CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority from United States Provisional Patent Application Numbers 61/552,106, filed on October 27, 2011, and 61/439,887, filed on February 6, 2011, the contents of each of which are hereby incorporated by reference in their entirety.

FIELD OF THE INVENTION

[0002] The present invention generally relates to biocidal zeolites, methods of preparation and uses thereof, and articles of manufacture containing them. It relates specifically to biocidal zeolites that are essentially free of antibiotics, antimicrobial metals, and entrapped biocidal substances.

BACKGROUND OF THE INVENTION

[0003] Zeolites are crystalline aluminosilicate minerals with a structure characterized by a framework of linked tetrahedra, each consisting of four O atoms surrounding a wide variety of cations, such as Na ⁺ , K ⁺ , Ca ²⁺ , Mg ²⁺ , etc. These positive ions are rather loosely held and can readily be exchanged for others. The zeolite structure comprises a regular framework surrounding pores that are generally of molecular dimensions. These molecular-sized pores give zeolites the ability to sort molecules selectively based primarily on a size exclusion process, and hence, one of the primary uses of zeolites is as "molecular sieves." The maximum size of the molecular or ionic species that can enter the pores of a zeolite is controlled by the dimensions of the channels. These are conventionally defined by the ring size of the aperture, where, for example, the term "8-ring" refers to a closed loop that is built from 8 tetrahedrally coordinated silicon or aluminum atoms and 8 oxygen atoms. These rings are not always perfectly symmetrical due to a variety of effects, including strain induced by the bonding between units that are needed to produce the overall structure, or coordination of some of the oxygen atoms of the rings to cations within the structure.

[0004] Zeolites comprising cations, salts or oxides of metals such as zinc, silver, and tin, are known biocidal materials. For example, U.S. Pat. No. 4115130 discloses organo-tin zeolites suitable for use in marine anti-fouling coatings. U.S. Pat. No. 5256390 discloses a method of producing zeolite particles with reduced carbonate species so that the zeolite particles are ion exchangeable with biocidal transition metal ions, such as Ag ⁺ , Cu ²⁺ , and Zn ²⁺ . U.S. Pat. Appl. 20100221486 discloses a biocidal zeolite composition comprising an inorganic biocide and at least one organic biocidal compound, wherein the inorganic biocide consists of at least one nanoscale metal oxide selected from ZnO, BaTiC>3, SrTiOs, Ti ⁽ ¾, WO3, AI2O3, CuO, NiO, Zr0 ₂ and MgO. Korean Pat. Nos. KR970000303 and KRO 154964 disclose anti-microbial zeolites that contain a mixture of biocidal metal ions and hydrogen ions.

[0005] These metal-containing zeolites, despite their effectiveness as biocides, possess disadvantages such as the possibility of leaching of the toxic metal ion or salts into the body. These materials may cause skin, eye and respiratory irritations. Silver zeolite, for example, is considered to be a toxic material; the MSDS of commercially available silver-exchanged zeolite, Ag ₈₄ Na ₂ [(A102)86(Si02)io6l · Λ¾0, lists the material as harmful by inhalation and irritating to the eyes and respiratory system.

[0006] European Pat. Appl. No. EP0597695 discloses antimicrobial food packaging containing an ion-exchange material or a percursor to an ion-exchange material that acts to lower the pH of a solid foodstuff.

[0007] Because zeolites are widely found in nature, they tend to be relatively inexpensive. A method for using zeolites as biocides that does not involve incorporation into to the zeolite a biocidal material such a heavy metal or ion or salt thereof would thus be both economically advantageous and of increased safety relative to methods known in the art. A method for using zeolites that are free of such biocidal materials and that also do not significantly alter the pH of the environment in which they are in contact would be of further economic and safety benefit, as well as of benefit in applications such as the food industry in which it is important to maintain the organoleptic properties of the material in which control of the population of microorganisms is desired. Development of such methods, biocidal compositions, and articles of manufacture thus represents a long-felt yet unmet need.

SUMMARY OF THE INVENTION

[0008] Methods are provided herein for controlling the population of microorganisms within a predetermined volume that use zeolites that do not contain significant amounts of leachable heavy metals or ions or salts thereof or other biocidal materials such as antibiotics. It is therefore an object of the present invention to provide a method for controlling the population of microorganisms within a predefined volume, said method comprising: exposing said microorganisms to a biocidal zeolite distributed about at least a portion of the boundary of said predefined volume, wherein the amount of antimicrobial material chosen from the group consisting of heavy metals, ions and salts thereof, antibiotics sequestered within said biocidal zeolite, and antibiotics bound to said biocidal zeolite that can be released into said predefined volume is insufficient to affect the population of microorganisms within said predefined volume, and further wherein said zeolite is substantially free of any material comprising a substituent that acts to kill microorganisms by disruption of the cell membrane following insertion into or binding thereto. In certain embodiments, the methods further comprise the step of distributing said biocidal zeolite about at least a portion of the boundary of said predefined volume. In certain embodiments, the step of distributing comprises a step of disposing said biocidal zeolite on a surface in contact with at least a portion of said predefined volume.

[0009] It is a further object of this invention to disclose such methods, wherein said biocidal zeolite is characterized in that it does not contain an effective amount of any of (a) any exchangeable biocidal ions, (b) any antimicrobial, antifungal, or antiviral substance leachable into said predefined volume, and (c) any surface-bound chemical moiety that can kill a cell by insertion into and consequent disruption of the membrane of said cell.

[0010] It is a further object of this invention to disclose such methods as defined in any of the above, wherein said biocidal zeolite is characterized in that it is substantially free of (a) any exchangeable biocidal ions, (b) any antimicrobial, antifungal, or antiviral substance leachable into said predefined volume, and (c) any surface-bound chemical moiety that can kill a cell by insertion into and consequent disruption of the membrane of said cell.

[0011] It is a further object of this invention to disclose such methods as defined in any of the above, wherein the concentration of antimicrobial material chosen from the group consisting of heavy metals, cations of heavy metals, salts of heavy metals, and antibiotics leached from said biocidal zeolite in said predefined volume does not exceed 1 ppm at any time during the course of said step of exposing said microorganisms to said biocidal zeolite.

[0012] It is a further object of this invention to disclose such methods as defined in any of the above, wherein said biocidal zeolite is an acid form zeolite. In some embodiments, the acid form zeolite has an H ⁺ concentration greater than about 2.5 x 10 ^"4 mol L ^"1 . In some embodiments, the H* concentration within said acid form zeolite is greater than or equal to about 1 meq/g. In some embodiments, the surface pH of said acid form zeolite, as measured by contact with the surface of said acid form zeolite immersed in water, is less than or equal to about 3. In certain embodiments, said acid form zeolite is chosen from the group consisting of mordenite and acid form zeolites prepared from zeolites chosen from the group consisting of β-zeolite, ZSM-23, ZSM-5, zeolite A, and zeolite Y.

[0013] It is a further object of this invention to disclose such methods, further comprising a step of preparing said acid form zeolite by deammoniation of an NH4 ⁺ -form zeolite. In some embodiments, said step of deammoniation of an NH4 ⁺ -form zeolite comprises a step of heating said NHZ-form zeolite at a temperature of between 500 °C and 550 °C until substantially all of the N¾ ⁺ ions within said zeolite have been converted to H ⁺ and NHj, and substantially all of said N¾ has been driven off.

[0014] It is a further object of this invention to disclose such methods as described in any of the above, wherein said biocidal zeolite is a biocidal zeolite in which at least 50% of the exchangeable cations are protons.

[0015] It is a further object of this invention to provide methods for controlling the population of microorganisms within a predefined volume, said method comprising: exposing said microorganisms to a base form biocidal zeolite distributed about at least a portion of the boundary of said predefined volume, wherein the amount of antimicrobial material chosen from the group consisting of heavy metals, ions and salts thereof, antibiotics sequestered within said biocidal zeolite, and antibiotics bound to said biocidal zeolite that can be released into said predefined volume is insufficient to affect the population of microorganisms within said predefined volume, and further wherein said zeolite is substantially free of any material comprising a substituent that acts to kill microorganisms by disruption of the cell membrane following insertion into or binding thereto. In certain embodiments, the methods further comprise the step of distributing said base form biocidal zeolite about at least a portion of the boundary of said predefined volume. In certain embodiments, the step of distributing comprises a step of disposing said base form biocidal zeolite on a surface in contact with at least a portion of said predefined volume. In some embodiments, the H ^" concentration within said basic biocidal zeolite is less than about 10 ^"8 mol L ^"1 .

[0016] It is a further object of this invention to provide methods for controlling the population of microorganisms within a predefined volume, said method comprising: exposing said microorganisms to a biocidal zeolite comprising a mixture of acid form and base form zeolites disposed on at least a portion of a surface in contact with at least a portion of predefined volume, wherein the amount of antimicrobial material chosen from the group consisting of heavy metals, ions and salts thereof, antibiotics sequestered within said biocidal zeolite, and antibiotics bound to said biocidal zeolite that can be released into said predefined volume is insufficient to affect the population of microorganisms within said predefined volume, and further wherein said zeolite is substantially free of any material comprising a substituent that acts to kill microorganisms by disruption of the cell membrane following insertion into or binding thereto. In certain embodiments, the methods further comprise the step of distributing said acid form and base form biocidal zeolites about at least a portion of the boundary of said predefined volume. In certain embodiments, the step of distributing comprises a step of disposing said acid form and base form biocidal zeolites on a surface in contact with at least a portion of said predefined volume. In some embodiments, the ratio of acid form zeolite to base form zeolite is chosen to yield a predetermined total H ⁺ concentration. In some embodiments, said predetermined H ⁺ concentration is about 10 ^"7 mol L ^"1 .

[0017] It is a further object of this invention to disclose the foregoing methods, wherein said biocidal zeolite comprising a mixture of acid form and base form zeolites is distributed about at least a portion the boundary of said predefined volume as macroscopic domains of biocidal zeolite, each of which comprises either an acid form zeolite or a base form zeolite. In some embodiments, the ratio of acid form zeolite to base form zeolite is chosen to yield a predetermined total H ⁺ concentration. In some embodiments, said predetermined H ⁺ concentration is about 10 ^"7 mol L ^"1 .

[0018] It is a further object of this invention to disclose the foregoing methods, wherein said biocidal zeolite comprising a mixture of acid form and base form zeolites comprises a mixture of particles of acid form zeolite and base form zeolite. In some embodiments, the ratio of acid form zeolite to base form zeolite is chosen to yield a predetermined total H ⁺ concentration. In some embodiments, said predetermined H ⁺ concentration is about 10 ^"7 mol L ^" '.

[0019] In some embodiments, the method further comprises a step of preparing a mixture of particles of acid form zeolite and particles of base form zeolite. In some embodiments, said step of preparing a mixture of particles of acid form zeolite and particles of base form zeolite further comprises steps of: preparing an aqueous suspension of a predetermined quantity of particles of zeolite chosen from the group consisting of (a) acid form zeolites and (b) base form zeolites; adding a sufficient quantity of particles of zeolite of the form not chosen in the previous step to bring the pH to a predetermined value, thereby forming a mixed acid/base zeolite suspension; and preparing a mixture of particles of acid form zeolite and particles of base form zeolite with the same weight ratio as found in said mixed acid/base zeolite suspension.

[0020] In some embodiments, the ratio of acid form zeolite to base form zeolite is chosen to yield a predetermined total H* concentration. In some embodiments, said predetermined H ⁺ concentration is about 10 ^"7 mol L ^"1 . In some embodiments, the ratio of acid form zeolite to base form zeolite is chosen to yield a predetermined surface pH. In some embodiments, said surface pH is between about 6 and about 8. In some embodiments, said surface pH is about 7.5.

[0021] It is a further object of this invention to disclose such methods as defined in any of the above, wherein said biocidal zeolite is characterized by a surface charge density of at least about It) ^"10 C/cm ² .

[0022] It is a further object of this invention to disclose such methods as defined in any of the above, wherein said step of exposing said microorganisms to said biocidal zeolite further comprises a step of exposing said microorganisms to said biocidal zeolite such that said microorganisms approach within about 50 nm of the surface of said biocidal zeolite. In some of the foregoing embodiments, said microorganisms approach within about 10 nm of the surface of said biocidal zeolite.

