WO2015084938A1 - Viral biocontrol formulations - Google Patents

Abstract

Provided herein are compositions which encapsulate and/or adsorb virus particles, such that the virus particles are stable for extended periods of time under ambient conditions, and uses of such compositions, e.g., in biocontrol films and food coatings.

Description

VIRAL BIOCONTROL FORMULATIONS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit under 35 U.S.C. § 119(e) of

U.S. Provisional Application No. 61/911,985, filed on December 4, 2013, which is hereby incorporated herein by reference in its entirety.

STATEMENT OF GOVERNMENTAL SUPPORT

[0002] This invention was made with government support under Grant No. 2011-

67021-20034 awarded by the United States Department of Agriculture - Agriculture and Food Research Initiative (USDA-AFRI). The government has certain rights in the invention.

FIELD

[0003] Provided herein are compositions which encapsulate and/or adsorb virus particles, such that the virus particles are stable for extended periods of time under ambient conditions, and uses of such compositions, e.g., in biocontrol films and food coatings.

BACKGROUND

[0004] Active packaging materials with antimicrobial properties have gained significant interest over the last decade. These material formulations include antimicrobial polymers such as chitosan as well as inert packaging materials with encapsulated antimicrobial agents (Hosseini, Razavi, & Mousavi, 2009; Royo, Fernandez-Pan, & Mate, 2010; Seydim & Sarikus, 2006). Whey protein isolate (WPI) edible films with encapsulated antimicrobial materials are a sub-class of active packaging materials that have been evaluated for diverse applications in food packaging. The unique advantage of these WPI films is their ability to utilize agri-based biomaterials for packaging applications (V alencia- Chamorro, Palou, del Rio, & Perez-Gago, 2011). Similarly, polysaccharide biomaterials derived from agricultural byproducts have been combined with acids, salts, oils, enzymes, and bacteriocins such as nisin to control and reduce the microbial load on food products (Pintado, Ferreira, & Sousa, 2010; Rossi-Marquez, Han, Garcia-Almendarez, Castano- Tostado, & Regalado-Gonzalez, 2009; Zinoviadou, Koutsoumanis, & Biliaderis, 2010). Review of prior studies demonstrate the effectiveness of edible antimicrobial films in reducing the growth of inoculated bacteria by 1-2.5 loglO as compared to the controls without antimicrobial agents (Joerger, 2007).

[0005] Currently, most of the antimicrobial active packaging materials are broad spectrum antimicrobials which do not target bacterial pathogenic species specifically.

There is a significant need to develop novel antimicrobial packaging materials that have high specificity to target only pathogenic organisms while maintaining commensal bacteria. The need for specificity in antimicrobial activity is important as pathogens may be a small fraction of the total microbial load present in food systems (Payment & Locas, 2011). Thus, developing pathogen-specific antimicrobial active packaging materials may improve the antimicrobial efficacy by reducing interactions with non-targeted microbes. Further, nonpathogenic microbes are necessary in the production of some dairy and fermented foods. Additionally, the commensal bacteria present may have benefits for human health such is the case with probiotics microbes or may even control the growth of pathogenic bacteria (Garcia, Martinez, Obeso, & Rodriguez, 2008).

[0006] Bacteriophages are viruses of bacteria that have high specificity for their host strain and multiply upon infecting their host for up to 1000 viral progeny per bacteria (Laskin, Gadd, & Sariaslani, 2011). Generating progeny upon infection is markedly different to most antimicrobial agents that are depleted when interacting with microbes. Based on these advantages and increases in antimicrobial resistance among common pathogens, phage therapy is gaining more attention as an alternative approach that can be used to control both human and food-borne pathogens (Coffey, Mills, Coffey, McAuliffe, & Ross, 2010; Garcia, et al., 2008; Hagens & Loessner, 2007, 2010; Monk, Rees, Barrow, Hagens, & Harper, 2010; Sulakvelidze, Alavidze, & Morris, 2001). For example, phages have been used to control pathogens in the guts of animals. Pathogen-specific phages may remain active during the digestive process, allowing for removal of E. coli 0157Ή7 from cows' digestive system (Callaway, et al., 2008). Further, Salmonella-specific phages have been shown to reduce pathogen numbers in poultry (Borie, et al., 2008; Higgins, et al., 2005). Additionally, phages have been directly applied to fresh foods contaminated with pathogenic microbes such as E. coli 01 7:H7 and Listeria monocytogenes and have been shown to be effective in controlling the growth of the pathogens. Phages have been shown to be successful in reducing pathogen numbers by 94% to 100% on tomatoes, spinach, broccoli, and ground beef (Abuladze, et al., 2008). Similarly, cocktails of phages have been spotted onto contaminated meat surfaces, and a 99%- 100% reduction of pathogen level was obtained (O'Flynn, Ross, Fitzgerald, & Coffey, 2004). Also, phages showed a similar capability of reducing pathogen numbers up to 99.9% on fresh cantaloupe slices and lettuce (Sharma, Patel, Conway, Ferguson, & Sulakvelidze, 2009).

[0007] In all these prior studies, microbes were inoculated on surface of the food materials, and then the inoculated surface was treated with phages, typically by directly spraying phages on the inoculated surface. In many situations, target pathogens can be localized or spread across the surface of food products and can also be present on either or both surfaces of food products.

SUMMARY

[0008] In one aspect, compositions edible by a mammal are provided. In varying embodiments, the compositions comprise a polymer, a plasticizer and viral particles, wherein the viral particles are stable or substantially stable in the composition at ambient conditions for at least 2 months. In varying embodiments, the viral particles are encapsulated in the composition. In some embodiments, oxidation, denaturation and/or loss of biological activity of the viral particles is reduced, inhibited and/or substantially eliminated. In varying embodiments, the plaque forming activity of the viral particles in the composition is at least about 50%, e.g., at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more, e.g., in comparison to the plaque forming activity at the time of initial application, after 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, weeks at ambient conditions. In some embodiments, at least a portion of the viral particles are released from the composition by contacting the composition with an external stimulus, e.g., an aqueous solution, moisture, light and/or biodegradation (e.g. , including digestion processes in the gut). In varying embodiments, the ambient conditions comprise a temperature in the range of about 4°C to about 20°C, 25°C, 30°C, 35°C or 40°C, e.g., at atmospheric pressure. In varying embodiments, the viral particles comprise one or more bacteriophages, e.g., lytic bacteriophages. In some embodiments, the one or more bacteriophages are a member of a viral family selected from the group consisting of Myoviridae, Siphoviridae, Podoviridae, Lipothrixviridae, Rudiviridae, AmpuUaviridae, Bicaudaviridae, Clavaviridae, Corticoviridae, Cystoviridae, Fuselloviridae,

Globuloviridae, Guttavirus, Inoviridae, Leviviridae, Microviridae, Plasmaviridae and

