Abstract:
A package providing a storage space with features capable of affecting atmospheric conditions therein, comprising a body having a first and at least one second storage space adjacent thereto, wherein the first storage space is defined by sidewalls, and the at least one second storage space is defined by sidewalls including at least a portion thereof which further defines the first storage space, the second storage space providing a reservoir for containing an antimicrobial agent therein, wherein the antimicrobial agent includes a precursor to chlorine dioxide gas and the portion of the sidewall which further defines the first storage space is configured to allow for the delivery of the chlorine dioxide gas from the second storage space into the first storage space.

Description:
CROSS-REFERENCE TO RELATED APPLICATION 
     The instant application claims priority to U.S. Provisional Patent Application Ser. No. 61/509,034 filed Jul. 18, 2011, the entire disclosure of which is expressly incorporated herein by reference. 
    
    
     STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT 
     The invention was made with government support under 2008-51110-04352 awarded by the U.S. Department of Agriculture. The government has certain rights in the invention. 
    
    
     BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     The present invention is directed to packaging, and more particularly, to packages and packaging systems used for shipping items, such as food products, that include one or more features for reducing harmful microbial contaminants which may be associated therewith. 
     2. Background of the Related Art 
     The following description of the background and embodiments of the invention thereafter is provided to aid in understanding the invention, but is not admitted to describe or constitute prior art to the invention. The contents of the articles, patents, and patent applications, and all other documents and electronically available information mentioned or cited in this application, are hereby incorporated by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference, including any references cited in the articles, patents, patent applications and documents cited herein. Applicants reserve the right to physically incorporate into this application any and all materials and information from any such articles, patents, patent applications, or other documents. 
     Disposable containers for packaging, shipping, displaying or otherwise housing perishable products are of significant economic importance. Many such packaging products have been developed with features that significantly improve or extend the life span of perishable products during shipping. These packages advantageously provide substantial savings to companies involved in the production and sale of perishable items in the marketplace. 
     While many packaging products include features for creating a “modified atmosphere” intended to extend the lifespan of a perishable product, these packaging products lack features which address potentially harmful contamination associated with the perishable products. 
     SUMMARY OF THE INVENTION 
     The invention is generally directed to methods and systems for product packaging having one or more features which facilitate the health and safety of products contained therein. In some embodiments, methods and systems of the invention are directed to packaging for food products which include one or more features that facilitate the health and safety of products contained therein by reducing or eliminating causes of foodborne illnesses. 
     A packaging system constructed in accordance with an exemplary embodiment of the invention provides a storage space and includes a feature for releasing an agent into the storage space, wherein the agent is configured to, or otherwise capable of, affecting food products therein in a manner which facilitates the elimination or retardation of the spread of diseases, pathogenic bacteria, viruses, toxins or parasites of the type which may exist on food products or result from the consumption of contaminated food. 
     The agent may therefore be an oxidizing agent, a disinfectant possessing antifungal properties, antibacterial (bacteriostatic or bactericidal) properties, insecticidal properties, and/or antimicrobial properties. Examples of agents include, but are not limited to, chlorine dioxide, silver nitrate, thiobendazole, zinc acetate, chlorpyrifos, and others, or combinations thereof, in any form, gaseous, liquid or solid. For convenience and ease in description, the agent having one or more of such properties is hereinafter referred to as an “antimicrobial agent.” 
     In some embodiments, the invention is directed to a package which includes a sealable storage space. The package and/or walls of the storage space may be fabricated of any suitable material. For example, the storage space may be constructed, either wholly or in part, by a plastic or polymer material such as polyethylene. The storage space walls may be substantially permeable, non-permeable or semi-permeable. 
     The package further includes a body containing an antimicrobial agent in an agent reservoir. The body or agent reservoir is constructed to release the antimicrobial agent into the storage space, either as a result of its properties or physical construction, or a combination thereof. For example, one or more body walls may be perforated, or constructed of a permeable or semi-permeable material or membrane, or a combination thereof, which ultimately provides for the desired release of the agent into the storage space. The body walls may include multiple locations from which agent is released. 