[0023] It is a further object of this invention to disclose such methods as defined in any of the above, wherein said step of exposing said microorganisms to said biocidal zeolite comprises a step of exposing to said biocidal zeolite at least one microorganism selected from the group consisting of Saccharomyces cerevisiae, Zygosucchacomycesrouxii, Byssochalamysfulva, Aspergillusniger, E. coli, Klebsiella pneumonia, Talaromycesflavus, Lactobacillus lactis, Bacillus subtilis, and Aspergillusochraceus.

[0024] It is a further object of this invention to disclose such methods as defined in any of the above, wherein said step of exposing comprises a step of exposing said microorganism to a biocidal zeolite, the properties of which are chosen to control the population of at least one predetermined microorganism.

[0025] It is a further object of this invention to disclose such methods as defined in any of the above, wherein said step of exposing comprises killing at least a portion of said microorganisms.

[0026] It is a further object of this invention to disclose such a method as defined in any of the above, wherein said step of exposing said microorganisms to said biocidal zeolite comprises exposing said microorganisms to said biocidal zeolite until the population of said microorganisms is reduced by a predetermined measure relative to the population of said microorganisms present in said volume prior to the commencement of said step of exposing. In some embodiments, said predetermined amount is at least a 2 log reduction. In some embodiments, said predetermined amount is at least a 5 log reduction.

[0027] It is a further object of this invention to disclose such methods as defined in any of the above, further including a step of maintaining the population of microorganisms within said predetermined volume to within a predetermined measure of its population prior to the commencement of said step of exposing.

[0028] It is a further object of this invention to disclose such methods as defined in any of the above, wherein said biocidal zeolite demonstrates antimicrobial activity as measured by a test method chosen from the group consisting of ISO 22196 and ASTM E2149.

[0029] It is a further object of this invention to disclose such methods, wherein said step of distributing comprises distributing no more than about 8 mg of zeolite per cm ³ of said predetermined volume. In some embodiments, said step of distributing comprises distributing between 0.5 mg and 8 mg of zeolite per cm ³ of said predetermined volume. In some embodiments, step of distributing comprises distributing between 2 mg and 4 mg of zeolite per cm ³ of said predetermined volume.

[0030] It is a further object of this invention to disclose such methods as defined in any of the above, wherein said step of distributing comprises distributing zeolite particles with an average particle diameter of between about 1 and about 3 μηι.

[0031] It is a further object of this invention to disclose such methods defined in any of the above, wherein said step of distributing comprises a step of distributing zeolite particles with an average particle diameter of between about 10 and about 20 μπι.

[0032] It is a further object of this invention to disclose such methods as defined in any of the above, wherein said step of exposing comprising exposing said microorganisms to zeolite with an internal surface area of at least 200 m ^" /g. In some embodiments, said zeolite has an internal surface area of between about 350 m ² /g and about 900 m ² /g.

[0033] It is a further object of this invention to disclose such methods as defined in any of the above, wherein said zeolite has an average pore size of between 0.3 nm and 0.8 nm. [0034] It is a further object of this invention to disclose such methods as defined in any of the above, comprising maintaining the pH to within a predetermined range relative to the pH within said predetermined volume prior to the commencement of said step of exposing, said maintaining occurring at least during part of the time during which said step of exposing takes place. In some embodiments, said range is ±1 pH units. In some embodiments, said range is +0.6 pH units. In some embodiments, said range is ±0.3 pH units.

[0035] It is a further object of this invention to disclose such methods as defined in any of the above, comprising maintaining the pH within said predetermined volume at substantially the same value as the pH within said predetermined volume prior to the commencement of said step of exposing, said maintaining occurring at least during part of the time during which said step of exposing takes place.

[0036] It is a further object of this invention to disclose such methods as defined in any of the above, wherein said biocidal zeolite has a Si/Al ratio of between about 3 and about 50. In some embodiments, said biocidal zeolite has a Si/Al ratio of between about 5 and about 20.

[0037] It is a further object of this invention to disclose such methods as defined in any of the above, further comprising a step of introducing an aqueous environment within said predefined volume. In some embodiments, the methods further comprise a step of buffering said aqueous environment. In some embodiments, the methods further comprise a step of buffering said aqueous environment to a pH of within about 0.5 pH units of the pH immediately prior to said step of exposing.

[0038] It is a further object of this invention to disclose such methods as defined in any of the above, wherein said step of exposing comprises a step of exposing said microorganisms indirectly to said biocidal zeolite.

[0039] It is a further object of this invention to disclose such methods as defined in any of the above, wherein said step of exposing comprises at least one step chosen from the group consisting of (a) shaking said predetermined volume; (b) inverting said predetermined volume; (c) stirring the material enclosed in said predetermined volume.

[0040] It is a further object of this invention to disclose such methods as defined in any of the above, further comprising a step of immobilizing said biocidal zeolite in a polymer matrix, thereby forming a zeolite/polymer matrix. In some embodiments, said step of immobilizing said biocidal zeolite in a polymer matrix is performed prior to distributing said biocidal zeolite about at least a portion of the boundary of predetermined volume. In some embodiments, said zeolite/polymer matrix contains at least 60% zeolite by weight. In certain embodiments, said zeolite/polymer matrix contains at least 70% zeolite by weight. In other embodiments, said zeolite/polymer matrix contains at least 75% zeolite by weight.

[0041] It is a further object of this invention to disclose such methods, wherein said step of immobilizing said biocidal zeolite in a polymer matrix comprises immobilizing said biocidal zeolite in a polymer matrix made from a polymer chosen from the group consisting of ethylene vinyl acetate; low density polyethylene; high density polyethylene; polypropylene; cellulose; cellulose derivatives; polyalkanoates; polyethylene terephthalate; polyvinyl alcohol; ethylene vinyl alcohol; polyethylene glycol; acrylics; polyesters; polyamides; polyacrylates; polycarbonates; other thermoplastic polymers; and copolymers and blends of any of the above.

[0042] It is a further object of this invention to disclose such methods, wherein said step of immobilizing said biocidal zeolite in a polymer matrix comprises immobilizing said biocidal zeolite in a polymer matrix such that said matrix at least partially covers said zeolite.

[0043] It is a further object of this invention to disclose such methods, wherein said step of immobilizing said biocidal zeolite in a polymer matrix comprises a step of forming, by a method chosen from the group comprising extruding, doping, coating, immersing, pressing, and encapsulating, a polymer matrix in which said biocidal zeolite is immobilized.

[0044] It is a further object of this invention to disclose such methods, wherein said step of immobilizing said biocidal zeolite in a polymer matrix comprises a step of forming by extruding a polymer matrix in which said biocidal zeolite is immobilized.

[0045] It is a further object of this invention to disclose such methods, further comprising a step of providing a second layer in contact with said zeolite/polymer matrix, said second layer comprising a polymeric material.

[0046] It is a further object of this invention to disclose such methods, wherein said step of providing a second layer comprises providing a second layer comprising a polymer chosen from the group consisting of ethylene vinyl acetate, low-density polyethylene, polyethylene terephthalate, and polypropylene.

[0047] It is a further object of this invention to disclose such methods, further comprising a step of coextruding a layer comprising said zeolite/polymer matrix with a second layer comprising a polymeric material. [0048] It is a further object of this invention tb disclose such methods, wherein step of immobilizing said biocidal zeolite in a polymer matrix comprises immobilizing said biocidal zeolite in a polymer matrix such that the resulting product is in the form of a film of a thickness of not more than about 200 μπι.

[0049] It is a further object of this invention to disclose such methods, wherein said step of disposing comprises a step of disposing the biocidal zeolite immobilized in a polymer matrix formed in said step of immobilizing.

[0050] It is a further object of this invention to disclose such methods, further comprising a step of incorporating said zeolite/polymer matrix into at least a part of the material enclosing said predefined volume.

[0051] It is a further object of this invention to disclose such methods as defined in any of the above, wherein said zeolite is immobilized in a zeolite/polymer matrix, and maintaining the pH within said predetermined volume to within a predetermined range relative to the pH within said predetermined volume prior to the commencement of said step of exposing, said maintaining occurring at least during part of the time during which said step of exposing takes place. In some embodiments, said range is +1 pH units. In some embodiments, said range is ±0.6 pH units. In some embodiments, said range is ±0.3 pH units.

[0052] It is a further object of this invention to disclose such methods as defined in any of the above, wherein said zeolite is immobilized in a zeolite/polymer matrix, and maintaining the pH within said predetermined volume at substantially the same value as the pH within said predetermined volume prior to the commencement of said step of exposing, said maintaining occurring at least during part of the time during which said step of exposing takes place.

[0053] It is a further object of this invention to disclose such methods, wherein said zeolite/polymer matrix is characterized by a predetermined value of at least one property chosen from the group consisting of (a) buffer capacity; (b) total¥ concentration; (c) rate of exchange of IT with ions within said predetermined volume; (d) rate of release of H ⁺ ions into said predetermined volume; (e) rate of uptake of H ⁺ ions from said volume, whereby said predetermined range is fixed by at least one of (a) said predetermined quantity and (b) said predetermined value.

[0054] It is a further object of this invention to disclose such methods as defined in any of the above, further comprising a step of disposing on at least a portion of the surface within said predetermined volume an ionomer. In some embodiments, said ionomer is chosen from the group consisting of polystyrenesulfonic acid, sulfonated tetrafluoroethylene copolymer, derivatives of sulfonated tetrafluoroethylene, polyacrylamide-immobilines, agarose- immobilines, cationic polyurethane, poly(diethylaminoethyl acrylate), ion exchange beads, and any polymer containing at least one functional group chosen from the group consisting of sulfonic acid, phosphonic acid, quaternary amine, tertiary amine, and derivatives thereof.

[0055] It is a further object of this invention to disclose such methods as defined in any of the above, further comprising disposing said biocidal zeolite on a substrate. In some embodiments, said step of disposing said biocidal zeolite on a substrate comprises disposing on a substrate made of a material chosen from the group consisting of cardboard, wood, plastic, metal, and glass. In some embodiments, said biocidal zeolite is disposed upon said substrate by a method chosen from the group consisting of doping, gluing, spraying, coating, immersing, and co-extruding.

[0056] It is a further object of this invention to disclose such methods, further comprising disposing said biocidal zeolite immobilized in a polymer matrix on a substrate. In some embodiments, said step of disposing said biocidal zeolite immobilized in a polymer matrix on a substrate comprises disposing said biocidal zeolite immobilized in a polymer matrix on a substrate made of a material chosen from the group consisting of cardboard, wood, plastic, metal, and glass. In some embodiments, said layer is disposed upon said substrate by a method chosen from the group consisting of doping, gluing, spraying, coating, immersing, and co-extruding.

[0057] It is a further object of this invention to disclose such methods as defined in any of the above, wherein said step of exposing comprises exposing microorganisms to a biocidal zeolite disposed about at least a portion of a surface of an insert placed or to be placed within said predefined volume.

[0058] It is a further object of this invention to disclose such methods, wherein said step of distributing said biocidal zeolite about at least a portion of the boundary of said predefined volume comprises a step of disposing biocidal zeolite on at least a portion of the surface of an insert placed or to be placed within said predetermined volume. It is a further object of this invention to disclose such methods, wherein said insert is placed or to be placed within said volume such that at least a portion of said biocidal zeolite is within a predetermined distance of said predetermined volume. In some embodiments, said predetermined distance is about 50 nm. In some embodiments, said predetermined distance is about 10 nm.

[0059] It is a further object of this invention to disclose such methods, wherein said step of exposing comprises a step of exposing said microorganisms indirectly to said biocidal zeolite.

[0060] It is a further object of this invention to disclose such methods wherein said biocidal zeolite is disposed on at least a portion of an insert, and maintaining the pH to within a predetermined range relative to the pH within said predetermined volume prior to the commencement of said step of exposing, said maintaining occurring at least during part of the time during which said step of exposing takes place. In some embodiments, said range is ±1 pH units. In some embodiments, said range is +0.6 pH units. In some embodiments, said range is +0.3 pH units.