Tectiviridae. In some embodiments, the one or more bacteriophages are lytic to a bacterial pathogen selected from the group consisting of Campylobacter, Helicobacter, Cholera, Cronobacter, Escherichia, Salmonella, Listeria, Lactococcus, Vibrio, Erwinia, Xanthomonas, Shigella, Staphylococcus, Streptococcus, Clostridium and Pseudomonas. In some embodiments, the bacteriophages are selected from the group consisting of T4 phage, T7 phage, bacteriophage SJ2, bacteriophage P100, phage LMP-102, phage LM-103, phage SP6, phage SP15, phage SP21, phage SP22, phage P7, phage KH1/SH1, phage KH 4, phage KH 11, phage KH5, phage el 1/2, phage e4/lc, phage PPOl, phage ECP-100, phage 29C, phage CP8, phage CP34, phage PPOl, phage NCTC s # 12669-12684, phage Al 11 , phage Felix-01 , phage PHL 4, phage P7, ECML-4, ECML- 117, ECML- 134, phage A511, phage P100, ATCC accession no. PTA-5372, ATCC accession no. PTA-5373, ATCC accession no. PTA-5374, ATCC accession no. PTA-5375, ATCC accession no. PTA-5376, ATCC accession no. PTA-5377, phage F01-E2, phage CJ6, phage φ88, phage φ35, and mixtures thereof. In some embodiments, the polymer is selected from the group consisting of whey protein isolate (WPI), soy protein isolate, corn proteins, mucins, rice proteins, wheat proteins, milk proteins, wheat gluten, pectin, collagen, gelatin, zein, sucrose esters, lipids, gums, alginates, chitosan, cellulose, cellulose-based polymers, starch, starch- based polymers, hydroxypropyl methyl cellulose (HPMC), Poly lactic acid, Poly Lactic-co- Glycolic Acid (PLGA), Polydioxanone, Polycaprolactone (PCL), polyanhydrides, polyester, polyphosphoesters, polyphosphazenes, polyhydroxybutyric acids, biodegradable copolymers (e.g. , AB diblock and ABA triblock polymers such as Poly(ethylene glycol) methyl ether-block-poly(D,L lactide), PEG-PLA; PLA-PEG-PLA, PLGA-PEG-PLGA, and mixtures thereof. In varying embodiments, the polymer is derived from whey protein isolate. In varying embodiments, the polymer comprises an average molecular weight in the range of from about 10 kDa to about 1000 kDa, e.g., from about 25 kDa or 50 kDa to about 750 kDa, 800 kDa or 900 kDa. In varying embodiments, the polymer is partially or wholly crosslinked. In some embodiments, the polymer is biodegradable. In some embodiments, the composition is not dried. For example, in varying embodiments, the composition comprises from about 0.1 wt. % to about 30 wt. % water, e.g., from about 0.5 wt. % or 1.0 wt. % to about 15 wt. %, 20 wt. % or 25 wt. % water. In some embodiments, the composition comprises from about 1 wt. % to about 50 wt. % polymer, e.g., from about 2 wt. %, 3 wt. % or 5 wt. % to about 35 wt. %, 40 wt. % or 45 wt. % polymer. In some embodiments, the wt. % ratio of polymer to plasticizer is from about 1 :4 to about 4:1, e.g., from about 1 :3 or 1 :2 to about 2: 1 or 3 : 1. In varying embodiments, the plasticizer has a molecular weight in the range of about 80 Da to about 2000 Da, e.g., from about 100 Da, 150 Da or 200 Da to about 1500 Da, 1750 Da, 1800 Da or 1900 Da. In some embodiments, the composition comprises from about 10 wt. % to about 70 wt. % plasticizer, e.g., from about 15 wt. % or 20 wt. % to about 50 wt %, 60 wt. % or 65 wt % plasticizer. In varying embodiments, the plasticizer is selected from the group consisting glycerol, sorbitol, ethylene glycol, polyethylene glycol (PEG), a sugar alcohol, a dextran, and mixtures thereof. In varying embodiments, the composition comprises a mechanical strength in the range of from about 0.1 MPa to about 250 MPa. In some embodiments, the composition further comprises an oil phase. In varying embodiments, the oil phase comprises from about 0.1 wt. % to about wt. 10% oil, e.g., from about 0.5 wt. % or 1.0 wt. % to about 7.5 wt. %, 8 wt. % or 9 wt. % oil. In varying embodiments, the oil phase comprises lipids selected from the group consisting of fatty acids, waxes, solid fats, liquid fats, and mixtures thereof. In varying embodiments, the composition is in the form of an emulsion (e.g., a water-in-oil emulsion or a water-in-oil-in- water emulsion), a film, a spray coating, a dip coating, or a combination thereof. In some embodiments, the film, spray coating or dip coating comprises multiple layers. In varying embodiments, the emulsion, film, spray coating or dip coating has a thickness in the range of about 100 nm to about 1 mm, e.g. , from about 150 nm or 200 nm to about 750 nm, 800 nm or 900 nm. In varying

embodiments, the composition is transparent. In varying embodiments, the composition is a film comprising whey protein isolate polymer, glycerol plasticizer and one or more bacteriophages.

[0009] In a further aspect, food edible by a mammal is provided. In varying embodiments, the food is wholly or partially coated with a composition as described above and herein. In varying embodiments, the composition forms a continuous barrier coating on the food. In some embodiments, the food comprises fresh produce or meat. The fresh produce can include intact or cut fresh produce. In varying embodiments, the produce is selected from apples, cucumbers, lettuce, tomatoes, spinach, broccoli, cantaloupe, strawberries and onions. In varying embodiments, the meat is selected from beef, pork, lamb, chicken, turkey and seafood, including fish. In varying embodiments, the meat can be prepared cuts, ground or entrails.

[0010] In a related aspect, provided is a medical device or a bandage. In varying embodiments, the medical device or bandage is wholly or partially coated with a composition as described above and herein.

[0011] In another aspect, a container wholly or partially coated with a composition as described above and herein is provided. In some embodiments, the container is a food container. In some embodiments, the container is a beverage container. In varying embodiments, the container is a plastic container or a paper container (e.g., a waxed paper container).

[0012] In a further aspect, methods of reducing or eliminating bacterial pathogens on surfaces are provided. In varying embodiments, the methods comprise contacting the surface with a composition as described above and herein. In varying embodiments, the surface is on food, a food container, a medical device or bandage. In varying embodiments, the methods further comprise the step of contacting the food with an external stimulus to release the viral particles from the composition. In some embodiments, the external stimulus is selected from the group consisting of an aqueous solution, moisture, light and biodegradation (e.g., including digestion processes in the gut).

DEFINITIONS

[0013] Viral particle (e.g. , bacteriophage) stability refers to the retention of structural integrity and biological function of viral particles encapsulated in or adsorbed onto the polymer matrices of the present compositions over a predetermined period of time. Viral particle (e.g. , bacteriophage) stability can be measured using any method in the art, including the ability of viral particles (e.g., bacteriophages) to form plaque forming units (PFU). Stability can be determined by measuring and comparing the biological function of viral particles in the composition at two time points (e.g., retention of at least about 50% PFU in comparison to the PFU at the time of initial application after 8 weeks), or by determining whether a unit measure of the composition has a minimum amount of viral particle activity (e.g., at least about lxlO⁴ PFU per mL).

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 illustrates stability of phages in WPI Films as function of time under both ambient (light and 22°C) and refrigerated storage conditions (4°C). The stability of phages in edible WPI films was also compared against phages deposited on a plastic surface under ambient conditions.

[0015] Figure 2 illustrates release of phages from phage encapsulating WPI films upon exposure to an aqueous environment and lettuce leaf surface. The concentration of released phages as a function of time reported in PFU. [0016] Figure 3 illustrates a) Reflectance imaging of the WPI films at 0 and 1 hour after incubation in water. The images were acquired approximately 20 μηι from the surface. Scale bar is 50 μιη. b) Line scans over 150 μιη at 0 and 1 hour images.

[0017] Figure 4 illustrates release of glycerol from edible WPI films upon incubation with an aqueous environment. The released glycerol content in aqueous solution was calculated as a percentage of total glycerol content of the film.

[0018] Figure 5 illustrates antimicrobial activity (growth inhibition assay) of phages encapsulated in WPI film over a 24 hour period as compared to the control film without phages. Asterisk indicates significance as compared to the control (p<0.05).

[0019] Figure 6 illustrates a) 3D Reconstruction of fluorescently labeled T4 bacteriophages in WPI film; and b) 3D reconstruction non-labeled T4 bacteriophages (control) in WPI film.

[0020] Figures 7A-B. Figure 7A illustrates a schematic of dipping cucumber slices into a phage WPI coating. Figure 7B provides an image of cucumber slices dip coated in phage WPI-based coating.

[0021] Figure 8 illustrates phage stability in WPI based coatings over a period of

7 days on cucumbers. The WPI-based dip coated and water dip coated cucumber slices were stored under refrigerated conditions (4°C) during the 7 day incubation period.

[0022] Figures 9A-B. Figure 9A illustrates phage loading efficiency at 5, 7, and 9 log PFU loading into WPI-based dip coating and water on cucumber. Figure 9B illustrates phage loading efficiency at 5, 7, and 9 log PFU in WPI dip coating and water on apple slices.

[0023] Figures 10A-B illustrate phage stability in WPI based coatings over a period of 6 days on apples. The WPI-based dip coated apple slices and water coated apple slices were stored under refrigerated conditions (4°C) over the 6 day incubation period.

[0024] Figures 1 1A-B illustrate phage distribution in WPI-based coatings. Figure

11 (a) shows the background fluorescence from phage encapsulating WPI films and Figure 11(b) shows the fluorescence signal from Syto 9 stained phages encapsulated in a WPI film.

[0025] Figure 12 illustrates reduction in the growth of bacteria on a cucumber slice after coating with a WPI-based film with T7 phages. The results are compared with a control WPI film (without T7 phages). The cucumber samples were incubated at refrigerated conditions (4°C) after coating with the WPI films.

[0026] Figure 13 illustrates the loading efficiency of silica stabilized water-in-oil emulsions. Before reflects the phage concentrations the emulsions were loaded with at 91og, 71og, and 51og PFU/mL. After reflects the recovered phage concentration from the emulsions.

[0027] Figure 14 illustrates confocal images of water-in-oil silica emulsions with a) fluorescently tagged T7 phage, b) fluorescently stained oil, c) white light image of the emulsion, and d) combined image of a, b, and c.

[0028] Figure 15 illustrates a schematic of encapsulating phages in a water-oil- water

(wi/o/w₂) emulsion. The wl phase contains phage and surfactant, in this example WPI. The oil phase contains oil and surfactant, in this example 6% PGPR. The w2 phase contains surfactants, in this example bile salt, lecithin, and Tween 20.

[0029] Figure 16 illustrates an example of a method to obtain the water-oil-water (wi/o/w₂) emulsion.

[0030] Figure 17 illustrates shape and size of the water-oil-water (wi/o/w₂) emulsion droplets. The white light images show size and some inner droplet encapsulation. The graph on the right depicts the size distribution of the water-oil-water (w_t/o/w^ emulsion.

DETAILED DESCRIPTION

1. Introduction

[0031] Provided herein are compositions comprising one or more polymers, one or more plasticizers and adsorbed and/or encapsulated viral particles, wherein the viral particles are stable or substantially stable in the compositions for extended periods of time at ambient conditions.

