorded slopes: [mOD54o/min]/[slope standard curve (mOD/mg M. lysodeikticus]/[ lysozyme]. a pg/mL = ppm [0176] The M. lysodeikticus assay as described can detect lytic activity down to the fractional to low ppm range. The rate of lysis, in suspension, is 32% (about 8.0 x 10 7 cells) of the M. lysodeikticus suspension per pg lysozyme. [0177] Example 9: This Example demonstrates a spectrophotometric assay for antimicrobial paper with a lytic additive. Lysozyme was used as the lytic additive. Equipment and reagents that were used are shown in the table below. [0178] Micrococcus lysodeikticus cell suspension was made by adding 9 mg M. lysodeikticus to 25 mL 10 mM Tris-HCI, pH 8.0 and mixing well. Lysozyme solution was prepared by adding 10 mg lysozyme in 1 mL 10 mM Tris-HCI, pH 8.0, and mixing well. Reaction buffer was 10 mM Tris-HCI, pH 8.0, with an alternative reaction buffer being 0.1 M KPO4 pH 6.4. Antimicrobial paper coated with a coating comprising lysozyme and control paper was prepared in accordance with Example 7.

[0179] A standard curve of the M. lysodeikticus was prepared. The lysozyme stock solution was diluted with the reaction buffer to create the following series: 10 mg/mL (undiluted); 5.0 mg/mL; 2.5 mg/mL; 1 mg/mL; 0.5 mg/mL; 0.1 mg/mL; 0.05 mg/mL; 0.01 mg/mL; 0.005 mg/mL; 0.001 mg/mL; 0.0005 mg/mL; 0.0001 mg/mL and 0 mg/ml. The controls included 3 replicates of 194 pL M. lysodeikticus cell suspension plus 6 pL buffer; and 3 replicates of 200 pL buffer. Pipet tips used fitted the pipette ( e.g multichannel pipettes). The liquid level was correct in the tips, as air bubbles, etc. may alter volume. Quality control and safety procedures were as described in Example 5.

[0180] Antimicrobial paper was cut into appropriately sized strips from both the antimicrobial and control paper. For a 5 mL assay in a 15 mL tube, standard sizes included 5 x 10 mm, 5 x 20 mm, and 5 x 40 mm. These strips could be combined to provide a desired step series. [0181] Analysis of samples included determining activity by monitoring the clearing of the cell suspension at OD570 and determining the best fit to a standard curve. For a 5 mL assay, M. lysodeikticus was added in reaction buffer to an OD600 of 0.5. The reaction was started with the addition of the stripes. The tubes were immediately placed at 28°C for a designated time {e.g., 4 hr and 24 hr). The absorbance at 570 nm was recorded. [0182] Analysis of the data included calculating the initial velocities for the recorded slopes:

[OD6oomin]/[slope standard curve (OD/mg M. lysodeikticus]/[\ysozyme]

[0183] Example 10: This Example demonstrates a biological assay for antimicrobial activity of paper strips comprising an antimicrobial enzyme additive against a microorganism. The antimicrobial enzyme additive comprised lysozyme, the microorganism used was vegetative, gram-positive M. lysodeikticus. The assay was adapted from ASTM 02020-92, Method A, Standard Test for Mildew (Fungus) Resistance of Paper and Paperboard (Reapproved 2003). Equipment and reagents that were used are shown in the table below.

[0184] Micrococcus lysodeikticus cell suspension was made by adding 9 mg Micrococcus lysodeikticus to NBY and mixing well, with OD6oo about 0.5. Antimicrobial paper coated with a latex coating comprising lysozyme and control paper was prepared in accordance with Example 7.

[0185] The assay includes cutting appropriated sized strips of both antimicrobial and control papers ( e.g ., a. 10 x 10 mm, 20 x 20 mm, 40 x 40 mm, or 50 x 50 mm). 100 pL of the prepared M. lysodeikticus suspension was transferred to 15 mL tube containing 5 ml_ NBY Soft Agar, held molten at 55°C, and mixed well. Pipet tips used fitted the pipette {e.g., multichannel pipettes). The liquid level was correct in the tips, as air bubbles, etc. may alter volume. The mixture was immediately poured over a prepared sterile agar plate, rotating the dish to completely cover the agar with the M. lysodeikticus overlay. The dish was covered and allowed to solidify on level surface. The prepared antimicrobial paper(s) were placed (face down) on the soft agar overlay. Coupon(s) up to 20 x 20 mm were able to be paired with a control on a single petri dish. The dishes were left at 28°C overnight, and visually evaluated for a zone of clearance around the antimicrobial coupon(s) relative to the control. Quality control and safety procedures were as described in Example 5.

[0186] Example 11 : This Example demonstrates a biological assay for the antimicrobial activity of a paper strip comprising ProteCoat® against fungal spores. The assay was adapted from ASTM 02020-92, Method A, Standard Test for Mildew (Fungus) Resistance of Paper and Paperboard (Reapproved 2003). Equipment and reagents that were used are shown in the table below. [0187] Fusarium oxysporium spores were prepared by maintaining cultures of Fusarium oxysporum f. sp. lycoperici race 1 (RM-1)[FOLRM-1 on Potato Dextrose Agar (PDA) slants.

Microconidia of the Fusarium oxysporum f. sp. lycoperici, were obtained by isolating a small portion of an actively growing culture from a PDA plate and transferring to 50 ml a mineral salts medium FLC (Esposito and Fletcher, 1961). The culture was incubated with shaking (125 rpm) at 25°C. After 960 h the fungal slurry consisting of mycelia and microconidia were strained twice through eight layers of sterile cheese cloth to obtain a microconidial suspension. The microcondial suspension was then calibrated with a hemacytometer. All fungal inocula were tested for the absence of contaminating bacteria before their use in experiments. Antimicrobial paper coated with a latex coating comprising ProteCoat® and control paper was prepared in accordance with Example 7.

[0188] The assay procedure included: cutting appropriated sized strips of both antimicrobial and control papers ( e.g 40 x 40 mm or 50 x 50 mm); centering the strips on a sterile Potato Dextrose Agar plate, treated side up; diluting spores to 2 x 10 3 per ml_ Potato Dextrose broth; transferring to a calibrated preval sprayer (i.e., dispense 50 pL per single pump action); dispersing spores in a hood onto the agar and paper surface with a single pump action (delivers approximately 100 spores to the area); covering and leaving at ambient conditions; and observing growth over several days, though time of assay will depend on organism. Pipet tips fitted the pipette ( e.g multichannel pipettes). The liquid level was correct in the tips, as air bubbles, etc. may alter volume. Quality control and safety procedures were as described in Example 5.

[0189] Example 12: This Example demonstrates a paper coating comprising an antimicrobial enzyme additive. The antimicrobial enzyme comprised a lysozyme. Assay standardization and data are shown in the following tables.

[0190] The rate of lysis upon contact with a coupon cut from antimicrobial treated paper, is approximately 0.5% (1 .35 x 10 7 cells) per pg lysozyme. This corresponds to a reduction in activity, per pg of lysozyme, of approximately 65% over that observed in suspension. Treated papers of identical size with antimicrobial loadings of 0.2%, 1 .0% and 2.0%, demonstrated antimicrobial function. The antimicrobial concentration on a per unit of area for those loadings, is provided in the following table.

[0191] Example 13: This Example qualitatively demonstrates an antimicrobial enzyme additive combined with an antimicrobial peptide additive to provide antimicrobial functionality to a paper coating formulation. An adaptation of ASTM 02020-92 was used as the assay to demonstrate the growth of a microorganism in a petri dish was inhibited by contact with the treated paper. The antimicrobial enzyme additive comprised lysozyme, and the antimicrobial peptide additive comprised ProteCoat ® Reactive Surfaces, Ltd.; also described in U.S. Patent application nos. 10/884,355; 11/368,086; and 11/865,514, each incorporated by reference).

[0192] The spectrophotometric lysozyme assay uses Micrococcus lysodeikticus bacterial cells as a substrate, and measures the change in the turbidity of the cell suspension as described in Example 8 and Example 9. The efficacy of an antimicrobial peptide ( e.g ProteCoat™) may be monitored biologically. Though the contemplated mechanism of action for an antimicrobial or antifouling peptide is similar, i.e. disruption of the structural components of the microbial cell, the cell wall may remain relatively intact. As an antifungal or antimicrobial peptide's biocidal or biostatic activity inhibits the cell, the cell may not lyse for detection of a change in turbidity. Biological assay conditions are shown in the table below. [0193] A zone of clearing was seen around the antimicrobial paper in contact with a petri dish covered by M. lysodeikticus, whereas the control paper had no such zone. The coupon of paper was about half the size of the smallest coupons in the quantitative M. lysodeikticus assay, yet growth inhibition was seen. Assay conditions for Fusarium oxysporum is shown at the table below.

[0194] Overgrowth of both test and control ProteCoat ® paper by the fungus, Fusarium oxysporium, was observed. The developmental state of the mycelium on the antimicrobial paper was retarded over that seen in the control paper, indicative of biostatic, and possibly biocide activity.

[0195] Example 14: This Example demonstrates synergism between an antimicrobial enzyme additive combined with an antimicrobial peptide additive in a coating applied to papers, and to demonstrate antimicrobial activity of a paper comprising the antimicrobial peptide. The antimicrobial enzyme additive comprised lysozyme, and the antimicrobial peptide additive comprised ProteCoat ® (Reactive Surfaces, Ltd.; also described in U.S. Patent application nos. 10/884,355; 11/368,086; and 11/865,514, each incorporated by reference). Assay conditions are shown at the tables below.

[0196] The concentration of lysozyme in the papers corresponded to between 2 and 50 ppm, whereas ProteCoat® was between 0.5 and 12 ppm. The comparison of lysis between the 2% lysozyme paper, and the combined paper which contained 2% lysozyme and 0.5% ProteCoat® indicates synergism between the additives. For example, the 100 mm 2 coupon size exhibited 44% lysis, whereas the combined paper exhibited 93%. This is an observed/expected (93/44+0) of 2.1 , indicative of significant synergism. To further demonstrate this activity, the assay was repeated by titrating the 2% lysozyme paper with individual swaths of 2.5% ProteCoat® paper. 5 x 10, 5 x 20, and 5 x 40mm 2 lysozyme paper strips with increasing amount of Protecoat ® paper were added to tubes in 4m I total volume 2.5 x 10 8 Micrococcus cells/ml. The assay conditions are shown at the tables below.

[0197] An example of a calculation for the lysozyme content in 2% lysozyme paper was: 23.2 x 2% g/m 2 = 0.464 g/m 2 = 0.464 pg/mm 2 . An example of a calculation for the Protecoat ® content in 2.5% Protecoat ® paper was: 23.9 x 2.5% g/m 2 = 0.60 g/m 2 = 0.60 pg/mm 2 .

[0198] The assay was repeated by titrating the 2% lysozyme paper with individual swaths of 2.5% ProteCoat® paper. Lysozyme in technical papers added to an assay at concentrations greater than 10 ppm exhibited antimicrobial activity in the M. lysodeikticus assay. Lysozyme at approximately 5 ppm in the assay did not exhibit significant antimicrobial activity over the course of the assay (20 hrs). The addition of ProteCoat® papers, with between 3 and 60 ppm ProteCoat® to the assay significantly enhanced the lytic activity of lysozyme, or possibly the reverse. This was also true with the 5-ppm lysozyme, in which the lytic activity was doubled by the addition of between 3 and 60 ppm ProteCoat ® to the assay. The peptide additive may be enhancing the activity of the enzyme, or the enzyme enhancing the activity of the peptide, or both, to produce these results.

[0199] Example 15: This Example demonstrates a spectrophotometric assay for an antimicrobial coating with a lytic additive. The lytic additive comprised a lysozyme. The antimicrobial coatings were created using acrylic latex, commercially available paints. Equipment and reagents that were used are shown in the table below.

