AU2015261647B2 - Generation of microbiocide inside a package utilizing a controlled gas composition - Google Patents

Abstract

An apparatus and method of producing an atmospheric non-equilibrium plasma (ANEP) in a sealed container having a selected working gas and an object to be treated is described. A variety of working gas mixtures including air, 02, N2, C0 2, He and Ar, in combination with a 5 range of ionization gradients, voltages and ANEP column lengths was investigated so as to establish effective ranges of the variables using the sterilization of a sample as a measure of effectiveness. Certain combinations of working gas, voltage gradient, voltage and ANEP column length were found to have greater effectiveness. The approach may be used for food products, medical equipment, or other objects where treatment with reactive gas atmospheres is 0 effective.

Description

2015261647 26Nov2015 ρ/00/011 Regulation 3,2 Australia

Patents Act 1990

COMPLETE SPECIFICATION STANDARD PATENT

Invention Title:

Generation of mlcrobloclde Inside a package utilizing a controlled gas composition

The following statement is a full description of this invention, including the best metliod of performing it known to US: 1 2015261647 26 Nov 2015 GENERATION OF MICROBIOCIDE WSIDE A PACKAGE UTILIZING A CONTROLLED GAS COMPOSITION The present application claims priority to US provisional application No.: 61/451,975, filed on March 11,2011, which is incorporated herein by reference, and is a continuation in part ofus application 12/861,106, filed on August 23, 2010, which is incorporated herein by reference and which is a continuation in part of us application No.: 12/726,097, (now abandoned), filed on March 17,2010 which claims priority to US provisional application No.: 61/306,774,. filed on Februaty 22, 2010, and US provisional application No.: 61/162,785, filed on March 24, 2009, each of which is incorporated herein by reference.

TECHNICAL FIELD

[0001] The present application relates an appa-rahrs and method for teeating packaged products to reduce undesirable contamination from virases, bacteria, yeast, and mold, including spores and toxins, or for other tteatment using a reactive gas atnrosphere.

BACKGROUND

[0002] Biological decontamination and surface sterilization is crucial throughout society: in militaty applications such as the decontamination of equipment and facilities exposed to deadly biological agents, or in a broad array of civilian applications including medical applications, food production and consumer goods. Chemical, heat, high-energy electron beams, χ-ray or gamma-ray irradiation sys'tems are presently used in commercial treatments؛ however, utilization of these systems may not be practical due to the cost, efficiency, immobility, electtic power requirements, toxic waste, personal hazard and the time required to decontajninate items.

[0003] Over the last decade, considerable researc.h has been conducted in using atmospheric plasmas as a decontamination method of surfaces. Atmospheric plasmas have the ability to generate unique radiolytic profiles. Research has 1Α 2015261647 26 Nov 2015 shown that biological contaminants exposed to atmospheric plasmas can be sterilized in seconds to minutes. Atmospheric plasmas are fairly easy to produce؛؛ and, the equipiuent needed is relatively inexpensive. There are no hazardous wastes and the gaseous by-products can be locally conttolled. Up to this time, utilization of atmospheric plasmas has been through sealed chambers and jets.

[0004] Atmospheric, non-equilibrium plasma (ANEP) is an exanrple of a non-thennal processing method. There is a wide variance in the tenninology for the process to produce such a plasma. In the literature, a variety of terminology is used to describe the phenomenon including atmospheric glow discharge, surface barrier discharge (SBD), dielectric barrier discharge (DBD), Single Dielectric Barrier Discharge (SDBD) and Surface Plasma Chemistty Process (SPCP). For convenience herein, the term dielectric barrier discharge (DBD) is used, without intending to exclude any of the ANEP plasma generating mechanisms implied by choosing a specific terminology for description ofthe technique herein.

[0005] FIG. 1 shows simplified examples of DBD configurations that may be used to produce an ANEP in an ambient air environment. A high voltage generator 10 applies an alternating current potential to a pair of metallic plates 20, 30, spaced apart fronr each other to fonn a region 50 in which an object may be placed. At least one dielectric layer 40 is disposed between a first plate 20 and the second plate 30. In this manner, the effect ofthe dielectric layer is to limit the current of any filamentaty discharge that is formed between the plate 20, 30 so as to prevent the formation of a high current arc. The discharge in region 50 is thus limited in energy and results in an ANEP where variety of reactive species may be formed from the gas (He, .2, N2, C02 and water vapor) and/or interaction with the packaged product. FIG. 1Α shows a configuration with one dielecttic layer 40 laid against an elecfrode 20. FIG. IB shows an example where a dielectric plate 40 is laid against an elecriode 20 and another dielectric plate 60 is laid against a second electrode 30. The charge accumulation on the plates which may be used in conjunction with the voltage waveform to estimate the power consumption may be measured by detenniningthe voltage developed across a conventional capacitor 75. FIG. 1C illustrates a sirtration where a single dielectric layer 50 is disposed 2 2015261647 26 Nov 2015 between the electrodes 20, 30, so that there are two regions 50 in which an ANEP maybe produced. 00061؛ As the possibility of an arc forming directly between the plates 20, 30 exists, by air paths around the dielecttic, at least one electrode is often ftilly enclosed in an insulating material؛, and, the exposed electtode may be pounded. The insulating material may be the same material as used for the dielectric 40, 60؛ however, the two materials may have differing properties. For example, the dielectric plate may be quartz and the insulating material may be a moldable material.

SUMMARY 0007؛] A system for treating an object is disclosed, including an apparahrs confined to create an atmospheric non-equilibrium plasma (ANEP) using a working gas in a closed storage volume sized and dimensioned to contain an object to be treated. The voltage gradient applied to the working gas maybe ..eater than about 1.4 times an ionization voltage gradient of the working gas.

[0008] In an aspect the ANEP column length is greater than about 2.0 cm. In another aspect, the voltage applied to electrodes of the apparahrs may be greater than about 50 kv RMS. 0009؛] The working gas may be selected from air, 02, Ν2, C02, He, Ar, or a combination of these gasses, depending on the specific object to be treated. The object may be disposed either inside or outside the ANEP column.

[0010] This technology generates reactive gas species in a sealed package. If the package is designed from a low permeability film then minutes to hours of contact time between the generated reactive gas species and the object can be realized, resulting in very large reductions in patlrological microbialspecies. The technique may also be used to tteat objects where the desired effect is a reaction ofthe ionized species with surface contaminants or with the surface. 0011؛] Many common packaging materials, used as the package, work well with this technology including: LDPE, HDPE, pp, PET, cardboard, Kraft paper, TYVEK (high density polyethylene fibers) and glass. 3 [0012] A method of treating an object is disclosed including the steps of: providing a dielectric bareier discharge (DBD) device؛ providing a package suitable for substantially completely enclosing the object; inserting the object into the package; filling the package with a working gas at substantially atmospheric pressure; disposing a portion of the package with respect to the 2015261647 22 Mar 2016 5 DBD device such that reactive species are produced in the package by the DBD apparatus; and, activating the DBD device for a first period of time by applying a voltage gradient.

[0013] The voltage gradient applied to the DBD device is greater than approximately 1.4 times an ionization voltage gradient of the working gas. In an aspect first period of time may be less than about 15 seconds. In another aspect, the first period of time may be less than about 60 10 seconds. The object may be retained in the treatment volume for a second period of time so as to permit the generated reactive species to interact with the object being treated.

[0014] In an aspect'the container may be manipulated so as to provide more even application of the reactive species to the object being treated.