[0032] In certain embodiments, described herein are compositions (e.g., emulsions

(e.g., a water-in-oil emulsion or a water-in-oil-in- water emulsion), films, spray coatings, dip coatings and combinations thereof) that expand the use of viral particles (e.g.,

bacteriophages) beyond direct spraying of phages on food products, providing material formulations that can encapsulate virus particles (e.g., bacteriophages) for improved stability and release at target sites for delivery. Thus, viral particles (e.g., bacteriophages) can have significant applications as additives to packaging material formulations. [0033] The herein described formulations provide a unique approach for the release of phages upon contact with diverse food materials. We have tested these formulations in coatings on fresh produce, meat and liquid food products. The application of the present compositions includes, without limitation, antimicrobial food packaging materials including edible coatings, coatings for medical devices, delivery of antimicrobial agents for wounds, etc. In addition to antimicrobial advantages, these coatings also extend the shelf life of the food product.

[0034] To develop viral particle (e.g., bacteriophage)-based antimicrobial edible coatings, the ability of WPI films to encapsulate, stabilize, and release bacteriophages to the surrounding environment was evaluated. WPI films were selected as a model edible coating material because these films have been used for incorporating diverse antimicrobial agents such as essential oils, lactoperoxidase, and nisin (Min, Harris, & Krochta, 2005; Pintado, et al., 2010; Seydim, et al., 2006). In addition, WPI films have excellent mechanical properties and oxygen barrier properties which make them ideal edible packaging materials (Janjarasskul & Krochta, 2010). For this study, Escherichia coli and T4 bacteriophage were used as a model bacteria and its corresponding bacteriophage. The stability of encapsulated bacteriophages in WPI-based edible films was examined in ambient and refrigerated conditions over an extended period of time. To characterize the release of phages from the films, the activity of released phages from WPI films was measured in water and on lettuce leaves. The antimicrobial activity of WPI films with phages was determined using a microbial growth inhibition assay. In this assay, the microbes were inoculated on surface of phage encapsulating WPI films and the results were compared with control WPI films without phages. Confocal imaging was used for characterizing the distribution of fluorescently labeled encapsulated phages in WPI films.

[0035] The invention is based, in part, on the discovery and development of pathogen-specific antimicrobial packaging materials. These materials improve efficacy of antimicrobial treatment and retention of commensal and probiotic microbes on food materials. To achieve these goals, a representative virus particle, T4 bacteriophage, was encapsulated in whey protein isolate (WPI)-based edible protein films. Phage encapsulated WPI films were characterized for stability and release of encapsulated phages.

Antimicrobial efficacy of phage encapsulating edible films was characterized using a microbial growth inhibition assay. Distribution of phages in edible film was measured using a confocal fluorescence microscopy. The results demonstrate that the WPI films are able to stabilize phages at ambient (22°C and light) and refrigerated (4°C and dark) conditions without significant loss in phage infectivity over a period of one month.

Additionally, the WPI films are able to release a significant concentration of phages in an aqueous environment and leaf surface within 3 hours of incubation. Antimicrobial activity measurements demonstrate that the phage encapsulating WPI film can effectively inhibit the microbial growth. The results of microbial growth analysis showed an approximately 5 log difference in microbial levels between the control and the treatment samples. Confocal imaging measurements show that fluorescently labeled phages are homogenously distributed within the WPI film matrix. Overall, this study demonstrates integration of phages with edible packaging materials to develop novel active packaging materials for biocontrol applications.

[0036] The present films provide pathogen-specific antimicrobial edible films by combining edible films with phages that are host-specific. The specific edible films were evaluated for stability, distribution, release, and efficacy of the phage as the antimicrobial material.

2. Compositions

a. Generally

[0037] In varying embodiments, the compositions can be in the form of emulsions

(e.g., a water-in-oil emulsion or a water-in-oil-in-water emulsion), films, spray coatings, dip coatings and combinations thereof. For example, in some embodiments, the composition can be an emulsion applied in the form of a film, a spray coating and/or a dip coating. Generally, the compositions provided herein are edible by a mammal, comprise a polymer, a plasticizer and virus particles, wherein the virus particles within the compositions are stable at ambient temperatures and pressures for extended periods of time. These edible compositions have an extended shelf life (e.g., over 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months) under ambient conditions (atmospheric pressure, ambient temperature). In varying embodiments, the ambient conditions comprise a temperature in the range of about 4°C to about 20°C, 25°C, 30°C, 35°C or 40°C, e.g., at atmospheric pressure. The extended stability of the virus particles is a surprising and unexpected discovery because prior to the present application, research has shown that viruses, including bacteriophages, are not stable in dry ambient conditions. In some embodiments, the compositions have not been dried, and can contain water. For example, in varying embodiments, the composition can comprise from about 0.1 wt. % to about 30 wt. % water, e.g., from about 0.5 wt. % or 1.0 wt. % to about 15 wt. %, 20 wt. % or 25 wt. % water. In varying embodiments, the compositions can be in the form of an emulsion (e.g., a water-in-oil emulsion or a water-in-oil-in- water emulsion), a film, a spray coating, a dip coating, or a combination thereof. In varying embodiments, the films or coatings can have a thickness in the range of about 100 nm to about 1 mm, e.g., from about 150 nm or 200 nm to about 750 nm, 800 nm or 900 nm. In varying

embodiments, the compositions are transparent.

[0038] In varying embodiments, the virus particles within the compositions retain biological activity (e.g., the ability to form plaque forming units) for at least 6 weeks, e.g., at least 7 weeks, 8 weeks, 9 weeks, 10 weeks, 1 1 weeks, 12 weeks or more, at ambient conditions. In varying embodiments, the viral particles retain at least about 50% biological activity, e.g., at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% biological activity, e.g., in comparison to the biological activity at the time of initial application, over the time period that they are encapsulated or adsorbed into or onto the present compositions, e.g., for at least 6 weeks, e.g., at least 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 4 months, 5 months, 6 months, 7 months, 8 months,

9 months, 10 months, 1 1 months, 12 months, or longer. The biological activity of the viral particles can be determined and measured using any method known in the art. In varying embodiments, the biological activity of the viral particles is measured by the ability to form plaque-forming units (PFU) (e.g. , the ability of bacteriophages to form plaque- forming units (PFU) in a bacterial culture). In varying embodiments, the compositions can deliver or release active viral particles (e.g., bacteriophages) at a concentration of at least about 1 x 10⁴ plaque forming units (PFU) per mL, e.g., at least about 5 x 10⁴, 1 x 10⁵, 5 x 10⁵, 1 x 10⁶ or 5 x 10⁶ plaque forming units (PFU) per mL. The present compositions improve both the loading of viral particles (e.g., bacteriophages) and extended stability of viral particles (e.g., bacteriophages) on food materials, including stability against acidic pH conditions.

b. Virus Particles

[0039] In varying embodiments, the viral particles are absorbed or encapsulated in the compositions. In varying embodiments, the viral particles are adsorbed or encapsulated in the compositions at a concentration of at least about 10¹ PFU/ml, e.g., in the range of about 10¹- 10¹² PFU/ml, e.g., in the range of about 10²- 10¹² PFU/ml, 10³- 10¹² PFU/ml, 10⁴- 10¹² PFU/ml, 10⁵- 10¹² PFU/ml, 10⁵- 10¹² PFU/ml or 10⁷- 10¹² PFU/ml. In some embodiments, the viral particles comprise one or more bacteriophages, e.g., lytic bacteriophages. In some embodiments, the one or more bacteriophages are members of a viral family selected from the group consisting of Myoviridae, Siphoviridae, Podoviridae, Lipothrixviridae, Rudiviridae, Ampullaviridae, Bicaudaviridae, Clavaviridae,

Corticoviridae, Cystoviridae, Fuselloviridae, Globuloviridae, Guttavirus, Inoviridae, Leviviridae, Microviridae, Plasmaviridae and Tectiviridae. In varying embodiments, the compositions comprise viral particles from more than one viral family. In some embodiments, the one or more bacteriophages are lytic to a bacterial pathogen selected from the group consisting of Campylobacter, Helicobacter, Cholera, Cronobacter, Escherichia, Salmonella, Listeria, Lactococcus, Vibrio, Erwinia, Xanthomonas, Shigella,

Staphylococcus, Streptococcus, Clostridium and Pseudomonas.

[0040] Illustrative bacteriophages lytic to Escherichia coli include without limitation, e.g. , T4 phage, T7 phage, ECML-4, ECML-1 17 and ECML-134. Illustrative bacteriophages lytic to Listeria include without limitation, e.g. , phages A51 1 and phage PI 00. Illustrative bacteriophages lytic to Salmonella include without limitation, e.g. , phage Felix-Ol, phage PHL 4 and phage P7. Illustrative bacteriophages lytic to Staphylococcus include without limitation phage φ88, phage φ35. Illustrative bacteriophages lytic to Campylobacter include without limitation, e.g., CJ6.