     The body may be rigid or flexible or include portions which are rigid and portions which are flexible. The body may further include multiple layers of material and one or more chambers with one or more antimicrobial agents contained therein. 
     In some embodiments, the antimicrobial releasing body is fabricated as a reservoir associated with the package. For example, the package may include a seam in which the body may be incorporated. The body may be configured with the package in other natural-appearing configurations, such as within a side wall of the package itself. 
     In some embodiments, the antimicrobial agent releasing body may be inserted into an existing package. The outer wall of the body may contain an adhesive which facilitates being adhered to an interior wall of the storage space of a package. 
     In some embodiments, the body construction is used to affect the rate of release of the agent into the storage space. The body construction or agent may differ depending on the desired rate of release of the agent and/or the food product contained in the package. 
     In some embodiments, the body includes a releasing tab  22  which may be adhered to the body in a position covering a permeable or semi-permeable wall. The tab can be removed upon sealing the package to effectuate the release of the agent. 
     In some embodiments, the body may include one or more releasing tabs  22  which can be selectively removed upon sealing the package depending on the food product contained therein. 
     It is envisioned that the invention may be used with any product in which it would advantageous to reduce harmful microbial contaminants, such as surgical equipment, tools, sensitive materials, medicines, and food products including, but not limited to, fruits (e.g., cut and/or whole fruit), vegetables (e.g., cut and/or whole vegetables), meats and/or seafood, or any other non-comestible product which may harbor harmful contaminants, such as flowers or plants. 
     A packaging system of the invention may be incorporated with other atmospheric modifiers, such as modifiers which improve the lifespan of a product or a desiccant. 
     Some embodiments are directed to a package providing a storage space with features capable of affecting atmospheric conditions therein, comprising a body having a first and at least one second storage space adjacent thereto, wherein the first storage space is defined by sidewalls, and the at least one second storage space is defined by sidewalls including at least a portion thereof which further defines the first storage space, the second storage space providing a reservoir for containing an antimicrobial agent therein, wherein the antimicrobial agent includes a precursor to chlorine dioxide gas and the portion of the sidewall which further defines the first storage space is configured to allow for the delivery of the chlorine dioxide gas from the second storage space into the first storage space. 
     The aforementioned package may be configured so that the at least one second storage space is positioned at a first end of the package body or alternatively in the middle area. There may be multiple second storage spaces, such as two defined at opposing or substantially distal ends of the package body. The second storage space may be substantially tubular in shape. 
     In some embodiments, the portion of the sidewall which further defines the first storage space is constructed of a material which is permeable to chlorine dioxide gas. In other embodiments, the portion of the sidewall may be perforated. 
     In some embodiments, the chlorine dioxide gas precursor is configured to deliver chlorine dioxide gas in dosage which is less than 10 milligrams per day. 
     The invention is also directed to a package providing a storage space with features capable of affecting atmospheric conditions therein, comprising a body divided into a first and at least one second storage space adjacent thereto, wherein the first storage space is defined by sidewalls, and the at least one second storage space includes a shared sidewall with the first storage space, the second storage space providing a reservoir for containing an antimicrobial agent therein, wherein the antimicrobial agent includes a precursor to chlorine dioxide gas and the shared portion of the sidewall is configured to allow for the delivery of the chlorine dioxide gas from the second storage space into the first storage space. 
     In some embodiments, the aforementioned package is configured such that the chlorine dioxide gas precursor is configured to deliver chlorine dioxide gas in dosage which is less than 10 milligrams per day from at least two distally positioned second storage spaces. 
     These and other aspects of the system of the invention will become more readily apparent to those having ordinary skill in the art from the following detailed description of the invention taken in conjunction with the drawings. 