In one aspect of the invention there is an apparatus for in-package processing of a 15 product, the apparatus comprising: a pair of electrodes, spaced apart, at least one dielectric layer disposed between the pair of electrodes, an alternating current power source, wherein a voltage greater than 50k٧ is applied between the electrodes so as to form an 20 atmosplreric non-equilibrium plasma (ANEP) in at least a portion of a closed container disposable, at least in part, between the pair of electrodes, the closed container having a working gas fill at substantially atmospheric pressure and the voltage, the working gas and the spacing between the electrodes selected such that the ANEP is formed only within the closed container.

As used herein, except where the context requires otherwise, the term "comprise" and 25 variations of the term, such as "comprising", "comprise-s" and "comprised", are not intended to exclude other additives, components, integers or steps.

BRIEF DES.CRIPTION OF THE DRAWINGS

[0015] FIG. 1 (prior art) shows (A) a DBD apparatus having a single dielectric barrier; (B) a DBD apparatus having two dielectric bartiers and an auxiliary capacitor for measuring the DBD 30 charge; and, (C) a DBD apparatus with the dielectric disposed between two conducting plates; [0016] FIG. 2 shows (A) a portion of a DBD apparahrs where a container having an object to be treated disposed between the plates of the apparatus; (B), a portion of a DBD apparatus where 4 a container having an object to be treated is disposed between the plates of the apparatus, such and, (c) a top ؛that the object to be treated is not disposed between the plates of the apparatus 2015261647 22 Mar 2016 ؛view of a portion of the apparatus .of FIG. 2Α 0017J FIG. 3 shows data for gas concentrations generated using the ΡΚ-1 DBD Ionization]

؛(5 System(13.5 kVRMS 0018] FIG. 4 shows data for gas concentoations generated using ΡΚ-2 DBD Ionization System؛

؛((80 kV RMS 4Α 2015261647 26 Nov 2015 [0019] FIG. 5 shows data for spore reductions resulting from treatment by the using ΡΚ-1 DBD Ionization System (13.5 kv RMS); and [0020] FIG. 6 shows data for spore reductions generated using ΡΚ-2 DBD Ionization System (80 kv RMS).

DESCRIPTION

[0021] Exemplary embodiments may be better understood with reference to the drawings. Like numbered elements in the same or different drawings perfonn equivalent firnctions.

[0022] In the interest of clarity, not all the routine features of the examples herein are described. It will of course be appreciated that in the development of any such actaal implenrentation, numerous implementation-specific decisions must be made to achieve a developer's specific goals, such as consideration of system, regulatoty and business related consfraints. These goals will vary from one implementation to another.

[0023] Atmospheric pressure “cold” plasmas have been shown to be effective in reducing or eliminating surface bacterial contamination of food samples. The temr “cold plasma" is meant to describe a plasma discharge, which may be a non-equilibrium plasma, occurring at a pressure of about one-atmosphere and at near ambient temperature (ANEP). This is to distinguish the ANEP plasma from a thermal plasma discharge operating at a bulk gas temperanrre ofhundreds or thousands of degrees above the ambient temperature. In a "cold plasma” at ahnospheric pressure the electrons nray have a significantly higher temperature than the ion and neufral species; however, the bulk temperanrre of the working gas is not significantly increased, with respect to the anrbient temperanrre In this context, the term “cold” should not be interpreted to require refrigeration or other cooling to perform the decontamination or treaunent functions described herein; however, this does not exclude the freating or the subsequent storage ofthe treated object at an appropriate temperanrre, which may include refrigeration or cooling. Keeping the gas at a near-ambient temperature may contribute to avoidance of heat damage to the object bei'ng freated. 5 2015261647 26 Nov 2015 [0024] One technique of creating an atmospheric non-equilibrium plasma is to apply a high voltage to the volume to be ionized, while inhibiting the transition from a glow discharge to an arc discharge by limiting the discharge current. This may be done, fo.r example, by covering at least one ofthe electrodes ofthe apparahis with a dielectric layer; resistive layers have also been used. The discharge current is selflinrited by charge build up on the dielecttic surface. Typically, the excitation voltage frequency is in the kHz range, but may range from power line frequencies to radio frequencies. The experinrental data presented herein used a 60Hz frequency due to the availability of high voltage ttansformers, whose output voltage could be easily be adjusted by controlling the input voltage thereof witli a variable voltage transformer.

[0025] Dielectric-barrier discharges (DBD) are a type of alternating-current high-voltage gaseous discharges that may be fonned in a nonrinally atmospheric pressure environment. The presence of a dielectric layer behveen the electrodes prevents the charge generated in the gas by the discharge from reaching at least one ofthe conducting electrode surfaces. Often the dielecttic layer is applied to both ofthe electrodes. Within each halfcycle ofthe driving voltage wavefonn, when the voltage gradient applied across the gas exceeds that required for breakdown, the formation ofnarrow ionized discharge filaments initiates the conduction of electrons toward the nrore positive electtode, and ions towards the more negative electtode, although the mobility ofthe electrons is greater than that ofthe ions. An electrical charge accumulates on the dielectric layer(s) at the end(s) of each ionized filament; and, the voltage drop across the ionized filament reduces until the voltage falls below the discharge-sustaining level, so that the discharge is extinguished. The duration ofthe filamentaty discharge is believed to be quite short: ofthe order of 100 nanoseconds or less. However, the resultant reactive species may have a significantly longer lifetime. The low charge mobility along the surface ofthe dielecttic also limits the lateral region over which the gap voltage is diminished, so that a plurality of filaments may fomi in close proximity to one another. 6 2015261647 26 Nov 2015 (0026] Production of ozone and other reactive species in a DBD occurs between the two electrodes when operated at a particular voltage, frequency, and geometry. In air, mixtares of .2 and, Ν2, or .2 alone, reactive oxygen species are generated which react with each other as well as oxygen molecules resulting in the formation of ozone. Other reactive species are created when Ν2, or other gases such as CO2, ¾0 or Cl are present. The most oxidative species in air and oxygen gas include ozone (Ο3), singlet oxygen (0 or 0), superoxide (Ο2), peroxide (02.2 or Η2Ο2), and hydroxyl radicals (OH). Most of these species have very short half lives (in the range ofmilliseconds)؛ however, ozone has a much longer halflife ranging from minutes to days depending on conditions. The effects of gaseous ozone on foods has previously been shrdied with promising results and ozone has been shown to be more efficient at lower concentrations and treatment times than more standard sanitizers, including chlorine. Presently, the use of ozone has been limited to the treatment of unpackaged products. (0027] The effectiveness of the systenr and method described herein is due to an extent on the ability to generate reactive gas species in a sealed package. If the package is fabricated fiom a low permeability film, then minutes to hours of contact time bertveen the reactive gas species and the bacteria can be realized, resulting in veity large reductions in microbial populations. Over the duration of the storage time, the ozone and nifrogen oxides in the package will convert back to simple oxygen and nittogen molecules;, and upon reaching a final destination (e.g.١ grocery store or medical supply store), the reactive gas species in the package will have been converted back to original gas composition (air or modified ahnosphere). (0028] In particular, the object to be tteated may be enclosed in a sealed or substantially sealed container. The container need notbe herjnetic unless the level of decontamination desired is such that subsequent contamination from another source is to be avoided. Low penneability container may retain long-lived reactive species, which may extend the effective freatmenttime and improve the resultant decontamination results. Νοη-hermetic containers may be used in applications where subsequent re-contamination ofthe sample is prevented by the ٦ 2015261647 26 Nov 2015 characteristics of the packaging. Νοη-hermetic containers may be penneable to sonre extent to air, and to the other constituent gases or the radicals or reactive species produced by the ANEP. That is, the packaging may be porous to gases, but prevent spoilage or pathogenic material from entering the package. The composition of the container may be a plastic such as TYGON, low-density polyethylene (LDPE), high density polyethylene (HDP), polypropylene (pp), polyethylene terapthalate (PET), -EK, or polystyrene; however, various other substantially dielectric materials can be used, including, glass, wax, cardboard, paper, foil, eggshells,, l,0W dielectric constant materials, or the like. The foil may be a plastic having a thin nretallic coating. This may pemit the heatment of objects stored in a foil package, or having a foil liner.