[0061] It is a further object of this invention to disclose such methods wherein said biocidal zeolite is disposed on at least a portion of an insert, and maintaining the pH within said predetermined volume at substantially the same value as the pH within said predetermined volume prior to the commencement of said step of exposing, said maintaining occurring at least during part of the time during which said step of exposing takes place.

[0062] In some embodiments, the zeolite/polymer matrix is characterized by a predetermined value of at least one property chosen from the group consisting of (a) buffer capacity; (b) total H ^" concentration; (c) rate of exchange of H ⁺ with ions within said predetermined volume; (d) rate of release of H ⁺ ions into said predetermined volume; (e) rate of uptake of H ⁺ ions from said volume.

[0063] It is a further object of this invention to disclose the use of a zeolite for the control of the population of microorganisms within a predetermined volume, wherein said zeolite is characterized in that it does not contain an effective amount of (a) any exchangeable biocidal ions, (b) any antimicrobial, antifungal, or antiviral substance leachable into said predefined volume, and (c) any surface-bound chemical moiety that can kill a cell by insertion into and consequent disruption of the membrane of said cell.

[0064] It is a further object of this invention to disclose the use of a zeolite as defined above, wherein said zeolite is substantially free of (a) any exchangeable biocidal ions, (b) any antimicrobial, antifungal, or antiviral substance leachable into said predefined volume, and (c) any surface-bound chemical moiety that can kill a cell by insertion into and consequent disruption of the membrane of said cell. [0065] It is a further object of this invention to disclose antimicrobial zeolite compositions, comprising at least one zeolite, wherein said composition is not contain an effective amount of any of (a) any exchangeable biocidal ions, (b) any antimicrobial, antifungal, or antiviral substance leachable into said predefined volume, and (c) any surface-bound chemical moiety that can kill a cell by insertion into and consequent disruption of the membrane of said cell.

[0066] It is a further object of this invention to disclose such compositions, wherein said composition is substantially free of (a) any exchangeable biocidal ions, (b) any antimicrobial, antifungal, or antiviral substance leachable into said predefined volume, and (c) any surface- bound chemical moiety that can kill a cell by insertion into and consequent disruption of the membrane of said cell.

[0067] It is a further object of this invention to disclose such compositions as defined in any of the above, wherein said antimicrobial zeolite comprises an acid form zeolite. In some embodiments, the H ⁺ concentration within said composition is greater than or equal to about 2.5 x 10 ^'4 mol L ^'1 . In some embodiments, the H ⁺ concentration is greater than or equal to about 1 meq/g. In some embodiments, the surface pH of said composition, as measured by contact with the surface of said acid form zeolite immersed in water, is less than or equal to about 3. In some embodiments, wherein said acid form zeolite is chosen from the group consisting of mordenite and acid form zeolites prepared from zeolites chosen from the group consisting of β-zeolite, ZSM-23, ZSM-5, zeolite A, and zeolite Y.

[0068] It is a further object of this invention to disclose such compositions, wherein said acid form zeolite is prepared by heating an NH form zeolite at a temperature of between 500 °C and 550 °C until substantially all of the NH ions within said zeolite have been converted to H ⁺ and Ν¾, and substantially all of said N¾ has been driven off.

[0069] It is a further object of this invention to disclose such compositions, wherein at least 50% of the exchangeable cations within said zeolite are protons.

[0070] It is a further object of this invention to disclose antimicrobial zeolite compositions, comprising at least one base form zeolite, wherein said composition is not contain an effective amount of any of (a) any exchangeable biocidal ions, (b) any antimicrobial, antifungal, or antiviral substance leachable into said predefined volume, and (c) any surface- bound chemical moiety that can kill a cell by insertion into and consequent disruption of the membrane of said cell. It is a further object of this invention to disclose such compositions, wherein said composition is substantially free of (a) any exchangeable biocidal ions, (b) any antimicrobial, antifungal, or antiviral substance leachable into said predefined volume, and (c) any surface-bound chemical moiety that can kill a cell by insertion into and consequent disruption of the membrane of said cell. In some embodiments, the H ⁺ concentration within said composition is less than or equal to about 10 ^"8 mol L ^"1 .

[0071] It is a further object of this invention to disclose antimicrobial zeolite compositions, comprising at least one acid form zeolite and at least one base form zeolite, wherein said composition is not contain an effective amount of any of (a) any exchangeable biocidal ions, (b) any antimicrobial, antifungal, or antiviral substance leachable into said predefined volume, and (c) any surface-bound chemical moiety that can kill a cell by insertion into and consequent disruption of the membrane of said cell. It is a further object of this invention to disclose such compositions, wherein said composition is substantially free of (a) any exchangeable biocidal ions, (b) any antimicrobial, antifungal, or antiviral substance leachable into said predefined volume, and (c) any surface-bound chemical moiety that can kill a cell by insertion into and consequent disruption of the membrane of said cell. In some embodiments, the ratio of acid form zeolite to base form zeolite is chosen to yield a predetermined total H ⁺ concentration. In some embodiments, said predetermined H ⁺ concentration is about 10 ^"7 mol L ^"1 . In some embodiments, the ratio of acid form zeolite to base form zeolite is chosen to yield a predetermined surface pH. In some embodiments, said predetermined surface pH is between about 6 and about 8. In some embodiments, said predetermined surface pH is about 7.5.

[0072] It is a further object of this invention to disclose such compositions, wherein said composition is formed from a mixture of acid form zeolite particles and base form zeolite particles. In some embodiments, the mixture of acid form zeolite particles and base form zeolite particles is prepared by a method comprising steps of: preparing an aqueous suspension of a predetermined quantity of particles of acid form zeolite; adding a sufficient quantity of particles of base form zeolite to raise the pH to a predetermined value, thereby forming a mixed acid/base zeolite suspension; preparing a mixture of particles of acid form zeolite and particles of base form zeolite with the same weight ratio as found in said mixed acid/base zeolite suspension.

[0073] It is a further object of this invention to disclose such compositions as defined in any of the above, wherein said antimicrobial zeolite has a surface charge density of at least about 10 ^'10 C/cm ² . [0074] It is a further object of this invention to disclose such compositions as defined in any of the above, characterized, when in contact with a medium containing microorganisms by a surface electric field that produces an electric field gradient of at least 10 ⁴ V/cm extending at least 50 nm into said medium.

[0075] It is a further object of this invention to disclose such compositions as defined in any of the above, comprising a zeolite chosen from the group consisting of (a) acid form zeolites, (b) base form zeolites, and ^' (c) mixtures thereof, wherein said zeolite has an average particle diameter of between 1 μηα and 3 μπι.

[0076] It is a further object of this invention to disclose such compositions as defined in any of the above, comprising a zeolite chosen from the group consisting of (a) acid form zeolites, (b) base form zeolites, and (c) mixtures thereof, wherein said zeolite has an average particle diameter of between 10 μπι and 20 μπι.

[0077] It is a further object of this invention to disclose such compositions as defined in any of the above, comprising a zeolite chosen from the group consisting of (a) acid form zeolites, (b) base form zeolites, and (c) mixtures thereof, wherein said zeolite is characterized by an average pore size of between 0.3 nm and 0.8 nm.

[0078] It is a further object of this invention to disclose such compositions as defined in any of the above, comprising a zeolite chosen from the group consisting of (a) acid form zeolites, (b) base form zeolites, and (c) mixtures thereof, wherein said zeolite has an internal surface area of at least 200 m ² /g. In some embodiments, said zeolite has an internal surface area of between about 350 m ² /g and about 900 m ² /g.

[0079] It is a further object of this invention to disclose such compositions as defined in any of the above, wherein at least one property chosen from the group consisting of (a) buffer capacity; (b) total H ⁺ concentration; (c) rate of exchange of H ⁺ with ions within said predetermined volume; (d) rate of release of H ⁺ ions into said predetermined volume; and (e) rate of uptake of H ⁺ ions from said volume is chosen such that when said composition is in contact with an aqueous environment, the pH of said aqueous environment remains within a predetermined range for at least a predetermined time. In some embodiments, said range is ±1 pH unit. In some embodiments, wherein said range is ±0.6 pH unit.

[0080] It is a further object of this invention to disclose such compositions as defined in any of the above, wherein said zeolite has a Si/Al ratio of between about 3 and about 50. In some embodiments, the Si/Al ratio is between about 5 and about 20. [0081] It is a further object of this invention to disclose such compositions as defined in any of the above, wherein said compositions comprised a zeolite/polymer matrix formed by immobilizing said biocidal zeolite in a polymer matrix. In some embodiments, said zeolite/polymer matrix is at least 60% zeolite by weight. In some embodiments, said zeolite/polymer matrix is at least 70% zeolite by weight. In some embodiments, said zeolite/polymer matrix is at least 75% zeolite by weight. In some embodiments, said polymer matrix is made from a polymer chosen from the group consisting of ethylene vinyl acetate; low density polyethylene; high density polyethylene; polypropylene; cellulose; cellulose derivatives; polyalkanoates; polyethylene terephthalate; polyvinyl alcohol; ethylene vinyl alcohol; polyethylene glycol; acrylics; polyesters; polyamides; poly aery lates; polycarbonates; other thermoplastic polymers; and copolymers and blends of any of the above. In some embodiments, said zeolite/polymer matrix is in the form of a film of thickness of not more than about 200 μπι. In some embodiments, wherein said polymer matrix is formed by a method chosen from the group consisting of extruding, doping, coating, immersing, pressing, and encapsulating.

[0082] It is a further object of this invention to disclose such compositions as defined in any of the above, further comprising a second layer comprising a polymeric material. In some embodiments, said second layer comprises a polymer chosen from the group consisting of ethylene vinyl acetate, low-density polyethylene, polyethylene terephthalate, and polypropylene. In some embodiments, said composition is formed by coextrusion of said layer comprising said zeolite/polymer matrix with said second layer comprising a polymeric material.

[0083] It is a further object of this invention to disclose such compositions as defined in any of the above, further comprising an ionomer. In some embodiments, said ionomer is chosen from the group consisting of polystyrenesulfonic acid, sulfonated tetrafluoroethylene copolymer, derivatives of sulfonated tetrafluoroethylene, polyacrylamide-immobilines, agarose-immobilines, cationic polyurethane, poly(diethylaminoethyl acrylate), ion exchange beads, and any polymer containing at least one functional group chosen from the group consisting of sulfonic acid, phosphonic acid, quaternary amine, tertiary amine, and derivatives thereof.

[0084] It is a further object of this invention to disclose such compositions as defined in any of the above, wherein said biocidal zeolite is disposed on a substrate. In some embodiments, said substrate comprises a material chosen from the group consisting of cardboard, wood, plastic, metal, and glass. In some embodiments, said biocidal zeolite is disposed upon said substrate by a method chosen from the group consisting of doping, gluing, spraying, coating, immersing, and co-extruding.

[0085] It is a further object of this invention to disclose such compositions, wherein said biocidal zeolite is immobilized in a polymer matrix and is disposed on a substrate. In some embodiments, said substrate comprises a material chosen from the group consisting of cardboard, wood, plastic, metal, and glass. In some embodiments, said biocidal zeolite is disposed upon said substrate by a method chosen from the group consisting of doping, gluing, spraying, coating, immersing, and co-extruding.

[0086] It is a further object of this invention to disclose such compositions as defined in any of the above, wherein said biocidal zeolite is disposed on an insert adapted to be placed inside a container.

[0087] It is contemplated that whenever appropriate, any embodiment of the present invention can be combined with one or more other embodiments of the present invention, even though the embodiments are described under different aspects of the present invention.

[0088] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

[0089] Exemplary embodiments of the invention will now be described with reference to the drawings, wherein:

[0090] FIG. 1 illustrates the surface electric potential at a charged zeolite surface according to one embodiment of the invention;

[0091] FIG. 2 presents a graph showing the dependence of the antimicrobial activity of a zeolite powder as a function of the deammoniation temperature; and, [0092] FIG. 3 presents a histogram showing the relative efficacies of untreated and treated LDPE bottles in controlling the microbial concentration in a sample of milk.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0093] In the following description, various aspects of the invention will be described. For the purposes of explanation, specific details are set forth in order to provide a thorough understanding of the invention. It will be apparent to one skilled in the art that there are other embodiments of the invention that differ in details without affecting the essential nature thereof. Therefore the invention is not limited by that which is illustrated in the figures and described in the specification, but only as indicated in the accompanying claims, with the proper scope determined only by the broadest interpretation of said claims.