[0041] In some embodiments, the bacteriophages are selected from the group consisting of T4 phage, T7 phage, bacteriophage SJ2, bacteriophage PI 00, phage LMP- 102, phage LM- 103, phage SP6, phage SP 15, phage SP21, phage SP22, phage P7, phage KH1/SH1, phage KH 4, phage KH 1 1 , phage KH5, phage el 1/2, phage e4/lc, phage

PPOl, phage ECP- 100, phage 29C, phage CP8, phage CP34, phage PPOl, phage NCTC s # 12669-12684, phage A1 1 1 , phage Felix-Ol , phage PHL 4, phage P7, ECML-4, ECML-117, ECML-134, phage A 1 1 , phage PI 00, ATCC accession no. PTA-5372, ATCC accession no. PTA-5373, ATCC accession no. PTA-5374, ATCC accession no. PTA-5375, ATCC accession no. PTA-5376, ATCC accession no. PTA-5377, phage F01-E2, phage CJ6, phage φ88, phage φ35, and mixtures thereof.

[0042] Additional bacteriophages useful for inclusion in the compositions provided herein include those described in Sulakvelidze, J Sci Food Agric (2013) 93:3137-3146; in U.S. Patent Nos. 8,003,323; 7,745,194; 7,674,467; 7,635,584; 7,625,741 ; 7,625,740;

7,625,739; 7,625,556; 7,622,293; 7,507,571; 7,459,272 and 6,699,701; and in U.S. Patent Publication Nos. 20130164374, 20120148505, 20100297086, 20100075398, 20100068787, 20090297561 , 20090155329, 20090155217, 20090087831 , 20090053144, 20090047727, 20090047726, 20080311643, 20080299641 , 20080194000, 200801 18468, 20070248724, 20070054357, 20050244383, 20050175991 , and 20040247569. All of the foregoing references are hereby incorporated herein by reference in their entirety for all purposes.

i. Polymers

[0043] Generally, the polymer or polymers used in the compositions provided herein are edible by a mammal. The polymer or polymers can be naturally occurring or synthetic. In varying embodiments, the polymer or polymers are biodegradable. In varying embodiments, the polymer or polymers have good oxygen barrier properties and prevent, reduce, inhibit and/or eliminate oxidation of the viral particles. The polymer component of the composition can be a homopolymer or a heteropolymer. The polymers provide a matrix for adsorbing or encapsulating the viral particles.

[0044] In some embodiments, the polymer is a food protein polymer, e.g. , a polymer derived from milk protein (e.g., whey, casein), soy protein, corn protein (e.g., zein), rice protein and/or wheat protein. In varying embodiments, the polymer is derived from plant proteins, e.g., soy protein, corn protein (e.g., zein), rice protein or wheat protein. In varying embodiments, the polymer is a naturally occurring polymer, e.g., derived from whey protein isolate (WPI), soy protein isolate, corn proteins, rice proteins, wheat proteins, milk proteins, wheat gluten, pectin, collagen, gelatin, zein, mucins, sucrose esters, lipids, gums, alginates, chitosan, cellulose, cellulose-based polymers, starch, and/or starch-based polymers. In varying embodiments, the polymer is a synthetic polymer, e.g. , hydroxypropyl methyl cellulose (HPMC), Poly lactic acid, Poly Lactic-co-Glycolic Acid (PLGA), Polydioxanone, Polycaprolactone (PCL), polyanhydrides, polyester, polyphosphoesters, polyphosphazenes, polyhydroxybutyric acids, biodegradable copolymers (e.g., AB diblock and ABA triblock polymers such as Poly(ethylene glycol) methyl ether-block-poly(D,L lactide), PEG-PLA; PLA-PEG-PLA, PLGA-PEG-PLGA, and mixtures thereof. In some embodiments, the polymer is selected from the group consisting of whey protein isolate (WPI), soy protein isolate, corn proteins, mucins, rice proteins, wheat proteins, milk proteins, wheat gluten, pectin, collagen, gelatin, zein, sucrose esters, lipids, gums, alginates, chitosan, cellulose, cellulose-based polymers, starch, starch-based polymers, hydroxypropyl methyl cellulose (HPMC), Poly lactic acid, Poly Lactic-co-Glycolic Acid (PLGA), Polydioxanone,

Polycaprolactone (PCL), polyanhydrides, polyester, polyphosphoesters, polyphosphazenes, polyhydroxybutyric acids, biodegradable copolymers (e.g., AB diblock and ABA triblock polymers such as Poly(ethylene glycol) methyl ether-block-poly(D,L lactide), PEG-PLA; PLA-PEG-PLA, PLGA-PEG-PLGA, and mixtures thereof. In varying embodiments, the polymer is derived from whey protein isolate. Lipid-based materials with film forming ability include lipids derived from animal and plant products, including oils and waxes, and mixtures thereof. Oils of use include, e.g., peanut, coconut, cocoa, palm kernel, palm oil, milk butters, lard, tallow, and mixtures thereof. Waxes of use include, e.g., candelilla, jojoba, carnauba, beeswax, whales wax, paraffin, microcrystalline wax, and mixtures thereof. See, e.g. , Morillon, et al., Critical Reviews in Food Science and Nutrition (2002) 42(l):67-89.

[0045] In varying embodiments, the polymer comprises an average molecular weight in the range of from about 10 kDa to about 1000 kDa, e.g., from about 25 kDa or 50 kDa to about 750 kDa, 800 kDa or 900 kDa. In some embodiments, the polymer {e.g., a protein-derived polymer, e.g., whey protein isolate), comprises an average molecular weight in the range of from about 10 kDa to about 100 kDa. In some embodiments, the composition comprises from about 1 wt. % to about 50 wt. % polymer, e.g., from about 2 wt. %, 3 wt. % or 5 wt. % to about 10 wt. %, 15 wt. %, 20 wt. %, 25 wt. %, 30 wt. %, 35 wt. %, 40 wt. %, 45% or 50 wt. % polymer. The compositions generally contain polymer concentrations to have a viscosity sufficient to form a film on a desired surface but not too viscous to impede depositing material or forming a film on a surface.

[0046] In varying embodiments, the polymer or polymers can be crosslinked. In some embodiments, the crosslinks are covalent bonds (e.g., disulfide bonds) or hydrogen bonds. For example, protein-based or protein-derived polymers may utilize disulfide bonds for crosslinking and polysaccharide-based or polysaccharide-derived polymers may utilize hydrogen bonds for crosslinking. The crosslinks can also be introduced by chemical crosslinking. In varying embodiments, the chemical cross linking materials may include small ions such as chemicals or small molecular weight chemical cross linkers such as glutaraldehyde or enzymatic cross linkers such as transglutaminase. Higher levels of crosslinking typically reduce the solubility of polymeric materials and increase the polymer resistance against various solvents including water. Crosslinked and non-crosslinked polymer can be combined to adjust for the level of porosity of the polymer matrix and the level of release of the viral particles upon contact of the compositions with an external stimulus (e.g. , an aqueous solution, moisture, light, biodegradation (e.g., including digestion processes in the gut)). Relatively lower levels of crosslinking allow for higher levels of viral particle release. Conversely, higher levels of crosslinking allow for lower levels of viral particle release. The level of crosslinked polymer in the compositions can be controlled using any method known in the art. For example, the length of time a crosslinking reaction is allowed to proceed can be lengthened for increased crosslinking or shortened for reduced crosslinking. Levels of crosslinking can also be controlled by combining different levels of crosslinked and non-crosslinked polymer in the compositions.

[0047] In addition to forming stand-alone films, the edible polymer compositions can also be deposited as an emulsion (e.g., a water-in-oil emulsion or a water-in-oil-in-water emulsion), a dip coating, a spray coating, or a combination thereof. In those applications, it is desired that these polymers form a continuous barrier coating on the surface (e.g. , food materials such as deli meat, leafy greens, cut vegetables and fruits). For forming an effective dip coating, it is desired that the surface contact properties (contact angle, affinity for bonding with surface of food material— determined by weak bonding such as hydrogen bonding) is favorable. The favorable properties include that the material interface has similar contact angles and ability of coating material to form bonding with the surface, e.g. protein coatings on meat product surfaces and carbohydrate-based polymers on the fresh produce surfaces.

[0048] The polymer or polymers included in the composition are selected appropriate for the desired context, for example, depending on the release mechanism or the coating method. In varying embodiments, the polymers used for emulsions, film-based, dip-coating, spray-coating formulations, and combinations thereof, include without limitation proteins such as whey protein isolate, sugar-based dextran; cellulose-based polymers such as HPMC and alginates; and lipids (including oils and waxes). Furthermore, the release of viral particles from the compositions can be adjusted by controlling the hydrophilicity of the composition. For example, polymers can be selected based on their extent of wetting properties to control the release. Polymers with wetting properties of protein and sugar-based polymers are useful for rapid release of viral particles from the compositions.

ii. Plasticizers

[0049] The compositions further comprise a plasticizer component that serves as a humectant and functions to preserve the stability of the viral particles. The plasticizer increases flexibility and decreases brittleness of the compositions. The plasticizer further functions to extend the shelf life (e.g., biological activity, stability) of viral particles encapsulated in or adsorbed on the compositions. Furthermore, release of the plasticizer from the polymer matrix upon exposure to an external stimulus (e.g. , an aqueous solvent, moisture, light, biodegradation (e.g., including digestion processes in the gut)), also facilitates release of the encapsulated or adsorbed viral particles. The plasticizers are typically a low molecular weight biomaterial; the low MW provides flexibility for these materials to diffuse within the polymer matrix— and enables slipping of macromolecular chains-required for flexibility.