[0029J An apparatus for treating a sanrple is shown in FIG. 2. An object to be treated 200 is placed in a substantially closed dielectric container or package 100. The container may be rigid or flexible and may be sealed by a ZIPLOC closure, by heat, by a close-fitting cap, or any other mechanism that has a similar effect. The container should have an ability to substantially retain the reactive species that are the residual of the generated ANEP plasma for a period of time that is sufficient for a particular treatment process. The working gas, which may be air, or a modified attnosphere packaging (MA) mixture, may be introduced into the container 100 prior to treatment. The container 100 may be purged prior to charging with the working gas so as to control he re-sulting gas mixhrre. The container may be sealed either pennanently or temporarily prior to treatment. ل0030ء A region within the container is selected where an ANEP trray be generated. This may be a specific formed region of a semi-rigid or rigid container, or may be formed by manipulation of a flexible container where the gas pressure gives the container a defonnable shape. In rigid containers, the gas pressure may be less than an atoosphere, while the gas pressure in a flexible container is an atmosphere or greater. This does not exclude sihrations, for exairrple, where vacuum packing is used, and a working gas may be introduced for the putposes of treatment. 2015261647 26 Nov 2015 [0031) FIG. 2Α illusttates a sittiation where the object being treated is disposed between the plates of the apparatus, while FIG. 2Β illustrates the sihration where the object being teeated is disposed so diat a small thickness of the storage bag having a gap between the opposing surfaces is disposed between the plates of the apparatus. For the sihration ofFIG. 2Β, the ANEP is CTeated inside a portion of the storage container; however, the object to be treated may not be directly exposed to the active ANEP (“out-of field” configuration). Rather, the residual reactive species may be diffirsed or circulated within the volume of container having the object to be treated. This configuration may reduce the voltage needed to establish the ANEP as the distance between the electrodes may be reduced compared with the thickness of the object. In addition, where the tennination of tire plasnra filairrents on the object itself trray be undesirable, that sihration is avoided. 1.032) In conhast, the arrangenrent of FIG. 2Α disposes the object to be heated behveen the electrodes;, and the olrject itself may behave as a dielechic, similar to that used on one or nrore ofthe elechodes. In this circumstance, the filanrents creating the ANEP nray extend ftom the electrode, or the dielectric barrier on an electrode, or an electrode without a dielectric barrier, to a surface ofthe object to be treated; and an active ANEP may also surround the object (“in-field” configuration). The electrons and the ions created in the ANEP may directly impinge on the surface ofthe object. Similarly to the arrangement ofFIG. 2Β, the object may continue to be exposed to the ANEP byproducts after the active phase of ANEP generation has been conrpleted. Each ofthe process.es may be repeated, if needed, where the object or the storage bag or container is manipulated to better distribute the active byproducts or expose other portions ofthe object to the plasma or the ANEP products. Conductive objects may also be fteated.

[0033) As shown in FIG. 2Α, the container 100 having a working gas 300 and an object to be tteated 200 luay be disposed bettveen the plates of a DBD apparattis 2. The plates 20, 30 are spaced apart so as to admit at least portion of the container 100 containing the object to be treated 200. The distance beftveen the plates may be confrolled by mechanical nteans, if desired, so that the container 9 2015261647 26 Nov 2015 100 may conveniently be placed between the plates 20, 30, and the spacing between the plates subsequently adjusted so as to partially compress the container 100, so as to achieve an appropriate gap spacing for the creation ofthe ANEP within the container 100. In this configuration, filamentary discharges may occur between the dielecttic surface 40 ofthe top plate 20 and the opposing surface of the object 200 being treated, and nray also occur between the bottom plate 30 and the object being treated 200. The ANEP may also be created by electrical currents flowing directly from one plate to another, as mediated by the dielectric layer on the plate. Other mechanical arrangements may also be used.

[0034J Where the object to be treated has the general characteristics ofa dielectric material, the filaments will exhibit a behavior similar to that which would occur in a DBD apparatus without an infroduced object, except that the filaments may tenninate one end thereof on the object. So, the object will be directly exposed to the filamentary discharges creating the ANEP, as well as to the shorter lasting and longer lasting reactive species that are generated during the active tteatment phase. As the surface density of filaments is governed by the electrical field distribution, and the shape and electrical -properties ofthe object to be tteated, the entire surface ofthe object may not be subject to the same intensity of direct treatment. Should more unifonn treatment be desired, the object to be treated 200 may be manipulated to expose other parts ofthe object to direct treatment.

[0035J The high voltage is often sinusoidal and may be produced by a high-voltage fransformer connected to the power grid, a signal generator connected to an airrplifier, or the like. Other voltage waveform shapes may be used, including sawtooth, trapezoidal, pulsed, symmetrical, asymmetrical, or displaced from DC. The amplitude ofthe voltage may be controlled during operation ofthe apparatus by, for example, a VARIAC fransformer, or by confrolling the signal generator amplitude output, or the amplifier gain. The frequency of operation may be fixed or variable. In the experiirrents described herein, the local power line frequency (60Hz) was used for convenience in configuring the experimental apparatus and 10 2015261647 26 Nov 2015 cost considerations. ANEP plasmas can also be created using DC where a resistive layer is used as a a current limiter or ballast. ل0036ء The voltage gradient at which a glow discliarge is formed is a firnction of the constitutive gases present between the electrodes, various geometrical considerations, and the gas pressure. The constituent gases may be modified so as to achieve a desired concentration and species of ionized particles. In addition to air, ٥2, Ν2, c٥2, ¾0, Cl, and other mixbrres, or pure gases, including inert and noble gases, are usable, depending on the application.

[0037] As shown in FIG. 2Β when a flexible container 100, which may be a plastic storage bag, is used, the gas fill level may selected so a that a portion ofthe container may be compressed behveen the plates 20,30 so as to form a smaller gap to facilitate creation ofthe ANEP at a lower voltage. Here, the container is shown in a state where a portion 110 ofthe container 100 is positioned between the elecfrodes ofthe DBD apparahis 3, so that a portion ofthe container 100 may be temporarily fonned into a region where the ANEP may be created. The filaments creating the ANEP are formed between the surface ofthe dielecrtic 40 and the other elecfrode plate 30, such that the object 200 to be treated is not disposed therebettveen. Portions ofthe container surface disposed so as to form the region in which the ANEP is to be formed may be held against the dielectric 40 and the plate 30 by the internal gas pressure. The effect ofthe dielecttic layer ofthe container surface may be small,, as the charge distributions are likely to be dominated by those ofthe electrodes and the dielectric 40.

[0038] FIG. 2C shows a top view ofthe DBD apparahis 3 ofFIG. 2Β. The dielectric material extends so as to inhibit sttay discharges, and, the elecrtodes may be disposed, opposite only a portion ofthe storage volume.