[0094] As used herein, with reference to zeolites used in embodiments of the invention herein disclosed, the term "biocidal zeolite" refers to a zeolite that exhibits biocidal properties but that does not incorporate substances other than protons that can act to kill or slow the reproduction of microorganisms. Non-limiting examples of such substances (i.e. examples of substances not found in the "biocidal zeolites" used in the present invention) include heavy metals, ions or salts thereof, charged substituents (e.g. cationic amino groups) that act to disrupt the cellular membrane, and antibiotics.

[0095] As used herein, the term "microorganism" refers hereinafter to any organism of microscopic size. While preferred embodiments of the invention are directed specifically to means for killing pathogenic microorganisms, the term as used herein is not limited to any particular type of microorganism. Non-limiting examples of microorganisms as the term is used herein include both prokaryotic and eukaryotic microorganisms, such as bacteria, protozoan, fungi, molds, yeasts, etc., as well as to viruses.

[0096] As used herein, the term "substantially free of a particular substance is used herein to define the substance as being present in a concentration of less than 1 ppm. Similarly, the term "substantially all" refers to a form is 99.99999% pure, i.e., one that comprises less than 1 ppm impurities.

[0097] As used herein, the term "about" refers hereinafter to a value of ±30% of the defined measure.

[0098] As used herein, the term "heavy metal" refers hereinafter to any metallic or semi- metallic element other than the alkalis, alkaline earths, aluminum, or silicon. The term also refers to mixtures, compounds, and alloys of such metals. Non-limiting examples of "heavy metals" as the term is used herein that are typically used in biocidal compositions include silver, zinc, tin, and copper.

[0099] Unless the form is specifically described otherwise, the term "metal" refers hereafter to a metal in any form, including but not limited to metal atoms, particles of any size comprising a metal, macroscopic pieces of metal, metal ions, metal complexes, organometallic compounds, and metal salts.

[0100] As used herein, with reference to the approach of a microorganism to the materials disclosed in embodiments of the present invention, the term "surface" refers to any part of the material to which the microorganism can approach sufficiently closely (in preferred embodiments, within about 50 nm; in the most preferred embodiments, within about 10 nm) that the biocidal effect of the material is observed. In this context, the term does not necessarily refer to the internal surface of the zeolite, since in most embodiments of the invention, the pores and interior channels of the material are too small to allow microorganisms to enter within.

[0101] As used herein, with reference to an interaction between a microorganism and one of the compositions of the present invention, the terms "contact" and "exposure" refer to any interaction by which the microorganism is affected by the surface charge of the composition. The contact may be direct physical contact, but it can also be indirect. As a non-limiting example of an indirect contact, the zeolite may be enclosed within a polymer, but as long as the microorganism can approach sufficiently closely such that its intracellular processes are affected by the electric field created by the surface charge, it is considered to have "contacted" or "been exposed to" the zeolite in this sense. Typically, a microorganism is affected by the surface electric field at distances on the order of tens of nanometers. Indirect contact may also be made by ion exchange or proton transfer to or from the surface.

[0102] As used herein, the term "ionomer" refers to a polymer made from monomers at least some of which carry a charge.

[0103] As used herein, the term "effective amount" refers to the minimum amount of a substance necessary to produce a desired effect. As a non-limiting example, an "effective amount" of an antibiotic would be the minimum needed to control a bacterial population to within some preset limit.

[0104] As used herein, with respect to a biocidal zeolite, the term "distributed about the boundary" of a volume within which the population of microorganisms is to be controlled refers to placement of the zeolite at a location sufficiently close to the volume that a microorganism located within can approach sufficiently close to the zeolite to experience a biocidal effect. Non-limiting examples of "distribution" of the zeolite as the term is used herein include disposing zeolite on the surface bounding the volume, disposing zeolite on or within the material enclosing the volume, and disposing zeolite on or within a matrix in contact with the boundary of the volume. The zeolite is considered to be "distributed about the boundary of the volume" even in the presence of material intervening between the zeolite and the volume, as long as microorganisms within the volume can approach sufficiently closely to experience the biocidal effect of the zeolite. The volume within which the population of microorganisms is to be controlled may be defined by the physical boundary surfaces of the container that holds the medium in which the microorganisms are found. It may also be defined by any other method that is convenient to the particular application to which the methods and materials herein disclosed are to be put.

[0105] Embodiments of the present invention provide methods for control of the population of microorganisms within a predefined volume that uses biocompatible and highly effective biocidal zeolites. The methods comprise exposing the microorganism to a biocidal zeolite disposed on at least a portion of a surface in contact with at least a portion of the predefined volume. In some embodiments, the methods further comprise disposing the biocidal zeolite the portion of the surface within the predetermined volume in which control of the microorganisms is desired. In some embodiments of the invention, the population of at least one species of microorganism is eradicated from within the predefined volume. In other embodiments, the population of at least one species of microorganism is controlled such that it does not exceed a predetermined level within a predetermined amount of time following the initial exposure of the medium within the volume to the biocidal zeolite.

[0106] Without wishing to be bound by theory, one explanation for the activity of the biocidal zeolites and methods for using them disclosed herein is as follows. When the microorganism approaches the highly charged surface of the biocidal zeolite, it experiences a local electrical potential gradient that disrupts at least one internal cellular process, thereby killing the cell. The electric field at the surface of the highly charged zeolite is created by ions in the vicinity of the surface. Reference is now made to FIG. 1A, which presents a schematic illustration of the origin and nature of this electric field. Region 1 in the figure represents a negatively charged polymer submerged in a beverage. A negative charge will develop on the polymer surface. This charged surface will attract oppositely charged ions (positive), which will be immobilized near the surface (layer 2, known as the "Stern layer" or double layer). This charged layer is followed by a transition layer in which the net charge is still positive (layer 3, diffusion layer) and gradually converts into the electrically neutral net composition of the bulk material contained within the predetermined volume (layer 4).

[0107] Reference is now made to FIG. IB, which presents the associated surface electric potential. The horizontal axis in FIG. IB represents the distance from the polymer surface. The electric potential and the associated electric field extend to a range of ~ 50 nm. It is this region in which the electric field will interact by Coulombic interaction with the electrical charges on the cellular membrane of microbes at close distances. The magnitude of the surface electric field can exceed values of on the order of 10 ⁴ V/cm, depending on bulk charge density concentration. Electric fields of this magnitude are known to generate irreversible changes and damage when acting on a cellular membrane.

[0108] Since the methods involve physical interactions between the microbe and the zeolite surface, the methods and materials disclosed herein demonstrate significant biocidal activity and effects even in the absence of a biocide such as an antibiotic or Ag ⁺ that is released from the zeolite into the environment; sequestration of material (e.g. biologically important ions such as Fe ³⁺ ) by the zeolite; or of reactive substituents (e.g. -NR.3 ⁺ groups) that insert into and thereby disrupt the cell membrane. The biocidal zeolites used in the methods herein disclosed are thus substantially free of leachable heavy metals, in particular copper, zinc, and silver, and cations or salts thereof. They are also free of other antimicrobial material such as antibiotics, as well as of substances containing substituents that disrupt the cell membrane by inserting into it or binding to it (e.g. such as cationic amino groups).

[0109] Furthermore, since the methods involve physical interactions between the microbe and the zeolite surface rather than chemical interactions between the zeolite and the microbe or the medium in which it is found, in preferred embodiments of the invention, the zeolite can be chosen for a specific use such that the pH of the environment or medium within the predetermined volume is not significantly affected during the time that it is exposed to the zeolite. In typical embodiments of the invention, the pH remains within 1 pH unit of its initial value (i.e. the pH before the zeolite was added) during the time that the exposure of the medium within the volume to the zeolite takes place. In preferred embodiments of the invention, the pH remains within 0.6 pH units of its initial value during this time. In more preferred embodiments of the invention, the pH remains within 0.3 pH units of its initial value during this time. In the most preferred embodiments of the invention, during the time of exposure to or contact with the biocidal zeolite, the pH of the environment exposed to or in contact with the zeolite does not change substantially from its value prior to the beginning of said exposure or contact.

[0110] The material contained within the volume can be any material capable of contact with the biocidal zeolites. As non-limiting examples of typical applications, the predefined volume may contain a liquid or suspension (e.g. an aqueous solution or suspension such as a beverage); a semi-liquid (e.g. a comestible such as yogurt or sour cream); a solid (e.g. a perishable comestible such as meat); or a powder or other particulate material. The volume can be bounded by a container made of any appropriate material known in the art. Non- limiting examples of acceptable container materials or linings providing the boundary surface to the predetermined volume include plastic, glass, metal, wood, cardboard, and paper. The particular container or lining material used will depend on the nature of the materials constituting the environment within the predetermined volume. One of ordinary skill in the art will readily recognize that the methods and materials herein disclosed may be adapted for use with any container or lining material known in the art.

[0111] Non-limiting examples of methods for performing a step of distributing biocidal zeolite about the boundary of the volume inside of which the population of microorganisms is to be controlled include disposing the zeolite on the interior of the surface bounding the volume or a part thereof, incorporating the zeolite into a polymer matrix and attaching the matrix to the interior surface, incorporating a zeolite/polymer matrix into the surface itself (e.g. the wall of the container is constructed at least in part of the zeolite/polymer matrix), incorporating the zeolite or zeolite/polymer matrix into or onto an insert that is then placed in the predetermined volume, etc.

[0112] In embodiments in which the zeolite is disposed on the interior of the surface containing the volume in which the bacterial population is to be controlled, the preferred thickness of the coating of zeolite depends on the material on which it is disposed. In embodiments in which the container is made from PET, the thickness of the zeolite layer is preferably -100 μπι; for HDPE containers, the thickness of the zeolite layer is preferably between about 30 and about 100 μηα; for cardboard containers, the thickness of the zeolite layer is preferably between about 20 and about 100 μπι; and for inserts in bottle caps, the thickness of the zeolite layer is preferably between about 2 and about 3.5 mm. [0113] In typical non-limiting embodiments in which the zeolite is incorporated into a polymer matrix, it is incorporated into a polymer film which in typical embodiments is -200 μπι thick. Methods for making zeolite/polymer films (e.g. by extrusion) are well-known in the art. Because the methods disclosed herein depend on the exposure of the microbe to a surface electric field gradient, the methods require higher concentrations of zeolite in the zeolite/polymer films in order to produce a sufficiently intense electric field gradient at the surface than are commonly used in the art. In typical embodiments, the zeolite/polymer films are at least 60% by weight zeolite. In preferred embodiments, the zeolite/polymer films are at least 70% by weight zeolite. In the most preferred embodiments, the zeolite/polymer films are at least 75% by weight zeolite.

[0114] The microorganisms are then exposed to the biocidal zeolite. In general, "exposure" consists of the microorganism closely approaching the zeolite, at which point the microorganism is killed by interaction with the charged surface of the zeolite. In typical embodiments, exposure consists of the microorganism approaching to within about 50 nm of the surface. In preferred embodiments, exposure consists of the microorganism approaching to within about 10 nm of the surface. This step may be performed by allowing the natural motions of the microorganisms to carry them into proximity of the zeolite. Alternatively, in order to lessen the time needed to expose a significant fraction of the microbial population to the zeolite, it is possible to speed up the time necessary to achieve the desired level of control of the microbial population by physical manipulation of the container enclosing volume (by shaking, inverting, etc.) or of the material within the volume (e.g. by stirring), thus increasing the likelihood that a microorganism within any given sub- volume will be brought sufficiently near to the biocidal zeolite to be affected by it.