    
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
       So that those having ordinary skill in the art to which the invention pertains will more readily understand how to make and use the invention, embodiments thereof will be described in detail below with reference to the drawings, wherein: 
         FIG. 1  is a schematic view of an embodiment of a package design constructed in accordance with some embodiments of the invention; 
         FIG. 2  is a schematic view of another embodiment of a package design constructed in accordance with some embodiments of the invention; 
         FIG. 3  is a schematic view of yet another embodiment of a package design constructed in accordance with some embodiments of the invention; 
         FIG. 4  is a graph illustrating profiles of gas release for fast- and slow-release chlorine dioxide precursors used in two studies, study  1  and study  2 ; 
         FIG. 5  provides Table 1 containing data relating to a randomized complete block design information for study  1 ; 
         FIG. 6  provides Table 2 containing data relating to the effect of package design and ClO 2  gas level treatments on  Escherichia coli  O157:H7 populations on cut lettuce, among other things; 
         FIG. 7  provides Table 3 containing data relating to the effect of sampling location on  E. coli  O157:H7 populations in packages of some embodiments of the invention, among other things; 
         FIG. 8  provides Table 4 containing data relating to the effect of chlorine dioxide gas (ClO 2 ) level on  E. coli  O157:H7 populations in packages of some embodiments of the invention, among other things; and 
         FIG. 9  provides Table 5 containing data relating to the effect of sampling location on  E. coli  O157:H7 populations in packages of some embodiments of the invention, among other things. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     Reference is now made to  FIG. 1 , wherein there is illustrated a package in accordance with some embodiments of the invention referred to herein and generally designated by the reference numeral  10 . 
     Package  10  generally includes a body  12  having a modified atmosphere storage space  14  with a closable opening  16  allowing for re-sealable and/or non-re-sealable entry thereto, and an antimicrobial agent reservoir (“GR”)  18  separated from modified atmosphere storage space by a wall  20 . Body  12  may be any size or shape, and constructed of a firm or flexible material, as may be desired for the contents to be contained in modified atmosphere storage space  14 . One or more antimicrobial agents are stored in GR  18 . Wall  20  is configured to allow for the migration of the antimicrobial agent from GR  18  into modified atmosphere storage space  14 . 
     In some embodiments, wall  20  is constructed of a material which is permeable, thus allowing for migration of antimicrobial agent into modified atmosphere storage space  14 . In other embodiments, wall  20  is perforated or passageways between GR  18  and modified atmosphere storage space  14  are provided to allow for the migration of antimicrobial agent from GR  18  to modified atmosphere storage space  14 . 
     In some embodiments, package  10  is further configured to include a feature which inhibits migration of the antimicrobial agent from GR  18  to modified atmosphere storage space  14 . This feature may be a mechanical feature, such as a removable cover constructed of a material which inhibits migration of the antimicrobial agent which is disposed over all or a portion of wall  20  or passageways therethrough. In such embodiments, the removable cover may be removed prior to filling modified atmosphere storage space  14  and closing opening  16 . 
     In some embodiments, the antimicrobial agent is selected based on properties which allow for the gradual or time-released migration thereof into modified atmosphere storage space  14 . For example, the migration of antimicrobial agent may require the occurrence of one or more reactions or phase changes prior to migration occurring. The antimicrobial agent may be in a precursor form or provided as an intermediate which will form the agent thereafter. Alternatively, the microbial agent may be encapsulated or otherwise controlled by a time-release substance or mechanism. The antimicrobial agent may also be selected based on properties which inhibit migration when package  10  is not in use. For example, the antimicrobial agent may be of a type or in a form which is relatively dormant or otherwise does not migrate during certain storage conditions, such as when package  10  is stored in a lowered temperature environment. 
     In some embodiments, GR  18  is prefilled with antimicrobial agent, whereas in some embodiments GR  18  is further configured to be filled with antimicrobial agent after package  10  is formed, which may include a receiving port with a closure, such as a twist off cap or patch with adhesive material. 
       FIG. 2  illustrates a package  110  constructed in accordance with another embodiment of the invention in which there are multiple antimicrobial agent reservoirs  118   a  and  118   b  separated from modified atmosphere storage space  114  by corresponding walls  120   a  and  120   b . It should be understood that the various alternatives and embodiments as discussed above with regard to package  10  and wall  20  and GR  18  may be similarly employed with respect to package  110 , and in connection with one or both GRs  118   a,b  and/or walls  120   a, b.    