[0039] The electrodes may be planar, as shown; however, other geometries may be used to conform to a container such as a box, pill bottle, jar, or other shape. Shaped elechodes may be used to encourage the formation of a plasmajet, or better disttibute the reaction products using induced convection. For example, large cardboard containers may be processed by using a pair ofelecttodes oriented 11 2015261647 26 Nov 2015 at a 90° angle and placed along one or more of the edges. Similar configuration may be used for large packages of other materials and shapes.

[0040J The term package has been used to represent the enclosure, bag, container, tteattnent volume or storage volunre in which the object is treated and subsequently stored. At least parts of the package are fabricated from a dielectric material compatible with the tteatnrent process, and could be, for example, a bottle, a vial, an opaque plastic food fray sealed with a thin fransparent film, or the like. The objects to be processed need not be dielectric, as metallic objects could be exposed as well. The apparaurs and technique described herein may be used to sterilize or otherwise decontaminate objects such as medical supplies, including surgical instruments, syringes, consumer products, or other treatable objects and materials. They do not need to be reinoved from the packaging after treatment and until inrmediately prior to use. One may repeat the sterilization process in the hospital or physician’s office or at a point of sale or distribution prior to opening the packaging for filrther suppression ofcontaminante or pathogens. It should be noted that the dielecttic characteristics of the material forming the container may be used as the dielectric barrier ofthe DBD, providing that the electrical characteristics thereof prevent dielectric breakdown.

[0041] The inventors have discovered unexpected results where process or apparahrs parameters such as relative humidity, voltage gradient, elecfrode geometry, and voltage, in addition to the gas composition and package type, may have a significant effect on perfonnance in a sterilization or decontamination application.

[0042] The data presented herein illustrates the use of an apparafrrs and method ofkilling Bacillus subtilis spores, as a representative ofbiological contaminants, under a variety of plasma generation voltages (~13 kv, 50kV, 80 kv RMS), electric field gradients (12.5- 20 kv/cm), gap distances (1.0, 2.5 and 4.5cm) and gas compositions (air, MA) where the object to be treated is disposed within a sealed package and either inside and outeide ofthe plasma field. Unexpected improvements in performance obtain when certain process parameters are adjusted. 12 2015261647 26 Nov 2015 [0043] An apparatus (ΡΚ-1), is based on a dielectric barrier discharge (DBD) process., with plate electrodes contprised of insulated conductors connected to a power unit with specifications of 18 kv RMS (max) @ 30 mA @ 60Hz. The sample package is in disposed such that opposing sides thereof are in contact with the insulated high voltage electrodes, providing a dielectric barrier bebveen the electrodes, thereby limiting curcent flow through the sample package and controlling the power requirements for treatment. Only 40-50 w of power was needed to ionize an air atmosphere inside a4L (nominal) re-sealable plastic (LDPE) bag. Other means of insulating the electrodes, which may be a flat plate, flat wound coil, or the like, include a dielectric sheet disposed between an electrode and the package, or a dielecflic layer formed around the electrode.

[0044] The high-voltage applied to the electrodes may ionize a gas, which may be a mixhrre of gasses, within the electric field inside the package containing the sample. The sample lttay be, for example, a food or a medical device, or other object to be sterilized, decontaminated or otherwise plasma treated. Ionization produced by the DBD process can result in the production of significant concentoations of reactive molecules, including ozone concentrations above 1% in a few minutes, without a noticeable increase in the sample surface temperabrre. Specific treattnent times for targeted spore or bacterial reductions are dependent on sample contamination, packaging material, gas composition, and package/electrode configuration. The in-package ionization process has been demonstrated in a number common packaging nraterials including, cardboard, glass, various plastics such as LDPE, HDPE, PET, polystyrene, TYGON, rubber and others.

[0045] A s.econd similar apparams (ΡΚ-2) was also bu.ilt and has specifications of 130 kv RMS (max) at 20 mA @ 60Hz, so as to enable exploration of different parameters. The ΡΚ-2 system can ionize a sealed package of air with an electrode gap of up to about 10 cm.

[0046] The ΡΚ-1 and ΡΚ-2 systems were comparatively evaluated for reduction in pathological organisms by studying the reduction oiBacillus subtilis 13 2015261647 26 Nov 2015 spores in packages containing either air or a variety ofMA (modified atmosphere) gases, where the sample was disposed either inside or outside of a plasma field.

[0047] A 2x3xlxlx2x3 experinrental series design was selected that utilized two voltage conditions: 13.5kV RMS/44W/1.0 cm gap (ΡΚ-1 ionization system) and 80kV RMS/150W/ 4.5 cm gap (ΡΚ-2 ionization system)3 ؛ treatment conditions: infield ionization, out-of field ionization, and no ionization؛ a treatment time of 300 s (ΡΚ-1) and 120 s (ΡΚ-2), respectively؛ room temperature; two package gas types: air (78% Ν2.22% 02) and modified atmosphere, MA (65% ٥2, 30% c٥2, 5% N2)؛ and replicated in friplicate.

[0048] Air (78./. Nj, 22./0Ο2) and modified atmosphere (MA) gas (65% Oj, 30% CO2, 5% Ν2) were purchased from a local gas supplier at specified concenfrations with a certificate of analysis. These gas composition(s) were then metered into sealed package at a rate of2.1 L/min. using a shielded flow meter with stainless steel ball (Gilmont Instruments, Inc., Barrington, IL, USA) yielding final fill volume of 1.5 L with average fill time of 45 s.

[0049] Clear, 3.78 L Ziploc™ (SC Johnson and Son, Inc., Racine, WI, USA) heavy-duty freezer bags were obtained from a local grocery S'tore. The bags were made of low-density polyethylene (LDPE) and had a 1.6 mm thick wall.

[0050] Bacillus subtilis var. niger (5. atrophaeus) spore sfrips (NAMSA, Northwood, OH, USA) with size of3.2 cm X 0.6 cm, each conteining Bacillus populations of 1.5-2.5 X 10®/strip or 6.18-6.40 logiowere loaded into an open sterile petri dish inside the treatment package and then used in the experiments.

For in-field ionization with ΡΚ-1 system, one end of each spore ship was secured with transparent tape to the inside of the storage bag within elecfrode gap space prior to freatment.

[0051] The ΡΚ-1 system was operated 13.5 kv RMS at 44W ,and 60 Hz generating a 13.5kV RMS/cm gradient between the electrodes (1.0 cm gap). The electrodes consisted of coils of wire wound around a flat dielectric object with a treataent area of 51 cm2 (8 5 cm X 6 cm). The ΡΚ-2 system was operated at 80 kv RMS at 150 w and 60 Hz across circular stainless steel electrodes (15 cm dia, 4.5 14 2015261647 26 Nov 2015 cm gap, 17.8 kV RMS/cm voltage gradient). The high voltage transformer of the ΡΚ-2 was obtained from Phenix Technologies, Accident, MD.