[0200] A Micrococcus lysodeikticus cell suspension was made by adding 1.5 mg Micrococcus lysodeikticus to 1 ml_ 10mM Tris pH 8.0 and mixing well. A lysozyme solution was prepared by adding 10 mg lysozyme in 1 ml_ ddH20, and mixing well. [0201] The lysozyme stock solution was mixed into Sherwin Williams Acrylic (SW) or

Glidden latex paint (1-part water : 7-part paint). 4 mil, 6 mil, and 8 mil free films were created from Sherwin Williams paint comprising a lysozyme, a Glidden paint comprising a lysozyme, and controls for both. The plate controls included 3 replicates of 50 pL M. lysodeikticus cell suspension plus 50 pl_ buffer; and 3 replicates of 100 mI_ buffer. Pipet tips used fitted the pipette ( e.g multichannel pipettes). The liquid level was correct in the tips, as air bubbles, etc. may alter volume. Quality control and safety procedures were as described in Example 5.

[0202] The antimicrobial films were cut into appropriately sized strips from both the antimicrobial and control coating. For a 5 ml_ assay in a 15 mL tube, standard size was 1 x 1 cm. [0203] Analysis of samples included determining activity by monitoring the clearing of the cell suspension at OD405 and determining the best fit to a standard curve. The reaction was started with the addition of 5 ml of the M. lysodeikticus stock. The tubes were immediately placed on a rocker for 3 hr; 100 pi samples were taken at 3 hr, and the absorbance at 405 nm was recorded.

[0204] Analysis of the data included calculating the initial velocities for the recorded slopes: [OD405 min]/[slope standard curve (OD/mg M. lysodeikticus]/[ lysozyme], [0205] Example 16: This Example demonstrates a biological assay for antimicrobial activity of coatings comprising an antimicrobial enzyme additive against a microorganism. The antimicrobial enzyme additive comprised lysozyme, the microorganism used comprised vegetative, gram-positive M. lysodeikticus. The assay was adapted from ASTM 02020-92, Method A, Standard Test for Mildew (Fungus) Resistance of Paper and Paperboard (Reapproved 2003). Equipment and reagents that were used are shown in the table below. [0206] A Micrococcus lysodeikticus cell suspension was made by adding 1.5 mg M. lysodeikticus to 10 mM Tris, pH 8.0, and mixing well. A lawn of M. lysodeikticus was generated by spreading 200 mI of this suspension onto an LBA plate, using a glass spreading rod. An antimicrobial latex coating comprising lysozyme and a control film was prepared in accordance with Example 15. [0207] The assay includes cutting appropriated sized strips of both antimicrobial and control latex films ( e.g a 1 x 1 cm). In triplicate the free films are carefully placed onto the surface of the petri dishes spaced out equally. This procedure was repeated for each of the paint film types/thicknesses.

[0208] The paint films comprising a lysozyme were active in lysing M. lysodeikticus, producing circular zones of clearing. The difference in Zone of Clearing Diameter between the different thicknesses of film was deemed negligible.

[0209] Example 17: This Example demonstrates a qualitative biological assay for survivability of an antimicrobial latex coating comprising an antimicrobial enzyme additive against a microorganism. The antimicrobial enzyme additive comprised lysozyme, the microorganism used comprised vegetative, gram-positive M. lysodeikticus. The assay was adapted from ASTM 02020-92, Method A, Standard Test for Mildew (Fungus) Resistance of Paper and Paperboard (Reapproved 2003). Equipment and reagents that were used are shown in the table below.

[0210] A Micrococcus lysodeikticus cell suspension was made by adding 1.5 mg M. lysodeikticus to 10 mM Tris, pH 8.0, and mixing well. A lawn of M. lysodeikticus was generated by spreading 200 pi of this suspension onto an LBA plate, using a glass spreading rod.

[0211] The paint formulations that were prepared included a Sherwin-Williams Acrylic Latex or a Glidden Acrylic Latex as controls (no additive), and both a Sherwin-Williams Acrylic Latex or a Glidden Acrylic Latex comprising 10 mg/mL Lysozyme (ddH20). Each paint was made by adding 1-part additive to 7 parts paint, and then mixed with a glass stirring rod and a paint mixer. Each film was immediately drawn onto polypropylene surfaces with a thickness of 4 mil, 6 mil, and 8 mil. Cure time was 24 days. Materials for assay were generated from the polypropylene surface as 1 cm 2 free films.

[0212] The assay includes cutting appropriately sized strips of both antimicrobial and control latex films ( e.g a 1 x 1 cm). In triplicate the free films were carefully placed onto the surface of the petri dishes spaced out equally. This procedure was repeated for each of the paint film types/thicknesses.

[0213] After 24 hrs incubation, the diameter of the zones of clearing was measured for each film. Using sterile tweezer, the films were removed and transfer to a new LBA plate spread with M. lysodeikticus in the same orientation as the plates the films were removed from. Repeat the procedure of measuring the zones of clearing through transfer to a new plate every day for 5 days.

1 N/A in this chart just means not available/ not applicable.

[0214] There were no 4 mil or 6 mil controls tested due to a limited LBA plate supply, though 8 mil control films were tested. The standard deviations for the 8 mil controls to 0, because all 3 controls produced a 0cm zone of clearing in each case.

[0215] The paint films comprising lysozyme were active in lysing M. lysodeikticus, producing circular zones of clearing, for five cycles of contaminant control. The difference in Zone of Clearing Diameter between the different thicknesses of each film appeared negligible.

[0216] Example 18: This Example demonstrates a sulfatase's activity in free-films using a plate reader. Equipment and reagents used are shown in the table below.

[0217] Samples preparation procedure included preparing: 14.5mM potassium 4- nitrophenyl sulfate in isopropyl alcohol; and 200mM TRIS, adjusted to pH 7.1 with HCI. [0218] The paint formulations that were prepared included a Sherwin-Williams Acrylic Latex control (no additive), and a Sherwin-Williams Acrylic Latex comprising sulfatase. 63 enzyme units of sulfatase was admixed with 1-part water, then added to 7 parts paint.

Each paint was mixed with a glass stirring rod and a paint mixer. Each film was immediately drawn onto polypropylene surfaces with a thickness of 8 mil. Cure time was 24 hours. Materials for assay were generated from the polypropylene surface as 3 cm 2 free films.

[0219] The plate reader assay included: cutting free films into appropriate size pieces; adding 1350uL 200mM TRIS into each microtube; adding 150uL of 14.5mM potassium 4- nitrophenyl sulfate to each tube; taking the 0-time sample; then adding the free films to the tubes, with the control sample being free film with no sulfatase. Quality control and safety procedures were as described in Example 5, including use of a hood for material handling as appropriate. [0220] Analysis included: taking 10Oul at the appropriate time points from each microtube and reading the absorbance at 405nm; and determining the initial rate slope by plotting absorbance vs. time to calculate sulfatase activity.

[0221] Example 19: This Example demonstrates a phosphodiesterase I assay using a plate reader. The equipment and reagents used are shown in the table below.

[0222] Samples prepared included: 14.5mM Thymidine 5-monophosphate p-nitrophenyl ester sodium salt in ddhteO; a 124U/ml ddhteO enzyme solution; and 200mM TRIS (adjusted to pH 7.1 with HCI). [0223] The plate reader assay comprised: diluting enzyme solution 1:1 and 1 :3; adding

16ul of each enzyme dilution in triplicate into a 96-well plate, with a control sample prepared by adding 16ul ddH20; adding 24ul ddH20 into each well; adding 50ul 200mM TRIS to each well; and adding 10ul_ of 14.5mM Thymidine 5-monophosphate p-nitrophenyl ester sodium salt in ddH20 to each well. Quality control and safety procedures were as described in Example 5, including use of a hood for material handling as appropriate.

[0224] The analysis included: taking 500 readings every 10 seconds at 405nm; and determining the initial rate slope by plotting absorbance vs. time to calculate phosphodiesterase I activity. Summary results are below.

[0225] Example 20: This Example demonstrates a phosphodiesterase I activity assay in free-films using a plate reader.

[0226] Samples prepared included: 14.5mM Thymidine 5-monophosphate p-nitrophenyl ester sodium salt in ddh O; and 200mM TRIS (adjusted to pH 7.1 with HCI).

[0227] The paint formulations that were prepared included a Sherwin-Williams Acrylic Latex control (no additive), and a Sherwin-Williams Acrylic Latex comprising phosphodiesterase I. 113 enzyme units of phosphodiesterase I was admixed with 1 -part water, then added to 7 parts paint. Each paint was mixed with a glass stirring rod and a paint mixer. Each film was immediately drawn onto polypropylene surfaces with a thickness of 8 mil. Cure time was 24 hours. Materials for assay were generated from the polypropylene surface as 1 cm 2 , 2 cm 2 and 3 cm 2 free films.

[0228] The plate reader assay comprised: cutting free films into appropriate sized pieces and place them into microtubes, though blank samples have no paint film inside the microtube; adding 600ul ddhteO into each microtube; adding 750ul 200mM TRIS into each microtube; and adding 150ul_ of 14.5mM Thymidine 5-monophosphate p-nitrophenyl ester sodium salt in ddhteO into each microtube. Quality control and safety procedures were as described in Example 5, including use of a hood for material handling as appropriate [0229] Analysis included: taking out 10Oul from each microtube at the appropriate time points, and reading the absorbance at 405nm; and determining the initial rate slope by plotting absorbance vs. time to calculate phosphodiesterase I activity.

[0230] Example 21: This Example describes identification and isolation of additional proteinaceous sequence(s) that may be used, such as a sequence possessing an antibiological activity. [0231] Although a synthetically obtained peptidic agent (/. e. , a peptide, polypeptide, a protein, an antifungal peptide) identified and produced as described herein (e.g., SEQ ID Nos. 1 to 47) may be used, it is also possible to employ suitable naturally produced peptidic agent (e.g., a microbe that produces a peptidic agent), as a component of a material formulation (e.g., an additive in a paint, a coating additive). A proteinaceous molecule, such as one possessing an antibiological activity, may be identified using an assay as described herein and/or the art. A number of such naturally occurring peptides are listed in the Table below, with reference citations often including activity assay(s) used in identification.

[0232] A natural source may provide additional sequences to be used for a material formulation (e.g., a coating additive). In some embodiments, the use of a natural antifungal products isolated in commercial quantity from a microorganism may use a large-scale cell culture (e.g., culture of an antifungal agent-producing microorganism) for the production and purification of the peptidic (e.g., an antifungal) product. In some aspects, the cultural isolate responsible for the production of the endogenously produced proteinaceous molecule (e.g., an antifungal peptidic agent) may be batch-cultured. In some facets, a purification technique and/or strategy, such as those described herein and/or in the art, may be used purify the active product to a reasonable (e.g., desired) level of homogeneity. However, in some aspects, a naturally derived peptidic agent (e.g., an antifungal agent) may co-purify with an unwanted microbial byproduct, especially a byproduct which may be undesirably toxic. Purification of an endogenously produced proteinaceous composition may result in a racemized mixture wherein one or more stereoisomer(s) are active, and/or wherein a disulfide linkage may occur (e.g., a disulfide linkage between peptide monomers). When a desirable naturally occurring proteinaceous molecule (e.g., an antifungal protein, an antifungal polypeptide, an antifungal peptide) may be isolated, for example, and the amino acid sequences at least partially identified, synthesis of the native molecule, or portions thereof, may use a specific disulfide bond formation, a high histidine requirement, and so forth. Of course, once a proteinaceous molecule is sequence is identified, and/or a nucleotide sequence for a proteinaceous molecule is isolated, it then may be recombinantly expressed using techniques described herein and/or in the art.

[0233] Example 22: This Example describes assay protocols for evaluating antifungal coatings. It is contemplated that such assays may be adapted to also assay other types of material formulations comprising various biomolecule composition(s) and activity against other types of biological cells.

[0234] A suitable assay protocol for evaluating a coating comprising an antifungal agent which may be applied in assaying an antifungal peptide is described by the American Society for Testing and Materials (ASTM) in D-5590-94 ("Standard Test Method for Determining the Resistance of Paint Films and Related Coatings to Fungal Defacement by Accelerated Four-Week Agar Plate Assay"). The assay method may be modified as indicated below, and generally comprises: preparing a set of four 1 x 10 cm aluminum coupons approximately 1/32 in thick will be prepared as follows: (1 ) blank Al coupon; (2) Al coupon coated with an aqueous solution of a peptide produced and identified as described herein, and allowed to dry; (3) Al coupon coated on both sides with a base paint composition, allowed to dry, and then the paint film will be coated with a like amount of the same test peptide solution as applied to coupon 2; and (4) Al coupon painted with a paint mixture comprising the same base paint composition as for coupon 3 and a like amount of the peptide, as for coupons 2 and 3. Duplicate or triplicate sets of these specimens may be prepared. Optionally, a conventional biocide may be included as a positive control. The base paint composition may be any suitable water-based latex paint, without biocides, which is available from a number of commercial suppliers.