[0050] In varying embodiments, the plasticizer has a molecular weight in the range of about 80 Da to about 2000 Da, e.g., from about 100 Da, 150 Da or 200 Da to about 500 Da, 1000 Da, 1500 Da, 1750 Da, 1800 Da or 1900 Da. In some embodiments, the composition comprises from about 10 wt. % to about 70 wt. % plasticizer, e.g., from about 15 wt. % or 20 wt. % to about 50 wt. %, 60 wt. % or 65 wt % plasticizer. Plasticizers of use in the present compositions include without limitation, e.g. , glycerol, sorbitol, ethylene glycol, polyethylene glycol (PEG), a sugar alcohol, a dextran, lipids, and mixtures thereof. Furthermore, wax-based lipids can function as a plasticizer. Illustrative waxes of use include, without limitation, e.g., candelilla, jojoba, camauba, beeswax, whales wax, paraffin, microcrystalline wax, and mixtures thereof.

[0051] In some embodiments, the wt. % ratio of polymer to plasticizer is in the range of from about 1 :4 to about 4: l , e.g., from about 1 :3 or 1 :2 to about 2: l or 3: 1.

[0052] In varying embodiments, upon contact of the compositions with an external stimulus (e.g. , an aqueous solution, moisture, light, biodegradation (e.g. , including digestion processes in the gut)), the plasticizer is released from the composition, also allowing for release of viral particles from the composition.

3. Applications

[0053] The present compositions find use as a biocontrol material formulation that is stable under ambient conditions but provide an efficient antimicrobial activity against specific pathogens in complex environments including food and agriculture.

a. Biocontrol Materials

i. Emulsions, Films, Spray Coatings and Dip Coatings

[0054] In varying embodiments, the edible compositions for encapsulation and controlled release of viral particles (e.g., bacteriophages) can be provided in the form of an emulsion (e.g., a water-in-oil emulsion or a water-in-oil-in- water emulsion), a film, a spray coating, a dip coating, or a combination thereof. The emulsions, films, spray coatings, dip coatings, and combinations thereof find use in protecting desired surfaces from bacterial contamination (e.g., biocontrol). In varying embodiments, the surfaces desired to be protected are on food, on food containers (including beverage containers), and on wipes, sanitary products, wound dressings, and medical devices.

[0055] In one embodiment, the edible composition is a dip coating comprising whey protein isolate (WPI), glyercol and beeswax. Illustrative concentrations of these components are as follows: 10% w/v (total solution) whey protein isolate (WPI), glyercol at 33% w/w WPI, and beeswax at 20%> w/w of WPI+glycerol.

ii. Food Coatings and Food Container Coatings

[0056] Accordingly, further provided is food edible by a mammal and food containers wholly or partially coated with a composition as described above and herein. In varying embodiments, the composition forms a continuous barrier coating on the food or food container. In some embodiments, the food comprises fresh produce or meat.

Illustrative produce that can be protected using the present compositions includes, without limitation, e.g. , apples, cucumbers, lettuce, tomatoes, spinach, broccoli, cantaloupe, strawberries and onions. The fresh produce may include intact food material or cut fresh produce. In varying embodiments, the meat is selected from beef, pork, lamb, chicken, turkey, and seafood, including fish.

[0057] Also provided is a container wholly or partially coated with a composition as described above and herein is provided. In some embodiments, the container is a food container. In some embodiments, the container is a beverage container. In varying embodiments, the container is a glass container, a metal container, a plastic container or a paper container (e.g., a waxed paper container). In varying embodiments, the container is Styrofoam. In some embodiments, the container is a personal products container (e.g., containing a product for topical application, e.g., shampoo, lotion, cream, toothpaste, etc.).

iii. Medical Device and Wound Dressing Coatings

[0058] Further provided are medical devices or bandages wholly or partially coated with a composition as described above and herein. In particular, the present compositions can be coated onto the wound dressing of a bandage or the exterior surface of a medical device. The compositions are generally coated onto an external plastic and/or metal surface of a medical device. Illustrative medical devices that can be coated with the present compositions include, suture thread, wound closure tape, catheters, tubes, stents, atheroscopic balloons, pace makers, replacement joints (e.g., hip, knee), valves, chips (e.g. , information storage media, computer chip, computer-readable media), etc.

iv. Methods of Application

[0059] Further provided are methods of reducing or eliminating bacterial pathogens on surfaces. In varying embodiments, the methods comprise contacting the surface with a composition as described above and herein. In varying embodiments, the surface is on food, a food container, a medical device or bandage. In varying embodiments, the methods further comprise the step of contacting the food with an external stimulus (e.g. , an aqueous solution, moisture, light and biodegradation (e.g., including digestion processes in the gut)) to release the plasticizer and viral particles from the composition. Concurrent release of non-crosslinked material (e.g., non-cross-linked polymer and plasticizer) and viral particles (e.g. , bacteriophages) from the compositions (e.g. , in the form of films or coatings) can be achieved by swelling, degradation and/or erosion of the polymer matrix. In varying embodiments, swelling of film matrix can be induced, triggered or promoted by exposure to surface moisture or an aqueous solvent (e.g., water). The swelling of the matrix and concurrent release of non-crosslinked material (e.g., non-cross-linked polymer and plasticizer) induces and promotes release of encapsulated viral particles (e.g. ,

bacteriophages). Biodegradation of the polymer matrix also induces and promotes release of encapsulated viral particles (e.g., bacteriophages). Biodegradable properties of the polymer can be particularly useful for antimicrobial coatings on medical implants. In varying embodiments, the polymer matrix can be degraded by external stimuli such as light.

EXAMPLES

[0060] The following examples are offered to illustrate, but not to limit the claimed invention.

Example 1

Encapsulation of Bacteriophages in Whey Protein Films

for Extended Storage and Release

MATERIALS AND METHODS [0061] Bacteriophage T4 and Host Strain. Coliphage T4 was purchased from

Carolina Biological Supply and used without modification. Escherichia coli DH5ot with a tetracycline resistance selection maker was a gift from Dr. Glenn Young (University of California, Davis) and was used as a model bacterium for all PFU counting experiments. E. coli BL21 was purchased from ATCC (#BAA-1025) and used in efficacy experiments. Both E. coli strains were stored in liquid nitrogen. Prior to experimental use, each liquid nitrogen stored strain was streaked onto an agar plate and grown overnight in a 37°C incubator. A colony from the plate was used to inoculate Luria Broth (LB) media and allowed to grow overnight in a 37°C shaking incubator. For use in experiments, each strain was subcultured from the initial culture and grown to log phase.

[0062] Bacteriophage Activity Assay - Plaque Forming Unit Counting. Phage activity was measured with a top agarose overlay plating method and reported in plaque forming units per milliliter (PFU/mL). Briefly, top agar (0.7% agar) was melted, and 3 mL aliquots were kept at 45°C in a water bath until used. Phage samples were serially diluted into SM buffer, and 100 μΐ of each sample was combined with 250 μΐ of fresh log phase bacteria culture with an optical density (O.D.) at 600 nm of 1.5. The samples were incubated for 10 minutes and then combined with an aliquot of molten agar. The molten agar mixture was poured onto a pre-warmed LB agar plate and allowed to solidify. Once the plates solidified, they were inverted and placed into a 37°C incubator. The plates were allowed to incubate for 12-18 hours and then counted.

[0063] WPI Film Preparation. Whey Protein Isolate (WPI, Davisco Foods International, Inc. (Eden Prairie, MN)) was a gift from Professor John M. Krochta

(University of California, Davis), and glycerol was purchased from Fisher Scientific. WPI films were prepared based on a previously reported method (Yoo & Krochta, 201 1).

Briefly, 5% w/v WPI was added to deionized (DI) water and stirred vigorously for 20 minutes. Glycerol at a 2: 1 WPI to glycerol weight ratio was added to the solution and then stirred for an additional 20 minutes. The solution was placed in a water bath at 90°C for 30 minutes to denature the whey protein and promote cross linking of the protein to form the film matrix. After cooling to room temperature in an ice bath, the WPI film solution was then degassed with a Welch Vacuum Pump (model 2567B-50 A). Phages in a 1 mL aliquot were then added to the solution and gently mixed to ensure an even distribution into the WPI film solution. The solution was then poured into 16 cm diameter circular cast molds and allowed to dry at 22°C and 18% relative humidity.