[0115] Since the activity of the zeolite does not depend on its liberating antimicrobial substances (e.g. Ag ⁺ ) into the volume, nor does it depend on an interaction between a cell and a substance found within the zeolite or bound to its surface (e.g. charged substituents bound to the surface that can disrupt a cellular membrane upon insertion), the exposure can consist of indirect contact. As long as the cell approaches the charged surface of the zeolite to within a certain necessary distance (typically on the order of tens of nm), the zeolite will act to kill the cell. Thus, in some embodiments of the invention, rather than exposing the microorganisms to the zeolite directly, the zeolite is immobilized in a polymer matrix such that at least a portion of the zeolite is within this distance of the surface of the matrix. A microorganism that approaches the surface of the matrix will thus experience the charged surface of the zeolite and is thus killed by this indirect exposure. Non-limiting examples of microorganisms that can be treated in this manner include Saccharomyces cerevisiae, Zygosacchacomycesrouxii, Byssochalamysfulva, Aspergillusniger, E. coli, Klebsiella pneumonia, Talaromycesflavus, Lactobacillus lac t is, Bacillus subtilis, and Aspergillusochraceus.

[0116] In some preferred embodiments of the invention, the biocidal zeolites distributed about the boundary of the volume inside of which the population microorganisms is to be controlled are in the "acid form," in which at least some of the cations outside the zeolite framework have been exchanged by protons. "Acid form" zeolites include such naturally- occurring zeolites as mordenite. Acid form zeolites are readily commercially available, and methods for preparing them (e.g. by ion exchange of Na ⁺ in Na ⁺ -form zeolites with N¾ ⁺ followed by heating to drive off NH3) are well-known in the art. In preferred embodiments in which the biocidal zeolite is in the acid form, the H ⁺ concentration within the zeolite is outside the range of viability of most pathogenic microorganisms. In preferred embodiments of the invention, at least 50% of the exchangeable ions in the zeolite used are protons. In more preferred embodiments in which the biocidal zeolite is in acid form, the H ⁺ concentration within the zeolite is at least about 2.5 x 10 ^"4 mol L ^"1 (pH < ~3.6); in yet more preferred embodiments in which the biocidal zeolite is in acid form, the H ⁺ concentration is at least about 10 ^"3 mol L ^"1 (pH < ~3). In the most preferred embodiments in which the biocidal zeolite is in the acid form, the H ⁺ concentration is equal to or greater than about 1 meq/g.

[0117] It is once again emphasized that in these embodiments, the pH of the medium itself is not necessarily lowered by contact with the acid zeolite, and that the biocidal effect does not depend on any changes in the bulk pH within the volume.

[0118] In preferred embodiments in which the acid form zeolite is prepared by heating of an NH4 ⁺ -form zeolite to drive off N¾, deammoniation is essentially complete, as can be determined by NMR. In the most preferred embodiments in which the acid form zeolite is prepared by heating of an NH ₄ ⁺ -form zeolite, the zeolite is heated to between 500 and 550 °C. Reference is now made to FIG. 2, which presents a graph of the survival rate of S. cerevisiae exposed to an acid-form zeolite prepared by heating of NI¾ ⁺ -zeolite-beta-25 powder as a function of the temperature at which the zeolite was heated to drive off the NH ₃ . As can be seen in the figure, the maximum effectiveness as measured by the lowest survival rate occurred when acid-form zeolite was used in which the NHV-form zeolite was heated to between 500 and 550 °C. Without wishing to be bound by theory, it appears that heating to lower temperatures is insufficient to convert all of the NH ₄ ⁺ to N¾ + H ⁺ , while heating above the optimum temperature causes structural changes in the zeolite that reduce is effectiveness.

[0119] The concentration of protons at the surface of a biocidal zeolite can be estimated by making surface pH measurements at contact performed on the zeolite immersed in doubly distilled water. In preferred embodiments of the invention in which acid form zeolites are used and the medium with which the zeolite is in contact is not strongly acidic (e.g. for beverages such as milk, which has a typical pH of about 6.5), the surface pH of the acid zeolite, as measured by this method, is less than or equal to about 4.5. In the most preferred embodiments of the invention in which acid form zeolites are used and the medium with which the zeolite is in contact is significantly acidic (e.g. tea or juice in which the pH is typically about 3), the surface pH of the acid form zeolite, as measured by this method, is less than 3.

[0120] In other preferred embodiments of the invention, the biocidal zeolites are in the "base form," in which the zeolite is a Lewis base. Base form zeolites are also readily commercially available. Means for preparing base form zeolites are also well-known in the art, e.g. via reaction of a zeolite with a Lewis base that acts to remove surface-bound protons (i.e. protons bound to the oxygen atom of a surface Si-O-Si linkage). In preferred embodiments, the base form zeolites are prepared by reaction with an alkali or alkaline earth hydroxide, and typically comprise Cs ⁺ -substituted zeolites. Reaction with other Lewis bases such as alkali or alkaline-earth oxides, hypochlorite, etc., can also be used to produce base-form zeolites. In the most preferred embodiments that include base-form zeolites, the H ⁺ concentration is less than about 10 ^"B mol L ^"1 (pH > ~8), i.e. outside the range of viability of most pathogenic microorganisms.

[0121] In other preferred embodiments of the invention, the biocidal zeolite disposed about the interior surface enclosing the predetermined volume is characterized by a mixture of domains, each of the domains being in a form chosen from the group consisting of acid form and base form. Such a form is referred to herein as being a "mixed acid/base form." In these embodiments, the ratio of acid form to base form domains (and hence overall H ⁺ concentration in the material) may be set to any predetermined value, e.g. lO ^"7 mol L ^"1 (i.e. neutral pH). By combining acid form and base form zeolites, the pH of the zeolite composition used can be adjusted to match that of the environment with which the composition is in contact while retaining the high surface charge necessary to control the microbial population. The domains may be of any size from macroscopic to individual zeolite particles. As a non-limiting example of preparation of macroscopic domains, mixed acid/base form biocidal zeolites may be prepared by disposing acid form zeolite about predetermined areas of the inside surface of the container and base form zeolite about other areas. At the other extreme, a mixture of acid-form zeohte particles and base-form zeohte particles may be prepared and disposed about the surface of the container, leading to microscopic "domains" due to the intimate mixture of the acid-form and base-form particles.

[0122] The surface pH of mixed acid/base form zeolites can be measured according to the method described above. In preferred embodiments of the invention in which mixed acid/base form zeolites are used, the surface pH is within about 6 and about 8. In more preferred embodiments of the invention, the surface pH is about 7.5. The surface pH can be controlled by the relative amounts of acid form and base form zeolite used. In some embodiments, the desired ratio of acid form to base form zeolite can be determined by producing a suspension of one form of zeohte (i.e. acid form or base form) in water and adding the other form until the pH reaches a predetermined value. The weight ratio of acid form to base form zeolite in this suspension can then be used to produce products (e.g. a zeolite/polymer matrix) with a mixed acid base form zeolite with desired properties. In particular, the H ⁺ concentration within the acid base form zeolite can be chosen to match that of the medium in which control of the population of microorganisms is desired, or the H ⁺ concentration or surface pH can be fixed so that the zeolite affects one or more predetermined species of microorganisms, while leaving other predetermined species of microorganisms wholly or partially unaffected.

[0123] In some embodiments of the invention, the biocidal composition is chosen from the biocides group defined in Biocidal Products Directive 98/8/EC (BPD).

[0124] In preferred embodiments of the invention, the zeolites used are those in which channels within the zeolite structure are large enough to allow the passage of guest species. In preferred embodiments of the invention, the channels within the zeolite must have a minimum width greater than that of 6-membered rings (i.e., rings consisting of six tetrahedra) in order to allow zeolitic behavior at normal temperatures and pressures. The zeohte forms with properties most appropriate to embodiments of the present invention include, but are not limited to, mordenite, clinoptilite, and acid form zeolites prepared by means well-known in the art from β-zeolite, ZSM-23, ZSM-5, zeolite A, and zeolite Y. In preferred embodiments of the invention, the zeolite used is chosen from these forms. In typical embodiments, the particle sizes used are about 10 - 20 μηι in diameter. In preferred embodiments of the invention, the zeolites used have pore diameters in the range of about 0.3 - 0.8 nm, and an internal surface area of between 350 and 925 m ² /g. Dehydration of hydrated phases of the biocidal zeolites disclosed herein is achieved by heating; generally, heating to a temperature below about 400 °C is sufficient. Dehydration of the biocidal zeolites disclosed herein is largely reversible.

[0125] The zeolite framework of the zeolites used in the present invention may be interrupted by OH groups or by F atoms; these occupy a tetrahedron apex that is not shared with adjacent tetrahedra.

[0126] In preferred embodiments of the invention, biocidal zeolites comprise a zeolite that is in the acid form, a zeolite that is in the base form, or a mixture of acid form and base form domains. In most preferred embodiments, the surface charge density is at least 1 x 10 ^"10 C/cm ² , which is sufficient to produce a surface electric field gradient strong enough to kill a microorganism that approaches sufficiently closely (typically to within about 50 nm; in preferred embodiments, to within about 10 nm) to the surface.

[0127] Acid form zeolites are generally produced by ion exchange between cations located within the pores of the zeolite and H ⁺ . In preferred embodiments of the invention, the acid- form zeolites are produced from zeolites that have a Si Al ratio of between 3 and 50. In most preferred embodiments, the Si/Al ratio is between 5 and 20. In some embodiments of the invention, the biocidal zeolite comprises a mixture of acid-form and base-form domains. By appropriate preparation of the domains and of the proper mixing ratio between them, a biocidal zeolite of any desired pH can be prepared. This specially prepared biocidal zeolite can be chosen to be effective against a particular microorganism of interest, as shown in the examples given below.

[0128] The method herein disclosed uses biocidal zeolites to control the population of microorganisms within a given volume. In some embodiments, rather than eliminating the microorganisms entirely, the zeolites prevent the population from increasing above a predetermined amount, e.g. the population of microorganisms present in the volume prior to contact with the biocidal zeolite. That is, in these embodiments, the rate of killing of microorganisms is in a predetermined ratio to the rate of reproduction of the microorganisms. In some embodiments, controlling the population of microorganisms comprises preventing its increase from its initial value (i.e. the material is bacteriostatic). The population regulation is essentially a balance between the rate of reproduction of the microorganisms and the rate at which they are killed by contact with the biocidal material. The rate of killing of the microorganisms can be regulated by the amount of surface upon which the biocidal material is disposed, the specific material chosen, the H ⁺ concentration in the biocidal material, etc.

[0129] In preferred embodiments of the invention, the step of exposing microorganisms to the biocidal zeolite kills those microorganisms exposed. If the rate of exposure is greater than the rate of reproduction, the population of microorganisms will thus decrease with time. In some embodiments of the invention, the step of exposing the microorganisms to the zeolite is performed until a 2-log decrease in the population of microorganisms is achieved. In some embodiments of the invention, the step of exposing the microorganisms to the zeolite is performed until a 5-log decrease in the population of microorganisms is achieved. In some preferred embodiments of the invention, the step of exposing the microorganisms to the zeolite is performed until the population of microorganisms is observed to have been eliminated entirely. In some embodiments, the volume may be exposed to the external environment, and the step of exposing microorganisms to the biocidal zeolite will thus include exposure to the zeolite of microorganisms introduced into the predetermined volume from the external environment. In these embodiments, the net observed effect of the method disclosed herein will be to control or prevent entirely the growth of the microbial population following exposure to, and contamination from, the external environment.