[0235] Each of the specimens from (a) will be placed on a bed of nutrient agar and uniformly inoculated with a fungal suspension. An example test organism comprises a Fusarium oxysporum. The fungal suspension may be applied by atomizer or by pipet, however a thin layer of nutrient agar mixed with the fungal inoculum may be used. The specimens are incubated at about 28°C under 85 to 90% relative humidity for 4 weeks. Fungal growth on each specimen is often rated weekly as follows: None = 0; traces of growth (<10% coverage) = 1; light growth (10-30%) = 2; moderate growth (30-60%) = 3; and heavy growth (60% to complete coverage) = 4.

[0236] Another suitable assay protocol for testing the antifungal properties of a coating or paint film containing an antifungal peptide is described by the ASTM in D-5590-94 ("Standard Test Method for Resistance to Growth of Mold on the Surface of Interior Coatings in an Environmental Chamber"). The testing protocol generally includes:

[0237] Preparation of the Coated Surface. Duplicate or triplicate sets of approximately 1/2 in. thick, 3 x 4 in. untreated wooden or gypsum board panels will be prepared as follows:

(1) blank panel; (2) coated with an aqueous solution of a peptide produced and identified as described herein, and allowed to dry; (3) coated on both sides with a base paint composition, allowed to dry, and then the paint film is coated with a like amount of the same test peptide solution as applied to panel 2; and (4) painted with a paint mixture containing the same base paint composition as for panel 3 and a like amount of the peptide, as for panels 2 and 3. Optionally, a conventional biocide may be included as a positive control.

[0238] Contamination: The panels will be randomly arranged and suspended in an environmental cabinet above moist soil that has been inoculated with the desired fungus, usually a Fusarium oxysporum. Enough free space is provided to allow free circulation of air and avoiding contact between the panels and the walls of the cabinet. [0239] Incubation: The panels will be incubated for two weeks at 30.5 - 33.5°C and 95 - 98% humidity.

[0240] Scoring: A set of panels (test, control, and, optionally, a positive control) will be removed for analysis at intervals, usually weekly. The mold growth on the specimen panels may be rated as described above.

[0241] Alternatively, one or more equivalent testing protocols may be employed, and field assays of coating compositions containing laboratory-identified antifungal peptide(s) and/or candidate peptide(s) may be carried out in accordance with conventional methods of the art. [0242] Example 23: This Example describes assay protocols for evaluating a latex paint comprising an antifungal peptidic agent. It is contemplated that such assays may be adapted to also assay other types of material formulations comprising various biomolecule composition(s) and activity against other types of biological cells.

[0243] Both the interior latex (Olympic Premium, flat, ultra white, 72001) and acrylic paints (Sherwin Williams DTM, primer/finish, white, B66W1 ; 136-1500) appeared to be toxic to both Fusarium and Aspergillus. Therefore, eight individual wells (48-well microtiter plate) of each paint type were extracted on a daily basis with 1 ml of phosphate buffer for 5 days (1-4 & 6) and then allowed the plates were allowed to dry before running the assay. Each well contained 16 ul of respective paint. [0244] Extract testing: The extract from two wells each of the two paints for each day was tested for toxicity by mixing the extract 1:1 with 2X medium and inoculating with spores (10 4 ) of Aspergillus or Fusarium. The extracts had no effect on growth of either test fungus. [0245] Well testing: The extracted and non-extracted wells for each of the paints were tested with a range of inoculum levels in growth medium using the two different fungi. For Fusarium the range was 10 1 -10 4 and for Aspergillus 10 2 -10 5

[0246] Well Testing of Acrylic Paint Plates: Both Fusarium and Aspergillus grew in all extracted wells at all inoculum levels. Only Aspergillus grew in non-extracted wells at the 10 5 level and not at lower levels indicative of an inherent biocidal capability.

[0247] Well Testing of Latex Paint Plates: Fusarium grew in the extracted wells only at the 10 4 inoculum level but not at 10 1 - 10 3 . Aspergillus grew in all extracted wells showing an inoculum level effect. No growth was observed for either Fusarium or Aspergillus in non- extracted wells. [0248] Conclusion: Extraction of the toxic factor(s) found in both paints was possible. However, it appeared that it may be less extractable from the latex paint.

[0249] Evaluation of peptide activity in presence of acrylic and latex paints: It was established that it was possible to extract both acrylic and latex paints dried in a 48-well format to make them non-toxic to the test microorganisms - Fusarium and Aspergillus.

Using that information an experiment was designed to determine the effect the paint has on peptide activity against two test organisms.

[0250] Experimental design: Coat 48-well plastic plates with 16pl of acrylic or latex paint. Dry for two days under hood. Extract designated wells with 1 -ml phosphate buffer changing the buffer on a daily basis for 7days. Control wells were not extracted to confirm paint toxicity. Add 20mI of peptide series in duplicate to designated dry paint coated wells. Peptide, SEQ ID No. 41 , series were added in a two-fold dilution series to wells and allowed to dry. The concentration of peptide added ranged from 200pg/20pl to 1 5pg/20pl. [0251] Inoculated paint-coated plates as follows: Extracted control wells received 180mI of medium + 20mI of spore suspension (10 4 spores/20pl of medium). Inoculum was either Fusarium or Aspergillus in each case. Non-extracted control wells received 180mI of medium + 20mI of spore suspension (10 4 spores/20pl of medium). Extract wells with dried peptide series received 180mI of medium + 20mI of spore suspension (10 4 spores/20pl of medium). In duplicate. Extract wells that did not have dried peptide series received 160mI of medium + 20mI of spore suspension (10 4 /20mI of medium) + 20mI peptide series as above. In duplicate. Plates were observed for growth over a 5-day period.

[0252] Growth and peptide controls: Use sterile non-paint coated 48 well plastic plates. Growth control wells for each test fungus received 180mI of medium + 20mI of spore suspension (10 4 spores/20pl of medium). Peptide activity controls received 160mI of medium + 20mI of spore suspension (10 4 spores/20pl of medium) + 20mI peptide series as above. Peptide series were added in a two-fold dilution series to wells and range from 200pg/20pl to 1 5pg/20pl. Therefore, the range of peptide tested was 200pg/200pl or 1.Opg/mI (1000pg/ml) to 0.0075pg/pl (7.5pg/ml). Uninoculated medium served as blank for absorbance readings taken at 24, 48, 72, 96 and 120h. [0253] Results: Unextracted wells containing either latex or acrylic paint inhibited growth of both Fusarium and Aspergillus. Extracted wells containing either latex or acrylic paint allowed growth of both Fusarium and Aspergillus. The calculated MIC for Fusarium in peptide activity control experiments was 15.62 pg/ml. For Aspergillus the calculated MIC was 61.4 pg/ml.

[0254] For extracted acrylic-coated plates the following results were obtained. Controls as stated in above. For Fusarium with dried peptide, inhibition was seen at 1000 and 500 pg/ml after 5 days. Spores exposed to liquid peptide added to dry paint wells were inhibited at 1000, 500 and 250 pg/ml after 4 days, and 1000 and 500 pg/ml after 5 days. For Aspergillus with dried peptide, inhibition was seen at 1000 pg/ml after 5 days. Spores exposed to liquid peptide added to dry paint wells were inhibited at 1000 and 500pg/ml after 5 days. [0255] For extracted latex-coated plates the following results were obtained. Controls as stated above. For Fusarium with dried peptide, inhibition was seen at 1000 pg/ml after 5 days. Spores exposed to liquid peptide added to dry paint wells were inhibited at 1000 pg/ml after 5 days. For Aspergillus with dried peptide, inhibition was seen at 1000 pg/ml after 5 days. Spores exposed to liquid peptide added to dry paint wells were inhibited at 1000 pg/ml after 5 days.

[0256] Example 24: This Example describes combinations of an antibiological proteinaceous composition and an antibiological chemical such as a standard preservative. [0257] A material formulation (e.g., a paint composition) comprising one or more conventional antibiological chemical(s) (e.g., a preservative, an antimicrobial agent, an antifungal substance) may be modified by addition of one or more of the antibiological proteinaceous composition(s) (e.g., an antifungal peptide) described herein. For example, combining a non-peptidic antibiological agent (e.g., antifungal agent) with one or more antibiological proteinaceous molecule(s) (e.g., an antifungal peptide) may provide antifungal activity over and above that seen with either the proteinaceous or the non- peptidic agent alone. The expected additive inhibitory activity of the combination is calculated by summing the inhibition levels of each component alone. The combination is then assayed on the assay organism to derive an observed additive inhibition. If the observed additive inhibition is greater than that of the expected additive inhibition, synergy is exhibited. For example, a synergistic combination of a proteinaceous molecule (e.g., an aliquot of a peptide library, a peptide) comprising at least one antibiological proteinaceous molecule (e.g., an antifungal peptide) occurs when two or more cell (e.g., fungal cell) growth-inhibitory substances distinct from the proteinaceous molecule are observed to be more inhibitory to the growth of an assay organism than the sum of the inhibitory activities of the individual components alone.

[0258] An example of an assay method for determining additive or synergistic combinations comprises first creating a synthetic peptide combinatorial library. Each aliquot of the library represents an equimolar mixture of peptides in which at least the two C-terminal amino acid residues are known. Using the testing methods described in one or more of U.S. Patent No. 6,020,312, U.S. Patent No. 5,885,782, and U.S. Patent No. 5,602,097 it is possible to determine for each such aliquot of the synthetic peptide combinatorial library, a precisely calculated concentration at which it will inhibit an assayed fungus in a coating. Next, the aliquot of the synthetic peptide combinatorial library is mixed with at least one non-peptide antifungal compound to create an assay mixture. As with the peptide component of the mixture, the baseline ability of the non-peptide antifungal substance to inhibit the test fungus is determined initially. Next, the assay fungus is contacted with the assay mixture, and the inhibition of growth of the assay organism is measured as compared to at least one untreated control. More controls are desirable, such as a control for each individual component of the mixture. Similarly, where there are more than two components being tested, the number of controls to be used must be increased in a manner in the art of growth inhibition assays. From the separate assay results for the peptidic and the non- peptidic agent(s) the expected additive effect on inhibition of growth is determined using standard techniques. After the growth inhibition assay(s) are complete for the combination of peptidic and the non-peptidic agent(s), the actual or observed effect on the inhibition of growth is determined. The expected additive effect and the observed effect are then compared to determine whether a synergistic inhibition of growth of the test fungus has occurred. The methods used to detect synergy may utilize non-peptide antimicrobial agents in combination with the inhibitory peptides described herein.

[0259] Example 25: This Example describes coating a surface to inhibit fungus infestation and growth.

[0260] When anchorage, food and moisture are available, a cell such as a microorganism (e.g., a fungus) are able to survive where temperatures permit. Susceptible surfaces may include a porous material such as a stone, a brick, a wall board (e.g., a sheetrock) and/or a ceiling tile; a semi-porous material, including a concrete, an unglazed tile, a stucco, a grout, a painted surface, a roofing tile, a shingle, a painted and/or a treated wood and/or a textile; or a combination thereof. Any type of indoor object, outdoor object, structure and/or material that may be capable of providing anchorage, food and moisture to fungal cells is potentially vulnerable to infestation with mold, mildew or other fungus. Moisture generally appears due to condensation on surfaces that are at or below the dew point for a given relative humidity.

[0261] To inhibit or prevent fungus infestation and growth, one or more antifungal peptidic agents described herein (e.g., approximately 250 - 1000 mg/L of the hexapeptide of SEQ ID No. 41 ), may be dissolved or suspended in water and applied by simply brushing and/or spraying the solution onto a pre-painted surface such as an exterior wall that may be susceptible to mold infestation. Conventional techniques for applying or transferring a coating material to a surface in the art are suitable for applying the antifungal peptide composition. The selected peptide(s) have activity for inhibiting or preventing the growth of one or more target fungi. The applied peptide solution is then dried on the painted surface, preferably by allowing it to dry under ambient conditions. If desired, drying can be facilitated with a stream of warm, dry air. Optionally, the application procedure may be repeated one or more times to increase the amount of antifungal peptide that is deposited per unit area of the surface. As a result of the treatment, when the treated surface is subsequently subjected to the target mold organisms or spores and growth promoting conditions comprising humidity above about typical indoor ambient humidity, presence of nutrients, and temperature above about typical indoor ambient temperature and not exceeding about 38°C, the ability of the surface to resistance fungal infestation and growth is enhanced compared to its pre-painted condition before application of the antifungal peptide.