[0052J The storage bags containing spore samples were filled with the working gas (air or i) and purged three times to ensure purity of the gas in the bag. A small, uniform amount of gas was expelled from the bag to allow for orientation of plasma electrodes if needed to achieve desired gap distance. The electrodes opposed each other, with the bag disposed therebetween and having an approximate gap distance of 1.0 cm (ΡΚ-1) and 4.5 cm (ΡΚ-2). Each system was activated for treatment times of 300s (ΡΚ-1) or 120s (ΡΚ-2). The gas volume in the bag was agitated, manually (by pressing lightly back-and-forth on the bag) once treatment was complete to allow for a more uniform distribution of gas inside the bag prior to double-bagging for 24h storage at room temperature (22.C). 10053] The temperahire of the electrodes and treated storage bags was measured prior to and immediately after treatment using an infrared thennometer (Omega Engineering, Inc., Stamford, CT, USA). All storage bag temperamres of treated samples registered at room temperature after tteatment for both systems. Ozone and nitric oxide concenfrations were measured immediately following the 300s or I20s tteatments as well as after 24h storage using DRAEGER Short-Tenn Detector tubes (Draeger Safety AG & Co., Luebeck, Germany). Carbon monoxide concentrations were also measured after the 24h storage period. The tubes were chosen for ease of use with the given experimental setup and for their rapid measurement capabilities. The hibes contain a reagent which changes color upon coming into contact with the specified gas and are calibrated for specific sampling volumes. Tubes were connected to a bellows hand pump, Accuro Gas Detector Pump (Draeger Safety AG & Co., Luebeck, Gennany), and calibrated such that one pump action equals 100 mL of gas. The Ozone tabes (part no. CH21001) had an indicated range of20-300 ppnr. Nitrous oxides (part no. 24001) tubes had an indicated range between 20-500 ppnr. A cross-sensitivity of 50 ppnr NOx per 1,000 ppnr ozone was identified. Carbon monoxide tabes (part no. 33051) had an indicated range between 25-300 ppm. 15 2015261647 26Nov2015 [0054) It was noted that carbon monoxide tubes had an interference with ozone. Thus, no carbon nronoxide measurements could be taken with ozone present. In order to detenuine ozone values when measuring very high concenfrations, smaller gas sample volumes were collected in 5 mL or 20 mL syringes. The syringe was connected to the detection tube by means of flexible tubing. A syringe volume was expelled into the detection tube and then removed allowing total flow volume of 100 ml to occur. The observed gas concenttation was then multiplied by the volume ratio of the detection rnbe volume over the syringe volume. The DRAEGER portable gas detection system had a precision of ± 15% (Draeger Safety AG & Co., Luebeck, Germany).

[0055] Spore recoveries and aseptic methods were in accordance with manufachtrer (NAMSA, Northwood, OH, USA) instructions for population verification oiBacillus subtilis spore strips. After ionization treatment and 24h storage, each strip was aseptically removed from bag and transferred into sterile 20x 150 mm test tube containing 10 mL of 0.1 % sterile peptone. Seven to ten sterile 6mm glass beads were ftien added to each test mbe. Each test tube was vortexed (model vortexer 59, Denville Scientific, Inc., Metuchen, NJ, USA) on high speed for I20s or until the spore sftip was fully macerated into loose fibers. Test tubes were then heat shocked by placing into a 500 mL beaker with 300 mL of water heated to 90.C and maintained at 80-85.C for 10 minutes. Test hrbes were transferred to a cold tap water bath momentarily (2 min), and then to ice water bath to rapidly cool test hibes to 0-4.C. Test tubes were then removed ftom ice bath and firrther serial dilutions were perfonned including 10"2, 1.-3, 10.4, and/or 10"5 based on tteatments or recoveries of positive (t) controls (Bacillus populations of 1.5-2.5 X 10®/strip, 6.18-6.40 logio). The required aliquot volumes fronr corresponding serial dilutions were then plated into respective petri dishes (100 X 15 mm) containing sterile Tryptic Soy Agar (TSA) prepared per Difco Manual specifications for spore colony enumeration [5]. TSA plates were incubated at 30-3 l.c and colony growth and recoveries were monitored at 24h, 48h, and 72h. 16 2015261647 26 Nov 2015 (0056) Relative humidity and temperahjres inside the storage bags were measured using a Springfield® Precise Temp™ relative humidity sensor (Taylor Precision Products, Oak Brook, IL, USA) recorded at Oh and 24 h storage. (0057( FIG. 3 and FIG. .4 document the reactive oxygen species generation during in-package ionization at the specified times for both 13.5 kv and 80 kV. It can be seen from these data that high levels ofreactive oxygen species can be generated for both air and MA gas. At 13.5 kv, an ozone generation rate of 1,200 and 1,500 ppm per minute were observed for air and MA gas, respectively. At 80kV, an ozone generation rate of 3,750 ppm and 6,250 ppm per minute were observed for air and MA gas, respectively. These results suggest that increased ionization voltage increases the generation rate ofreactive oxygen species .In air, the nifrous gas concentrations did not significantly change with ionization voltage. Both voltages (13.5 kv and 80 kV) achieved maximum nitrous gas concentration of approxinrately 1,000 ppm with an air atmosphere. However, the MA gas nitrous gas concenttations reached a significantly higher level with increased ionization voltage. Nitrous gas concentrations at 80 kv reached over 4,000 ppm at 120 seconds freatment time. (0058( At least some of the increase in the ozone generation rate, and the resultant concentrations at the higher voltages may be atttibuted to the longer ionization path resulting from die 4.5cm elecfrode spacing when using 80kV in some ofthe experiments. However, some ofthe increase may also be due to the higher voltage gradients, which may also generate other reactive species that have not yet been measured. Each ofthe constibient gases has a different ionization potential at atmospheric pressure. These factors interact, and thus a different set of experiments would, be perfomied to optimize these parameters. (0059( Both ozone, and nittous oxides levels decayed to zero within 24 hours of treatment. However, there was a measureable carbon monoxide concentration in MA gas at 24-hours post-treatment with levels 200 ppm and 400 ppm for the 13.5 kv and 80 kv at freabnent times of 300 s and 120 s, respectively. The current carbon monoxide measurement method did not allow measurement in the presence of ozone (e.g., time zero). 17 2015261647 26 Nov 2015 [0060] FIG. 5 and FIG. 6 illustrate the spore reductions achieved with ANEP treatment. In-package ionization both inside and outside of the ionization field at 13.5 kv and 80 kv may eliminate Bacillus subtilis spores. At 13.5 kv, tteatment times for MA gas spore elimination were 180 s and 300 s for outside and inside field positioning, respectively. At 13.5 kv, treatment times for air ahnosphere, spore elimination occurred at 300 s inside ionization field with insignificant spore reductions (< 1.2 log) outside ofthe ionization field.

[0.61] However, at 80 kv, complete elimination of spores was obtained in 15 s or less with no measureable difference in spore reduction rates between air and MA gas. When the samples were disposed inside the field, high voltage treatment times showed increased spore populations (>2 log) recoveries at 48 h conrpared to 24 h; however, no addition organisms were recovered at 72 h. These results demonstrate that using an 80 kv in-package ionization process, air or MA gas can provide complete elimination of Bacillus subtilis spores in 15s or less. For these studies, dry air was used and all santples were maintained at between 20% and 30% relative humidity at room temperature. Elevated humidity may provide an even greater spore reduction rate.

[0062] Atmospheric or MA plasma may be advantageous to quickly remove microorganisms from surfaces. These experimental resulte clearly demonsttate the sterilization capability of in-package ionization for Bacillus subtilis spores, and would be indicative of results that should be obtained with other microorganisms. Using in-package ionization processes with higher ionization voltages, voltage gradients and MA gas resulting in shorter sterilization tinres. A complete elimination of spores was observed in less than 15 s or les.s for air and MA gas at 80 kV. In addition, at 13.5 kv spore elimination can be achieved with MA and air in 300 s or less.