[0064] Phage Stability in WPI Films. Stability was tested by storing WPI films in the dark under refrigerated conditions (4°C), and on a lab bench in ambient conditions (22°C and light). At specified time points, a quarter of the circular WPI film was mechanically minced and placed in 50 mL of water. The WPI film was then placed in a shaker on moderate speed at 22°C for 4 hours to release phages from the minced pieces of the WPI film. The WPI film was then removed, and the water was assayed with PFU counting to determine the number of active phages released from the film. Three independent samples for each storage condition were evaluated at 0, 1 , 3, and 5 weeks of storage. As a comparison control, 1 mL of lxlO⁹ PFU/mL T4 phages in buffer was dried on a petri dish. At each time point, the phage coated petri dishes (two coated petri dishes per time point) were incubated with sterile water at 22°C for one hour to recover phages from the surface. The water was recovered and tested for remaining active phage particles.

[0065] Release Profile from WPI Films into Aqueous Environment . To determine the release profile of phages from the WPI film in an aqueous solution, 1.5 cm diameter disks were cut randomly from a WPI film and placed in water without any mechanical shaking to measure the passive release of phages from WPI films. The WPI film was removed from the water at 1 , 2, 3, 4, and 5 hours, and the resulting solution assayed with PFU counting to determine the number of phages released in the water. Each sample was done in triplicate. For phage release on lettuce leaves, 1.5 cm disks of flat lettuce leaves and WPI films were cut. The lettuce leaves were inoculated with 10 \iL sterile water to ensure contact between the leaf surface and the WPI film disk. At 1 , 2, 3, 4, and 5 hours, the lettuce leaves were separated from their film disks and washed in 5 mL of water by shaking for 10 minutes at 250 rpm. The wash water was sampled to count for active phage released from the films. Each time point was measured in triplicate. In order to calculate the percentage of phages released from the disks, the total phage loading per square centimeter was calculated first for the cast molded film. The total phage loading per centimeter was then multiplied by the area of the disks used in the release assay to yield the total phage loading per disk. The cumulative percentage of released phages over a five hour period was then calculated by dividing the total phage concentration measured for each time point with the total phage loading per disk.

[0066] Antimicrobial Growth Inhibition Assay. The antimicrobial efficacy of WPI films was measured based on a growth inhibition assay, similar to other antimicrobial edible packaging film studies (Min, et al., 2005; Rossi-Marquez, et al., 2009). In a 24 well plate (Falcon, 35- 1 147), WPI films with T4 phages and WPI films without phages were placed in duplicate. One milliliter of approximately 3 log CFU E. coli BL21 diluted in LB media from a log phase culture was added to each well. The plate was then sealed to prevent evaporation of the media. The well plate was placed on a rotating platform at room temperature (22°C) for 24 hours. Samples of the initial inoculum at 0 hour and samples of the wells at 24 hours were plated on LB agar and incubated for 18 hours at 37°C. After the colonies became visible, the plates were counted and reported as CFU/mL.

[0067] Determining Glycerol Concentration. Glycerol release from WPI films in water was measured using a coupled enzymatic reaction system kit (Cayman Chemicals, Item No. 10010755). The final product of the coupled reaction system is a brilliant purple product, Quinoneimine, with an absorbance maximum at 540 nm. Glycerol concentration is linearly correlated with Quinoneimine concentration and therefore the absorbance intensity at 530-550nm.

Glvc rsl Kinase

Glycerol 4- ATP ~ > Glycerol^■■■■ 3 - Phosphate - ADP

ΰ iy r oJ Pk asp h « te

Glycerol - 3 - Phosphate -f 0₂ *Dihydroxy cet<me Phosphate + <¾0

Peroxidase

2H2<¾ + 4— + ESPA ^tQuinoneimine dye - H₂#2

[0068] Briefly, 1.5cm disks cut out from WPI films were placed in 1 mL deionized water. Samples were taken at 0, 2, 3, and 6 hours and serial diluted to 10-3. The 0 hour time point reflects soaking a disk in water for 5 minutes. Per protocol instructions, 10 μί of samples and glycerol standards were added to a 96 well plate and incubated with Glycerol Assay Kit solutions for 15 minutes. Absorbance measurements were taken at 550nm using an absorbance/fluorescence plate reader (Molecular Devices, Model: M2, Sunnyvale, CA). Each sample was done in triplicate. The standard curve in the range of 0 to 20 mg/L of glycerol was fitted using linear equation with R2 value equals to 0.9953: y = 360.04*x - 0.1694, with x being the difference of absorbance values of each standard sample with respect to the absorbance of standard 0 mg/L glycerol and y being the glycerol

concentration (mg/L). Each sample of interest was serial diluted so that the glycerol concentration falls into the standard curve range. The glycerol concentrations of the diluted sample were calculated based on the glycerol standard curve, and from that value, the concentration of glycerol in the original sample was determined. [0069] Bacteriophage Imaging within the WPI Films. T4 bacteriophages were conjugated to Alexa Fluor 647 carboxylic acid, succinimidyl ester fluorophores (excitation: 647 nm; emission: 665 nm) purchased from Invitrogen. T4 bacteriophages were purified from the storage buffer provided by Carolina using PEG purification. A PEG solution (40% w/v Polyethylene Glycol 8000 in 2.5M NaCl, both purchased from Fisher Scientific) was added to the stock T4 solution and kept on ice for 30 minutes to several hours. The solution was then centrifuged at 1 l ,000g and 4°C for 10 minutes. After draining off the supernatant, the pellet was suspended in 100 iL of sterile water. In order to fluorescently tag the phages, 20 of Alexa 647 was added to 500 μί of phage solution and incubated at room temperature for 3 hours to allow for the dye to conjugate to the reactive amines on the protein capsid. The phage solution was then spun down in a Zeba Desalt Column (Fisher Scientific) equilibrated with SM buffer to remove excess fluorophores. A Genesys lOuv Scanning UV-vis spectrophotometer was used to scan the phage solution for an absorbance peak from 640 nm - 660 nm to confirm presence of the fluorophore. Once confirmed, the conjugated phages were placed into a prepared WPI film solution and immediately poured in cast molds. The molds were covered to prevent photobleaching of the fluorophore. Control WPI films were prepared using non-labeled T4 phages. Samples of the WPI films were mounted in oil on glass slides and imaged with a Zeiss LSM 510 Meta Microscope with a 633 nm Helium-Neon laser and bandpass filter set to 640 nm to 670 nm. Z image stacks were taken in 1 μηι intervals were acquired for 3D reconstruction of the image stack.

[0070] Near Infrared Reflectance Confocal Imaging of WPI Films. In order to assess the structural changes in the WPI film upon exposure to an aqueous environment, the films were imaged using near infrared reflectance confocal microscopy. NIR reflectance confocal microscopy takes advantage of the biological material's ability to scatter and absorb NIR light in order to image the structure of the material (Carlson, Coghlan,

Gillenwater, & Richards-Kortum, 2007). To prepare for imaging, films were placed in water for 1 hour in triplicate, with a set of films that had no contact with water as a control. These films were mounted on slides and imaged with the same Zeiss microscope and a 633 nm laser as in the imaging assay used in this study. The emission filter was set to a 683 nm low pass filter to image in reflectance mode. The WPI films were imaged at 9 μιη intervals from the surface to the end of the film. Line scans of confocal z stack images within the WPI film that were selected for this analysis were generated using Metamorph image analysis software to characterize the reduction in reflectance signal intensity after incubating WPI films in water.

[0071] Statistical Analysis. Statistical analysis of the data was completed in Excel

(Microsoft, Redmond WA). In order to determine significance, the data were tested using a T-test assuming unequal variances with an alpha of 0.05. Results are denoted with an asterisk or noted in the text where appropriate.

RESULTS AND DISCUSSION

[0072] Stability of phages in edible WPI films. One of the key challenges in developing phage-based antimicrobial packaging materials is the stability of phages in material formulations. Most of the current understanding of phage stability is based on the analysis of phages in an aqueous environment while there is very limited understanding of the stability of phages in material formulations (Jepson & March, 2004; Schaper, Duran, & Jo fire, 2002). The stability of encapsulated phages in active packaging material is a critical requirement for the successful integration of phages with packaging materials and their application in food systems. Figure 1 shows the results of stability testing of T4 phages encapsulated in WPI-based edible coatings under both refrigerated (4°C and dark) and ambient conditions (22°C and light) over a five week period. Overall, phages encapsulated in WPI films that were stored under ambient conditions show no significant loss in phage activity after 5 weeks of storage, and the phages stored under refrigerated conditions had an approximately 1 log decrease in phage activity over a five week period. This decrease in phage activity in refrigerated samples was only detected between week 3 and week 5, and there was no significant loss in phage activity during the first three weeks of storage under refrigerated conditions (p>0.05). Excellent storage stability of phages in ambient conditions is highly significant as previous studies have shown that encapsulation of phages in various biopolymer matrices lead to significant loss in activity within a similar time period to this study. The stability of Felix Ol-E.coli targeting phage after encapsulation in alginate microspheres showed significant loss in phage activity within a five week period. After air drying at 22°C, only 12% and 6% of the total encapsulated phages were active when stored at 4°C and 22°C for six weeks respectively. The dried microspheres with encapsulated phages were combined with trehalose, a common stabilizing agent for viruses, to increase the phage stability after drying (Bieganski, Fowler, Morgan, & Toner, 1998). Similar encapsulation of phages in synthetic PVA (polyvinyl alcohol) polymer using the electrospinning process has shown a significant loss in number of viable phages (-1-6% viable phages) immediately after electrospinning (Salalha, Kuhn, Dror, & Zussman, 2006). In summary, comparison of stability of phages in other biomaterial system highlights the ability of WPI films to maintain stability of phages in ambient and refrigerated storage conditions.