[0262] A simple spray-coated surface may provide sufficient durability for certain applications such as surfaces that are exposed to weathering, though longer-term protection may be provided against adhesion and growth of mold by mixing one or more of the antifungal peptides with a base paint or other coating composition, which may be any suitable, commercially available product in the art. The base composition may be free of chemicals and other additives that are toxic to humans or animals, and/or that fail to comply with applicable environmental safety rules or guidelines. The typical components, additives and properties of conventional paints and coating materials, and film-forming techniques, of the art, described herein, and/or described in U.S. Patent Application No. 10/655,345 filed September 4, 2003, U.S. Patent Application No. 10/792,516 filed March 3, 2004, and U.S. Patent Application No. 10/884,355 filed July 2, 2004, may be used.

[0263] If additional, long-term protection against growth and adhesion of a mold, a mildew and/or a fungus is desired, the paint or other coating composition may include a barrier material that resists moisture penetration and also prevents or deters penetration and adhesion of the microorganisms and the airborne contaminants which serve as food for the growing organisms. Some typical water repellent components are an acrylic, a siliconate, a metal-stearate, a silane, a siloxane and/or a paraffinic wax. The user will preferably take additional steps to deter mold infestation include avoiding moisture from water damage, excessive humidity, water leaks, condensation, water infiltration and flooding, and taking reasonable steps to avoid buildup of organic matter on the treated surface.

[0264] Example 26: This Example describes a method of treating a fungus-infested surface.

[0265] In situations where existing fungal growth is present, the mold colonies and/or spores may be removed and/or substantially eliminated before application of one of an antifungal coating, it is expected that in some situations an antifungal composition may be applied to existing mold infected surfaces. In this case, the composition, comprising one or more antifungal peptides, may inhibit, arrest the growth of, or substantially eradicate the mold. Early detection and treatment are highly preferred in order to minimize the associated discoloration or other deterioration of the underlying surface due to mold growth. The treatment procedure may comprise applying one or more coats of an antifungal peptide solution and/or a coating composition (e.g., a paint) as described herein. [0266] Example 27: This Example describes an impregnating a porous substrate to inhibit cell (e.g., fungus) growth.

[0267] A porous and/or a semi-porous object and/or a material, such as a paper, wood, a fabric, a carpet, some types of stone, and many other items that are employed indoors and/or outdoors, have internal surface areas that can be susceptible to infestation e.g., mold infestation) and are very difficult to treat effectively by conventional methods. A surface treatment (e.g., a coating material, a non-coating surface treatment, a liquid component comprising an antibiological agent) comprising one or more antibiological agents (e.g., an antifungal peptide, an enzyme) may be used to impregnate such an object, as described herein. The liquidity of the composition may be such that it is capable of penetrating into the pores of the object. In this way, an effective amount of the antibiological agent (e.g., an antifungal peptide) becomes deposited on the internal surfaces as well as the exterior ones. Circumstances requiring treatment of a porous surface may benefit from using a relatively thin material (e.g., a thin surface treatment, a thin coating material, a liquid component comprising an antibiological agent) rather than a thick, pigmented paint, in order to facilitate penetration of the pores.

[0268] Example 28: This Example describes a coating a fruit or grain storage vessel to inhibit mold.

[0269] The interior wall(s) of a grain silo, a fruit storage, a grain storage, and/or a transportation tank may be coated with a peptidic antifungal material described herein (e.g., a peptidic antifungal composition, a coating comprising an antifungal peptide) to deter the attachment and growth of mold organisms inside the container. By selecting antifungal peptides that target specific organisms, and that may be non-toxic to a human and/or an animal, mold contamination of a wide variety of agricultural products may be deterred.

[0270] Example 29: This Example is directed to the assay for active phosphoric triester hydrolase expression in cells. Routine analysis of parathion hydrolysis in whole cells is accomplished by suspending cultures in 10 milli-Molar ("mM") Tris hydrochloride at pH 8.0 comprising 1.0 mM sodium EDTA ("TE buffer"). Cell-free extracts are assayed using sonicated extracts in 0.5 milliliters ("ml") of TE buffer. The suspended cells or cell extracts are incubated with 10 microliters ("pi") of substrate, specifically 100 pg of parathion in 10% methanol, and p-nitrophenol production is monitored at a wavelength of 400 nm. To induce the opd gene under lac control, 1.0 pmol of isopropyl-p-D-thiogalactopyranoside (Sigma) per ml is added to the culture media.

[0271] Example 30: This Example is directed to the preparation of an enzyme powder. In a typical preparation, a single colony of bacteria that expresses the opd gene is selected and cultured in a rich media. After growth to saturation, the cells are concentrated by centrifugation at 7000 rotations per minute ("rpm") for 10 minutes for example. The cell pellet is then resuspended in a volatile organic solvent such as acetone one or two times to desiccate the cells and to remove a substantial portion of the water contained in the cell pellet. The pellet may then be ground or milled to a powder form. The powder may be frozen or stored at ambient conditions for future use, or may be added immediately to a surface coating formulation. Additionally, the powder may be freeze dried, combined with a cryoprotectant ( e.g cryopreservative), or a combination thereof.

[0272] Example 31 : This Example is directed to the formation of an OPH powder and latex coating. In an example of use of the powder prepared as described in Example 9, 3 mg of the milled powder was added to 3 ml of 50% glycerol. The suspension was then added to 100 ml of Olympic ® premium interior flat latex paint (Olympic ® , One PPG Place, Pittsburg, PA 15272 USA). This paint with biomolecule composition was then used to demonstrate the activity of the paint biomolecule composition in hydrolysis of a pesticide or a nerve agent analog. [0273] Example 32: This Example demonstrates, in a first set of assays, a paint product as prepared in Example 31 was applied to a hard, metal surface. The surface used in the present Example was a non-galvanized steel surface that was cleaned through being degreased, and pretreated with a primer coat. A control surface was painted with the identical paint with no biomolecule composition. Paraoxon, an organophosphorus nerve gas analog was used as an indicator of enzyme activity. Paraoxon, which is colorless, is degraded to form p-nitrophenol, which is yellow in color, plus diethyl phosphate, thus giving a visual indication of enzyme activity. In multiple assays, the surface with control paint remained white, indicating no production of p-nitrophenol, and the surface painted with the paint and biomolecule composition turned yellow within minutes, indicating an active OPH enzyme in the paint. This demonstration has shown that the surface remains active for more than 65 days, which was the maximum duration of the protocol.

[0274] In a further demonstration, the surfaces were treated as described above and each surface was then treated with paraoxon, an OP insecticide. Approximately 100 flies were then placed on each surface under a plastic cover. In each procedure, within three hours, virtually all the flies on the control surface with no paint biomolecule composition were killed by the paraoxon. In contrast, approximately 5% of the flies on the enzyme comprising surface had died.

[0275] In a demonstration of enzyme stability in the paint, a series of wood dowels were dipped into the paint comprising OPH enzyme composition. The dowels were then placed in tubes containing paraoxon to indicate enzyme activity as described above. In each case, a positive yellow color was seen except in those dowels painted with no biomolecule composition as controls. The control solution remained clear in every case. [0276] To demonstrate the shelf life of both the dry biomolecule composition and the paint with biomolecule composition, the biomolecule composition was aged from 0 to 20 days prior to mixing in the paint. The mixed paint and biomolecule composition were then also aged from 0 to 20 prior to painting individual dowels. The enzyme composition retained strong activity after 20 days aging prior to being mixed in the paint, and for 20 days after mixing the maximum time used in the assay.

[0277] Example 33: This Example relates to a NATO demonstration of Soman detoxification using an OPH coated surface. At the September 22, 2002, meeting of the NATO Army Armaments Group in Cazaux, France, painted metal surfaces were assayed with soman using standard NATO procedures and protocols. For the assays, 10 cm x 10 cm metal plates primed with standard NATO specification paints were coated with paint containing OPFI. Control plates plus two different versions of the OPFI enzyme composition differing in soman detoxification specificity were used. These surfaces were allowed to dry for several hours at room temperature and then assayed according to standard NATO assay protocol (described below), modified to account for the character of the surfaces treated with a paint comprising OPFI.

[0278] The form of OPFI in the biomolecule composition contains both the changes of the previously described FI254R mutant and the FI257L mutant, and is corresponding designated the "FI254R, FI257L mutant." The FI254R, FI257L mutant demonstrates a several-fold enhanced rates of R VX catalysis relative to either the FI254R mutant or the FI257L mutant, and a 20-fold enhancement of activity relative to wild-type OPFI. This version of the OPFI biomolecule composition has been assayed in paints treated with soman or R-VX, and are described below.

[0279] Following standard protocols, OPD painted surfaces were uniformly contaminated with an isopropanol solution containing the chemical warfare agent soman. The concentration of soman on each contaminated surface was 1.0 mg/cm2. The contaminated plates were maintained at or slightly above room temperature (>20°C) without any forced air-flow for various periods of time. A zero-time, 15 minutes, 30 minutes, and 45 minutes sample was taken for each control and biomolecule composition-containing plate series.

To terminate the reaction and isolate residual soman on the plate surface, each plate was submerged in a container of isopropanol at the end-point and placed on a shaker to thoroughly extract any residual nerve agent. The solubilized portions were then quantified for soman. These assays showed that both the forms of OPH biomolecule composition were effective in detoxifying soman on metal surfaces. The two different OPH biomolecule compositions assayed detoxified the soman at levels over 65% and 77% after 45 minutes (NATO Army Armaments Group Project Group 31 on Non-Corrosive, Biotechnology-Based Decontaminants for CBW Agents, 2002). Additional assays with a CWA simulant indicated that had the NATO assay run for one to two hours, substantially all of the soman would have been detoxified.

[0280] Example 34: This Example relates to a demonstration of an OPH biomolecule composition at Aberdeen Proving Ground (SBCCOM) in Aberdeen, MD. In these assays, a primed wooden stick was coated with paint containing OPH biomolecule composition. The painted sticks used were 2 millimeters ("mm") in diameter x 15 mm in length. By estimating that the paint layer was 0.25 mm thick, the resulting surface area was approximately 125 mm2. After coating the stick with paint containing OPH biomolecule composition and allowing the paint to dry, the coated stick was inserted into a microfuge tube containing 100 ml of 3.24 mM Russian VX agent in saline and 900 ml phosphate buffer at pH 8.3. The tubes containing R-VX and the painted sticks were allowed to sit overnight in a hood at room temperature. Appropriate controls were run simultaneously.

[0281] The following morning, the contents of the microfuge tubes were assayed for free thiols by the Ellman method. 10 mM DTNB [molecular weight ("MW) 396.3] was prepared in 10 mM phosphate buffer at pH 8.0 for use as the indicator of enzyme activity. OPH paint’s cleavage of R VX releases a free thiol that reacts with DNTP to produce a colored product detectable spectrophotometrically at 405 nm. Ten ml of the microfuge tube contents, 100 ml DTNB solution and 890 ml phosphate buffer at pH 8.3 were read for thiol release at 405 nm using a Varian Carey 300 Spectrophotometer. The spectrophotometer was blanked with an unpainted stick control reaction. The molar equivalent of the R-VX hydrolyzed was determined using an extinction coefficient of 14,150 and the Beer-Lam bert equation to calculate the product concentration. Results indicated that overnight exposure to OPH paint coated sticks resulted in decontamination of Russian VX from 32.4 mM in the original tube to less than 1 mM.

[0282] Example 35: This Example demonstrates the use of a coating comprising a lipase, and the enzymatic activity conferred to the coating comprising the lipase by detection of triglyceride breakdown through monitoring pH. [0283] The equipment/reagents were as follows: pH meter; shaker; Lightin Lab Master paint mixer; phenol red (Sigma-Aldrich; Catalog # - P3532), 1.128mM in distilled water, pH = 7.0; lipase (Sigma-Aldrich; Catalog # - L3126), Sherwin Williams acrylic latex paint; sodium hydroxide; hydrochloric acid; isopropyl alcohol; and vegetable oil. The solutions used in measuring pH changes included a phenol red stock solution, 1.128 mM in distilled water, pH = 7.0.