[0130] In some embodiments of the invention, the biocidal zeolite is at least partially enclosed in a polymer matrix such that the contact with the microorganism of interest is only indirect. The enclosure of the zeolite within the polymer matrix to form a zeolite/polymer matrix may be performed by any method known in the art. Non-limiting examples of such methods include doping, gluing, coating, immersing, ionically bonding, covalently bonding, pressing, and co-extruding. Any technique known in the art may be used. Non-limiting examples of polymers suitable for use in these embodiments include ethylene vinyl acetate (EVA); low density polyethylene (LDPE); polypropylene (PP); cellulose; cellulose derivatives; polyalkanoates; polyethylene terephthalate (PET); polyvinyl alcohol; ethylene vinyl alcohol; polyethylene glycol; acrylics; polyesters; polyamides; polyacrylates; polycarbonates; other thermoplastic polymers; and copolymers and blends of any of the above. In these embodiments, the surface charge on surface of the biocidal material (i.e. the material that comprises both a zeolite and a polymer) is produced substantially entirely by the zeolite. [0131] In some embodiments of the invention, the zeolite/polymer matrix comprises a zeolite powder (acid form, base form, or a mixture of the two) ground to or sieved in order to limit the maximum particle size to a predetermined limit. The zeolite powder is then incorporated into a polymer matrix. In some embodiments, the zeolite/polymer matrix is formed by dissolving a polymer such as EVA in an organic solvent, adding zeolite powder to the polymer solution and the resulting matrix coated onto a substrate such as paper or cardboard. In other embodiments, the zeolite/polymer matrix is formed by extrusion according to methods known in the art to form a composition that is at least 60% by weight zeolite. In some embodiments, the extruded zeolite/polymer matrix is further treated by pressing at elevated temperature to form zeolite/polymer sheets.

[0132] In yet other embodiments, an extruded zeolite/polymer matrix (e.g. those in which the polymer is one such as LDPE that is used to manufacture bottles) is used in one layer of blow-molded two-layer bottles. The internal layer comprises the zeolite/polymer matrix, while the external layer comprises polymer to which zeolite has not been added. Methods for manufacturing such bottles are well-known in the art.

[0133] In yet other embodiments, the zeolite/polymer matrix is formed into inserts that can be placed in regular containers, e.g. by placement under the caps of bottles. These inserts can be formed, e.g. by placing zeolite and polymer (or a zeolite/polymer matrix formed as described above) into a press and pressing the mixture to a desired shape and size.

[0134] In another embodiment of the invention, the step of distributing the zeolite about the boundary of the predefined volume of interest includes distributing an ionomer about at least a part of the boundary of the predefined volume (e.g. by disposing ionomer about at least a portion of the interior of the surface bounding the volume). As ionomers comprise charged monomers, they too have a surface charge that imparts to them biocidal properties. Thus, exposing the microorganisms to at least one ionomeric species in addition to the zeolite further allows fine-tuning of the population control, e.g. by preparing a biocidal material with a predetermined desired H ⁺ concentration and/or surface charge density. This fine-tuning can allows, for example, design of a system that provides biocidal activity against specifically chosen microorganisms. Non-limiting examples of ionomers that can be used in the present invention include polyvinyl alcohol, polystyrenesulfonic acid, sulfonated tetrafluoroethylene copolymer, derivatives of sulfonated tetrafluoroethylene, polyacrylamide-immobilines, agarose-immobilines, cationic polyurethane, poly(diethylaminoethyl acrylate), ion exchange beads, and any polymer containing at least one functional group chosen from the group consisting of sulfonic acid, phosphonic acid, quaternary amine, tertiary amine, and derivatives thereof.

[0135] In some embodiments of the invention, the zeolite/polymer material is provided in contact with a second, not necessarily biocidal, polymer layer. Non-limiting examples of polymers appropriate for production of this second layer include EVA, LDPE, and PET. This second layer is primarily used to support the biocidal zeolite/polymer layer, and in practice will be placed external to the biocidal layer relative to the volume being treated by the method. In preferred embodiments, the two layers are produced by coextrusion, but any method known in the art may be used.

[0136] Similarly, in some embodiments of the invention, the biocidal zeolite or zeolite/polymer material is disposed onto a not necessarily biocidal substrate. Non-limiting examples of suitable substrate materials include cardboard, wood, plastic, metal, and glass. In preferred embodiments, the zeolite or zeolite/polymer material is disposed on the substrate by a method chosen from the group consisting of doping, gluing, spraying, coating, immersing, and co-extruding. Any method known in the art for disposing the biocidal material on the substrate may be used. The primary purpose of the substrate is structural; that is, the predetermined volume is actually contained by the substrate. In some embodiments of the invention, disposing the biocidal material on the substrate provides a means for fixing the total amount of biocidal material used in a particular volume.

[0137] It is well-known that many pathogenic microorganisms produce foul-smelling gases and vapors (e.g. mercaptans) as by-products of their metabolism or as by-products of chemical breakdown of the substances on which the microorganisms feed. Likewise, as is well-known, the large internal surface area of zeolites makes them excellent high-capacity absorbents for gases and vapors. Thus, in some embodiments of the present invention, the absorbent properties of the zeolites are used in addition to their biocidal properties by including a step in which at least some products of microbial metabolism are adsorbed or absorbed by the zeolite. Upon contact with a volume in which the microorganisms are enclosed, the foul-smelling products either diffuse (in embodiments in which there is no mass fluid flow) or are carried with a mass fluid flow (in embodiments in which there is such a flow) to the zeolite, in which they are entrapped.

[0138] In some embodiments of the invention, the step of exposing the microorganisms to the biocidal zeolite comprises a step of indirectly exposing the microorganisms within the volume to the biocidal zeolite. Such embodiments are produced, for example, in cases in which the zeolite is enclosed in a polymer, or in which a layer of material is placed between the zeolite or biocidal material and the volume in which the microorganisms are found. A non-limiting example of such an embodiment would be the placement of a membrane or other ion-selective barrier (in preferred embodiments, less than 50 nm; and in the most preferred embodiments, less than about 10 nm) through which only water and selected ions (e.g. H ⁺ or OH ^" ) can pass between the biocidal material and the volume in which the microorganisms are found.

[0139] In some embodiments, the material is chosen to kill one or more specific species of microorganisms. These embodiments are created by careful regulation of the relevant properties of the biocidal material, such as the H ⁺ concentration, the surface charge density, the pore size, etc.

[0140] The following examples are given to illustrate various embodiments of the invention and to assist one of ordinary skill in the art to be able to use it. No limitation of the invention and the modes for enabling it is to be implied from the examples.

EXAMPLE 1

[0141] As a non-limiting example of a method used to increase the surface H* concentration, and hence surface charge, of a zeolite, the following procedure was employed. NH ₄ -ZSM-5- 15 ammoniated zeolite (pH = 5.8) was purchased from ZEOlyst (cat. No. CBV-3024E). 50 g of the zeolite was poured into a crucible and placed in a furnace (Electro therm model MS-8). The zeolite was then heated according to the following sequence: (1) the temperature was raised from room temperature to 120 °C at a rate of 15 °C/min, and held at 120 °C for 60 min; (2) the temperature was then raised to 300 °C at a rate of 5 °C/min, and held at 300 °C for 120 min; (3) the temperature was then raised to 480 °C at a rate of 5 °C/min, and held at 480 °C for 360 min. The pH of a 1% suspension of the treated zeolite stirred at room temperature for 1 hour was determined to be 3.5, i.e. a ~200-fold increase in the H ⁺ concentration relative to the untreated zeolite.

EXAMPLE 2

[0142] The antibacterial activity of an acid-form zeolite - EVA film on a paper matrix was tested using the ISO 22196 method. The zeolite - EVA material was prepared according to the following protocol. First, 5 g of EVA (EVA EVATANE 40-55, obtained from Arkema) was put into a 50 ml polycarbonate tube, and 40 ml of methylene chloride (CP, obtained from Gadot) were added. The mixture was stirred for 4 h until the EVA fully dissolved. The resulting solution was then divided into two equal parts. 7.5 g of zeolite (commercially available H-Mor-17 zeolite obtained from ZeoChem AG) was sieved through a 250 μηι mesh sieve and added to the EVA solution and shaken until a homogeneous suspension was formed. The resulting suspension was then vigorously shaken and stirred for an additional 30 min. The liquid was then poured into a Pyrex container.

[0143] Standard white A4 paper was cut into 5 cm x 5 cm squares, held by a pin or tweezers and soaked in a 1M HCl solution for 2 min in order to remove CaCOa filler from the paper matrix. After bubbling, indicating release of CO ₂ , was observed to have stopped, the squares were removed from the HCl solution and dried for 0.5 h in a fume hood. The dried paper squares were then dipped into the zeolite suspension, held there for 1 s, and removed, thus coating both sides of the paper. The coated paper was then dried for several minutes in a fume hood.

[0144] Microbiological experiments were performed according to the ISO 22196 protocol using E. coli as the test organism and the pour plate sampling method. The results are presented in the Table 1.

Table 1 Results for ISO 22196 protocol: E. coli

Time after introduction of Control Zeolite Acid-form zeolite

E. coli

0 3.75 x 10 ³ 3.75 x 10 ³ 3.75 x 10 ³

4.38 x 10 ³ 4.38 x 10 ³ 4.38 x 10 ³

(average, 0 h) 4.06 x 10 ³ 4.06 x 10 ³ 4.06 x 10 ³

2.38 x 10 ⁵ 0 3.81 x 10 ¹

24 h 1.81 x 10 ⁵ 0 2.31 x 10 ²

2.00 x 10 ^s 0 4.69 x 10 ¹

(average, 24 h) ^' 2.06 x 10 ^s 0 1.05 x 10 ²

EXAMPLE 3

[0145] The antimicrobial activity of various Zeolite EVA sheets was evaluated. The seven compositions listed in Table 2 were prepared. In all cases, a total of 24 g of starting material (zeolite + EVA) was used to prepare the sheets.

Table 4 Results for ASTM method 2149: E. Coli (concentrations in CFU/ml)

Time Negative Positive _A B C D E F G control control

^~~ 0 5 x 10 ³ 5 x 10 ³ 5 x 10 ³ 5 x 10 ^s 5 x 10 ³ 5 x 10 ³ 5 x 10 ³ 5 x 10 ³ 5 x 10 ³

2.0 x 10 ^s 0 0 2.8 x 10 ^s 2.5 x 10 ^s 2.3 x 10 0 2.7 x 10 ^s 2.6 x 10 ^s

24 h 2.5 x 10 ⁸ 0 0 2.7 x 1Ό ⁸ 2.6 x lO ⁸ 2.1 x 10 ^s 0 2.4 x 10 ^s 2.7 x 10 ⁸

2.0 x 10 ⁸ 0 0 2.6 x 10 ⁸ 2.4 x lO ⁸ 2.4 x 10 ⁸ 0 2.8 x 10 ^s 2.3 x 10 ^s avg 1.5 x 10 ^s 0 0 2.7 x 10" 2.5 x lO ⁸ 2.3 x 10 ^s 0 2.6 x 10 ^s 2.5 x 10 ^s

Table 5 Results for ASTM method 2149: S. cerevisiae (concentrations in CFU/ml)

Negative Positive

Time A B C D E F G control control

0 4.1 x 10 ^s 4.1 x 10 ³ 4.1 x 10 ³ 4.1 x 10 ³ 4.1 x 10 ³ 4.1 x 10 ³ 4.1 x 10 ³ 4.1 x 10 ³ 4.1 x 10 ³

1.1 x 10 ⁶ 8.0 x 10 ³ 4.0 x 10 ² 5.0 x 10 ⁵ 6.0 x lO ⁵ 6.0 x lO ⁵ 6.0 x 10 ⁵ 6.0 x 10 ⁵ 8.0 x 10 ⁵

24 h 4.0 x lO ⁵ 1.2 x lO ⁴ 4.0 x 10 ¹ 6.0 x 10 ⁵ 5.0 x 10 ⁵ 5.0 x 10 ⁵ 5.0 x 10 ⁵ 6.7 x 10 ⁵ 3.0 x 10 ⁵ 1.3 x 10 ⁶ 4.0 x 10 ³ 1.0 x 10° 4.2 x 10 ⁵ 4.0 x lO ⁵ 5.0 x lO ⁵ 4.2 x 10 ⁵ 5.5 x 10 ⁵ 3.0 x 10 ⁵ avg 9.3 x 10 ³ 8.0 x 10 ³ 1.5 x 10 ^z 5.1 x 10 ³ 5,0 x lO ³ 5.5 x lO ³ 5.1 x 10 ³ 5.8 x 10 ³ 4.7 x 10 ³

[0149] As can be seen from the results summarized in the tables, composition "A" (acid-form zeolite in EVA) provided the most active biocide against all three microorganisms. Composition "E" (comprising base-form zeolite and acid-form zeolite in a 1:3 ratio) was effective against K. pneumoniae and E. coli, but not against S. cerevisiae.