[0073] To further evaluate the role of WPI -based edible film environment in maintaining this excellent stability, the stability of WPI film encapsulated phages was compared with phages that were deposited on the surface of a polystyrene plastic as a control. Polystyrene was chosen as it is a common plastic packaging material. The results show that the deposited phages on a plastic surface have a significant loss in activity during storage under ambient conditions. Within the first week, the phages deposited on plastic surface had an approximately 3 log decrease in phage activity. This trend in loss in activity of phages continued over a five week period during which a 7 log decrease in the activity of encapsulated phages was observed. Overall, these results indicate the edible WPI films provide an excellent environment to encapsulate phages and maintain the bioactivity of phages over an extended storage period of time under ambient conditions.

[0074] Most of the current understanding of factors that contribute to the stability of viral particles is predominantly based on empirical observations. Based on these observations, the key factors that may enhance stability of viral particles include: prevention and/or limiting oxidative damage to viral capsid and DNA/RNA components and stabilization of the structure of viral capsid proteins (Wigginton, Pecson, Sigstam,

Bosshard, & Kohn, 2012). There are many unique factors in the design of WPI -based edible films that can contribute to enhanced stability of phages and potentially other viral particles. These factors include: (a) excellent oxygen permeation barrier properties of WPI films that may limit oxidative damage (Janjarasskul, et al., 2010), (b) the presence of a significant fraction of the plasticizer, glycerol, that may aid in stabilizing viral capsid upon drying of WPI films (Mendez, et al., 2002), and (c) the role of the WPI protein environment in stabilizing viral capsid proteins. Prior studies have shown the significance of proteins in solutions, surfactants, and organic matter in stabilizing viral particles (Johnson, Hooker, Francis, & Clark, 2007; Mylon, et al., 2009; Puapermpoonsiri, Ford, & van der Walle, 2010).

[0075] Release and Activity of Encapsulated Phages from WPI films . For antimicrobial activity of phage encapsulated WPI films, it is important that phages encapsulated in WPI films are in physical contact with the target microbes that may be present in the environment. Phage release from edible coatings upon contact with a food product can enable phage delivery to target microbes. To assess the release of phages from WPI films, WPI edible films with encapsulated phages were brought in contact with water and lettuce as model aqueous and food surface systems. Figure 2 shows the quantitative measurement of release of phages into an aqueous environment and a food surface. The release of phages from WPI films in an aqueous environment was measured by sampling the water fraction at selected time points. Upon contact with water, phage encapsulated WPI film showed a burst pattern release of phages in an aqueous solution. Within a three hour period, approximately 3.3% of the encapsulated phages were released from the film into an aqueous system. For measuring the released phages on the leaf surface, the leaf samples were rinsed with water and the phage concentration in the wash water was measured at selected time points. Similarly to the aqueous system, the WPI films released 0.37% of encapsulated phages to the leaf surface in 3 hours. After 3 hours of contact, no significant increase in number of released phages was observed (p>0.05) during the total six hour incubation period for either aqueous system or leaf surface. It is important to note that the number of phages released immediately upon contact with the food product is highly significant for microbial control in food materials, and these edible films can deliver phages that will be an order of magnitude higher than the typical number of pathogenic microbes (10-1000 CFU/mL) found in most applications. The overall trend of release of phages in this study is similar to the release of phages from alginate microspheres upon contact with water (Ma, et al, 2008).

[0076] In addition to release of phages from edible films, significant swelling of the edible films after contact with water was observed as shown in Table 1.

TABLE 1

Swelling of WPI film after 1 hour of incubation in an aqueous environment

[0077] The hydration induced swelling of films can adversely influence the mechanical and barrier properties of the WPI films. These results are in agreement with the current literature in which studies have shown that WPI edible films have limited barrier properties against permeation of water (Janjarasskul, et al., 2010). Confocal reflectance imaging of the films was used to further demonstrate film swelling after being in contact with water for 1 hour. Representative reflectance images are shown in supplementary Figure 3a. The image on the left depicts a WPI film that has no contact with water, and the image on the right is of a WPI film after being immersed in water after 1 hour. Figure 3b shows the intensity line scans across the center of each image to quantify the gray scale intensity as a function of distance in microns. The WPI film that had no contact in water exhibited higher intensity values than the WPI film that had been in contact with water. The images show significant changes in the structure of film based on the representative images and the reflectance intensity line scan (Figure 3b). Hydration induced swelling of films appear to decrease the intensity of the reflected light indicating a decreased packing density of the polymer in the film.

[0078] To further understand the physical process that induces the release of phages from edible coatings, the release of glycerol from WPI films was characterized. Glycerol was chosen because it has a small molecular weight (92.09 g/mol) as compared to individual WPI proteins (14,000 to 69,000 molecular weight depending on protein composition) and is highly soluble in water. Furthermore, the WPI polymers are crosslinked by thermal treatment during the film formation process that can significantly decrease the solubility of individual WPI polymers in water. The release of glycerol from WPI edible films upon contact with water is shown in Figure 4. Approximately 22% of the total glycerol loaded in the WPI film was released upon immediate contact with water. Over the next six hours of incubation, the percentage cumulative release of glycerol from

WPI edible films in contact with water increased to approximately 33%. While the majority of the glycerol is released in the first hour, phage release continues to increase after the first hour. In comparison, only 1 1.78% of the total glycerol from the film was released to the leaf surface. This is in part due to the limited amount of water available on the leaf surface for glycerol to diffuse as compared to a 100% aqueous system. Based on these results, it is thought that phage transport is facilitated through the release of glycerol and swelling of the WPI film matrix. [0079] Antimicrobial Efficacy ofT4 encapsulated WPI Films. The antimicrobial efficacy of T4 encapsulated WPI films was tested through a growth inhibition assay. Figure 5 shows the results after 24 hours of incubation at room temperature. After 24 hours, the T4 WPI films show an approximate 2 log CFU decrease from the initial inoculation level while the negative control shows an increase of 3 log CFU. Thus, a total of 5 log CFU reduction in microbial count with respect to the negative control sample was measured. These results illustrate the antimicrobial efficacy of phages in not only reducing the growth rate of microbes but also effectively reducing the level of microbes below the initial inoculum level. These results are comparable to previous studies that have observed a similar reduction in microbial count using broad spectrum antimicrobial agents on food materials. For example, a 2-3 log reduction in growth of Pseudomonas spp. on a meat surface was observed upon coating meat samples with WPI films encapsulating sodium lactate or ε-polylysine as compared to the meat samples treated with control films (Zinoviadou, Koutsoumanis, & Biliaderis, 2010). Similarly, another study reported a 4.2 log reduction of Listeria monocytogenes on smoked salmon samples coated with WPI films encapsulating lactoperoxidase, an antimicrobial enzyme, compared to the control films (Min, 2005). The choice of antimicrobial in WPI films is inherently flexible as these films may be tailored to target different pathogens by including different phage types or including a cocktail of phages that target a single pathogen for more efficient elimination. [0080] Imaging Distribution of Phages within WPI Films. To develop a comprehensive understanding of spatial distribution of phages in WPI edible films, the phages were tagged with Alexa 647 as described in the materials and methods section. Fluorescently labeled phages were encapsulated in the WPI films and imaged using confocal microscopy. Figure 6 shows the 3-D reconstruction and axial projection of individual confocal z- stacks of WPI films with fluorescently labeled phages. Imaging results show that the phages are distributed relatively uniformly across the entire field of view both along the depth and surface of the WPI edible film. The fluorescent signal corresponding to the distribution of phages in WPI films can be categorized in two distinct groups- diffuse and specular staining. The diffuse staining pattern is expected of individual phages which are significantly smaller than the optical resolution, around 300-400 nm, while the specular staining pattern can correspond to small aggregates of phages approximately 0.5 μηι to 3 μιη in size. The aggregation of phages within WPI edible films can be induced by many factors including the effect of the film drying process and the presence of net opposite charges in head and tail regions of the phage. Similar observations of organization of phages due to net dipole movement have been demonstrated in prior studies (Steinmetz, et al, 2008). In addition, studies have also reported aggregation of phages even in certain buffer and pH conditions (Floyd & Sharp, 1979; Langlet, Gaboriaud, Duval, & Gantzer, 2008; Langlet, Gaboriaud, & Gantzer, 2007) .