       FIG. 3  illustrates a package  210  constructed in accordance with another embodiment of the invention which includes an antimicrobial agent reservoir tube  218  thus defining a peripheral wall  220  allowing for the migration of antimicrobial agent into modified atmosphere storage space  214 . GR  218  is shown in a substantially central location of modified atmosphere storage space  214 , but may be located elsewhere. Multiple reservoir tubes such as tube  218  may be employed as well as the various alternatives discussed above with respect to package  10  and reservoir  16  may also be employed with respect to package  210 , reservoir tube  218  and wall  220 . 
     The following detailed description provides exemplary non-limiting embodiments of the invention in which ClO 2  was employed as the antimicrobial agent in packaging containing fresh-cut lettuce and tests were conducted. It should be readily apparent that other agents may be used in connection with the same or other products in accordance with other embodiments of the invention. 
     Studies 
     Introduction 
     Embodiments of the invention as shown in  FIGS. 1 ,  2  and  3  were used in these studies as described herein. ClO 2  gas was generated in the GR at either 4 or 8 mg ClO 2  per kg lettuce per day.  Escherichia coli  O157:H7-artificially inoculated fresh-cut lettuce was packed in the bags and stored at 4° C. for up to seven days. Increasing the ClO 2  gas dose resulted in greater log 10  CFU/g reductions of  E. coli  O157:H7 for the package  10  and package  210  designs. For the package  110  design, the same degree of antimicrobial effect could be achieved with the lower ClO 2  dose. Significantly greater (p&lt;0.05) log in  CFU/g reductions of  E. coli  O157:H7 were observed in lettuce samples taken from locations adjacent to the reservoirs. 
     Materials and Methods 
     Flexible polyolefin packages were formed as shown in  FIGS. 1 ,  2  and  3  (i.e., packages  10 ,  110  and  210 ) and employed herein. The antimicrobial agents consisted of ClO 2  gas precursors (Special Mix with linear release) which were obtained from ICA TriNova LLC (Newnan, Ga.). Two types of precursor were used in the study to provide different levels of ClO 2  gas release: the fast-release type released 8 mg ClO 2  per kg lettuce per day (high level) and the slow-release type released 4 mg ClO 2  per kg lettuce per day (low level). 
     Package  10  for this study included modified atmosphere storage space  14  separated from GR  18  by wall  20  formed by a seal or seam in body  12  having six small openings therein. Package  110  for this study included walls  120   a  and  120   b  formed by two seams each of which having three small openings therein, thus forming the two GRs  118   a  and  118   b . Each reservoir ( 18  and  118   a,b ) contained the ClO 2  precursor. Packages  10  and  210  configured as described were used in study  1  as discussed herein. 
     Package  210  for this study incorporated a reservoir tube  218  constructed of poly(ethylene) (LDPE) for holding the ClO 2  precursor inserted therein, with cut lettuce was placed around tube  218 . The permeability coefficient (P) of the LDPE was 66.0±1.09 kg ClO 2  m/m 2 *s*Pa (Netramai, Rubino, Auras, &amp; Annous, 2009); ClO 2  gas was released from the precursor by permeation through the LDPE tube. Package  210  as described herein was used in study  2 . 
     Chlorine Dioxide Treatment 
     The high level of ClO 2  gas release used in the studies was determined in preliminary experiments by exposing cut Romaine lettuce to different levels of ClO 2  gas in a closed chamber for 1 h. The 8 mg ClO 2 /d level resulted in significant reductions in  E. coli  O157:H7 populations (data not shown) and did not cause noticeable changes in lettuce appearance. The 4 mg ClO 2 /d release level was selected to quantify the antimicrobial effects of a lower ClO 2  dose. 
     To generate the ClO 2  gas, ClO 2  precursor was placed in paper tubes that were inserted into the reservoirs or reservoir tube for each package. A total of 8 g of ClO 2  precursor was added to each package: for reservoir  18  and reservoir tube  218 , this total amount was placed in one paper tube; for the reservoirs  118   a,b , two paper tubes containing 4 g of precursor each were used in each reservoir  118   a  and  118   b , respectively. The high or low level of ClO 2  gas release per kg lettuce (8 mg ClO 2 /d or 4 mg ClO 2 /d, respectively) was determined by the type of precursor used. The profiles of gas release were determined using a titration procedure provided by ICA TriNova and are shown in  FIG. 4 . 