[0063] In yet another aspect, to further understand the results ofthe voltage gradient and the different MA packaging atmospheres on the efficacy of in-package plasma-based sterilization, a further rivo-phase series of experiments was perfonned. In phase I, in-package ionization was performed on empty, sealed packages for sixteen gas blends and the concenfrations ofreactive gas species 2015261647 26 Nov 2015 measured. The composition ofthese gas blends were selected to encoirrpass a wide range ofcoirrmon gases (oxygen, nitrogen, carbon dioxide, helium, and argon) and shown in Table 1.

[0064J Thee data were used to identify three gas blends hat yielded high concenttations of measured reactive gas species (e.g., ozone, nitric oxides, carbon monoxide) and, along with air, were then used for sporicidal treatment in phase II. The selection of the particular gas mixhires was appropriate for a survey experiment where a large range of valid data was being collected, rather than in an experiment exploring one or more of the mixtures in detail. As such, the selection of the gas mixhjres, and the voltages and voltage gradients that were used should be understood as providing for comparable sporicidal treatarent data behveen the differing parameters, rather than limiting the scope of the MA mixtures and processing parameters that may be desirable in a particular situation.

[0065] Phase I ofthis experinrent series comprised a 16 X 7 X 2 experinrent: 16 gas blends of02, N2, C02, He, andAr were configured (Table 1) and placed inside of packages sealed in a Cryovac Β2630 high barrier package. The sealed packages (22 cm X 30 cm) were filled with 1.76 L of the selected gas blend using a calibrated flow meter and stored at room temperature (22.C). All of packages were treated in duplicate with the ΡΚ-2 ionization system at 50 kv RMS (65-75 w @ 0.5-0.8 mA) with a depth of2.5 cm. Ionization electrodes consisted of rectangular wrappings of wire coils approximately 7.5 cm X 11.5 cm placed directly above and below the center of the package. Underneath the package was a TYVEK layer (0.1905 mm) and a layer of red polypropylene (1.94 mnr) sandwiched between the package and the bottom electrode The TYVEK layer was intended to simulate a bag that had layers ofrtvo different materials, as while TYVEK is a preferred material for use in medical instrument packaging, the material is not gas tight, so it would likely be conrbined with a gas tight polypropylene or other such bag as used in these experiments.

[0066] Treattnent times used were: Os, 15 s, 30 s, 60s, 150 s, 300 s, and 600 s. Ozone and nitrogen oxide gas measurements were taken using the DRAEGER gas analysis system immediately after treatment and at 24 h room temperature storage. 19 2015261647 26 Nov 2015

Carbon monoxide measurements using the DRAEGER system cannot be taken in the presence of high ozone concentrations due to interference and were only taken after 24 h. Relative humidity and temperature were also recorded. 0067ء] In summary, the results ofphase I were that all of the selected gas blends could be ionized to generate bactericidal molecules (e.g., ozone, nitric oxides, and carbon monoxide). In general, a greater concenftation of ozone was observed for gas blends with higher oxygen content, except when a noble gas (in these experiments, argon or heliunr) was added to the gas blends. When a noble gas was added to the gas blend, the minimum voltage needed for ionization was reduced; however, the benefit of adding noble gas to generate increased reactive gas species was mixed. Some gas blends showed increased ozone concentratio'ns while others showed reduced ozone concenftations. Maximum ozone concenftations were obtained in gas blend # 12 " 16,000 ppm at 150 s an.d 18,750 ppm at 600 s. Maxinrum nitric oxide concentrations of4,500 ppm were also generated in gas blend # 12 with a number of other gas blends (#10, #11, and #16) having maximmn nifric oxide concenftations between 1,500 and 2,000 ppm. Carbon monoxide measurement is only available after 24 h due to measurement interference from high concentrations ofnifric oxide and ozone. After 24 h storage, maximum carbon monoxide levels of375 ppm were obtained from gas blend #9 at 600 s treatment. (0068] In phase II of this experiment series a4x5x2x2 experiment was performed. Four gas blends identified in phase I with significant concentration of reactive gas species were selected (shown in bold in Table 1). Active plasma treatment times used were: Os, 15 s, 30 s, 60 s, and 120 s. Single spore sfrips (1.5- 2.5 X 10٥ cfij) of Bacillus subtilis var. niger were placed in open pefti dishes at the center (direct exposure to the ionizing field) inside the sealed bag and at the right edge (indirect exposure) inside of the bag. The packages (22 cm X 30 cm) were then sealed and filled with two liters of the selected gas (#7, #9, #12, #16) using a calibrated flow meter and stored at room tenrperature (22.C). All packages were treated in duplicate with the ΡΚ-2 ionization system at 50 kv RMS (65-75 w @ 0.5-0.8 mA) with a depth of2.5 cm. All treated packages were stored for 24 h and 20 2015261647 26 Nov 2015 then bacterial spore recoveries were conducted using standard microbiological methods as previously described. In addition, a 72 hour recovery was also performed to ensure no regrowth.

[0069] In summaiy, for phase II, the results documented complete elimination ofbacterial spores with all treatmente for both direct and indirect exposure after 24 h storage. The tinre required for complete elimination (greater than 6 log reduction) varied with the gas blend. The shortest times for spore elimination were 60 s for both direct and indirect tteatment in gas blend # 9 and #16. The longest times were 120 s for gas blend # 7 (air) and # 12. Additional reductions in treatment times may likely be achieved by fiirther adjustnrent of processing parameters such as increasi'ng electric field voltages, reducing electrode gap, and electrode geometty. The results demonstrate that in-package ionization can eliminate bacterial spores, whether under direct or indirect exposure, from inside medical packages and potentially provides an alternative ηοη-thermal sterilization method for these products.

Table 1. Selected gas blends used in Phase I of experiments. Gas blends used in Phase II are shoi in bold.

Gas Blend ئ0 ν2 .: , : At .He. 1 .'5% 80% 10%,::',,., ' —.-:-;-'إ 5%-.., 2 5% : 80% .'ا؛..%0ا؛إ:-ا,'.:) ' 5% ثم ؛ %1.0 : 25% 45%) 1-:./.:: -- 20% ه/لا10 25%؛ 45% 20% ؛ 5 20% ثم %10 60% 10% 6 20% 10% 60% 10% (Air) 7 22% 78% ' ' 77 —ثم ) 8 22% ثم %30 ؛%40 -:.--.-.-8% 9 22% 30% 40% 8% -- 10,, 50% 10% 20% -- 20% .لآ:::': 11 50:./. 10% 20% 20% 21 12 65% 5% 30./. ٠٠ ..٠٠ 13' 65% 5% 20% -- 10¾ 14' 65% ; 5% 20% 10% .. 15 ;؛ : %80 5./. 10./. 5% 16 80% 5./. 10./. 5./. 2015261647 26 Nov 2015 [0070] The protocol for this second series ofexperimente was similar to that of the first set of experiments and only salient differences in the protocol are presented.

[0071] Gas tanks with 16 different compositions were purchased from a local gas supplier at specified concentrations, each with a certificate of analysis. These gas coinposition(s) were then metered into sealed package at a rate of 2.112 L/min using a flow meter (Model 2260, Gilmont Instruments, Inc., Barrington, IL, USA) yielding final fill, volume of 1.76 L with average fill time of 50 s. The gas compositions were verified using an oxygen analyzer to verify oxygen concentrations.