EXAMPLE 4

[0150] The biocidal properties of several different zeolites were compared. Rates of killing of four different species of microorganisms (E. coli, Staphylococcus Aureus, Candida, and B. Fulva) were measured for six types of zeolites (Clinoptilolite was obtained from Incal Materials). The activities are summarized in Table 6. As can be seen from the results summarized in the table, naturally occurring forms of Clinoptilolite and an Na ⁺ -form zeolite showed no biocidal activity whatsoever, while all of the charged forms (acid, base, and mixed) showed significant ability to reduce the populations of pathogenic microorganisms. Table 6 Biocidal properties of different types of zeolite (rates of killing in CFU h)

Commercial Yeast

Zeolite form E. Coli S. Aureus B. Fulva name (Candida)

Na ⁺ (pH ~ 8) Zeoflair 300 Not active Not active Not active Not active

Mordenite

acid (pH -3.2) 10 ² 10 ³ 10 ¹ - 10 ² 10 ¹

17-H

base (pH -11.8) Zeoflair 100 10 ¹ 10 ¹ 10 ¹ 0 (static)

Mg ²⁺ Clinoptilolite Not active Not active Not active Not active

Ca ²⁺ Clinoptilolite Not active Not active Not active Not active mixed acid +

base form 10 ¹ - 10 ² 10 ¹ - 10 ³ 10 ¹ - 10 ² 10 ¹ - 10 ² zeolites

EXAMPLE 5

[0151] The biocidal activity of various zeolite/EVA sheets against Staphylococcus aureus was evaluated.

[0152] Zeolite/EVA sheets were prepared according to the Cath-5-141 sheet preparation method. The sheets were prepared in a polymer mixer. The mixer was set to 80 °C for 30 min prior to introduction of material into the mixer. First, 6 g of EVA were fed into the mixer. A few minutes later, after the EVA had melted, 18 g of zeolite were slowly added. The components were mixed and the bulk then transferred to the press. The bulk was placed between 2 silicone sheets and pressed at 10 tons and 80 °C to form a sheet. After the sheets were formed, they were placed on a marble table top to cool. After cooling, the silicone sheets were peeled from the zeolite/EVA sheet. The zeolite/EVA sheet was then cut into 1.5 x 2 cm rectangles. Each rectangle was weighed and then placed in a 50 ml test tube. Twenty 1.5 x 2 cm samples of each of the seven compositions were prepared.

[0153] The antimicrobial activity of the zeolite/EVA sheets was then tested by the ASTM E2149 method using TSA as the growth medium. All samples were incubated at 30 °C The pour plate sampling method was used. Three independent replications were performed both for the control studies (no biocide) and the experimental runs. The results are summarized in Table 7. Table 7 Results for ASTM method E2149: S.aureus (concentrations in CFU/ml)

„ „ , , . Clinoptohte Chnoptolite Zeoflair 300 Zeoflair 100

Control Mordemte ,, , _{2+ ί Λ} i+ , _λ , _{I T 0 >} , „ , , ₀ ,

(Mg form) (Ca form) (pH 8 (pH -1 1.8

7.0 x 10 ⁴ 7.0 x 10 ⁴ 7.0 x 10 ⁴ 7.0 x 10 ⁴ 7.0 x 10 ⁴ 7.0 x 10 ⁴

1.7 x 10' 2.1 x 10 ⁴ 2.9 x lO ⁵ 1.4 x lO ⁶ 6.3 x 10 ⁶ 3.4 x lO ⁴

24 h 2.7 x 10 ⁷ 7.8 x 10 ³ 9.0 x lO ³ 1.2 x 10° 2.5 x 10 ⁶ 2.5 x 10 ⁵

1.8 x 10 ⁷ 3.7 x 10 ⁴ 2.1 X 10 ⁶ 1.9 x 1Ό ⁶ 1.9 x 10 ⁶ 6.4 X 10 ⁴ avg

2.1 x lO ⁷ 2.2 x 10 ⁴ 1.1 x lO ⁶ 1.5 x lO ⁶ 3.6 x lO ⁶ 1.2 x 10 ^s (24 h)

[0154] As can be seen from the results presented in the table, the acid form zeolite (Mordemte) was the most effective biocide, with commercially available Zeoflair 100 (the more highly basic of the two basic forms tested) showed biocidal activity to a lesser extent.

EXAMPLE 6

[0155] The biocidal activity of various zeolite/EVA sheets against E. coli was evaluated.

[0156] Zeolite/EVA sheets were prepared according to the same protocol used in the previous example. Their effectiveness against E. coli was determined using the ASTM E2149 method. The experimental conditions were as in the previous example. The results are summarized in Tables 8 A and 8B.

Table 8A Results for ASTM method E2149: E. coli

(concentrations in CFU/ml)

Time Control Mordenite

0 2.1 x 10 ⁵ 2.1 x 10 ⁵

2.7 x lO ⁸ 5.0 x 10°

24 h 3.3 x 10 ⁸ 5.0 x 10°

3.4 x 10 ⁸ 5.0 x 10°

avg (24 h) 3.1 x 10 ^s 5.0 x 10 ^u

Table 8B Results for ASTM method E2149: E. coli (concentrations in CFU/ml)

Clinoptolite Clinoptolite Zeoflair 300 Zeoflair 100

Time Control

(Mg ²⁺ form) (Ca ²⁺ form) (pH 8) (pH ~1 1.8)

0 2.1 x 10 ⁵ 2.1 x 10 ^s 2.1 x lO ⁵ 2.1 x lO ⁵ 2.1 x 10 ³

1.9 x 10 ⁸ 5.4 x lO ⁴ 2.2 X lO ⁸ 8.0 x lO ⁴ 5.9 x 10 ³

24 h 3.1 x 10 ⁸ 3.0 x 10 ⁴ 2.6 x lO ⁸ 5.3 x lO ⁴ 4.4 x 10 ⁴

1.7 x 10 ^s 2.4 x 10 ⁴ 2.l x l0 ⁸ 6.2 x lO ⁴ 4.1 x 10 ⁴ [0157] As can be seen from the results summarized in the tables, once again, the acid form form of the zeolite (mordenite) showed excellent biocidal activity against E. coli. Although except for the Ca ²⁺ form of clinoptolite, the other zeolite forms showed some biocidal activity, the acid form form reduced the concentration of the bacteria more effectively than the others by some four orders of magnitude.

EXAMPLE 7

[0158] In a separate series of experiments, the biocidal activity of various zeolite/EVA sheets against E. coli and 5. cerevisiae was evaluated. In this set of experiments, the biocidal activity of an acid-form zeolite prepared as described above was compared with that of an acid-form zeolite prepared in such a way as to provide a significantly more porous surface.

[0159] Zeolite EVA sheets were prepared according to the same protocol used in the previous example. The antimicrobial activity of the sheets was tested by ASTM method E2149. The pour plate method was used. Tests were done in a 10 ml container. Microorganisms were incubated at 30 °C for 24 hours. For E. coli, the liquid was TSB diluted 1: 100, TSA medium was used for the plates, and the initial concentration was 3.5 x 10 ⁵ CFU/ml. For S. cerevisiae, the liquid was PDB diluted 1:100, MEA medium was used for the plates, and the initial concentration was 8 x 10 ⁶ CFU/ml. Test results are presented in tables 9A and 9B.

Table 9A ASTM E2149 results for antimicrobial activity against E. coli

„ .. . c , . Initial concentration, Concentration after 24 h,

Zeolite type Sample No. „„ _T , , .

^J CFU/ml CFU/ml

1 3.5 x lO ³ 7.0 x 10 ⁷

2 3.5 x 10 ³ 2.7 x 10 ⁸

Control (no zeolite)

3 3.5 x 10 ⁵ 2.1 x 10 ^s average 3.5 x 10 ³ 1.8- x lO ⁸

1 3.5 x 10 ⁵ < 1

2 3.5 x lO ⁵ < 1

Acid-form zeolite

3 3.5 x 10 ³ < 1 average 3.5 x lO ³ < 1

1 3.5 x 10 ³ < 1

1 1 c m5 ^ 1

"Porous surface" acid- 2 3.5 x 10

form zeolite 3 3.5 _x 1 o ⁵

average 3.5 x 10 ⁵ < 1 Table 9B ASTM E2149 results for antimicrobial activity against S. cerevesiae

Initial concentration, Concentration after 24 h,

Zeolite type Sample No.

CFU/ml CFU/ml

1 8 x 10* 1.0 x 10 ^b

2 8 x 10 ^s 2.0 x 10 ⁶

Control (no zeolite)

3 8 x 10 ⁵ 1.3 x 10 ⁶ average 8 x 10" 1.4 x 10°

8 x 10 ⁵ 5.1 x 10 ¹ 8 x 10 ⁵ 3.0 x 10 ²

Acid-form zeolite

8 10 ⁵ 3.1 x 10 ² average 8 l0 ³ 2.2 x 10

1 8 x 10 ³ 1.0 x 10 ²

"Porous surface" 2 8 x 10 ⁵ 4.8 x 10 ¹

form zeolite 3 8 x l0 ⁵ < 1

average 8 x l0 ⁵ 5.1 x 10 ¹

[0160] Both the acid-form and the porous acid-form zeolites showed excellent activity against E. coli, producing a population reduction of 6 orders of magnitude relative to the control, and effectively eliminating the entire population. Against 5. cerevisiae, the acid- form zeolite produced a population reduction of 4 orders of magnitude relative to the control, while the porous acid-form zeolite was on average 4 times more effective than the plain acid- form zeolite. Without wishing to be bound by theory, it appears that the presence of additional pores on the surface produces a larger effective surface and hence a larger effective surface charge, increasing the effectiveness of the "porous surface" acid-form zeolite relative to that of the acid-form zeolite.

EXAMPLE 8

[0161] An antimicrobial zeolite immobilized by extrusion in an EVA matrix was manufactured and its efficacy for control of the population of E. coli was tested.

[0162] Compounding of the zeolite/EVA formulation was performed as follows. The temperature of a BUSS MDK-46 extruder was set to 90°C. Due to friction, the temperature rose to 134°C. The temperature of the secondary extruder was set to 126°C. A 1: 1 by weight mixture of EVATANE 40-55 EVA, obtained from Arkema (France), and CP 811C-300 zeolite (H-Beta-360), obtained from Zeolyst (USA), was fed through the first feeding zone. The second feeding zone was fed with zeolite. The total ratio of the weight of material fed into the first feeding zone to the weight of material fed into the second feeding zone was 60:40, to yield a granular zeolite EVA composition comprising 70% by weight zeolite.

[0163] The composition was then placed between two nylon sheets in a press that had been heated to 90°C. The press was closed but without pressure and the sample was heated for about 20 sec. A pressure of 350 bars was then applied. The resulting pressed sheet was then cooled on the metal plate of the press.

[0164] Six squares were cut from the cooled pressed zeolite EVA sheets. Scratches were made in three of the six squares in order to increase the surface area.

[0165] The antimicrobial activity of the sheets was tested by ASTM method E2149. The pour plate method was used. Tests were done in a 10 ml container. Microorganisms were incubated at 30 °C for 24 hours. For E. coli, the liquid was TSB diluted 1:100, TSA medium was used for the plates, and the initial concentration was 3.4 x 10 ⁵ CFU/ml. Results of the microbiology experiments are given in Table 10.

Table 10 ASTM E2149 results for antimicrobial activity of zeolite/EVA extruded sheets against E. coli

Initial concentration, Concentration after 24 h,

Zeolite type Sample No.

CFU/ml CFU/ml

3.4 x 10 ³ 1.8 x 10' 3.4 x 10 ⁵ 1.1 x lO ⁷

Control (no zeolite)

3.4 x 10 ⁵ 1.9 x 10 ⁷ average 3.4 x 10 ⁵ 1.6 x 10 ⁷

3.4 x 10 ³ < 1

70% H-Beta-360 in 3.4 x 10 ⁵ < 1

EVA, scratched surface 3.4 x 10 ⁵ < 1

average 3.4 x 10 ^s < 1

1 3.4 x lO ⁵ < 1

70% H-Beta-360 in 2 3.4 x 10 ⁵ < 1

EVA, surface untreated 3 3.4 x 10 ⁵ < 1

average 3.4 x l( < 1 [0166] As can be seen from the results reported in the table, 24 hours of exposure of the medium to the zeolite/EVA led to complete elimination of the E. coli population.