     Preparation of Inoculum 
       Escherichia coli  O157:H7 SEA13B88 (human feces, apple cider-associated disease outbreak), maintained at −80° C. in trypticase soy broth (TSB; Becton Dickinson, Sparks, Md.) and 10% (v/v) glycerol, was grown at 35° C. for 18-24 hours in TSB, transferred to a trypticase soy agar (TSA; Becton Dickinson) slant, and this working stock culture was stored at 4° C. for no more than 21 days. Inoculum was prepared by transferring a loopful (1 μl) of the working stock to 10 ml TSB, which was incubated at 35° C. in a shaking incubator for 6-8 hours. Following incubation, 180 μl of the culture was transferred to 1.8 L of TSB, and then incubated at 35° C. in a shaking incubator for 18-24 hours. The culture was then centrifuged (6740×g) at 4° C. for 15 min After decanting the supernatant, the resulting pellet was resuspended in sterile deionized water and centrifuged. The supernatant was decanted and the pellet was resuspended in 3.6 L of sterile deionized water. The concentration of the inoculum was determined by serially diluting the inoculum in 0.1% peptone water (PW; Becton Dickinson) and plating on TSA. 
     Inoculation of Lettuce 
     Romaine lettuce ( Lactuca sativa  L. var.  longifolia ) was purchased at a local supermarket (in Wyndmoor, Pa.) and stored at 4±2° C. for a maximum of 24 hours before use in experiments. Damaged outer leaves were removed from each head of lettuce, and the lettuce was cut into pieces approximately 4-6 cm 2  and immediately submerged into the  E. coli  O157:H7 inoculum suspension for 5 min Excess liquid culture on the lettuce was removed using a conventional salad spinner purchased at a retail store for 1 min. The prepared lettuce was placed into an open container and allowed to dry at 22±2° C. for 2 h in a biosafety cabinet prior to packaging. 
     Packaging of  E. coli  O157:H7-Inoculated Lettuce 
       E. coli  O157:H7-inoculated lettuce (283 g [about 10 oz]) was placed in each package and the appropriate paper tube(s) containing the ClO 2  precursor were placed in the reservoirs as described herein. The food storage spaces of the packages were sealed, photographed (day  0 ), and stored upright at 4±1° C. for up to 7 days. Following the 1st, 4th, or 7th day of storage, samples were photographed and visually inspected for changes in appearance and color, and then evaluated for residual microbial populations. 
     Microbial Enumeration 
     Results from preliminary experiments suggested that there was no significant difference in  E. coli  O157:H7 cell reductions on lettuce samples taken from the top or bottom of the single reservoir  18  package  10  and dual reservoir  118   a,b  package  110  designs. Thus, sampling locations used with each bag design were based on dividing the bag into lengthwise areas, substantially parallel to the gas reservoirs, and taking samples therefrom. Microbial enumeration and comparison was focused on these sampling locations. 
     For sampling, one 25-g sample of lettuce was removed from each location within each bag and homogenized with 75 mL Dey-Engley (DE) neutralizing broth (Becton Dickinson) for 1 min, using a stomacher blender. Undiluted DE broth from the homogenate was serially diluted in sterile neutralizing broth to the desired dilutions and then spread-plated in duplicate onto TSA. The plates were incubated at 37±0.5° C. for 2 hours (for injured cells to recover), overlaid with Sorbitol MacConkey agar (SMAC; Remel, Lenexa, Kans.), supplemented with cefixime and potassium tellurite (CT; Invitrogen, Dynal AS, Oslo, Norway), and then incubated at 37±0.5° C. for an additional 22 hours; colonies were enumerated as  E. coli  O157:H7 (Keskinen, Burke, &amp; Annous, 2009). 
     Statistical Analyses 
     The experimental design followed a randomized complete block design, as outlined in Table 1 shown in  FIG. 5 , in which the experiments were repeated twice (2 blocks) and each experiment involved three replicates. The results obtained were statistically evaluated by analysis of variance (ANOVA) using the Statistical Analysis System (SAS) software (SAS Institute Inc., Cary, N.C.) at the confidence level of 95% (a=0.05) with Tukey&#39;s adjustment for comparison of means. 