[0284] The procedure for preparation of the surfaces coated with paint either comprising lipase or not (control paint) was as follows: first, 100 mg/ml, 50 mg/ml, and 0 mg/ml lipase solutions in paint were made; second, solutions were mixed for 3 minutes; third, paints were spread to 8 mils thickness and allowed to dry for 96 hours, and fourth, 1cm X 4cm coupons were cut from the paint film.

[0285] The pre-experimental set-up included the following steps: first, a 1cm X 4cm piece of film of each lipase concentration was placed in a 15ml Eppendorf tube in triplicate; second, 10 ml ddH20 was added inside the Eppendorf tube; third, tubes on shaker were set for 24 hours, and fourth, after 24 hours, the water from the tube was removed and the film placed in a new 15ml Eppendorf tube. For measuring the control paint (no lipase) samples, the following steps were conducted: first, 5 ml of phenol red stock solution was added into a 15 ml Eppendorf tube; second, 5 ml of phenol red stock solution with 100 pi vegetable oil was added into a 15 ml Eppendorf tube; third, a 1cm X 4cm piece of paint film (no lipase) from both the washed and non-washed films was added into a 15 ml Eppendorf tube in triplicate; fourth, 5 ml of the phenol red stock solution was added into the 15 ml Eppendorf tubes along with 100 mI vegetable oil; and fifth, the tubes were set on a shaker for 24 hours. To measure the paint samples comprising lipase: first, a 1cm X 4cm piece of the 50 mg/ml paint film, both washed and unwashed, was added into a 15 ml Eppendorf tube; second, a 1 cm X 4cm piece of the 100 mg/ml paint film, both washed and unwashed, was added into a 15 ml Eppendorf tube; third, 5 ml of the Phenol Red stock solution was added into each tube along with 100 mI vegetable oil; and fourth, the tubes were set on shaker for 24 hours. For both the control paint and lipase paint samples, the pH of each sample was recorded at 24 hours.

[0286] Phenol Red comprises a pH indicator that is yellow in color below pH 6.8 and red in color above pH 8.2. Setting the pH at 7.0 right before the 6.8 end point would demonstrate a color change if the solution becomes slightly more acidic. If in fact the triglycerides are being broken down into free fatty acids by lipase, the pH of the solution should go down, thus exhibiting a color change. In the presence of a paint film with no lipase, the pH of the phenol red solution rose from 7 to almost 9. The pH of the tubes with lipase in them were both substantially lower than the control tubes, demonstrating that the triglycerides were broken down into fatty acids, decreasing the pH of the solutions. All lipase impregnated coatings demonstrated catalytic activity. Washing the coating films with water decreased their effectiveness but the films were still active. Further, vegetable oil was spread over panels that were either control (no lipase) or lipase impregnated. After a day, the lipase impregnated panels were dry while the control panels were still visibly full of oil. It is also contemplated that greater loads of lipase, such as, for example, 200 mg/ml, 100 mg/ml, and 50 mg/ml lipase, may be used. [0287] Example 36: This Example demonstrates the use of a coating comprising a lipase, and the enzymatic activity conferred to the coating comprising the lipase by detection of the hydrolysis of 4-nitrophenyl palmitate through monitoring pH.

[0288] The equipment/reagents were as follows: 40 mM CHES Buffer; bring to pH = 9.0 with NaOH; 4-nitrophenyl palmitate (Sigma Product# N2752), 14.5 mM solution in isopropyl alcohol; 4-nitrophenyl acetate; lipase from porcine pancreas (Sigma Product # L3126); Sherwin-Williams acrylic latex paint; 2 ml_ microtubes; paint spreader (1-8 mils); polypropylene blocks; Lightnin Labmaster Mixer; rotator shaker; pipettes and pipetteman; and centrifuge.

[0289] The following paint formulations were evaluated: Sherwin-Williams Acrylic Latex Control (no additive), and Sherwin-Williams Acrylic Latex with 100 mg/mL lipase. The paints were mixed in a plastic 50ml Eppendorf tube with a glass stirring rod for three minutes followed by a paint mixer for three minutes. The paints were spread with a mils spreader to 8 mils thickness onto polypropylene surfaces and were allowed to dry a minimum of 72 hours prior to assay. Coupons were generated as free films from the polypropylene surfaces. [0290] The procedure for the preparation of the blank (control) samples was: adding 500ul 40mM CHES, 400ul ddhteO, and 10Oul 14.5 mM p-nitrophenyl palmitate to a 2ml microtube. The procedure for preparation of the experimental (comprising lipase) samples was: cutting the following free film sizes for the 100 mg/ml lipase films - 1cm X 3 cm, 1cm X 2 cm, and 1 cm X 1 cm, and for the control film (no lipase) - 1 cm X 3cm; placing the free films into labeled 2m L microtubes, where each of the coupon sizes were tested in triplicate; adding 500ul 40mM CHES to each microtube; adding 400ul ddH20 to each microtube; adding 10Oul 14.5 mM p-nitrophenyl palmitate to each microtube; and setting microtubes on a shaker. At each time point, tubes were placed in a centrifuge for 5 minutes at 13,000 RPM. A 10Oul was removed from each tube and the absorbance of the reaction product p- nitrophenol read at 405nm in a 96-well plate.

[0291] The tables below show the activity of each sample. The measured rates of reaction for the free films without any lipase were essentially baseline, exhibiting no destruction of the 4-nitrophenol palmitate. All lipase impregnated coatings demonstrated catalytic activity. The specific activity per centimeter basis was consistent within the different sample sizes.

[0292] The reaction containing the 1cm X 3cm free-film with lipase went to 50% completion. This is due to the nature of the insolubility of 4-nitrophenyl palmitate. Particles of 4- nitrophenyl palmitate were present in all microtubes due to precipitation when it comes in contacts with water. The 1cm X 1cm free-film was likely too small a film size, although the microtube was visually yellow, the data did not support the fact that the reaction did in fact take place. 4-nitrophenyl palmitate was originally used, but it self-hydrolyzed in water. Further, vegetable oil was spread over panels that were either control (no lipase) or lipase impregnated. After a day, the lipase impregnated panels were dry while the control panels were still visibly full of oil. It is also contemplated that greater loads of lipase, such as, for example, 200 mg/ml, 100 mg/ml, and 50 mg/ml lipase, may be used.

[0293] Example 37: This Example demonstrates a lipase assay determining the efficacy of lipase in a coating ( e.g paint). Films of Sherwin-Williams Acrylic Latex comprising lipase were assayed 7 months after they were prepared. Materials used are shown in the table below.

[0294] The reaction procedure included: cutting 1cm X 3cm free film coupon sizes; placing individual coupons into labeled 2ml_ microtubes, with each of the coupon samples tested in triplicate; adding 750pl 200mM TRIS to each microtube; adding 600ul ddH20 to each microtube; adding 150ul 14.5mM p-nitrophenyl acetate to each microtube; preparing control samples that had 750ul 200mM TRIS, 600ul ddH20, and 150ul 14.5mM p-nitrophenyl acetate; taking out at each desired time point, 10Oul and reading the absorbance at 405nm in a 96-well plate; and plotting absorbance vs. time to calculate the slope. Data and calculate values are shown below, demonstrating lipase activity in a cured coating's film 7 months after preparation.

[0295] Example 38: This Example demonstrates lipase activity in a Glidden alkyd/oil solvent based coating. The materials used are shown in the Table below.

[0296] The assay procedure included: cutting appropriate coupon sizes; placing individual coupons into labeled 2ml_ microtubes, with each of the coupon sizes are tested in triplicate; adding 750ul 200mM TRIS to each microtube; adding 600ul ddH20 to each microtube; adding 150ul 14.5mM p-nitrophenyl acetate to each microtube; preparing control samples (no films) to have 750ul 200mM TRIS, 600ul ddH20, and 150ul 14.5mM p-nitrophenyl acetate; removing at each desired time point, 10Oul and reading the absorbance at 405nm in a 96-well plate; and plotting absorbance vs. time to calculate the initial rate slope.

[0297] Example 39: To provide a description that is both concise and clear, various examples of ranges have been identified herein. Any range cited herein includes any and all sub-ranges and specific values within the cited range, this example provides specific numeric values for use within any cited range that may be used for an integer, intermediate range(s), subrange(s), combinations of range(s) and individual value(s) within a cited range, including in the claims. Examples of specific values ( e.g %, kDa, °C, °F, pm, kg/L, Ku) that can be within a cited range include 0.000001 , 0.000002, 0.000003, 0.000004, 0.000005, 0.000006, 0.000007, 0.000008, 0.000009, 0.00001 , 0.00002, 0.00003, 0.00004, 0.00005, 0.00006, 0.00007, 0.00008, 0.00009, 0.0001 , 0.0002, 0.0003, 0.0004, 0.0005,

0.0006, 0.0007, 0.0008, 0.0009, 0.001 , 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008,

0.009, 0.01 , 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11 , 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21 , 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.30, 0.31 ,

0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.40, 0.41 , 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.50, 0.51 , 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.60, 0.61 , 0.62, 0.63,

0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.70, 0.71 , 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.80, 0.81 , 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.90, 0.91 , 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, 0.99, 1.00, 1.01 , 1.02, 1.03, 1.04, 1.05, 1.06, 1.07, 1.08, 1.09, 1.10, 1.11 , 1.12, 1.13, 1.14, 1.15, 1.16, 1.17, 1.18, 1.19, 1.20, 1.21 , 1.22, 1.23, 1.24, 1.25, 1.26, 1.27, 1.28, 1.29, 1.30, 1.31 , 1.32, 1.33, 1.34, 1.35, 1.36, 1.37, 1.38, 1.39, 1.40, 1.41 , 1.42, 1.43, 1.44, 1.45, 1.46, 1.47, 1.48, 1.49, 1.50, 1.51 , 1.52, 1.53, 1.54, 1.55, 1.56, 1.57, 1.58, 1.59,

1.60, 1.61 , 1.62, 1.63, 1.64, 1.65, 1.66, 1.67, 1.68, 1.69, 1.70, 1.71 , 1.72, 1.73, 1.74, 1.75, 1.76, 1.77, 1.78, 1.79, 1.80, 1.81 , 1.82, 1.83, 1.84, 1.85, 1.86, 1.87, 1.88, 1.89, 1.90, 1.91 ,

I .92, 1.93, 1.94, 1.95, 1.96, 1.97, 1.98, 1.99, 2.00, 2.01 , 2.02, 2.03, 2.04, 2.05, 2.06, 2.07, 2.08, 2.09, 2.10, 2.11 , 2.12, 2.13, 2.14, 2.15, 2.16, 2.17, 2.18, 2.19, 2.20, 2.21 , 2.22, 2.23, 2.24, 2.25, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1 , 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0,

4.1. 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1 , 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0,

6.1. 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1 , 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0,

8.1. 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1 , 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0,

I I , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58,

59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82,

83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 99.10, 99.20, 99.30, 99.40,

99.50, 99.60, 99.70, 99.80, 99.90, 99.91 , 99.92, 99.93, 99.94, 99.95, 99.96, 99.97, 99.98, 99.99, 99.999, 99.9999, 99.99999, 99.999999, 99.9999999, 100, 101 , 102, 103, 104, 105, 106, 107, 108, 109, 110, 111 , 112, 113, 114, 115, 116, 117, 118, 119, 120, 121 , 122, 123,

124, 125, 126, 127, 128, 129, 130, 131 , 132, 133, 134, 135, 136, 137, 138, 139, 140, 141 ,

142, 143, 144, 145, 146, 147, 148, 149, 150, 151 , 152, 153, 154, 155, 156, 157, 158, 159,

160, 161 , 162, 163, 164, 165, 166, 167, 168, 169, 170, 171 , 172, 173, 174, 175, 176, 177,

178, 179, 180, 181 , 182, 183, 184, 185, 186, 187, 188, 189, 190, 191 , 192, 193, 194, 195, 196, 197, 198, 199, 200, 201 , 202, 203, 204, 205, 206, 207, 208, 209, 210, 211 , 212, 213,