[0081] Conclusion. To enable development of pathogen- specific antimicrobial edible coatings, presented herein is the encapsulation of a model T4 bacteriophage in edible WPI films. The present results demonstrate that the WPI films stabilize phages at ambient and refrigerated conditions without significant loss in phage infectivity over a period of one month. Additionally, the WPI films are able to release significant concentration of phages in an aqueous environment within 6 hours of incubation. In contrast with the leaf surface, less phage was released to the surface. The results indicate that the release of phages from edible film is mediated by a rapid release of glycerol and swelling of WPI film in aqueous environment. Antimicrobial activity measurements using a growth inhibition assay demonstrate that WPI film encapsulating phages can effectively reduce the rate of growth of E. coli. In summary, this study demonstrates the potential of incorporating phages into active packaging materials.

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Example 2

Illustrative WPI Dip Coating Protocol

MATERIALS:

• Whey Protein Isolate (WPI)

• Glycerol

· Beeswax

• Water

• Phage

EQUIPMENT:

· Beaker/Flask

• Stir plate and stir bar

Hand disperser

• Hot Water Bath (90°C)

• Ice Bath

· Vacuum Pump PREPARATION METHOD:

1. Prepare a 10% w/w WPLwater solution and stir for 20 minutes or until WPI is

completely dissolved.

2. Place in hot water bath for 30 minutes.

3. Add 1 :3 w/w glycerol (glycerol: WPI). Swirl to mix evenly.

4. Add 20% dry basis (w/w) beeswax. Melt and mix evenly.

5. Bring solution to 15.6% total solid content with water.

6. Take solution out of hot water bath and homogenize with hand disperse for 4 minutes at 9500 rpm.

7. Place in ice bath for 5-10 minutes until room temperature. Swirl repeatedly to ensure even cooling.

8. Degas solution with vacuum pump, approximately 10 minutes.

9. Store at 4°C if not used immediately.

DIP COATING METHOD:

1. Add antimicrobial material and mix gently to prevent air bubbles in solution.

2. Dip coat samples by fully submerging them in dip coating. Remove after 5 seconds.

3. Allow for 30 minutes drying time.

[0082] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

Claims

CLAIMS What is claimed is:

1. A composition edible by a mammal comprising: a polymer, a plasticizer and viral particles, wherein the viral particles are stable or substantially stable in the composition at ambient conditions for at least 8 weeks.

2. The composition of claim 1, wherein the viral particles are encapsulated in the composition.

3. The composition of any one of claims 1 to 2, wherein oxidation, denaturation and/or loss of biological activity of the viral particles is reduced, inhibited and/or substantially eliminated.

4. The composition of any one of claims 1 to 3, wherein the plaque forming activity of the viral particles in the composition is at least about 50% after 8 weeks at ambient conditions.

5. The composition of any one of claims 1 to 4, wherein at least a portion of the viral particles are released from the composition by contacting the composition with an external stimulus selected from an aqueous solution, moisture, light and biodegradation.

6. The composition of any one of claims 1 to 5, wherein the ambient conditions comprise a temperature in the range of about 4°C to about 40°C.

7. The composition of any one of claims 1 to 6, wherein the viral particles comprise one or more bacteriophages.

8. The composition of claim 7, wherein the one or more bacteriophages are lytic bacteriophages.

9. The composition of any one of claims 7 to 8, wherein the one or more bacteriophages are a member of a viral family selected from the group consisting of

Myoviridae, Siphoviridae, Podoviridae, Lipothrixviridae, Rudiviridae, Ampullaviridae, Bicaudaviridae, Clavaviridae, Corticoviridae, Cystoviridae, Fuselloviridae, Globuloviridae, Guttavirus, Inoviridae, Leviviridae, Microviridae, Plasmaviridae and Tectiviridae.

10. The composition of any one of claims 7 to 9, wherein the one or more bacteriophages are lytic to a bacterial pathogen selected from the group consisting of Campylobacter, Helicobacter, Cholera, Cronobacter, Escherichia, Salmonella, Listeria, Lactococcus, Vibrio, Erwinia, Xanthomonas, Shigella, Staphylococcus, Streptococcus, Clostridium and Pseudomonas .

11. The composition of any one of claims 7 to 10, wherein the bacteriophages are selected from the group consisting of T4 phage, T7 phage, bacteriophage SJ2, bacteriophage PI 00, phage LMP-102, phage LM- 103, phage SP6, phage SP 15, phage SP21, phage SP22, phage P7, phage KH1/SH1, phage KH 4, phage KH 11, phage KH5, phage el l/2, phage e4/lc, phage PPOl, phage ECP-100, phage 29C, phage CP8, phage CP34, phage PPOl, phage NCTC s # 12669-12684, phage Al 511, phage Felix-Ol, phage PHL 4, phage P7, ECML-4, ECML-117, ECML-134, phage A511, phage P100, ATCC accession no. PTA-5372, ATCC accession no. PTA-5373, ATCC accession no. PTA-5374, ATCC accession no. PTA-5375, ATCC accession no. PTA-5376, ATCC accession no. PTA-5377, phage F01-E2, phage CJ6, phage φ88, phage φ35, and mixtures thereof.

12. The composition of any one of claims 1 to 11, wherein the polymer is selected from the group consisting of whey protein isolate (WPI), soy protein isolate, corn proteins, mucins, rice proteins, wheat proteins, milk proteins, wheat gluten, pectin, collagen, gelatin, zein, sucrose esters, lipids, gums, alginates, chitosan, cellulose, cellulose-based polymers, starch, starch-based polymers, hydroxypropyl methyl cellulose (HPMC), Poly lactic acid, Poly Lactic-co-Glycolic Acid (PLGA), Polydioxanone, Polycaprolactone (PCL), polyanhydrides, polyester, polyphosphoesters, polyphosphazenes, polyhydroxybutyric acids, biodegradable copolymers, PEG-PLA; PLA-PEG-PLA, PLGA-PEG-PLGA, and mixtures thereof.

13. The composition of any one of claims 1 to 12, wherein the polymer comprises an average molecular weight in the range of 10 kDa to 1000 kDa.

14. The composition of any one of claims 1 to 13, wherein the polymer partially or wholly crosslinked.

15. The composition of any one of claims 1 to 14, wherein the polymer is biodegradable.

16. The composition of any one of claims 1 to 15, wherein the composition is not dried.

17. The composition of any one of claims 1 to 16, wherein the composition comprises from about 0.1 wt. % to about 30 wt. % water.

18. The composition of any one of claims 1 to 17, wherein the composition comprises from about 1 wt. % to about 50 wt. % polymer.

19. The composition of any one of claims 1 to 18, wherein the wt. % ratio of polymer to plasticizer is from about 1 :4 to about 4:1.

20. The composition of any one of claims 1 to 19, wherein the plasticizer has a molecular weight in the range of about 80 Da to about 2000 Da.

21. The composition of any one of claims 1 to 20, wherein the composition comprises from about 10 wt. % to about 70 wt. % plasticizer.

22. The composition of any one of claims 1 to 21, wherein the plasticizer is selected from the group consisting glycerol, sorbitol, ethylene glycol, polyethylene glycol (PEG), a sugar alcohol, a dextran, and mixtures thereof.

23. The composition of any one of claims 1 to 22, wherein the composition comprises a mechanical strength in the range of from about 0.1 MPa to about

250 MPa.

24. The composition of any one of claims 1 to 23, wherein the composition further comprises an oil phase.

25. The composition of claim 24, wherein the oil phase comprises from about 0.1 wt. % to about wt. 10% oil.

26. The composition of any one of claims 24 to 25, wherein the oil phase comprises lipids selected from the group consisting of fatty acids, waxes, solid fats, liquid fats, and mixtures thereof.

27. The composition of any one of claims 1 to 26, wherein the composition is in the form of an emulsion, a film, a spray coating, a dip coating, or a combination thereof.

28. The composition of claim 27, wherein the film, spray coating or dip coating comprises multiple layers.

29. The composition of claim 27, wherein the emulsion, film, spray coating or dip coating has a thickness in the range of about 100 nm to about 1 mm.

30. The composition of any one of claims 1 to 29, wherein the composition is transparent.

31. The composition of any one of claims 1 to 30, wherein the composition is a film comprising whey protein isolate polymer, glycerol plasticizer and one or more bacteriophages.

32. Food edible by a mammal, wherein the food is wholly or partially coated with a composition of any one of claims 1 to 31.

33. The food of claim 32, wherein the food comprises fresh produce or meat.

34. The food of any one of claims 32 to 33, wherein the composition forms a continuous barrier coating on the food.

35. A medical device or bandage wholly or partially coated with a composition of any one of claims 1 to 31.

36. A container wholly or partially coated with a composition of any one of claims 1 to 31.

37. The container of claim 36, wherein the container is a food container.

38. The container of any one of claims 36 to 37, wherein the container is a beverage container.

39. The container of any one of claims 36 to 38, wherein the container is a plastic container or a paper container.

40. A method of reducing or eliminating bacterial pathogens on food, comprising contacting the food with a composition of any one of claims 1 to 31.

41. The method of claim 40, further comprising the step of contacting the food with an external stimulus to release the viral particles from the composition.

42. The method of claim 41 , wherein the external stimulus is selected from the group consisting of an aqueous solution, moisture, light and biodegradation.