EXAMPLE 9

[0167] In order to demonstrate that zeolites incorporated into a polymer matrix are capable of contacting the environment with which they are in contact, zeolite EVA sheets were prepared by the method described above using NH4 ⁺ -mordenite 20 as the zeolite. The zeolite/EVA sheets were then exposed to 1M aqueous HC1. The H ⁺ concentration of the zeolites was observed to increase, demonstrating that the zeolite is capable of interaction with the environment in contact with the zeolite/EVA matrix.

EXAMPLE 10

[0168] An antimicrobial zeolite/LDPE composition was prepared, and bottles manufactured from the composition. The efficacy of these bottles in controlling the microbial population in milk contained within them was tested.

[0169] Zeolite immobilized in an LDPE matrix was prepared as follows. The temperature of a BUSS MDK-46 extruder was set to 135°C. Due to friction, the temperature rose to 160°C. The temperature of the secondary extruder was set to 145 °C. A 1:1 by weight mixture of LDPE, obtained from Carmel Olefins (Israel), and CP 8 UC-300 zeolite (H-Beta-360), obtained from Zeolyst (USA), was fed through the first feeding zone. The second feeding zone was fed with zeolite. The total ratio of the weight of material fed into the first feeding zone to the weight of material fed into the second feeding zone was 60:40, to yield a stable granular zeolite/LDPE composition comprising 60% - 70% by weight zeolite. The process was performed for three different grades of LDPE (LDPE 111, LDPE 323, and LDPE 670), and similar results were obtained for all three grades.

[0170] Two-layer bottles were then prepared by extrusion blow molding. The 0.1 mm thickness internal layer consisted of a composition containing either 50% or 60% by weight zeolite immobilized in LDPE. The external layer was a standard 0.5 L, 27g HDPE bottle. The bottles were then filled with fresh milk (3% fat) and incubated at 30 °C or room temperature. As a control, was filled in Oplon active and control bottles and incubated at 30 °C or room temperature. Contamination levels were tested over the course of 21 days.

[0171] Reference is now made to FIG. 3, which presents a histogram summarizing the results of the test. Results are presented showing the microbial population (total concentration of microorganisms in CFU/ml on a logarithmic scale) at 0, 5, 14, and 21 days following introduction of milk into the bottles. The four bars at each time represent, from left to right, results for control bottles, bottles containing a layer of 50% zeolite in LDPE, and two independent sets of data for bottles containing a layer of 60% zeolite in LDPE. The bottles containing a layer of 50% zeolite in LDPE produced an approximately 1-log reduction in the microbial population relative to the controls, while those containing a layer of 60% zeolite in LDPE succeeded in completely preventing microbial growth during the time over which tests were made.

[0172] The pH of the milk samples was measured at the conclusion of the test, and was found to be between 6.4 and 6.5 in all cases, i.e. the pH remained essentially unaffected over the course of the experiments.

EXAMPLE 11

[0173] A series of experiments was performed to test the efficacy of the method disclosed herein to control growth of yeast and mold in various beverages. PET bottles (600 ml unless otherwise indicated) were filled with one of NESTEA brand iced tea, LIPTON brand iced tea, or GATORADE brand beverage. A cylindrical insert comprising an acid form zeolite in a EVA 40-55 matrix was attached to the underside of the bottle cap. Table 11 shows results for the use of acid-form mordenite (H-mordenite-20), obtained from Arkema, and Table 12 shows results for the use of CP 811C-300 zeolite (H-Beta-360), obtained from Zeolyst (USA). The tables give the weight percentage of zeolite (%Z) in the zeolite EVA matrix, whether CH ₂ C1 ₂ was added to the zeolite/EVA slurry, the weight in grams of zeolite in the insert, the results of the microbiology test including the population reduction of yeast and mold relative to a control experiment in which no zeolite was used, and the pH of the beverage at the conclusion of the experiment. The starting pH was approximately 3. In all cases, the beverage was inoculated with a known quantity of yeast and mold and then incubated at 30 °C.

Table 11 Control of yeast and mold in beverages by mordenite/EVA

Z wt

%z additive product result pH

(g)

77 CH ₂ C1 ₂ Lipton 2.15 total eradication after 7 days (3 log reduction) 2.98

77 CH ₂ C1 ₂ Lipton 2.15 total eradication after 7 days (5 log reduction) 3.02

2 96

77 none Gatorade 2.15 total eradication after 7 days (5 log reduction) ^ 43

77 CH2CI2 Gatorade 2.15 total eradication after 3 days (5 log reduction) 2.98 Table 12 Control of yeast and mold in beverages by CP811/EVA

%z Z wt

additive product result pH

(g)

88 CH ₂ C1 ₂ Nestea 3.55 total eradication after 3 days* 3.54

87 none Nestea 3.15 total eradication after 3 days (5 log reduction) 3.7

82 none Nestea total eradication after 3 days (5 log reduction)

82 none Nestea total eradication after 7 days (5 log reduction)*

82 none Nestea 3.46 total eradication after 3 days (5 log reduction 3.11

82 none Nestea 2.6 total eradication after 3 days (6 log reduction)* 3.51

82 CH ₂ C1 ₂ Nestea total eradication after 3 days (5 log reduction)

82 CH ₂ C1 ₂ Lipton total eradication after 7 days

82 CH2CI2 Gatorade total eradication after 3 days

82 CH2CI2 Lipton total eradication after 7 days (2 log reduction)

81 CH2CI2 Nestea 3.25 total eradication after 7 days (5 log reduction)* 3.54

80 none Nestea 2.2 total eradication after 14 days (4 log reduction) 3.67

*Total eradication was only observed in some of the bottles tested ^T 500 ml bottle

[0174] The results demonstrate the efficacy of both mordenite and Zeolite-β in their acid forms against mold and yeast growth in beverages. Moreover, the pH was not significantly changed; in all cases, the pH changed by less than 1 pH unit over the course of the experiment, and in most cases, the pH change was less than 0.6 pH units.

EXAMPLE 12

[0175] A series of experiments was performed to test the efficacy of the methods herein disclosed to prevent spoilage of ACTARA, an insecticide manufactured by Syngenta that contains thiamethoxam as its active ingredient, by control of the naturally occurring spoilage microorganisms.

[0176] 250 ml HDPE bottles were treated by coating with a zeolite/polymer composition containing acid form zeolite (either CP81 lC-300 or a mixture of CP81 lC-300 and H-ZSM-5- 23) and EVA 40-55 either the inside surface of the bottle or the inside surface of the bottle and the underside of the cap. An aqueous solution of Actara was then poured into the bottles and the bottles incubated at 30 °C. Table 13 shows the results of the experiments including the reduction in the population of microorganisms relative to control experiments in which the HDPE bottles containing the Actara were uncoated. Table 13 Efficacy of Zeolite/EVA 40-55 in control of spoilage of Actara

Zeolites used additive surfaces treated result

CP811

CH2CI2 inner surface, cap total eradication after 5 days (6 log reduction) H-ZSM-5-23

CP811

CH2CI2 inner surface, cap 5 log reduction after 3 days H-ZSM-5-23

CP811 CH2CI2 inner surface 5 log reduction after 3 days

CP811

bottle 5 log reduction

H-ZSM-5-23

[0177] As can be seen from the results summarized in the table, the acid form zeolite was effective in controlling the population of spoilage microorganisms in the Actara.

EXAMPLE 13A

[0178] A mixture of acid-form (mordenite 20) and base-form (4A) zeolites in an EVA 40-55 matrix was prepared as follows. 14.4 g of a 50:50 by weight mixture of mordenite 20-15a and 4A zeolites and 8.4 g of Evetane EVA 40-55 were introduced into a Brabender mixer heated to 80 °C over the course of between 1 and 1.5 min. The mixture was then ground for 1 minute. An additional 7.2 g of the zeolite mixture was then added. The mixer speed was increased, and mixing continued for an additional 3 minutes. The temperature remained below 92 °C during the mixing. A mill was heated to 80 °C. The mixture produced in the previous step was added to the mill and ground until the largest pieces could pass through a 6 mm sieve (approximately 30 s).

[0179] 0.5 g fractions of the mixture were then placed into a press and pressed into 0.5 mm thickness disks for 30 s at a pressure of 200 bar and a temperature of 80° C.

EXAMPLE 13B

[0180] Bottle-cap inserts comprising a mixture of acid-form and base-form zeolites were prepared as follows. 2.5 g of the mixture prepared as described in the previous example and 0.3 g of a 50:50 by weight mixture of mordenite 20- 15a and 4A zeolites were placed in a glass jar and shaken in a shaker for 30 s. The material was then mixed with a spatula to disperse any clumps. The material was then transferred to a press and pressed at 300 bar and 80 °C for 1 min. The inserts were then hot-glued to the bottom of standard plastic bottle caps. EXAMPLE 14

[0181] A series of tests was performed to assess the activity of mixed acid/base form zeolite compositions against L. lactis according to the methods herein disclosed.

[0182] A suspension of 0.5 g of CP-811 (acid form) zeolite powder in 25 ml of doubly distilled (18 ΜΩ) water was prepared in a 50 ml test tube. Sufficient base form Zeolite 4A powder (obtained from Argenol) was added to raise the pH to 9.33. It was found that the pH reached this value when the ratio of CP-811: Zeolite 4A was 3:1 by weight. The surface pH of the mixture, measured as described above, was found to be 7.5.

[0183] Inserts comprising a 3: 1 mixture by weight of CP-811 powder and Zeolite 4A powder in EVA were prepared according to the methods described above. Extended shelf-life milk (3% fat) was placed in a 50 ml tube and inoculated with L. lactis. The insert was placed under the cap of the tube. The tubes were then incubated at 30 °C. Samples were taken every day for three days. All results represent the average of samples taken from multiple independent tubes, and are reported relative to controls to which no zeolite was added. The results are summarized in Table 14.

Table 14 Efficacy of mixed acid/base Zeolite/EVA in control of L. lactis

Inoculation (CFU/ml) result

5 complete eradication in 1 day (8 log reduction), no growth in 3 days 5 complete eradication in 3 days (8 log reduction)

1 x 10 ⁴ 5 log reduction in 3 days

7.1 x 10 ² 6 - 8 log reduction in 1 day

EXAMPLE 15

[0184] A series of tests was performed to determine the efficacy of mixed acid/base form zeolite compositions in the control of E. coli. A series of mixed acid form/base form zeolite/polymer matrices containing Mordenite FM8 (acid form) and Zeolite 4A (base form) were prepared according to the methods described above. The antimicrobial activity of the matrices against E. coli was determined using the ISO 22196 protocol and an inoculation of 6500 CFU/ml. Results (reported relative to a control in which no zeolite was used) are summarized in Table 15. Results for control experiments in which one or the other of mordenite or Zeolite 4A was used are also given in the table. The results for the use of mordenite indicate that the matrix does not affect the antimicrobial activity, and that the mixed acid/base zeolites will therefore be effective in other matrices as well.

Table 15 Efficacy of mixed acid base form zeolites against E. Coli acid form: base surface pH

Matrix result

form ratio (w/w) (±1σ)

in ethylcellulose

5:4 spread on 5 cm x 5 cm 6.6 ± 0.7 3 log reduction in 24 hours cardboard sheets

in ethylcellulose

5:3 spread on 5 cm x 5 cm 6.5 ± 0.6 2.6 log reduction in 24 hours cardboard sheets

complete eradication in 24 hours acid form only EVA disk

(>5 log reduction) in ethylcellulose

complete eradication in 24 hours acid form only spread on 5 cm x 5 cm - (>5 log reduction) cardboard sheets

in ethylcellulose

base form only spread on 5 cm x 5 cm - ~4 log reduction in 24 hours cardboard sheets