214, 215, 216, 217, 218, 219, 220, 221 , 222, 223, 224, 225, 226, 227, 228, 229, 230, 231 , 232, 233, 234, 235, 236, 237, 238, 239, 240, 241 , 242, 243, 244, 245, 246, 247, 248, 249, 250, 260, 270, 275, 280, 290, 300, 310, 320, 325, 330, 340, 350, 360, 370, 375, 380,

390, .400, 410, 420, 425, 430, 440, 450, 460, 470, 475, 480, 490, 500, 510, 520, 525, 530, 540, 550, 560, 570, 575, 580, 590, 600, 610, 620, 625, 630, 640, 650, 660, 670, 675, 680,

690, 700, 710, 720, 725, 730, 740, 750, 760, 770, 775, 780, 790, 800, 810, 820, 825, 830,

840, 850, 860, 870, 875, 880, 890, 900, 910, 920, 925, 930, 940, 950, 960, 970, 975, 980, 990, 1000, 1025, 1050, 1075, 1100, 1125, 1150, 1175, 1200, 1225, 1250, 1275, 1300,

1325, 1350, 1375, 1400, 1425, 1450, 1475, 1500, 1525, 1550, 1575, 1600, 1625, 1650,

1675, 1700, 1725, 1750, 1775, 1800, 1825, 1850, 1875, 1900, 1925, 1950, 1975, 2000,

2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400,

3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800,

4900, 5000, 5250, 5500, 5750, 6000, 6250, 6500, 6750, 7000, 7250, 7500, 7750, 8000,

8250, 8500, 8750, 9000, 9250, 9500, 9750, 10,000, 25,000, 50,000, 75,000, 100,000, 250,000, 500,000, 1 ,000,000, or more. Additional examples of the use of this definition to specify sub-ranges are given herein. For example, a cited range of 25,000 to 100,000 would include specific values of 50,000 and/or 75,000, as well as sub-ranges such as 25,000 to 50,000, 25,000 to 75,000, 50,000 to 100,000, 50,000 to 75,000, and/or 75,000 to 100,000. In another example, the range 875 to 1200 would include values such as 910, 930, etc. as well as sub-ranges such as 940 to 950, 890 to 1150, etc.

[0298] In embodiments wherein a value or range is denoted in exponent form, both the integer and the exponent values are included. For example, a range of 1.0 x 10 17 to 2.5 x 10 7 , would include a description for a sub-range such as 1.24 x 10 17 to 8.7 x 10 11 .

[0299] Example 40: Nuclease Coating Demonstration

[0300] In this Example, 50pL Minwax was combined to 50pL DNase I 2998 U/mL (2.98 U/pL) or EcoRI 50 U/pL. A second EcoRI was combined to 90pL Minwax and 10pL EcoRI due to heavy cloudiness in coating (DNase I coating remained clear to eye). 2pL of coating was spread into bottom of 96-well plate. The coating was cured one triplicate set (with Minwax control) at 60°C for 30 minutes and the other at room temp. The first evaluation was 2 days following application. 5pL of precut plasmid DNA (cut with EcoRI so linear instead of circular) was added and incubated 20 minutes at 35°C. 10pL of sterile water loading buffer (10:1) was added and entire contents loaded into precast 0.8% agarose gel containing ethidium bromide. The samples were electrophoresed for 30 minutes in a gel kit, then illuminated using UV light box. DNase I incorporated into minwax produced no visible bands indicating the coating degraded the DNA.

[0301] Additionally, another verification of the DNA degrading coating’s activity was conducted. 25 pi TE buffer and 25 pi pAMP plasmid (0.2 pg/ml) was added to a microcentrifuge tube. The material was mixed and added 5 pi pAMP/TE solution into the following wells: 3 wells Minwax control, 3 wells EcoRI coating, and 3 wells DNase I coating. The coatings were incubated at 35°C for 20 minutes. 10mI_ TE containing 10% loading dye was added. Pipetting was done to evenly rinse wells and load entire contents into wells of a 0.8% agarose gel containing ethidium bromide. The materials were electrophoresed 30 minutes and visualized under UV light. The DNase coated wells had no visible bands, while all other samples did, demonstrating the coating degraded the DNA.

[0302] Example 41: Preservative Packaging Film Having Polyvinyl Alcohol and Chitosan (“Chito”)

[0303] This Example was an evaluation to 1 ) make substrate disks that did not curl up when coated with chitosan-based coatings (therefore making full contact with the agar surface); 2) to find which volume to apply to get a coating thick enough to demonstrate the killing effects of chitosan, but not so thick that the chitosan diffuses from the disk.

[0304] Disks of either 0.25-inch or 0.5-inch diameter were cut from a sheet of polyethylene substrate and coated with various volumes of either polyvinyl alcohol (“PVA”) or PVA:Chitosan 1:1. The coatings were allowed to cure at room temperature overnight. An overnight culture of E. coli K12 was diluted in sterile ddH20 so that A492 was in between 0.01 and 0.05 (corresponds to ~1.5 x 10 8 CFUs/mL), and 250 mI_ of this dilution were transferred to 5 ml_ sterile dH20. Sterile swabs were dipped into the E. coli suspension, tapped onto the inside of the tube to remove excess liquid, and streaked onto tryptic soy agar (“TSA”) plates. The disks were placed on top of the E. coli lawn with the coated-side facing down (toward the cells). The plates were incubated at 36°C overnight. Inhibition zones around the disks were measured, as well as colony counts and diameters, which could be seen through the transparent disks.

[0305] The disks did not curl up at the edges, and appeared to make full contact with the agar surface [0306] All samples with chitosan showed equal levels of killing, with the exception of the 50 mI-loaded 0.5-inch disk - NOTE: the liquid dispensed on the 50 mI samples tended to pull away from one edge and pool toward the other edge, so that the coating may not have been evenly distributed. This may be the case for this sample, since the colonies were mostly observed near one edge of the disk, particularly for 0.5-inch diameter coated disks. [0307] Samples loaded with ~200-250 mI of liquid coating per square inch of substrate did not show inhibition zones, but displayed about the same amount of killing power as coatings loaded as a larger volume (resulting in thicker cured coatings). The tables below show the data for micrographs of coated 0.25-inch an 0.5-inch diameter disks on prepared E. coli lawns and measurements of the inhibition zone.

[0308] Example 42: Bio-based Antimicrobial Food Packaging Coatings [0309] This Example demonstrates the antimicrobial properties of two disparate bio-based coating additives evaluated in a polyvinyl alcohol (“PVA”) food packaging coating for antimicrobial activity. Chitosan, a shrimp and crustacean shell derived polysaccharide, and an antimicrobial peptide were evaluated in a dissolvable food package coating for reductions in microbial growth after contacting agar “patties” serving as food simulants. [0310] Two quantitative assays were developed to analyze effectiveness of the coatings using modern microbiological methods and statistical software. The first used clear coated plastic disks through which bacterial colonies could be enumerated on the agar surface beneath. The second used the agar patties food simulants in a vacuum sealed food packaging system. The contaminated agar was contacted on one or more sides (e.g., flat surfaces, round edges) with an antimicrobial coating and the microbial growth inhibition evaluated, though one side was generally contacted during the studies of this Example. [0311] Polyvinyl alcohol (“PVA”) was obtained from Sigma-Aldrich (Cat# 348406, reported

Mw 13,000-23,000, 98% hydrolyzed; from Millipore Sigma, 400 Summit Drive, Burlington, MA 01803). Chitosan was obtained from Hard Eight Nutrition LLC, 7511 Eastgate Rd, Henderson, NV 89011 Doing Business as BulkSupplements.com. E. coli K12 was obtained from Presque Isle Cultures, 3804 W Lake Rd, Erie, PA 16505, and the peptide AMP7 (Seq Id no. 40) was obtained from Reactive Surfaces, Ltd., LLP, 300 West Ave. #1316 Austin TX. Isopropanol (91%) was purchased in Hattiesburg, MS. All growth media used Difco Tryptic Soy Agar (“TSA”) or tryptic soy broth from Becton, Dickinson, and Co., 7 Loveton Cir, Sparks Glencoe, MD 21152. MacConkey, Eosin Methylene Blue (“EMB”), and Luria- Bertani (“LB”) agar were obtained from Carolina Biological Supply Co., P.O. Box 6010,

2700 York Road, Burlington, NC 27216. 4-Methylumbelliferyl-p-D-glucuronide dehydrate (“MUG”) was obtained from Thermo Fisher Scientific, 1683rd Ave, Waltham, MA 02451. [0312] Disks of 0.5-inch diameter were cut using a 40W CO2 laser (Glowforge®, Seattle, WA) from a 3-mil thick sheet of clear Dura-Lar polyester (Grafix, Maple Heights, OH) substrate. For the vacuum-sealed food simulant experiment, 8.5 cm diameter disks were hand-cut from Dura-Lar sheets, or from commercial substrates from Stora Enso (Stockholm, Sweden), including Opalen, a clear, PET film and Trayforma, a PET-coated paperboard. A 5% (w/w) PVA solution, 1% (w/w chitosan in 2% (v/v) acetic acid solution, and 10% (w/w) AMP7 in 5% (w/w) PVA solution were prepared and mixed before application to create the final concentrations indicated below. The bio-based additive levels were based on the percentage by weight in the final, dry coating. All Dura-Lar films were coated by applying a specific volume of the liquid coating directly to the film, so that the final film thickness was approximately 1 mil. The coated films were left to dry at room temperature overnight before being used in any of the antimicrobial evaluations.

[0313] Traditional zone of inhibition testing uses paper disks infused with the target active, wherein the paper disks are incubated with microbes, and the zone of clearing seen around the disk (/.e., lack of microbial colony growth due to leaching of the active from the disk) measured to indicate the effectiveness of the active. Using the transparent plastic disks herein with a clear, dissolvable coating allowed rapid inspection of cell growth directly beneath the disk as well as any observable zone of inhibition. A cotton swab was dipped into a suspension of E. coli K12 (~5x10 6 CFUs/mL) and was spread over 15 cm diameter TSA plates (done in triplite). After the plates had dried approximately 15 minutes, the disks were placed, coated side down, on top of the E. coli layer. The plates were incubated at 30°C to 36°C overnight. Each disk was photographed using a dissecting microscope for magnification, and colonies were counted using ImageJ software from the National Institutes of Health, 9000 Rockville Pike Bethesda, Maryland 20892. The dose response of individual additives was evaluated using log-logistic regression model with the R package drc. (Ritz, C. et al., (2015). “Dose-Response Analysis Using R” PLoS One 10(12), e0146021). Where a combination of bio-based additive, chitosan and an antimicrobial peptide AMP7, were used in a food contact coating the interaction between the two bioadditives (/.e., synergistic, additive, antagonistic) was statistically evaluated using the zero interaction potency model with the R package synergyfinder. (He L. et al. (2018) Methods for High-throughput Drug Combination Screening and Synergy Scoring. In: von Stechow L, editor. Cancer Systems Biology: Methods and Protocols. New York, NY: Springer New York. P351-98).

[0314] To mimic packaged food, agar patties were cast into petri dishes and then gently removed from the dishes once firm to serve as food-patty simulants. These patties were vacuum sealed with plastic film inserts containing mixtures of PVA, chitosan, and AMP7 as studied in the small disk assays (Figure 1). Several selective and differential agar media were evaluated for visualization of E. coli colonies. These included MacConkey agar, LB agar with MUG, and EMB agar. MacConkey agar consistently produced clearly visible and easily observable E. coli colonies, and thus was primarily used. Each agar patty was placed in a vacuum bag, and an aliquot of diluted E. coli was spread over one surface so that approximately 200 CFUs were added (each evaluation done in triplicate). Coated films sized to match the agar patties were placed, coating side facing the bacteria, on the agar patties, and uncoated films were used as controls. The vacuum seal bags were sealed using the “Low” vacuum setting and the default setting on a Harvest Keepers Commercial vacuum sealer. The vacuumed samples were incubated 24-hours at 30°C, and colonies were counted using ImageJ software. For experiments measuring the effect of AMP7 and chitosan combinations, the interaction between the two bioadditives (i.e. , synergistic, additive, antagonistic) was evaluated using the R package synergyfinder, with the Bliss model being used to calculate predicted response because the number of combinations was low.