[0072] Treatments were carried out utilizing ΡΚ-2 system. The electrodes were made from coils of wire wound around a planar dielectric form with a treatment area of 86.25 cm2 (7.5 cm X 11.5 cm), and spaced apart by the treatment distance: in this case 2.5cm or 4.5 cm. The storage bags containing spore samples were filled with the working gas and purged three times to ensure purity of the gas in the bag.

[0073] The temperature of the electrodes was measured prior to and immediately after treatoent using an infrared thermometer (Omega Engineering, Inc., Stamford, CT, USA). The electrodes were allowed to cool to reach room temperahrre (23-25 °C) between treatments forunifonn treatment temperahire conditions. Relative humidity and temperatures inside the storage bags were measured using a Springfield® Precise TempTM relative humidity sensor (Taylor Precision Products, Oak Brook, IL, USA) recorded at 0 h and 24 h storage.

Relative humidity varied daily and ranged from 20-50% for all samples tested. 22 2015261647 26 Nov 2015

Ozone and nitric oxide concenfrations were measured inunediately following the treatment and after 24 h storage using the techniques previously described. ρπ4،λ Bacillus subtilis var, niger (B. atrophaeus) ا؟لأا٠ت١ة جاً0بل١ة Northwood, OH, USA) with size of 3.2 cm X 0.6 cm, each containing Bacillus populations of 1.5-2.5 X 10٥ colony fonning units per strip were loaded into open sterile petri dish inside freatment package and then used in ionization treatments. Spore recoveries and aseptic methods were followed per manufacturer (NAMSA, Northwood, OH, USA) for population verification ofBacillus subtilis spore strips as previously described [0075] Gas concenttations and Bacillus subtilis populations were analyzed in SAS Version 9.2 (Statistical Analysis Software, Caiy, NC). Mean comparisons were performed using the GLM Procedure and the Tukey Multiple Mean Conrparison with a ρ<0.05.

[0076] All of 16 gas blends could be ionized to generate measurable levels of ozone, nitric oxides, and carbon monoxide under the specified conditions, with the results shown in Table 2. In general, greater concentrations of ozone were observed for gas blends with higher oxygen content except when Ar or He gas were added into gas blends. These noble gases have low ionization energy requirements, and, when blended with other gasses reduce the minimum ionization voltage gradient required. When a noble gas was blended into 22% oxygen gas blends the maxinrum ozone concentration increased. This is shown in the results where gas blends #8 and #9 (8% noble gas) achieved 1125 ppm ozone at 15 s ionization whereas gas #7 (air - a similar (22%) oxygen composition without noble gas) took approximately 30 s. Further, gas # 7 reached a maximum ozone concentration of 2,750 ppm whereas gas mixtures # 8 and #9 reached a maximum 8,000 ppm.

Table 2. Concentration of ozone immediately after treattnent for specified gas blends. Results are color coded for noble gas additions (He addition in bold and Ar addition in italic). 23 2015261647 26 Nov 2015

Gas Os 41 0 #2 0 43 0 #4 0 45 ٥ #6 0 4٦ 0 48 ٥ <؟4 0 410 ٥ 411 0 #12 0 413 ٥ #14 0 415 ٥ #16 0 15 s 30 s 406.25 562.5 468.75 1375 275 625 375 1125 875 1625 500 1500 350 1500 1125 1500 1125 1875 2500 2000 2500 3000 2625 3250 625 1500 375 625 1125 2375 1375 2750

Treatment Time 60 s 625 I50s 625 1875 2000 1000 1500 1500 2875 2000 4250 2125 4000 2000 2750 3000 5000 3000 4750 5000 6250 4000 6125 4375 15000 2000 2750 3000 2750 3375 5000 3750 4625 300 s 600 s 1125 312.5 2000 750 2000 1500 2750 1500 6250 3750 6125 4000 2750 2750 10000 8125 11250 7875 7500 10000 9375 12500 16875 18750 3000 4000 3000 5500 10625 13125 10000 14375 0077؛] Interestingly, for all gas blends evaluated the maximum ozone concenttation was achieved for gas blend # 12 (65% .2-5% N2 -30% c٥2) which contained no noble gas. It achieved ozone concentrations of 15,000 ppm at I50s and a maximum 18,750 ppm at 600 s. This concentration of 15,000 ppm at I50s is 2.5 times greater than any other gas blend. When noble gas was blended into a 65% Ο2 gas (#13 and #14) reduced ozone concentrations were obtained. It is suspected that the helium ions are preferentially ionized creating lower energy electrons which in him create less ozone and nitric oxides. Further, in gases # 15 and #16 when the oxygen content is increased (80% 02) and noble gas is added the ozone concentration again increases to very high levels (> 10,000 ppnt). The details of the plasma dynamics are not yet fillly understood. However, it is clear that a range of gas and voltage parameters has been identified where efficacious results are obtained. ]00781 Nitric oxide concentrations immediately after treatment are shown in Table 3. The maximum nittic oxide concentration of4,250 ppm were generated in gas blend # 12 at 600 s with a number of other gas blends (#9, #11, and #16) 24 2015261647 26Nov2015 having maximum nitric oxide concentrations beftveen 1,500 and 2,000 ppm. There were no measurable concentrations of ozone or nitric oxide after 24 h. Carbon monoxide measurements were only available after 24 h due to measurenrent interference from high concentrations of ΝΟχ and .3. After 24 h storage, maximum carbon monoxide measurements of 375 ppm CO were obtained from gas blend #9 at 600 s treatment (Table 4).

Table 3. Concentration of nitric oxides immediately after treatment for specified gas blends. Results are coded for noble gas additions (He addition in bold and Ar addition in italic).

Gas Os 15s 30 s Treatment 60 s Time I50s 300 s 600 s 11 ٥ 3.5 6.25 11.25 18.75 25 72.5 ئ 0 12.5 56.,25 81.25 100 131.25 21.25 43 ٥ 12.5 18.75 225 200 200 100 44 0 7.5 22.5 %٦.5 112.5 100 50 45 ٥ 22.5 37.5 75 250 550 425 ة4 0 16.25 25 225 300 550 400 #7 (Air) 0 31.5 31.5 75 450 900 700 48 0 87.5 112.5 325 450 1000 ٥25 #9 0 50 93.75 350 550 1500 875 410 ٥ 37.5 137.5 500 1000 800 2000 #11 0 100 75 200 225 1550 1750 #12 0 160 270 300 2500 4250 4250 413 0 31.25 31.25 50 350 350 550 414 0 7.5 37.5 50 175 250 450 ة\4 0 37.5 100 325 400 450 1000 416 ٥ 43.75 250 375 650 1000 1560

Table 4. Concentration of carbon monoxide 24 hours after tteatment for selected gas blends.

Gas Os 15 s 30 s Treatment Time 60s I50s 300 s 600 s 4٦ (Α\ή 0 0 0 0 0 0 15 #9 0 31.25 50 112.5 150 325 375 #12 0 20 50 in .5 150 205 250 4\6 0 3 12.5 18.75 40 100 137.5 25 2015261647 26 Nov 2015 [0079] Results in phase II showed complete elimination of bacterial spores with all treatment parameters for both direct and indirect exposure of the sanrple and are presented in Table 5. The time required for complete elimination of the spores (greater than 6 log reduction) varied with the gas blend. The shortest times for spore elimination were 60 s for both direct and indirect treatment in gas blend # 9 and #16. The longest times were 120 s for gas blend # 7 (air) and #12.