     Results and Discussion 
     This work considered the packaging system as one safety component to be used in a hurdle strategy. Two consecutive studies were conducted to evaluate the impact of packaging design modifications on the efficacy of the ClO 2  gas. Study  1  compared the package  10  and package  110  designs in which the ClO 2  was released from one or two reservoirs or side chambers of the package into the modified atmosphere storage space, or for purposes of this embodiment, a food storage space for holding the lettuce. Study  2  was conducted with the  210  package, at least to evaluate the effect of minimizing the distance between the ClO 2  gas releasing reservoir and the lettuce. 
     Study  1 : Efficacy of ClO 2  in 1-GR and 2-GR Bags 
     The dimensions of the reservoir were determined in packages  10  and  110  by, among other things, by taking into consideration the flexible volume, ClO 2  dose, ClO 2  chemical characteristics, and lettuce weight per bag. The openings on the bags&#39; interior walls that formed the reservoirs were designed to distribute an equal amount of ClO 2  gas to the lettuce sample throughout the whole modified atmosphere storage space. The total numbers of openings in the walls of both designs were the same, but the locations of the openings in the walls were different. Gas distribution in the bags is presumed to be at least partially dependent on the reservoir location, that is, in the package  10  design the ClO2 gas enters the modified atmosphere storage space from one direction only, whereas in the package  110  design the ClO 2  gas enters from two directions. Unmodified packages were used as controls in the study. 
     A summary of the influence of package design and ClO 2  gas levels on reductions of the average populations of  E. coli  O157:H7 recovered from the bagged, inoculated lettuce after storage is provided in Table 2 shown in  FIG. 6 , wherein 1-GR refers to the package  10  design and 2-GR refers to the package  110  design. The log 10  CFU reductions were calculated from the difference of  E. coli  O157:H7 populations recovered from the control bags and those recovered from package  10  and package  110 , and represent the total reduction in  E. coli  O157:H7 populations by ClO 2  gas at each storage time. 
     For the package  10  design, increasing the ClO 2  gas dose (from 4 to 8 mg ClO 2 /d) resulted in a significant increase in the average log 10  CFU/g reductions for the packaged samples on day  1  and day  4  of storage. However, increasing the dose of ClO 2  gas per day by 100% did not double the reduction of the pathogen populations. For the package  110  design, increasing ClO 2  gas dose resulted in a significant increase in the reduction of  E. coli  O157:H7 cells only on day  1  of storage; in the package  110  design the ClO 2  gas level had no significant effect on population reductions after day  4  and day  7 . 
     The reservoir locations and openings affected the antimicrobial efficacy of the ClO 2  gas, as indicated by the significantly greater population reductions realized in the package  110  design compared to the package  10  design at the same ClO 2  dose level (See for example, Table 2 of  FIG. 6 ) at most storage times. Releasing the antimicrobial gas from both sides as in the package  110  design reduced  E. coli  O157:H7 populations on the lettuce more effectively than releasing the gas from only one side in the package  10  design, which may be due to the shorter distance that the ClO 2  gas has to travel to reach the target surfaces. In addition to the traveling distance for ClO 2  gas, another factor affecting gas distribution could be the absorption of ClO 2  by the lettuce. Though no data exist on the ClO 2  absorption behavior of lettuce, in the bagged lettuce systems studied here, the ClO 2  gas is being constantly released and could react and be absorbed by the lettuce, especially by pieces located adjacent to the reservoir. As a result, less ClO 2  gas would reach the lettuce located in the areas further away from the reservoir. 
     Differences in log 10  reductions of  E. coli  O157:H7 achieved by the package  110  designs compared to the package  10  designs were more pronounced at the 4 mg ClO 2 /d dose. At day  1 , these differences were 0.12 and 0.07 log 10  CFU/g at dose levels of 4 and 8 mg ClO 2 /d, respectively, as shown in Table 2 of  FIG. 6 . This finding suggests that there was interaction between design and ClO 2  level (design*concentration), as supported by statistical outcomes (p=0.042). 