[0315] The efficacy of the bio-based antimicrobials in food-safe coatings was assessed by placing the coated 0.5-inch diameter Dura-Lar disks, coating-side down, onto prepared lawns of E. coli. The disks were coated with PVA-based coatings dosed with AMP7 at concentrations from 5,000 (0.5% (w/w)) to 20,000 ppm (2% (w/w)), and with chitosan at concentrations from 10,400 (1.04% (w/w)) to 90,800 ppm (9.08% (w/w)), or combinations of these two additives, with evaluations done in triplicate. The dose response, as determined by percent reduction in colony numbers compared to the PVA negative control, was determined for AMP7 and chitosan. The effective dose to kill 50% of the bacterial population (“EDso”) of AMP7 was around 4,500 ppm and 30,000 ppm for chitosan (Figure 2A and 2B). The responses of the bacteria to various combinations are displayed in the heatmap (Figure 2C), which shows the relative response as a color from red (highest percent growth inhibition) to green (lowest percent growth inhibition). To evaluate whether antagonism or synergy exists between these two compounds, the responses from AMP7 and chitosan combinations were used to determine the synergy score using the zero interaction potency method, which returns a score based on the deviation of the actual response from the expected response. (Yadav B. et al., Computational and Structural Biotechnology Journal 13: 504-513, 2015). These scores were visualized for each combination in a contour plot (Figure 2D), which indicates that the two antimicrobial agents generally interact in an antagonistic manner, with the highest regions of antagonism existing for concentrations of AMP7 between 5,000 ppm (0.5% (w/w)) and 10,000 ppm (1.0% (w/w)) and for concentrations of chitosan ranging from 10,400 ppm (1.04% (w/w)) to 47,600 ppm (4.76% (w/w)). Beyond this region of antagonism, all other combinations of AMP7 and chitosan appear to interact with reduced levels of antagonistic behavior. This indicated that the AMP7 peptide and chitosan are interfering with each other’s function, but that the negative interaction can be overcome as the concentrations of both additives increase. The antagonistic response could be occurring because both additives target the cellular membrane of microbes to produce a biocidal effect.

[0316] To evaluate the scalability of the results from the clear disk assay, the common vacuum-sealed storage of moist foods (meats, fruits, vegetables, etc.) was simulated using a nutrient agar patty contacting a bio-based antimicrobial coated plastic disk while stored inside a vacuum sealed bag. Single additives were evaluated as well as combinations at corresponding concentrations. These results are summarized in the table below.

[0317] Addition of 1% (w/w) AMP7 to 2.17% (w/w) chitosan-PVA coatings markedly worsened the efficacy of chitosan at this concentration, indicating stronger antagonistic behavior in this concentration regime, which is consistent with the original model from the small disk study above. Interaction between the two additives was evaluated by calculating synergy scores, using the Bliss model to calculate predicted responses, since a limited number of combinations were evaluated. Although the combination of 0.5% (w/w) AMP7 with 2.17% (w/w) chitosan-PVA coating exhibited improved antimicrobial activity compared with the 0.5% AMP7 or 2.17% chitosan PVA coatings alone, the percent growth inhibition was less that predicted for an additive combination response, resulting in a negative synergy score. The contour plot of these scores is shown in Figure 3. These evaluations indicate that vacuum-sealed patty studies had agreement with the results seen in the disk assay, which confirmed that the disk assay is a method for screening the effectiveness of these bio-based additives alone and in combination. Overcoming the antagonistic effects of these two components was evaluated by increasing the final concentration in the PVA coating to 16% (w/w) chitosan and 1.5% (w/w) AMP7 and evaluating if cost-effective materials could be made by reducing the overall coating thickness on the films. Because the bioactive coatings are soluble, dosage of the active ingredients was varied both by their concentrations in the coating mixture and by the amount of the coating added to the films (higher volumes applied to the films resulted in thicker coatings and a higher dose of actives). This evaluation was conducted using disks of commercial packaging products from Stora Enso, Klarabergsviadukten 70, 111 64 Stockholm, Sweden: Opalen, a clear, PET film and Trayforma, a PET-coated paperboard. The Stora Enso disks were evaluated with either a 0.2 mil thick coating or a 0.6 mil thick coating (compared to the 1 mil thick coatings used in the earlier studies). It was confirmed that the bio-based additives could be used with commercial packaging materials to get efficient kill of E. coli contamination by controlling coating thickness to achieve enough of the chitosan and AMP7 to overcome their antagonistic effects (see the Table below).

[0318] Individually, and in combination, AMP7 and chitosan in a PVA coating demonstrated effective antimicrobial activity in reducing bacterial growth in a food simulant contacting the food packaging coating. The assays were successful in detecting antimicrobial activity against E. coli for both the bio-based additives that were analyzed, and the statistical methods used detected an antagonistic effect at most of the concentrations evaluated and only slightly antagonistic or additive effects at higher concentrations, rather than synergistic activity. These additives, though biochemically dissimilar with one being a polysaccharide and the other an amino acid oligomer, act upon the same cellular target (the external cell membrane of bacteria and other microorganisms). When combined, these additives may compete for physical interaction with the cellular membrane to disrupt the membrane and produce a biocidal effect, so it is possible that one interfered with the other’s effectiveness. The antagonistic effects were shown to be overcome at high concentrations of both additives. It is contemplated both additives can be used in antimicrobial food packaging systems because they have demonstrated antimicrobial activity and both possess low toxicity; for example, chitosan has previously been used in antimicrobial edible coatings (Elsabee, M. Z and Abdou, E. S. Materials Science and Engineering-. C. 33 (4): 1819-1841 , 2013; A. Cagri and Z. Ustunol, E.T., J. Food Protect. 67:833-848, 2004) and the antimicrobial peptide used has previously been demonstrated to exhibit no discernable toxicity in rodent oral administration evaluations. [Kuhn, J. O. ProteCoat Final Report Acute Oral Toxicity Study (UDP) in Rats OPPTS No. 870.1100 StillMeadow, Inc., Sept 1, 2010 (Unpublished Study)].

[0319] Example 43: Screening for Antimicrobial Bioadditives as Antibiological Agents [0320] The analytical methods used in Example 42 may be used in screening potential bio based actives for additive, antagonistic, or synergistic activity as antibiological agents upon incorporation in a food packaging material formulation. These methods proved useful in screening the candidate bio-based additives presented above herein and are contemplated for use to evaluate other bio-based additives for incorporation into food packaging. Other bio-based antimicrobials can be selected to act on different cellular targets, and combinations that target different cellular components would likely produce additive or synergistic antimicrobial effects. Selection of antimicrobials that interact synergistically in combination is preferred, because this increases the antimicrobial activity of a plurality of additives while decreasing the concentration needed, thereby reducing overall production costs in a commercial application. Enzymatic additives may be selected to catalyze destructive reactions on lipids, proteins, sugar, and cellular wall components that sustain microbial life. Enzymes can be selected to be specific to the biochemistry of target microorganisms, such as selecting an enzyme that preferentially degrades bacterial cell walls vs. the cell walls of fungi. Alternatively, some enzymatic additives could be selected to exert nonspecific antimicrobial effect, such as certain oxidases that produce reactive oxygen species that attack most microbial biomolecules, including DNA. Other non- enzymatic peptide bio-additives, such as nisin and AMP7, have varying modes of action, typically through disrupting microbial membranes and cell walls, but due to their small molecular sizes, may be more suitable for applications where diffusion from a food preservative coating may aid in getting better protective coverage of the food item during storage. Evaluations using different combinations of these types of bio-additives are contemplated to produce coatings to safely enhance the shelf-life of food, and in some cases, be tailored to protection of specific food items from microbes, particularly pathogens, that preferentially contaminate those products. [0321] The techniques and coatings developed herein indicates that improvements in food safety can be achieved using coatings containing non-toxic bio-based antibiological agents (e.g., biocides) upon incorporation into a food packaging material formulation. Bio-based additives often have low toxicity to humans. Where the antimicrobial components of such packaging coatings are chosen to be generally recognized as safe (“GRAS”) by worldwide regulatory agencies, it is contemplated that migration from the packaging into headspaces and food-contact surfaces can provide enhanced efficacy against foodborne pathogens, including viruses. Though not a prerequisite for an antimicrobial food contact coating, the selection of materials, whether polymeric coating components or bio-based additives, with GRAS notices previously filed can be used in selecting bio-based additives. For example, both polyvinyl alcohol and chitosan fall into this category. Other considerations that can be used is the likely efficacy of the additive against microorganisms commonly associated with foodborne illness (/.e., bacteria like Escherichia coli), known general lack of toxicity, and biodegradability. The numbers and types of foodborne pathogens are varied, and consideration may be given to bio-based biocides that are potentially capable of controlling not only bacteria, but also spores of Gram-positive species, fungi, algae, and viruses (e.g., antimicrobial peptides).

[0322] Packaging coatings that can reduce such contamination while the food is traveling to its point of sale can be created using such bio-based additives. Further, edible films and chemical or physical modifications to plastic packaging materials that are permanent and specifically may be designed to leach or not leach onto or into the packaged food into one product can be created by retaining the physical barrier properties afforded by plastic packaging film and gaining the effectiveness of dissolvable (but safe to eat) bio-based antibiological agent(s) [(e.g., antimicrobial additive(s)].

[0323] It is contemplated that the antibiological compositions, methods, articles and/or devices described herein may preserve food (e.g., food going to market), extend food shelf- life, reduce food microbial contamination during packaging, reduce economic losses due to contamination, improve public health by reducing contamination of packaged food, reduce recall(s) of food that may be contaminated, reduce outbreak(s) of foodborne illness, or a combination thereof. For example, there are several contamination points during food preparation and packaging and numerous examples of outbreaks of foodborne illness leading to the recall of food (e.g., E. coli 0157: H7 contamination in a United States restaurant chain, Canadian pork, beef products, alfalfa sprouts, and romaine lettuce; E. coli O104:H4 spread from German sprouts; listeriosis throughout Europe in frozen corn and process meat in South Africa; Listeria monocytogenes contamination of packaged salads and frozen vegetables; E. coli 0157:H7 on Canadian pork; Heptitus A on scallops and frozen strawberries; Salmonella ( e.g ., Salmonella Adelaide) contamination of papaya and precut melon; Cyclospora spp. contamination of vegetable trays; etc. [Carney, E. et al. , Food Microbiology, 23(1 ):52-59, 2006; Moretnzs, T. and Langsrud, S. Comprehensive Reviews in Food Science and Food Safety, 16(5): 1022-1041 , 2017; Jay, M.T. et al., Emerging infectious diseases, 13(12): 1908, 2007; Pennington, H. The Lancet,

376(9750): 1428-1435, 2010; Frank, C. et al., New England Journal of Medicine,

365(19): 1771 -1780, 2011 ; Honish, L. et al., Releve des maladies transmissibles au Canada, 43(1):21-24, 2017] It is contemplated that such contamination and the effects of contamination may be reduced by the antibiological compositions, methods, articles and/or devices described herein.