Table 5. spore reductions fox Bacillus subtilis var. tiiger after treatnrent and 24 h storage in sealed packages of selected gas blends. ‘D’ indicates direct field exposure and '1' indicates indirect field exposure, (logio)

Treatment Time

Gas D/I Os 15 s 30 s 60s I20s #7 (Air) D 0 0.398 0.408 2.39 6.17* #7 (Air) I 0 0.419 0.300 2.63 6.17* 49 D 0 0.365 3.11 6.40* 6 40* 49 I 0 0.450 3.81 6.40* 6.40* #12 D 0 0.345 0.645 2.80 6.26* #12 I 0 0.310 0.653 6.26* 6.26* #16 D 0 0.513 2.81 6.39* 6.39* #16 I 0 0.592 2.90 6.39* 6.39* * indicates no recoverable organisms found after 72 hrs recovery.

[0080] Additional reductions in treatment times may likely be achieved by fiirther adjustment ofprocessing parameters such as elecfric field voltages, electrode gap, and electrode geometry. The results from the sfttdies demonsfrate that with in-package ionization treatment, whether under direct or indirect exposure, bacterial spores can be eliminated from inside packages, potentially providing non-thennal sterilization for medical products.

[0081] Since the voltage gradient of about 12.5 kV/cm represents the about lowest value of ionization potential for other than the noble gasses, this value represents about a lower bound on the voltage gradient that could be effective. However, the relatively low rate ofproduction ofreactive species at the low voltage is reflected in the longer ANEP generation time to achieve an effective sporicidal effect. As many production processes place an emphasis on throughput, 26 2015261647 26 Nov 2015 the reduction in processing times hat can be achieved with higher voltages and voltage gradients may be beneficial. The type ofMA to be selected may depend on the particular object to be processed؛ and,, the sensitivity of the object to oxidation may place limits on the percentage composition of Ο2 that is desirable. Carbon dioxide MA packaging gasses may preferentially produce CO and this reactant nray be effective in processing certain food products.

[0082J The higher voltages and the longer ANEP column length between the electrodes contribute to both higher rates of generation and possibly to the generation of other reactants, whose effect may be seen in the reduction in processing times. Raising the processing voltage so that the voltage gradient is about 1.4 times the ionization potential of oxygen has been shown to be effective over a wide range ofMA gas conrpositions. The combination of increasing the voltage gradient and the length of the ANEP plasnra column with respect to the volume of the container has been shown to be efficacious.

Table 6. Brief Summary of Experimental Results

Voltage (kv) Voltage gradient (kV/cm) ANEP Path Length (cm) Ionization Volume (cm٨3) Total Package Volume (cm٨3) Ionization Volume Ratio (%) Active treatoent time for complete sterilization in MA (sec) 12.5 12.5 1.0 86 500- 3780 2.3-16.1 180 50 20 2.5 215 1760 12.2 ~60 80 Υ1.Ί 4.5 788 1500 52.5 15 J0083J While the methods disclosed herein have been described and shown with reference to particular steps perfonned in a particular order, it will be understood that these steps may be combined, sub-divided, or reordered to form an equivalent nrethod without departing ftom the teachings of the present invention. 27 2015261647 26 Nov 2015

Accordingly, unless specifically indicated herein, the order and grouping of steps is not a limitation of the present invention.

[0084J Although only a few exanrples of this invention have been described in detail above, those skilled in the art will readily appreciate that many modifications are possible without materially departing from the novel teachings and advantages of the invention. Accordingly, all such modifications are intended to be included within the scope of this invention as defined in the following clainrs. 28

Claims (28)

1. The claims defining the invention are as follows:

   1. An apparatus for in-package processing of a product, the apparatus comprising; a pair of electrodes, spaced apart, at least one dielectric layer disposed between the pair of electrodes, an alternating current power source, wherein a voltage greater than 50kV is applied between the electrodes so as to form an atmospheric non-equilibrium plasma (ANEP) in at least a portion of a closed container disposable, at least in part, between the pair of electrodes, the closed container having a working gas fill at substantially atmospheric pressure and the voltage, the working gas and the spacing between the electrodes selected such that the ANEP is formed only within the closed container.
2. 2. The apparatus of claim 1, wherein the dielectric layer is conformal to at least one of the pair of electrodes.
3. 3. The apparatus of claim 1, wherein the dielectric layer is a dielectric sheet.
4. 4. The apparatus of claim 1, wherein the pair of electrodes are planar.
5. 5. The apparatus of claim 1, wherein the pair of electrodes are shaped to be conformal with the closed container.
6. 6. The apparatus of claims 1 through 5, wherein the voltage is greater than 50kV and less than 80kV RMS.
7. 7. The apparatus of claims 1 through 4, wherein the voltage is greater than 80 kV and less than 130kV RMS.
8. 8. The apparatus of claims 1 through 7, further comprising the closed container, sized and dimensioned such that at least a portion thereof is positionable between the pair of electrodes.
9. 9. The apparatus of claims 1 through 8, wherein the spacing between the electrodes is adjustable so as to partially compress the container.
10. 10. The apparatus of claims 1 through 9, wherein a column length of the ANEP is greater than or equal to 2.0 cm.
11. 11. The apparatus of claims 1 through 10, wherein the pair of electrodes is configured such that an object in the container is outside of an ANEP column.
12. 12. The apparatus of claims 1 through 11, wherein the working gas comprises at least 5% and less than 20% of a noble gas.
13. 13. The apparatus of claims 1 through 12, wherein the working gas comprises at least one of C02,02, N2, or atmospheric air.
14. 14. The apparatus of 13, wherein the working gas further comprises a selectable amount of water vapor.
15. 15. The apparatus of claims 1 through 7, wherein the spacing between the electrodes is adjustable such that the closed container is captivated by the pair of electrodes.
16. 16. A method of treating a product, comprising: providing the apparatus of claim 1; providing an object to be treated using the apparatus; providing a sealable dielectric container; introducing the object into a sealable dielectric container; filling the container with a working gas at substantially atmospheric pressure; sealing the dielectric container; introducing at least a portion of the sealed dielectric container into a space between the electrodes of the apparatus; applying a voltage to the apparatus for a predetermined period of time; removing the voltage from the apparatus after the predetermined period of time; and removing the container from the apparatus.
17. 17. The method of claim 16, further comprising: storing the treated object in the sealed container.
18. 18. The method of claim 16 further comprising: performing the treatment steps of claim 16, for another predetermined time after a predetermined storage time and before opening the sealed container.
19. 19. The method of claim 16, where the container comprises a dielectric material.
20. 20. The method of claim 19, wherein the container comprises a plurality of dielectric layers.
21. 21. The method of claim 20, wherein one of the plurality of dielectric layers is formed of high-density polyethylene fibers.
22. 22. The method of claim 21, wherein the layer formed of high-density polyethelyene fibers is TYVEK.
23. 23. The method of claims 16 through 22 further comprising: agitating or manipulating the container to expose all surfaces of the object during the step of applying the voltage.
24. 24. The method of claims 16 through 22, further comprising: agitating or manipulating the container to expose all surfaces of the object after the step of removing the container.
25. 25. The method of claim 16 through 24, wherein the working gas comprises at least 5% and less than 20%of a noble gas.
26. 26. The method of claims 16 through 25, wherein the working gas comprises at least one of CO2, O2, N2 or atmospheric air.
27. 27. The method of 26, wherein the working gas further comprises a selectable amount of water vapor.
28. 28. The method of claim 16, wherein the spacing of the electrodes is adjusted such that the dielectric container is captivated against the electrodes by an internal gas pressure in the dielectric container.