     A comparison of log 10  CFU/g reductions between the package  10 /8 mg and the package  110 /4 mg treatments at 1 and 4 days of storage revealed no significant difference in population reductions, even though only half the amount of ClO 2  gas is released with the 4 mg/d dose. Furthermore, at 7 days of storage, a greater log 10  CFU/g reduction was achieved with the lower amount of ClO 2  gas in the package  110  than with the package  10  design (with 8 mg ClO2/d). 
     The package design had a significant impact on improving the antimicrobial efficacy of ClO2 gas. Therefore, by reducing the traveling distance of the gas to its target surfaces, it should be possible to reduce the dose of ClO 2  and still deliver the equivalent log 10  CFU/g reduction of  E. coli  O157:H7. Utilization of lower ClO 2  doses could also result in less appearance and texture changes to fresh food products (Ellis, et al., 2006; Gomez-Lopez, et al., 2009). 
     In addition to browning, which normally occurs at cut and bruised areas of fresh produce (Cantwell, Rovelo, Nie, &amp; Rubatzky, 1998; Lopez-Galvez, Saltveit, &amp; Cantwell, 1996) the lettuce samples exposed to ClO 2  gas also developed softer, watery and/or bleached tissues. Other researchers have observed similar changes in produce (Du, Fu, Li, &amp; Xia, 2007; Han, et al., 2000). These changes were more obvious in the lettuce pieces located in areas adjacent to the reservoir sections. Lettuce samples in package  10  designs with the 8 mg ClO 2 /d dose showed more changes in appearance than lettuce obtained from package  110  designs with the 4 mg ClO 2 /d dose, and these changes became more pronounced at day  4  and day  7 . 
     Sampling from specific locations generally parallel to the reservoir(s) within the bags was carried out to further evaluate how proximity of the lettuce to the antimicrobial gas source impacts the product throughout the package, as determined by log 10  CFU/g reductions of  E. coli  O157:H7. 
     Regardless of the level of ClO 2  gas used, the reductions of  E. coli  O157:H7 on the lettuce sampled from locations adjacent to the reservoir were significantly greater than on those samples collected further away from the reservoirs, as shown in Table 3 of  FIG. 7 . For package  10 , reductions of  E. coli  O157:H7 on the samples taken from the middle area (location  2 ) and the area most distant from the reservoir (location  3 ) were statistically equal. For package  10 , location  1  is the area adjacent to the reservoir. For package  110  location  1  is the area adjacent to the reservoirs and location  2  is the middle area of the package. For package  210 , location  1  is the middle area closest to the reservoir tube and location  2  is the side areas most distant from the middle area. These trends were observed at all storage times. These results confirmed that ClO 2  gas was most effective in the areas close to the reservoir. Furthermore, the softer and watery cut leaves could partially block gas flow to other areas of the bag, and further reduce accessibility to the target surfaces, resulting in low kills in the areas distant from the gas release (Du, et al., 2002; Ellis, et al., 2006). 
     Study  2 : Efficacy of ClO 2  Gas in Package  210  Designs (Aka “Mid-GR” Bags) 
     The LDPE tube placed in the center of the package  210  bag has a very low barrier to ClO 2  gas. The permeability of the LDPE polymer allows the gas to be readily released and available for decontamination within the package chamber. For this study, ClO 2  gas was released through the entire surface of the LDPE tube, whereas in the previous study the ClO 2  gas was mainly delivered through the six openings located along the interior seals. 
     Reductions of the average populations of  E. coli  O157:H7 recovered from the package  210  bagged lettuce after storage are shown in Table 4 of  FIG. 8 . With this design, increasing the ClO 2  gas in the package headspace significantly increased the log 10  CFU/g reductions of  E. coli  O157:H7. This dose effect with ClO 2  gas has been reported previously in green peppers and lettuce (Han, et al., 2000; Lee, Costello, &amp; Kang, 2004). As demonstrated in study  1 , reductions in  E. coli  O157:H7 populations on cut lettuce sampled from location  1  (adjacent to the reservoir) were significantly greater than from the other locations, as shown in Table 5 of  FIG. 9 . Changes in appearance, including bleached and watery tissues, were more noticeable from lettuce in the bags with the higher ClO2 dose, and these changes were most apparent in the lettuce pieces next to the reservoir. 