[0324] Example 44: Screening for Antimicrobial Bioadditives using an in vitro Metabolic Assay

[0325] E. coli strains K12 and HB101 were grown overnight in 5 ml M9 minimal media at 30°C with agitation. The cell cultures were diluted 1 ml into 9 ml M9 for a 1 :10 total dilution. XTT (2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2/-/-Tetrazolium- 5-Carboxanilide; InvitrogenTM, 1600 Faraday Avenue, PO Box 6482, Carlsbad, California 92008 U.S.A) cell viability assay measures the reduction of the tetrazolium dye to a colored product by living cells. The colored product is measured spectrophotometrically at a wavelength of 492 nm. Ten milligrams of XTT (Biotium, 46117 Landing Pkwy, Fremont, CA 94538) was dissolved into 10 ml phosphate buffered saline (“PBS”) that had been pre-warmed to 40°C, and filter- sterilized using a 0.45 pm nylon filter. Forty microliters of 1.7 mg/ml menadione in acetone was added to 5 ml of the XTT solution. Glucose oxidase (Sigma, cat # G7141-1 OKU), AMP7, lysozyme (Biocat, lot# LYS-RA17), nisin (Sigma, cat # N5764-1G), and acylase (TCI, cat # A0688) stock solutions were made by dissolving 10 mg into 1 ml sterile ddhteO. A 10 mg/ml stock of monolaurin (ground Lauricidin®) was made by dissolving 10 mg into 1 ml 200-proof ethanol. These 10 mg/ml stock solutions were also used to make 1 mg/ml solutions by diluting 100 pi into 900 pi sterile ddhteO (or 200-proof ethanol in the case of monolaurin). A 40 mg/ml dextrose solution (in sterile ddhteO) was also made. The diluted cells, the 10 mg/ml and 1 mg/ml additive solutions, the XTT + menadione solution, and M9 diluent was added to a 96-well microplate. The absorbance at 492 nm was measured immediately after adding all components (0 H timepoint), and 20 hours after incubation at 30°C (20 H timepoint). The percent reduction in cell metabolism for each treatment was calculated using the following procedure. First, the percent increase in absorbance was calculated for each treatment type (including the cells that received no treatment, which served as the negative control). The percent increase in absorbance was calculated as follows:

% increase absorbance = [(A20H - AOH))/AOH)] * 100

[0326] A20H is the absorbance after 20 hours incubation, and AOH is the absorbance before starting incubation. The reduction in cell metabolism compared to the untreated control (no additive, no additive with ethanol (for monolaurin control)) was calculated as follows:

% reduction cell metabolism = 100 - [(T/C)*100]

[0327] T is the percent increase absorbance for the cells under test, and C is the percent increase absorbance for the negative control. [0328] Both strains of E. coli showed similar levels of sensitivity or resistance to the additive tested, with some small differences. Both strains showed the greatest sensitivity to AMP7, monolaurin, and glucose oxidase. The K12 strain seemed to be more sensitive to both concentrations of AMP7 and to the lower concentration of monolaurin compared to the HB101 strain. However, the K12 strain was slightly more resistant to lysozyme and acylase compared to the HB101 strain. Both strains had similar responses to the higher concentration of monolaurin and both concentrations of glucose oxidase. The combination of glucose oxidase, AMP7, and lysozyme did not seem to be advantageous to the single additives, especially compared to AMP7 and glucose oxidase alone.

[0329] Example 45: In-film XTT Assay with E. coli K12 and HB101 using PVA:Chitosan 1:1 coatings with lysozyme, AMP7, glucose oxidase, acylase, nisin, and monolaurin.

[0330] The E. coli strain K12 was grown overnight in 5 ml M9 minimal media at 30°C with agitation. The cell cultures were diluted 1 ml into 9 ml M9 for a 1 : 10 total dilution. Ten milligrams of XTT was dissolved into 10 ml PBS that had been pre-warmed to 40°C, and filter-sterilized using a 0.45 pm nylon filter. Forty microliters of 1.7 mg/ml menadione in acetone was added to 5 ml of the XTT solution. The PVA:Chitosan 1:1 coatings were prepared with the following additives: 2% lysozyme, 0.2% lysozyme, 2% AMP7, 0.2% AMP7, 2% glucose oxidase (+ 2 mg/ml dextrose), 0.2% glucose oxidase (+ 2 mg/ml dextrose), 2% acylase, 0.2% acylase, 2% nisin, 0.2% nisin, 2% monolaurin, 0.2% monolaurin. Three wells in a 96-well microplate were painted with each coating; three wells were also left uncoated to serve as the negative controls. Twenty microliters of the XTT + menadione solution and 100 pi of the diluted E. coli cells were added to each well. The absorbance at 492 nm was measured immediately after adding all components (0 H timepoint), and 20 hours after incubation at 30°C (20 H timepoint). The percent reduction in cell metabolism for each treatment was calculated using the procedure described previously above.

[0331] All of the coated wells showed a similar level of growth inhibition as the PVA:Chitosan 1:1 control with no additives, indicating that the antimicrobial activity of the chitosan-based coating is responsible for most of the growth inhibition seen in the samples with additional antimicrobial compounds. The samples with AMP7 and glucose oxidase showed slight improvement compared to the PVA:Chitosan alone, while the samples with acylase, nisin, and monolaurin did not perform as well as the control. [0332] Example 46: Vacuum-sealed E. coli K12 challenges using PVA:Chitosan 1:1

Coatings with Various Bioadditives.

[0333] PVA:Chitosan coatings (with or without additives) were prepared, and drawn down onto Duralar films at 5 mil thickness. The following films were prepared as shown in the table below. _

[0334] Duplicate films were cut to the size of regular-sized petri dishes. A culture of E. coli K12 that had been incubated overnight at 30°C with agitation was serially diluted so that the final dilution was 1 : 10 5 . P re-sterilized plastic inoculation loops (~10 pi capacity) were dipped into the 1 : 10 5 dilution and spread onto 18 MacConkey agar plates that had been removed from the petri dishes into vacuum sealer bags (will be ~1 : 10 7 total dilution). The films were placed onto the agar surface so that the coating-side is on top of the bacteria that had been spread onto the agar. Two plates did not receive films, and served as controls. The agar pucks were vacuum-sealed in the bags using the “Low” vacuum setting and sealed using the default seal setting. The agar pucks were incubated at 30°C.

[0335] Nearly all of the films coated with PVA:Chitosan, with the exception of coatings containing nisin and monolaurin, showed no bacterial growth in the form of colonies. Some plates exhibited a color change to pink, but this could be due to wicking of the bacteria underneath the agar puck (where there is no antimicrobial coating). Since the PVA:Chitosan control without additives also showed complete growth inhibition, the antimicrobial additives contribution to overall growth inhibition was not quantified (see Table below).

[0336] Example 47: Preservative Packaging Film Having an Antimicrobial Peptide [0337] This Example demonstrates that ProteCoat™ that previously showed efficient kill of E. coli in a liquid laboratory test (XTT test); that PVA / chitosan coatings containing ProteCoat™ were successfully applied to Stora Enso Opalen PET films and Trayforma PET paperboard samples; and that thin coatings of PVA/chitosan with ProteCoat™ showed the ability to control E. coli on a vacuum-bagged, contaminated food-simulant at different challenge levels.

[0338] To demonstrate the ability to coat Stora Enso’s film and paperboard packaging materials (Stora Enso AB Head Office, World Trade Center, Klarabergsviadukten 70, C4, P.O. Box 70395, SE-10724 Stockholm, Sweden; “Stora Enso”) with a coating containing a bio-based antibiological agent to reduce microbial growth, the growth of one standard food contaminant ( Escherichia coli) was evaluated after contact with a food packaging, food container, and/or processing material coating comprising an antimicrobial peptide ProteCoat™ (AMP7, Reactive Surfaces, Ltd., LLP, Austin, Texas). Physical samples of the food-simulant agar patties evaluated as controls and evaluated with the active coatings.

[0339] The E. coli K12 was grown overnight in 5 ml of M9 minimal media at 30°C with agitation. The cell culture was diluted by adding 1 ml of the culture mixture to 9 ml of M9 for a 1 : 10 total dilution. [0340] The XTT assay measures the metabolic activity of the cultured cells using a quantitative, colorimetric method. This is a rapid viability assay that allows screening of candidate biocides as liquids and in-films using very large arrays (several concentrations, different bio-based molecule combinations, several microbial strains, coatings formulations, etc.). The assay measures the ability of living cells to reduce the tetrazolium dye XTT [2,3- bis-(2-methoxy-4-nitro-5-sulfophenyl)-2/-/-tetrazolium-5-car boxanilide] to a colored product that can be spectrophotometrically measured at 492 nm ( XTT Cell Proliferation Assay Kit Instruction Manual, American Type Culture Collection, Manassas, VA, 2011). The technique allows quick down selection of biomolecules that inhibit metabolic activity of the target contaminant, as visually apparent darker orange/red color indicates living (metabolically active) cells; lighter colors indicate reduced metabolic activity (e.g., dead cells).

[0341] The ProteCoat™ active component, AMP7 (Seq. Id. no. 40), was evaluated in solution against the E. coli K12 cell challenge and monitored after 20 hours of exposure using the XTT assay. The coating film components PVA/Chitosan and ProteCoat™ were also examined for in-film activity against E. coli K12 using the same assay conditions by coating the bottoms of the test wells before the XTT evaluation. Different loadings of ProteCoat™ in solution (0.5 mg/mL and 0.05 mg/mL) and in the PVA/Chitosan coating (2.0 wt% on solids and 0.2 wt% on solids) were evaluated. The AMP7 in solution showed approximately 80% reduction in cellular metabolism which has been shown to correlate to complete kill of cells (see Table below).

[0342] In the PVA/Chitosan/AMP7 coating, this response was nearly a 50% reduction. It is contemplated that optimization of the dosing of AMP7 may be done for the PVA/Chitosan coating.

[0343] A nutrient agar was used to create “food simulant patties” that were vacuum bagged with Stora Enso coated and uncoated films for evaluation. After allowing the agar to solidify in petri dishes, the agar patties were removed for use in the E. coli challenge and packaging procedure. MacConkey (“MAC”) agar was used as the nutrient agar for the growth of the selected E. coli strain. MAC agar has traditionally been used for the selective growth and enumeration of coliform bacteria, such as E. coli. P re-formulated MAC agar media (Alpha Bioscience, 3651 Clipper Mill Rd., Baltimore, MD 21211) was prepared according to package instructions, sterilized by autoclaving, and the cooled, molten agar was poured into sterile petri dishes. The solidified agar was removed from the plastic dish and used as a food simulant. As E. coli ferments lactose in the media, the pH drops and presence of the indicator, neutral red, causing the colonies to become dark pink. This allowed for easy visualization of colony growth.

[0344] A liquid coating comprised of 2.5 wt% polyvinyl alcohol (PVA) and 0.5 wt% chitosan was prepared in water containing acetic acid to assist with chitosan solubility. The bio- based active agent, ProteCoat™, was added to the PVA/Chitosan coating at 1.5 wt% of the coatings solids (3 wt% total). The final coating was applied to plastic films and paperboard at 5 mil and 20 mil wet thickness using a drawdown bar. The coated films (Stora Enso Opalen PET film) and paperboard (Trayforma PET paperboard) were allowed to dry at room temperature. Once dry, all the films appeared transparent on the coated surfaces, and circular samples of the films and paperboard were cut and used for the vacuum bagged sample evaluation.

[0345] The E. coli K12 culture was incubated in tryptic soy broth overnight at 30°C with agitation and was serially diluted to 1 : 10 5 before application to the food-simulant patties. [0346] For control studies, the food-simulant patties were contacted with a bottom Trayforma circular cut out, inoculated on the top of the agar surface with E. coli, and covered with a top Opalen circular cut out. The evaluation was conducted similarly for the ProteCoat™ studies with the addition of a coating layer on the Opalen and Trayforma surface.

[0347] Opalen film and Trayforma paperboard were coated with PVA/Chitosan/ProteCoat™ films at 5 mil wet thickness. Once completely dry, these films were evaluated in the vacuum bag setup previously described above. After 2 days of incubation at 30°C, the number of colonies were counted for the control films and the ProteCoat™ films. The table below provides colony count and size comparisons of agar plates for the plain, uncoated Stora Enso films and ProteCoat™ containing film samples. Colonies of E. coli that were challenged at 1 :5x10 6 dilution appeared as dark spots. A similar evaluation of the agar patties for the plain, uncoated Stora Enso films and ProteCoat™ containing thinner film samples was conducted where the challenge level of E. coli was reduced. The colonies of E. coli that were challenged at 1 : 10 7 dilution were observed as dark pink spots. The films containing ProteCoat™ at the selected thickness and loading levels were not able to completely kill the E. coli challenge; however, the colonies are fewer in number and smaller in size compared to the control sample.

[0348] Because the results with the 5-mil application of coating were not complete kill of the E. coli challenge, thicker films of the ProteCoat coating [increased dosing of antimicrobial bioadditive(s)] were evaluated. The coatings used for this evaluation were applied at a 20- mil wet thickness. The coatings at this thickness were able to show nearly complete kill (4 or fewer colonies remained at the edges of the sample plates) for multiple E. coli challenges. The table below shows the colony counts and colony sizes for one of the challenges of the agar patties for the plain, uncoated Stora Enso films and ProteCoat™ containing samples. Colonies of E. coli that were challenged at 1 : 10 7 dilution were observed as dark spots. The thicker ProteCoat™ coating showed nearly complete kill of the higher E. coli challenge.

[0349] It is contemplated that the identification of additional target food contaminants and typical contamination levels may be conducted, that optimization of the coating thickness and ProteCoat™ loading level against the selected contaminant panel may be conducted, and that specification determination and evaluation of coated films may be conducted.