     Comparison of 1-GR, 2-GR and Mid-GR Bag Designs 
     At the gas dose of 4 mg ClO 2 /d the package  110  and  210  designs provided similar levels of microbial inactivation, even though the gas release areas of the two designs were not equal. This finding could be attributed to the reactive nature of the ClO 2  gas. On the other hand, at the gas dose of 8 mg ClO 2 /d, comparisons of log 10  CFU/g reductions between lettuce samples showed that the efficacy of the gas was greater in the package  210  than in the package  110 , likely as a result of improved gas distribution.  E. coli  O157:H7 population reductions were greater in package  110  than in package  210  by 0.61, 0.37, and 0.27 log 10  CFU/g on day  1 ,  4 , and  7 , respectively. However, at this ClO 2  dose the appearance of the lettuce was compromised. 
     Increasing the gas release area from six small openings to the entire surface of a LDPE tube was likely the main factor in improving gas distribution in the package  210  design compared to the package  110  design. Also, a center position for the GR may have contributed to better gas distribution compared to the side positions in package  110  design. Given that the package  110  design showed comparable average log 10  CFU/g reductions at both gas doses (Table 2), it was likely that soft and watery lettuce pieces, adjacent to the reservoir openings, partly blocked the flow of ClO 2  gas and affected gas distribution, especially in the 8 mg ClO 2 /d treatment. This problem could lessen when the gas was distributed through the entire tube surface of package  210 . 
     At the testing conditions evaluated, utilization of ClO 2  as an antimicrobial gas in the packages provided only up to 1.24±0.09 log 10  CFU/g reductions of  E. coli  O157:H7 (package  210  with 8 mg ClO 2 /d, see Table 4). Although the average reductions represent the average microbial inactivation for a particular bag design and ClO 2  level, a closer examination of the inactivation at specific locations in the bags indicated that up to 2 log 10  CFU/g reductions could be achieved at the areas adjacent to the gas release areas. 
     Thus, it is believed that the interior characteristics of the flexible bag, i.e., the location and size of the gas releasing area, at least partially influenced the antimicrobial effects of ClO 2  on  E. coli  O157:H7 inoculated on Romaine lettuce. By minimizing the travel distance between the ClO 2  gas and the target surface, the ClO 2  gas was more effective with the package  110  and package  210  designs than with the package  10  design. Maximizing the gas releasing surface area also resulted in increased reductions of the pathogen populations. These findings are of significant interest, since antimicrobial gases are typically introduced in packaging either by modifying the headspace or using sachets. 
     The present study also suggested that package design optimization can result in applying a smaller dose of ClO 2  gas to achieve the same level of reduction of  E. coli  O157:H7 populations. The delivery of ClO 2  gas within a packaging system could also be adjusted and regulated to suit specific product requirements by (1) selecting a polymer film with a different permeability, or (2) by changing the location or number of openings along the seal of a reservoir. 
     Although exemplary aspects and embodiments of the invention have been described with a full set of features, it is to be understood that the disclosed systems and methods of use and manufacture may be practiced successfully without the incorporation of each of those features. The foregoing described embodiments of the invention and accompanying materials are provided as illustrations and descriptions. They are not intended to limit the invention to the precise forms described herein. 
     In particular, it is contemplated that a functional implementation of the invention described herein and in the accompanying materials may be constructed of varying materials and different packaging arrangements. For example, it should be readily apparent that a package and agent releasing body of the invention may be formed in a variety of sizes and shapes, with additional reservoirs placed in various locations, such as the corners of the package, or along the periphery of a side thereof. More than one modified atmosphere storage space may also be used in a package of the invention, such as a package design with two modified atmosphere storage spaces separated by a central reservoir space. Packages may be configured to hold a variety of products therein, such as surgical tools, medicines, or other items which would benefit advantageously from storage in a modified atmosphere space as described herein. Thus, variations and additional embodiments are possible in light of above teachings, and it is not intended that this description should limit the scope of invention. It is to be understood that modifications and variations may be utilized without departure from the spirit and scope of the invention and method disclosed herein, as those skilled in the art will readily understand, and such modifications and variations are therefore considered to be within the purview and scope of the invention as set for the by the claims and equivalents thereto.